Accepted Manuscript

lncRNA Gm10451 regulates PTIP to facilitate iPSCs-derived β-like cell differentiationby targeting miR-338-3p as a ceRNA

Yan Huang, Yang Xu, Yuhua Lu, Shajun Zhu, Yibing Guo, Cheng Sun, Lianchen Xu,Xiaolan Chen, Yahong Zhao, Bin Yu, Yumin Yang, Zhiwei Wang

PII: S0142-9612(19)30365-5

DOI: https://doi.org/10.1016/j.biomaterials.2019.119266

Article Number: 119266

Reference: JBMT 119266

To appear in: Biomaterials

Received Date: 28 January 2019

Revised Date: 3 June 2019

Accepted Date: 8 June 2019

Please cite this article as: Huang Y, Xu Y, Lu Y, Zhu S, Guo Y, Sun C, Xu L, Chen X, Zhao Y,Yu B, Yang Y, Wang Z, lncRNA Gm10451 regulates PTIP to facilitate iPSCs-derived β-like celldifferentiation by targeting miR-338-3p as a ceRNA, Biomaterials (2019), doi: https://doi.org/10.1016/j.biomaterials.2019.119266.

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lncRNA Gm10451 regulates PTIP to facilitate

iPSCs-derived β-like cell differentiation by targeting

miR-338-3p as a ceRNA

Yan Huang1,2#, Yang Xu2#, Yuhua Lu1,2*, Shajun Zhu1, Yibing Guo2,

Cheng Sun3, Lianchen Xu2, Xiaolan Chen4, Yahong Zhao3, Bin

Yu3,5, Yumin Yang3*, Zhiwei Wang1*

1 Department of Hepatobiliary and Pancreatic Surgery, Affiliated

Hospital of Nantong University, Nantong, 226001, China

2 Research Center of Clinical Medicine, Affiliated Hospital of

Nantong University, Nantong, 226001, China

3 Key Laboratory of Neuroregeneration of Jiangsu and Ministry of

Education, Co-innovation Center of Neuroregeneration, Nantong

University, Nantong 226001, China

4 Department of Nephrology, Affiliated Hospital of Nantong

University, Nantong, 226001, China

5 Jiangsu Clinical Medicine Center of Tissue Engineering and Nerve

Injury Repair, Affiliated Hospital of Nantong University, Nantong

226001, China

# These authors contributed equally to this work.

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* Correspondence and requests for materials should be addressed

to Y.Y. (email: yangym@ntu.edu.cn), Y.L. and Z.W. (email:

wzw3639@163.com).

ABSTRACT

iPSCs-derived insulin-producing cell transplantation is a promising

strategy for diabetes therapy. Although there have been many

protocols of mature, glucose-responsive β cells induced in vitro

over the past few years, many underlying problems remain to be

resolved. As a crucial regulator, long noncoding RNAs (lncRNAs)

participate in numerous biological processes, including the

maintenance of pluripotency, and stem cell differentiation. In this

study, we identified a novel lncRNA Gm10451 as a functional

regulator for β-like cell differentiation. Localized to the cytoplasm,

Gm10451 regulates histone H3K4 methyltransferase complex PTIP

to facilitate Insulin+/Nkx6.1+ β-like cell differentiation by targeting

miR-338-3p as a competing endogenous RNA (ceRNA).

miR-338-3p has also been shown to suppress Nkx6.1+ early-stage

β-like cell differentiation by targeting PTIP. Following

transplantation into streptozotocin (STZ)-mice, Gm10451 loss in

β-like cells prevented the expression of mature β-cell makers, such

as Insulin, Nkx6.1, and Mafa. Accordingly, hyperglycemia in the

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mice was not resolved. Taken together, this study provides an

efficient epigenetic target for generating more mature and

functional iPSCs-derived β-like cells. We anticipate that pancreatic

organoids, which are generated from human stem cells, biological

materials, and epigenetic modifications, can be used in the future

as a novel diabetes treatment option.

Keywords: iPSCs, β-like cells, differentiation, long noncoding

RNAs, competing endogenous RNA

1. INTRODUCTION

Type 1 diabetes (T1D), which is characterized by β cell deficiency

and functional disorder, results from an autoimmune attack

triggered by spontaneous or environmental stressors. In recent

years, extensive efforts have been made to generate functional

insulin-producing cells for substitution therapy to control

hyperglycemia[1-3]. Embryonic stem cells (ESCs) or induced

pluripotent stem cells (iPSCs), with the capacity to differentiate into

all cell types of the body, have been used as starting materials to

produce functional islet cells[2, 3]. However, all technical attempts

have been hampered by limitations in maturation and glucose

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sensitivity[4]. Additionally, a greater understanding of the genetic

and epigenetic control of the differentiation of pancreatic endocrine

cells is required to overcome such limitations. Most studies focused

on pancreatic cell lineage fate have focused on the regulatory roles

of key transcription factors, such as Pdx1, Gata6, Nkx6.1, Sox9[5],

and other protein-coding genes. Noncoding RNAs (ncRNAs),

including short and long noncoding transcripts, have significantly

affected the differentiation, histogenesis, and biological behavior of

cells and tissues[6-8]. Over the last decade, thousands of long

noncoding RNAs (lncRNAs) have been identified, which have been

characterized as transcripts larger than 200 nucleotides.

Functionally, lncRNAs have been shown to participate in numerous

biological events, including genomic imprinting, chromatin

remodeling, X chromosome inactivation, maintenance of

pluripotency, and cancer progression[9-12]. Transcriptomic

analysis of pancreatic islets and purified β cells revealed the

expression of more than 1,100 lncRNAs in both human and mouse

[13]. Recently, several lncRNAs were shown to affect insulin

secretion, cell cycle, and apoptosis in mouse pancreatic β cells,

including lncRNAsTUG1, Gas5, and Meg3[14-16]. Additionally, a β

cell-specific lncRNA PLUTO, which is downregulated in type 2

diabetes, controls a Pdx1-dependent regulatory mechanism in

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humans, suggesting an underlying role in diabetes

etiopathology[17]. Moreover, H19, a well-known long noncoding

RNA, was shown to be crucial for β-cell mass expansion in

neonatal rats[18]. Although several lncRNAs have been

demonstrated to affect pancreatic islet morphogenesis or mature β

cells, the molecular mechanisms underlying pancreatic β-like cell

differentiation have not been comprehensively investigated.

In this study, we used a 3-step protocol to generate pancreatic

β-like cells and utilized RNA sequencing to screen for differentially

expressed lncRNAs during mouse iPSC-derived pancreatic β-like

cell differentiation. Using biochemical and functional experiments,

our studies have identified an important regulatory lncRNA

Gm10451, which acts as a ceRNA of miR-338-3p, facilitated

Insulin+/Nkx6.1+ β-like cell formation, and enhanced insulin

production by upregulating Paxip1 (Pax transactivation

domain-interacting protein 1, also known as PTIP), a nuclear

protein that is a part of the histone H3K4 methyltransferase

complex[19]. Furthermore, interfering with the expression level of

PTIP caused a notable decrease in H3K4me3, a marker of

transcriptional activation[20] that is associated with several

transcription factors of stem cell differentiation and β-cell formation,

including Smyd3, Cnot2, and Nr4a1. Finally, transplantation

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experiments demonstrated that Gm10451-silencedβ-like cells

could not control hyperglycemia in STZ-induced diabetic mice,

suggesting that Gm10451 is a fundamental regulatory factor in

mature insulin-producing cell differentiation.

2. Methods

2.1. Cell Culture and Differentiation

Mouse GFP-iPSCs were obtained from Stem Cell Bank, Chinese

Academy of Sciences[21], cultured on feeders in mESC culture

conditions, and induced to differentiate into pancreatic-like cells

using a 3-step protocol as previously described[3, 22]. Briefly,

embryoid bodies (EBs) were formed from GFP+- iPSCs at step1. At

step 2, EBs were induced to multilineage progenitor cells (MPCs).

At step 3, MPCs were differentiated into β-like cells by using β-cell

selective differentiation medium.

2.2. RNA-sequencing

Total RNA was isolated at four time points during differentiation

using TRIzol (Invitrogen, Carlsbad, CA, USA). The quality of the

isolated RNA samples was determined using an Agilent

Bioanalyzer 2200 (Agilent Technologies, Santa Clara, CA, USA)

following the manufacturer’s protocol. Raw RNA-Seq samples were

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obtained from sequencing Poly-A+ fractions. The sequencing

library of each RNA sample was prepared with an Ion Total

RNA-Seq Kit v2.0 (Life Technologies, USA), following the protocol

recommended by the manufacturer. RNA-Seq was performed by

NovelBio Corp. Laboratory (Shanghai, China) using the Ion

ProtonTM instrument, resulted in an average of ~14 million reads

per sample with an average sequence length of 150 bp.

Raw reads was filtered sequencing adaptors, poly-A, as well as

poor-quality bases by FAST-QC

(http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) in

order to evaluate quality distribution of nucleotides, position specific

sequencing quality, GC content, the proportion of PCR duplication,

kmer frequency etc.

After QC, the clean reads alignment and gene-level read count

estimation were performed using MapSplice and HTSeq against

the mouse reference genome (GRCm38), respectively.

2.3. Series Test of Cluster (STC)

We selected differential expression genes at a logical sequence

according to RVM Random variance model corrective ANOVA.

In accordance with different signal density change tendency of

genes under different situations, we identify a set of unique model

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expression tendencies. The raw expression values were converted

into log2ratio. Using a strategy for clustering short time-series gene

expression data, we defined some unique profiles. The expression

model profiles are related to the actual or the expected number of

genes assigned to each model profile. Significant profiles have

higher probability than expected by Fisher’s exact test and multiple

comparison test[23, 24].

2.4. Differentially Expressed Genes and LncRNAs Analysis

We determined differentially expressed genes and lncRNAs using

EBseq algorithm for identification of transcripts from RNA-Seq data.

False-discovery rate (FDR) was used to determine the significance

threshold of the q-value for multiple tests. A fold change >2 or <0.5

and an FDR <0.05 represented the thresholds signifying genes that

were considered as being differentially expressed.

2.5. lncRNA-mRNA-network

We built lncRNA-mRNA-network to identify the interactions

between gene and lncRNA[25]. lncRNA-mRNA-network were built

according to the normalized signal intensity of specific expression

in gene and lncRNA. For each pair of gene-lncRNA, gene-gene.

We calculate the Pearson correlation and choose the significant

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correlation pairs with which to construct the network[26].

In a network analysis, degree centrality is the simplest and most

important measures of a gene or lncRNA centrality within a network

that determining the relative importance. Degree centrality is

defined as the link numbers one node has to the other[27].

Moreover, to study a variety of properties of networks.

2.6. GO analysis

GO analysis was applied to analyze the main function of the

differential expression genes according to the Gene Ontology

which is the key functional classification of NCBI, which can

organize genes into hierarchical categories and uncover the gene

regulatory network on basis of biological process and molecular

function[28].

Specifically, two-side Fisher’s exact test and test were used to

classifying the GO category, and the false discovery rate (FDR) [29]

was calculated to correct the P-value the smaller the FDR, the

small the error in judging the p-value. The FDR was defined as

, where refers to the number of Fisher’s test

P-values less than test P-values. We computed P-values for the

GOs of all the differential genes. Enrichment provides a measure of

the significance of the function: as the enrichment increases, the

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corresponding function is more specific, which helps us to find

those GOs with more concrete function description in the

experiment. Within the significant category, the enrichment Re was

given by:

where “ ” is the number of flagged genes within the particular

category, “ ” is the total number of genes within the same category,

“ ” is the number of flagged genes in the entire microarray, and

“ ” is the total number of genes in the microarray[30].

2.7. RNA Extraction and Quantitative RT‐PCR Analysis

Total RNA was isolated using a RNeasy Mini Kit (Qiagen).

First-strand cDNA synthesis for lncRNAs and mRNAs was

performed by using the RevertAid First Strand cDNA Synthesis Kit

(Thermo Scientific). miRNA 1st Strand cDNA Synthesis Kits (by

stem-loop) (Vazyme) were used to synthesize the first-stand cDNA

of miRNAs following the manufacturer's instructions. The relative

expression levels of each lncRNA, mRNA, and miRNA were

calculated by the 2 ∆∆Ct method. The expression levels of

miR-337-3p and miR-338-3p analyzed by qRT–PCR were

normalized to control values of U6. The expression levels of

lncRNA Gm10451 and mRNAs analyzed by qRT–PCR were

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normalized to control values of GAPDH. The qRT PCR primer

sequences for miR-337-3p, miR-338-3p,and U6 were designed and

synthesized by RiboBio (Guang Zhou, China). The qRT PCR

primer sequences for lncRNA Gm10451 and mRNAs were

designed and synthesized by GenScript Biotech Corp. (Nanjing,

China) (Supplementary table 1).

2.8. Flow cytometry

All cells were digested and resuspended as single cells. Then the

cells were incubated in Reagent 1 (Fixation) (Beckman Coulter)

and Reagent 2 (Permeabilization) (Beckman Coulter) for 20 min

respectively. Next, the cells were resuspended in PBS with primary

antibody, incubated in the dark for 20 min once and washed. The

cells were analyzed with a BD LSRFortessaTM X-20 (BD

Biosciences) and the results were analyzed using FlowJo (Ashland)

software. All the procedures were performed at room temperature.

The primary antibodies used were: anti-h/b/m Insulin

APC-Conjugated Rat IgG2A, Rat IgG2A Control APC-Conjugated

(R&D System), Alexa Fluor® 647 Mouse anti-Nkx6.1, and Alexa

Fluor® 647 Mouse IgG1k Isotype control (BD Biosciences).

2.9. Glucose-stimulated insulin secretion

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iPSCs-derived β-like cells were transferred into new 24-well plates

for 12h. After pre-incubation in Krebs-Ringer Bicarbonate buffer

(KRB) without glucose for 2h, the cells were stimulated with KRB

containing glucose 0, 5, 15, 30 mM for 2.5h. The supernatants

were then collected. Insulin content and secretion from β-like cells

were assessed by ELISA carried out using an ultrasensitive mouse

insulin assay kit (Mercodia) following the manufacturer’s

instructions.

2.10. Immunofluorescence

Cultured on glass coverslips, iPSC-derived β-like cells were fixed

with 4% paraformaldehyde. After being washed three times with

PBS, these cells were permeabilized with 0.5% (v/v) Triton X 100.

Next, cells were blocked with 5% donkey serum and incubated with

different primary antibodies at 4°C overnight. The cells were then

stained with corresponding fluorescent secondary antibodies for 2

h and DAPI (Solarbio) for 10 min at room temperature. Images

were captured with a Leica TCS SP8 confocal imaging system

(Leica, Germany). The primary antibodies used were: anti-insulin

antibody (Abcam), anti-Pdx1 antibody (Abcam), anti-Sox9 antibody

(Abcam), anti-Nkx6.1 (D804R) rabbit mAb (Cell Signaling

Technology), anti-Mafa antibody (Abcam), and PTIP Rabbit

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Polyclonal antibody (Proteintech). Secondary antibodies included

donkey anti-rabbit (Alexa Fluor® 647, Abcam), donkey anti-rabbit

(Alexa Fluor® 555, Abcam), goat anti-guinea pig (Alexa Fluor® 647,

Abcam), donkey F (ab,)2 anti-goat (Alexa Fluor® 594, Abcam),

donkey anti-goat (Alexa Fluor® 647, Abcam), and goat anti-mouse

(Alexa Flour®555, Abcam) antibodies.

2.11. Western Blotting

Cells were washed with cold PBS three times and lysed on ice for

35 min with RIPA buffer (high) (Solarbio). Protein concentrations

were detected using the BCA Protein Assay (Thermo Fisher

Scientific). Proteins were separated by SDS PAGE, transferred to

PVDF membranes (Millipore, Bedford, MA, USA), and incubated

with primary antibody in Antibody Dilution Buffer (Solarbio) at 4°C

overnight. After 3 washes in TBST, membranes were incubated

with HRP-conjugated secondary antibodies for visualization.

Primary antibodies and HRP-conjugated secondary antibodies

were anti-PTIP antibody (Proteintech), anti-H3K4me3 antibody

(Cell Signaling Technology), anti-beta actin antibody (Abcam), and

goat anti-rabbit HRP antibody (Abcam).

2.12. Cytoplasmic and Nuclear RNA Fractionation

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The nuclear and cytoplasmic fractions of mouse MPCs were

partitioned by PARIS Kit (Life technologies) following the

manufacturer’s instructions. Next, analysis of cytoplasmic and

nuclear RNA was carried out by quantitative RT PCR.

2.13. Fluorescence in situ hybridization (FISH)

In situ hybridization was carried out with a Fluorescent In Situ

Hybridization (FISH) Kit (RiboBio, Guangzhou, China). Cells were

washed with PBS and fixed in 4% paraformaldehyde for 10 minutes.

The cells were then permeabilized with PBS containing 0.5%

Triton-X 100 at 4°C for 5 min, washed with PBS thre e times for

5min, and pre-hybridized at 37°C for 30 min before hybridization.

An anti-lncRNA Gm10451, anti-U6, or anti-18S

oligodeoxynucleotide probe was added into the hybridization

solution at 37°Covernight in the dark. The cells we re incubated with

DAPI for10min. lncRNA Gm10451-cy3 FISH probes were designed

and synthesized by RiboBio Co., Ltd. Mouse U6 FISH probes and

mouse 18S FISH probes were used as the nuclear and cytoplasmic

controls respectively. Images were captured using a Leica TCS

SP8 confocal imaging system (Leica, Germany).

2.14. RACE (rapid-amplification of cDNA ends)

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cDNA for RACE experiments were obtained from MPCs (at day 4 of

step 3, defined as early stage). The primers were designed by

Primer Premier 5.0 and are listed below. The first 5’-NESTED

RACE was performed with lnc151 primer using the following cycle

conditions: 95°C for 5 mins, (95°C for 40 secs, 60°C for 30 secs,

and 72°C for 1 min 30 secs) ×35 cycles, and finally 72°C for 10

mins. The second 5’-NESTED RACE was reformed with lnc152

primer by the following cycles, 95°C for5 mins, (95°C for 40 secs,

60°C for 30 secs, 72°C for 1 min 30 secs) 20×cycles, 72°Cat 10

mins. The 3’-NESTED RACE was repeated using the steps above.

The sequences of primers used are as follows:

3RACEP1 5-CGAAAGCGACAAGGCCGTGATCCCGAAAGCTTT

TTTTTTTTTTTTTTTTTTTTTTTVN-3

3RACEP2: 5-CGAAAGCGACAAGGCCGTGATCCCGAAAGC-3

lnc131: 5-CAGGCTGATGCTGGAATGTTAATG-3

lnc132: 5-GAGGACATGACAAGCAAAATTCC-3

5RACEP1 5-GGCCACGCGTCGACTAGTACC CCCCCCCCCCC

C-3

5RACEP2 5-GGCCACGCGTCGACTAGT-3

lnc151 5-CTTTCTAGTTTAAATAGTTCCAATTAGCTTG-3

lnc152 5-GAAATTCCTGCAAGAGACCGTCAC-3

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2.15. lncRNA Gm10451 lentiviral transduction

Lentivirus production were done by BrianVTA (Wuhan, China)

lentivirus titer was determined by BrianVTA (Wuhan, China) too.

iPSC-derived β-like cells at day 4 of step 3 were seeded in 6-well

culture dishes, incubated overnight and allowed to reach 40% to 50%

confluence, and then transiently infected with 50 MOI

Lentivirus-CMV-LncRNA Gm10451 or 50 MOI

Lentivirus-CMV-Negative Control. After72 h transduction, the

expression of lncRNA Gm10451 was examined using qRT-PCR

analysis.

2.16. CHIP immunoblotting assay

CHIP assay was carried out using an EZ-Magna CHIP assay kit

(Merck Millipore, Darmstadt, Germany) following the

manufacturer’s instructions. In brief, iPSC-derived β-like cells were

cross-linked with 1% formaldehyde, collected, lysed, and sonicated

to shear DNA. The DNA-protein complexes were then isolated with

a Tri-Methyl Histone H3 (Lys4) antibody (Cell Signaling

Technology), or an isotype control IgG antibody (Santa Cruz

Biotechnology, SantaCruz, CA).

2.17. Dual‐luciferase Reporter Assay

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A luciferase reporter assay was carried out to assess the

interactions between lncRNA Gm10451 and

miR-337-3p/miR-338-3p, and between PTIP and

miR-337-3p/miR-338-3p. Wild-type lncRNA Gm10451

(WT-3’UTR-lncRNA Gm10451), mutant lncRNA Gm10451

(MUT-3’UTR-lncRNA Gm10451), wild-type PTIP (WT-3’UTR-PTIP),

and mutant PTIP (MUT-3’UTR-PTIP) were cloned into the reporter

vector for miR-337-3p/miR-338-3p targeting. WT-3’UTR or

MUT-3’UTR vectors were co-transfected with

miR-337-3p/miR-338-3p mimic or miRNA mimic control. Firefly and

Renilla luciferase activities were assayed with a Dual Luciferase

Assay (Promega, Madison, USA) at 2 days post-transfection

following the manufacturer’s instructions.

2.18. Pancreas harvest and decellularization

All the animal experiments were performed in accordance with the

Institutional Animal Care guidelines and were approved by the

Laboratory Animal Center of Nantong University. C57BL/6J mice

weighing 25–30g obtained from the animal center of Nantong

University and used for decellularized pancreatic scaffolds. Harvest

and decellularization of pancreas of C57BL/6J mice were

performed as previously described[31].

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2.19. In Vivo Implantation of Decellularized Pancreas

The sterile scaffolds were sectioned into 1×1mm2 sections. All the

groups of β-like cells at day 21 were digested and resuspended.

The β-like cells were co-cultured with sections (1×106cells/1section)

for 2 days on a 6-well plate. The sections (n=7) were then

implanted into dorsal subcutaneous tissue of anesthetized mice. All

of the operations were performed under sterile conditions and mice

were sterilized using iodophor. All the mice were carefully

monitored after the procedure.

2.20. Statistical Analysis

The statistical significance was analyzed by Student’s t test using

GraphPad Prism 7.0 (GraphPad Software, Inc.). All the error bars

represent the mean ± standard error of mean (s.e.m.). P-value <

0.05 was considered statistically significant.

3. RESULTS

3.1. Differentially Expressed lncRNAs Involved in iPSC-derived

β-like Cell differentiation

Using a 3 steps protocol, mouse GFP+-iPSCs were differentiated

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into β-like cells (Fig.1A). Approximately 31.63 ± 1.52% (n=3) of late

stage cells co-expressed Insulin and Nkx6.1, both were the

markers of mature β cells (Fig.1B). Comparing with the adult

mouse islets, although β-like cells express insulin and mature

beta-cell markers, such as Pdx1, Nkx6.1, Mafa, ISL, and Glut2, but

there is still an obvious gap (Fig.1C). Like adult mouse islets, β-like

cells can secrete insulin in response to different concentrations of

glucose (Fig.1D).

To identify the regulatory networks of lncRNAs and mRNAs during

iPSC-derived β-like cell differentiation in vitro, we performed

RNA-seq and collected differentiated cells at sequential time points,

including primitive iPSCs and early-stage iPSC-derived pancreas

β-like cells (at day 4 of step 3), middle-stage cells (at day 14 of step

3), and late-stage cells (at day 21 of step 3) (Fig.1A). We selected

the differentially expressed lncRNAs and mRNAs compared to

iPSCs at early stage, middle stage, and late stage in step 3,

respectively. In all, 302 lncRNAs and 2233 mRNAs were identified.

Hierarchical clustering was used to classify lncRNAs with

differential expression>2-fold (FDR<0.05) (Fig.1E). Interestingly,

the majority of differentially expressed lncRNAs and mRNAs were

downregulated (Supplementary Data1). These differentially

expressed lncRNAs, whether down or up-regulation, suggesting

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potential involvement in maintaining the pluripotency and

self-renewal capacity of iPSCs. To analyze and classify the

differentially expressed lncRNAs, Series Test of Cluster (STC) was

used to exhibit the corresponding changes of these underlying

functional lncRNAs, which acted as key regulators ofβ-like cell

differentiation. In total, 26 profiles were generated, and each profile

represented a cluster of multiple lncRNAs with coincident

expression patterns (data not shown). Among the 26 patterns,

6expression patterns, namely, profiles 1, 2, 4, 5, 22, and 23,

showed statistical significance (P<0.05) (Fig.1F).

Valuating the mRNA level of lncRNA in 6 profiles by qPCR (data not

shown), we selected profile 22 for further studies as it contained a

significant number (28 transcripts) of differentially expressed

lncRNA. The suitable number of lncRNAs and disciplinary

expression pattern make it better to further research. To

preliminarily evaluate the possible functions of these lncRNAs

contained in this profile, co-expression networks between the

lncRNAs and mRNAs were established (Supplementary Fig.1). We

found that 22 upregulated lncRNAs and 521 mRNAs included in the

co-expression networks of profile 22 appeared to be directly

correlated. Gene Ontology (GO) analyses of the co-expressed

genes of differentially expressed lncRNAs were used to predict the

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biological processes and molecular mechanisms of potentially

important lncRNAs. GO analyses revealed that the top biological

processes affected by the co-expressed genes of the enriched

lncRNAs included transcriptional regulation, cell proliferation,

migration, and apoptosis (Fig.1G).

3.2. Knockdown of lncRNA Gm10451 suppresses iPSC-derived

β-like cell differentiation

The results from RNA sequencing and bioinformatic analysis

indicated that differentially expressed lncRNAs in profile 22 may

play an important role in regulating the differentiation of mouse

iPSC-derived β-like cells. Therefore, we performed qRT-PCR to

validate the expression of these lncRNAs. As shown in the

histograms in Figure 2, lncRNAs Gm10451 shown obvious an

up-regulation expression in early and middle stage of differentiation,

which was selected for further functional studies (Fig.2A). To

determine if Gm10451 is a key regulator for β-like cell

differentiation, we generated Gm10451-targeting small interfering

RNA (siRNA) constructs. Quantitative RT-PCR analysis validated

that the siRNAs achieved approximately 75% knockdown of

Gm10451 in early-stage cells (Fig.2B). During cell cultured, we

transfected cells with siRNA every 7 days to insure the Gm10451

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expression at a relatively low level and detected the expression

level by qRT-PCR (Fig.2B). All essential transcription factors (TFs)

of pancreatic endocrine cells, such as Pdx1, Nkx6.1, Ngn3, Sox9,

Pax4/6, and Gata4/6, were downregulated in Gm10451-silenced

cells (Fig.2C). By immunofluorescent staining, we observed that

there were reduced in Pdx1, Nkx6.1, and Sox9 positive cells at

early-stage differentiation (day 6 of step 3) (Fig.2D). This data

suggested thatGm10451may be important in early differentiation of

β-like cells by regulating the expression of key TFs involved in

pancreas endocrine cell formation.

Then, Gm10451-silenced cells were cultured for 15 days, using

selective differentiation medium and collected for further functional

experiments. The interference of Gm10451led to a decrease in the

population of Insulin+/Nkx6.1+ β-like cells, from 37.2 ± 1.21% to

18.6 ± 1.51% (n=3) (Fig.2E). Immunofluorescence staining also

demonstrated a similar phenotype, where the NC cells were

Insulin+ and co-expressed higher levels of Nkx6.1 and Mafa than

the siRNA group (Fig.2F). We noted a statistically significant drop in

the mRNA expression of functional markers of pancreatic islets,

including Insulin, Glut2, ISL, GCG, and SST. The biomarkers of

mature β cells, such as Pdx1, Mafa, and Nkx6.1were also

downregulated at the transcriptional level in Gm10451-silenced

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cells in comparison to control (NC) cells(Fig.2G).ELISA showed

that exposure to different concentrations of glucose resulted in a

notable difference in the secretion of insulin from control and

Gm10451-silenced cells. In that, Gm10451-silenced cells showed

hyposensitivity to glucose and produced and secreted less insulin

(Fig.2H).

3.3. Identification and characterization of novel lncRNA

Gm10451

Mus musculus predicted gene 10451 (Gm10451) is a long

non-coding RNA. To date, there have been no reports of Gm10451

function in the literature. Our RNA-seq data showed that lncRNA

Gm10451 is polyadenylated. Meanwhile, our data could be viewed

by IGV here. Gm10451.gtf was the validated sequence modified

Sanger sequencing, while the ref_GRCm38.p2_top_level.gtf was

the current sequence recorded in the NCBI database. The Mapped

reads was displayed (Supplementary Fig. 2A).To viewing the

genomic and epigenomic landscape of Gm10451, the UCSC

custom track mode was used and Jaspar database which indicated

the TF binding region together with the 10ways species conserved

region track was showed on the supplementary figure

(Supplementary Fig.2B).The figure revealed that the certain region

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of Gm10451 in mouse genome was conserved to human and rat

genome including not only the exon region, but promoter region as

well according to the 10 ways data. Several promoters such as

TBX21, ELF and et.al could regulate the Gm10451 promoter region

by the Jaspar promoter binding data (Supplementary Fig. 2B).

Additionally, cell fractionation analysis indicated that Gm10451 is

mostly localized to the cytoplasm (Fig.3A). The results of

fluorescence in situ hybridization (FISH) were also in agreement

with the cell fractionation data (Fig.3B). The full length of Gm10451

(1246 nt) was amplified from 5’ and 3’ ends using rapid

amplification of cDNA ends (RACE) (Fig.3C). We then used

qRT-PCR to quantify the exact copy numbers of Gm10451. We

formulated standard curves with limit dilution approaches using

Gm10451 expressing vector pMD@19-T as standard templates,

and then the exact copy numbers of Gm10451 per cell were

calculated according to cell counts and molecular weights

(Supplementary Fig.2C).As a result, we found that the expression

level of Gm10451 was approximately 50 copies per cell

(Supplementary Fig.2D). We then found that Gm10451was

enriched in the pancreas and lungs of new born mice, suggesting a

potential functional role in pancreatic development (Fig.3D).

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3.4. lncRNA Gm10451 promotes Insulin+/Nkx6.1+ β-like cells

differentiation in vitro

To explore the effect of Gm10451 on pancreas β-like cell

differentiation, a lentivirus was used to upregulate

Gm10451inearly-stage (day 4 of step 3) MPCs (Supplementary

Fig.3). Seventy-two hours after transduction, there was a >3-fold

upregulation in the gene expression level of Gm10451 in the

Gm10451-overexpressing cells in comparison to NC cells (Fig. 4A).

The different MOI (multiplicity of infection) have been tested, such

as 2.5 ,5 ,10 ,15 and 20, showing an optimum MOI at 20 (data not

shown). Additionally, the population of Nkx6.1+ cells, which

represented endocrine progenitors that can differentiate into the

β-cell lineage[32], increased from 27.83 ± 0.76% to 36.03 ± 1.05%

(n=3) (Fig.4B). Furthermore, the key TFs of pancreas β-cell

differentiation were significantly upregulated in comparison to the

NC group (Fig.4C). We then compared the expression of key

markers of pancreatic islets in Gm10451-overexpressing and

control cells. We found that there was a significant difference in the

transcriptional levels of Insulin, Glut2, ISL, Pdx1, and Mafa

between the two groups (Fig.4D). As is shown,

Gm10451-overexpression promoted β-like cells to secrete insulin

rapidly in response to a low concentration of glucose (5 mM) (Fig.

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4E). Insulin secretion then plateaued at glucose concentrations of

15–30 mM (Fig.4E). Additionally, the population of Insulin+/Nkx6.1+

β-like cells showed a significant increase from 31.3 ±1.19% to

49.83 ± 2.02% (n=3), further suggesting that Gm10451 can act as a

modulator of mature insulin-secreting cell differentiation (Fig.4F).

3.5. miR-338-3p regulates PTIP expression and inhibits

early-stage β-like cell differentiation

Based on the cytoplasm localization of Gm10451, we used

bioinformatics prediction analysis to predict whether Gm10451

could act as a ceRNA to regulate pancreatic β-like cell

differentiation. The ceRNA network showed that Gm10451harbors

many miRNAs targeting sites, including miR-338-3p, miR-337-3p,

and miR-130b-5p. Meanwhile, paxip1 (PAX interacting protein 1,

also known as PTIP) may be a downstream target of all miRNAs

(Supplementary Fig.4).

PTIP protein is an omnipresent nuclear-localized chromatin

regulator. It has been reported to participate in several biological

processes, such as maintaining pluripotency of stem cells, and

controlling the development and activation of B cell subsets. Loss

of PTIP is also embryonically lethal at day 9.5[33]. To determine

which miRNA may be involved in regulating PTIP, we utilized

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immunoblotting to detect PTIP levels. The results showed that

miR-338-3p significantly downregulated the level of PTIP (Fig.5A).

To explore the mechanism, putative miR-338-3p binding sites in

PTIP were analyzed and a luciferase assay system was performed

to verify whether miR-338-3p could suppress the translation of

PTIP (Fig.5B). The luciferase reporter assay showed that the

wild-type 3’-UTR of PTIP was only translated at a low level in the

presence of miR-338-3p (Fig.5C). Meanwhile, the mutated 3’-UTR

did not show a statistically significant response to miR-338-3p

(Fig.5C). In summary, our data suggest that PTIP is a specific

target of miR-338-3p.

It has been observed that blockade of miR-338-3p can facilitate the

proliferation of β-cells and impede apoptosis in vitro[34]. We

attempted to ascertain the function of miR-338-3p in the early stage

of β-like cell differentiation by overexpressing it on day 4 after the

induction of differentiation. The results showed that there was a

significant decrease in the population of Nkx6.1+ cells from 31.06 ±

1.00% to 23.13 ± 1.03% (n=3) (Fig.5D). In addition to PTIP, several

key transcription factors of pancreatic endocrine progenitors, such

as Pdx1, Nkx6.1, Ngn3, Pax6, and Gata6, were notably

downregulated (Fig.5E). Immunofluorescence staining also

demonstrated that miR-338-3p overexpression reduced the PTIP,

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Nkx6.1, and Ngn3 positive cells in the early stage of differentiation

(Fig.5F). These results suggest that miR-338-3p can inhibit

pancreatic endocrine progenitor differentiation and regulate PTIP

expression. To confirm the relationship between miR-338-3p with

Gm10451, we transfected Gm10451 siRNA into MPCs and then

suppressed the miR-338-3p expression. The population of Nkx6.1+

cells of early stage differentiation is 7.14 ±1.03% (n=3) after

Gm10451 knockdown. Meanwhile, miR-338-3p inhibition increased

the ratio of Nkx6.1+ cells to 14.8 ± 0.8% (n=3). As data shown,

comparing with negative control (21.6 ± 1.51%, n=3), miR-338-3p

knockdown partly reversed the effects of Gm10451

under-expression on impairing differentiation (Figure.5G).

3.6. Gm10451 directly binds to miR-338-3p and regulates β-like

cell differentiation through a miR-338-3p/PTIP axis

To confirm the interaction between Gm10451and miR-338-3p, we

compared the sequences of Gm10451 with miR-338-3p using the

bioinformatics prediction website TargetScan Mouse and noticed

that Gm10451 contained binding sites for miR-338-3p from

484-495 (Fig.6A). We then constructed a luciferase vector of

Gm10451 (Luc-Gm10451-wt) and a mutated form

(Luc-Gm10451-mut) (Fig.6A). As the luciferase assay showed,

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miR-338-3p could suppress the luciferase activity of Gm10451, but

it had less effect on mutated Gm10451, which suggested that

Gm10451 can interact with miR-338-3p using the predicted binding

site (Fig.6B).

On this basis, we then investigated whether Gm10451 could

regulate PTIP expression, which had been confirmed as a

downstream target of miR-338-3p. Knockdown of

Gm10451resulted in the downregulation of PTIP (Fig.6C).

Conversely, the overexpression of Gm10451resulted in the

upregulation of PTIP (Fig.6D). Therefore, Gm10451 neutralized the

inhibitory effect of miR-338-3p on PTIP expression (Fig.6E). Taken

together, the data suggest that Gm10451 can act as a ceRNA of

miR-338-3p to regulate PTIP expression.

We then determined if PTIP is a functional target of Gm10451 in

β-like cell differentiation. Rescue experiments indicated that

overexpressed Gm10451 can reverse the inhibitory effect of

PTIP-siRNA in Insulin+ cells (Fig.6F). The qRT-PCR results showed

that overexpressed Gm10451 also rescued the transcriptional

levels of vital genes in PTIP-knockdown cells (Fig.6G), suggesting

that Gm10451 can regulate iPSC-derived β-like cell differentiation

through a miR-338-3p/PTIP axis. For further testify the conclusion,

we constructed a lentivirus contain the mutational binding sites of

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Gm10451. Compared with the negative control, the population of

Insulin+ cells decreased after PTIP interference and showed no

change after transfecting Gm10451 binding sites mutated lentivirus

(Supplementary Figure.5).

3.7. Reduced genome-wide H3K4me3 after PTIP knockdown in

pancreatic endocrine progenitors

To explore the role of PTIP in iPSC-derived β-like cell

differentiation, we silenced PTIP in MPCs by siRNA and this

caused a reduction in the expression of H3K4me3 (Fig.7A). Next,

chromatin immunoprecipitation coupled with Illumina sequencing

(ChIP-Seq) was used to compare genome-wide H3K4me3 profiles

(Supplementary Data 2). The distribution of enrichment changes in

H3K4me3 was displayed using a pie chart and most were localized

within intergenic or intron regions (Fig.7B). A Volcano plot was

generated, which showed that 101 genes were more associated

with the H3K4me3 fraction in the NC group than in the PTIP-siRNA

group (Fig.7C). GO analysis revealed that the differentially

expressed genes were enriched in several biological processes,

including cell biology and organism related processes, which are

important in β-cell differentiation and formation (Fig.7D). Since

PTIP is part of the histone H3K4 methyltransferase complex, a

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chromatin marker of transcriptional activation, deficiencies in PTIP

can lead to a decrease in H3K4me3. Identifying genes with lowered

levels of H3K4me3 will be allow us to evaluate the functional role of

PTIP in β-like cell differentiation in vitro. Several genes were

validated by qRT-PCR, such as Mrpl15, Smyd3, Cnot2, Nrf1, Nr4a1,

and Igf1r, and were shown to have lower levels of H3K4me3

enrichment by CHIP-seq (Fig.7E). These genes have been

reported to be involved in embryonic development, cell death,

glucose-stimulated insulin secretion, and β-cell proliferation[35-41].

Our analysis suggested that PTIP may regulate H3K4me3

modification of these genes and promote Nkx6.1+ early-stage β-like

cell formation and regulate β-like cell differentiation in vitro.

3.8. Gm10451-silenced β-like cells cannot reverse

hyperglycemia in streptozotocin (STZ)-induced diabetic mice

To compare the function of β-like cells from NC and

Gm10451-siRNA groups in diabetic mice, we subcutaneously

transplanted decellularized rat pancreatic scaffolds into the backs

of mice (Fig.8A). The decellularized scaffolds were prepared by

perfusing 10% Triton X-100/0.1% ammonium hydroxide solution

and PBS into native pancreases and cutting them into 1×1cm2

slices (Supplementary Fig.6A-C). The protocol we used has been

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reported in our previous study[41]. Approximately 1×106 β-like cells

from each group were implanted into pieces of scaffolds

respectively, using 6-well plates as containers. Following

transplantation of the scaffolds into STZ-diabetic mice (n=7)

(Supplementary Fig.6D), fasting blood glucose levels were

monitored for 59 days post-transplant (Fig.8B). At 29d

post-transplant, blood glucose levels were returned to baseline by

planted NC β-like cells. However, injection of Gm10451-siRNA

β-like cells could not completely reverse hyperglycemia. Finally,

returning to hyperglycemia was observed in NC β-like cells planted

mice within 5 days of grafts removal (Fig.8B).

The grafts were taken out 59 days after transplantation, and

immunofluorescence staining showed that the NC grafts contained

more Insulin+ cells and showed robust expression of Nkx6.1 and

Mafa in comparison to the Gm10451-siRNA grafts (Fig.8C). Our

results therefore support that Gm10451 regulates PTIP to facilitate

iPSC-derived β-like cell differentiation by targeting miR-338-3p as a

ceRNA in vivo and in vitro (Fig.8D).

4. DISCUSSION

Diabetes affects the health of millions people worldwide and is

becoming a huge burden to the healthcare system[42]. T1D is an

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autoimmune disease, leading to the selective destruction of β-cells

and absolute insulin deficiency. Additionally, recent genetic linkage

studies uncovered that β-cells in patients with T2D are also

suffering a significant decrease[43]. Standard of care treatments

rely on daily insulin injections or the transplantation of cadaveric

islets. However, current strategies are limited by drug tolerance,

donor shortage, and immunosuppressive treatments. Therefore,

the generation and transplantation of β-cells derived from

pluripotent stem cells (PSCs) is considered a promising alternative

for T1D and T2D patients.

Induced pluripotent stem cells (iPSCs), as a virtually unlimited cell

supply, have the capacity to form all of the somatic cells in the body,

including pancreatic β-cells[2, 3]. However, the generation and

production of iPSCs are hampered by several technical and

biological limitations, including the differentiation efficiency, the

amount of insulin released, and the sensitivity to glucose stimuli.

These impediments can result in imperfect differentiation and

immature insulin-producing cells. During cell differentiation, DNA

methylation, post-translational modification of histones, and the

expression of non-coding RNAs are crucial epigenetic events that

orchestrate the transformation of pancreas endocrine progenitor

cells into β-cells in vivo and in vitro[44, 45]. As important regulators,

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lncRNAs have been reported to participate in endoderm

differentiation, muscle differentiation, neural cell differentiation, and

cardiac development[46-49].

Previous studies have focused on the endogenous development of

β cells, and not on the differentiation if induced β-cells. In this study,

we performed a transcriptomic analysis of iPSCs-derived β-like

cells in different stages of differentiation. Akin to what was

observed in human endoderm and pancreatic lineage[50],

hundreds of differential expressed lncRNAs were identified as

potential functional regulators. We identified and characterized

Gm10451 as an effective promoter of Nkx6.1+ β-like cell formation

and Insulin+/Nkx6.1+β-like cell differentiation.

The expression of Nkx6.1 has been reported as necessary for the

development of the β-cell lineage from pancreatic endocrine

progenitors[51]. Our data revealed that Gm10451 can directly

regulate the population of Nkx6.1+ cells and the expression of

several early key TFs, including Pdx1, Nkx6.1, Sox9, and Gata4.

As expected, the quantity of Insulin+/Nkx6.1+cellsbeen regulated by

Gm10451 suppression or overexpression after 21 days culture.

Several studies have demonstrated that PSCs can generate both

polyhormonal and mono-hormonal insulin-expressing cells[1, 2] .

As a distinguishing feature of polyhormonal and mono-hormonal

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cells, Insulin+ cells co-expressing Nkx6.1 can differentiate into

functional β-cells following transplantation in vivo[52]. In vitro

results showing, Gm10451 can facilitate Nkx6.1+ progenitors and

act as an important regulator of Insulin+/Nkx6.1+ β-like cell

differentiation.

Numerous studies have shown that lncRNAs can function as

signals, molecular decoys, scaffolds for protein-protein interactions,

and guides to target elements in cis and in trans[53, 54]. In recent

years, several lncRNAs have been shown to contain miRNAs

binding sites and inhibit miRNA function effectively. Bioinformatics

analysis and dual luciferase reporter assays indicated that

Gm10451, a novel cytoplasmic lncRNA, can function as a ceRNA

to block miR-338-3p and control PTIP protein expression in vitro.

It has also been shown that the downregulation of miR-338-3p and

its host gene AATK can promote β-cell proliferation in vivo[34]. In

our study, we showed that miR-338-3p can also negatively regulate

the differentiation of iPSC-derived Nkx6.1+ progenitors. We also

showed that PTIP (a ubiquitously expressed nuclear protein, which

is a part of the histone H3K4 methyltransferase complex), can

serve as a target of miR-338-3p and knockdown of PTIP led to a

reduction in Insulin+ cell differentiation. In activated B cells, MLL3

(mixed-lineage leukemia 3)-MLL4 complex without PTIP displays

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impaired trimethylation of H3K4 (H3K4me3), leading to defective

immunoglobulin class switching[20]. In our study, CHIP-seq

similarly revealed that knockdown of PTIP led to a genome-wide

decrease in H3K4me3 in pancreatic endocrine progenitor cells.

Less H3K4me3 was evident in genes, such as Mrpl15, Smyd3,

Cnot2, Nrf1, Nr4a1, and Igf1r, which all participate in stem cell

differentiation, and β cell differentiation and function. As reported,

the nuclear receptor subfamily 4, group A, member 1 (Nr4a1) is

necessary and sufficient for Nkx6.1-mediated β-cell

proliferation[35]. Therefore, we speculate that PTIP-mediated

H3K4me3 recruitment may play an important role in lineage

differentiation of β-like cells by regulating Nkx6.1+ progenitor cells

formation.

Following transplantation, the immunofluorescence of grafts

indicatedthatGm10451-siRNA cells feebly expressed Insulin and

two markers of more mature β-cells, Nkx6.1 and Mafa. This implied

that these cells were immature and functionally deficient, which

was consistent with our in vitro results. It is suggests that the

impairment of Gm10451 expression led to defective formation of

mature β-like cells in vitro and in vivo.

As descried by many studies, stem cell differentiation or tissue

regeneration can produce Insulin+ and glucose responsive β cell or

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Nkx6.1+ pancreatic progenitors, both in mouse and human[2, 55].

As the recent seven-step protocol, the Insulin+/Nkx6.1+ cells

generated from human iPSCs at S7 are approximately 40.0%[2]. In

our study, over-expressed lncRNA Gm10451 can increase the

population of Insulin+/Nkx6.1+ cells to 49.83 ± 2.02%, which show a

similar differentiation efficiency with S7 cells. Meanwhile, the ability

of Gm10451 to regulate Nkx6.1+ cells formation during early

differentiation stage also implicit assumption about the important

role of it in epigenetic control.

5. Conclusion

In summary, our research constructs a regulatory network of

lncRNAs in iPSC-derived β-like cell differentiation in vitro and

identifies a novel lncRNA Gm10451 as a powerful promoter

facilitating Insulin+/Nkx6.1+ β-like cell formation. In early stage of

β-like cell differentiation, lncRNA Gm10451 can regulate the PTIP

expression by binding the miR-338-3p and accelerate the Nkx6.1+

pancreatic progenitor differentiation. Both in mouse and human,

expression of Nkx6.1 is essential for development of the β cell

lineage[51]. Meanwhile, Insulin+ cell express Nkx6.1 are glucose

responsive and can give arise to functional β-like cell[56].

Therefore, our data indicate that lncRNA Gm10451 can serve as an

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epigenetic target for improving mature β-like cell differentiation

efficiency and cell function. In addition, as a novel lncRNA

participating in functional β cell formation in vitro, lncRNA Gm10451

suggests that the long non-coding RNA may provide effective

strategies for diabetes cell therapy in future.

Acknowledgments

This work was supported by National Natural Science Foundation

of China (Grant Nos. 31830028, 81471801, 81671823, 81672903,

81701835) and National Key Research and Development Program

of China (Grant Nos. 2017YFA0104701, 2017YFA0701304),

Jiangsu Provincial Key Medical Center and Medical Innovation

Team Program of Jiangsu Province.

We thank LetPub (www.letpub.com) for its linguistic assistance

during the preparation of this manuscript.

We thank Novel Bioinformatics Ltd., Co. (Shanghai China) for the

support of bioinformatics analysis with their Novel Brain Cloud

Analysis Platform (www.novelbrain.com).

We thank Gene Denovo Biotechnology Co. (Guangzhou China) for

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their assistance with our chromatin immunoprecipitation coupled

with Illumina sequencing (ChIP-Seq).

Author Contributions

Y.H., Y.X. and S.Z. carried out all cell differentiation, biological

measurements, and biological assays, and drafted the manuscript.

Y.G., L.X., Y.Z. and B.Y. performed experiments, collected and

analyzed the data and wrote the manuscript. C.S. and X.C. edited

the manuscript. Y.L., Y.Y. and Z.W. designed the study, performed

data analysis, and reviewed the manuscript. All authors approved

the final manuscript.

Data Availability

The raw required to reproduce the findings from this study will be

made available to interested investigators upon request.

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Figure Legends

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Figure 1: Differentially expressed lncRNAs are involved in

iPSC-derived β-like cell differentiation. (A) Flow diagram

exhibiting a 3-step protocol for β-like cell differentiation and the 4

time points at which total RNA was isolated (iPSCs, early stage,

middle stage, late stage; EBs, embryoid bodies; MPs, multilineage

progenitors; IPCs, insulin-producing cells.) (B) Flow cytometry

analyses of Insulin+/NKX6.1+ β-like cells. Data are presented as

mean ± s.e.m. n=3. (C) Gene expression profile ofβ-like cells,

comparing with adult mice islets. Data are presented as mean ±

s.e.m. n=3. *P<0.05, **P<0.01, ***P<0.001, Student’s t test. (D)

Static glucose-stimulated Insulin secretion by β-like cells,

comparing with adult mice islets. Data are presented as mean ±

s.e.m. n=3. **P<0.01, ***P<0.001, Student’s t test. (E) Hierarchical

clustering analysis of lncRNAs that were differentially expressed at

the four time points. Expression levels are represented by red and

green, demonstrating expression above and below the median

expression value of all the time points, respectively. (F) The

expression patterns of differentially expressed lncRNAs were

analyzed, and six profiles with statistical significance (P<0.05) are

shown. Each box represents a model expression profile. The upper

annotation are the mode profile number and p-values. (G) Gene

ontology analysis of genes correlated with lncRNAs in profile 22 by

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bubble charts. The top 20 biological processes are listed.

Figure 2: Knockdown of lncRNA Gm10451 suppresses

iPSC-derived β-like cell differentiation. (A) qPCR analysis of

lncRNA Gm10451 at four time points during differentiation. (B)

qPCR analysis ofGm10451 after cells were transfected with short

interfering RNAs (siRNAs) during early, middle, and late stage. NC

represents a scramble siRNA as a negative control. Data are

presented as mean ± s.e.m. n=3. **P<0.01, ***P<0.001, Student’s t

test. (C) qPCR analysis of key transcription factors of pancreatic

endocrine cells of Gm10451 siRNA cells, comparing with NC cells.

n=3. Data are presented as mean ± s.e.m. n=3. *P<0.05, **P<0.01,

***P<0.001, ****P<1×10-4, Student’s t test. (D) Nkx6.1/Pdx1/Sox9

immunostaining of early stage cells at day 6. Scale bar represents

100µm. Data are calculated by ImageJ and presented as mean ±

s.e.m. n=3. **P<0.01, ***P<0.001, Student’s t test. (E) Flow

cytometry analyses of Insulin+/NKX6.1+Gm10451 siRNA cells,

comparing with NC cells. Data are presented as mean ± s.e.m. n=3.

****P<1 × 10-4, Student’s t test. (F)

Insulin/Mafa/Nkx6.1immunostaining of Gm10451 siRNA cells at

day 21, comparing with NC cells. Scale bar represents 100µm.

Data are calculated by ImageJ and presented as mean ± s.e.m.

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n=3. *P<0.05, ***P<0.001, Student’s t test. (G) qPCR analysis of

pancreatic islet functional genes and key transcription factors of

Gm10451 siRNA cells, comparing with NC cells. Data are

presented as mean ± s.e.m. n=3. *P<0.05, **P<0.01, ***P<0.001,

****P<1×10-4, Student’s t test. (H) Static glucose-stimulated Insulin

secretion byGm10451 siRNA cells, comparing with NC cells. Data

are expressed as concentration. Data are presented as mean ±

s.e.m. n=3. *P<0.05, **P<0.01, Student’s t test.

Figure 3: Identification and characterization of novel lncRNA

Gm10451. (A) The distribution of lncRNA Gm10451 in the

cytoplasm and nucleus of MPCs. (B) Fluorescence in situ

hybridization (FISH) of U6, 18S, and Gm10451 in MPCs. Like 18S,

Gm10451 is expressed in the cytoplasm. Scale bars, 50 µm. (C)

Gm10451 sequence (1264 nt). (D) Distribution of Gm10451 in the

organs of neonatal mice. Data are expressed as the fold change

relative to GAPDH.

Figure 4: lncRNA Gm10451 promotes Insulin+/Nkx6.1+ β-like

cell differentiation in vitro. (A) qPCR analysis of the

overexpression efficiency of Lentiviral-CMV lncRNA Gm10451

(Gm10451 LV-CMV) in comparison to NC. Data are presented as

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mean ± s.e.m. ***P<0.001, Student’s t test. (B) Flow cytometry

analyses ofNKX6.1+Gm10451LV-CMV cells, comparing with NC

cells. Data are presented as mean ± s.e.m. n=3. ***P<0.001,

Student’s t test. (C) qPCR analysis of pancreatic endocrine key

transcription factors of Gm10451 LV-CMV cells at day 6, comparing

with NC cells. Data are presented as mean ± s.e.m. n=3. *P<0.05,

**P<0.01, ***P<0.001, ****P<1×10-4, Student’s t test. (D) qPCR

analysis of pancreatic islet functional genes and key transcription

factors of Gm10451LV-CMV cells, comparing with NC cells. Data

are presented as mean ± s.e.m. n=3. *P<0.05, **P<0.01,

***P<0.001, Student’s t test. (E) Static glucose-stimulated Insulin

secretion byGm10451LV-CMV, comparing with NC LV-CMV cells.

Data are presented as mean ± s.e.m. n=3. **P<0.01, Student’s t

test. (F) Flow cytometry analyses of Insulin+/NKX6.1+Gm10451

LV-CMV cells, comparing with NC cells. Data are presented as

mean ± s.e.m. n=3. ***P<0.001, Student’s t test.

Figure 5: miR-338-3p regulates PTIP expression and inhibits

pancreatic endocrine progenitor cell differentiation. (A)

miR-338-3p suppresses the expression of PTIP. MPCs were

transfected with miR-338-3p-agomir (miR-188-3p) or negative

control-agomir (NC-agomir). The levels of PTIP were analyzed by

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immunoblot. n=3. (B) Putative binding site of miR-338-3p in the

3’UTR region of PTIP. PTIP wild-type (WT) 3’UTR and a mutated

3’UTR of the miR-338-3p-binding site are shown. (C) miR-338-3p

suppresses PTIP translation. HEK293 cells were transfected with

miR-338-3p, NC-agomir, the luciferase constructs of the wild-type

PTIP-3’UTR (PTIP-WT) or a mutated PTIP-3’UTR (PTIP-MUT).

Luciferase activity was analyzed and is shown as mean± s.e.m.

n=3. **P<0.01, Student’s t test. (D) Flow cytometry analyses of

Nkx6.1+ miR-338-3p cells at day 6, comparing with NC-agomir cells.

Data are presented as mean ± s.e.m. n=3. ***P<0.001, Student’s t

test. (E) qPCR analysis of PTIP and pancreatic endocrine key

transcription factors of miR-338-3p agomir cells, comparing wit NC

cells. Data are presented as mean ± s.e.m. n=3. **P<0.01,

****P<1×10-4, Student’s t test. (F) PTIP/Nkx6.1/Ngn3

immunostaining of miR-338-3p agomir cells at day 6, comparing

with NC cells. Scale bar represents 100µm. Data are calculated by

ImageJ and presented as mean ± s.e.m. n=3. **P<0.01, Student’s t

test. (G)Flow cytometry analyses of Nkx6.1+Gm10451 siRNA / NC

antagomir cells and Gm10451 siRNA/miR-338-3p antagomir and

NC siRNA/NC antagomir at day 6. Data are presented as mean ±

s.e.m. n=3. **P<0.01, ***P<0.001, Student’s t test.

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Figure 6: Gm10451 directly binds to miR-338-3p and regulates

β-like cell differentiation through miR-338-3p/PTIP axis. (A)

Gm10451 RNA contains a site complementary to miR-188-3p. The

wildtype and mutated form of the Gm10451 miR-338-3p binding

site (Luc-Gm10451-WT, andLuc-Gm10451-MUT) are shown. (B)

HEK293 cells were transfected with miR-338-3p or NC-agomir,

then transfected with the luciferase constructs of Gm10451-WT or

Gm10451-MUT. Luciferase activity was analyzed and is shown as

mean± s.e.m. n=3. *P<0.05, Student’s t test. (C) Knockdown of

Gm10451 reduced the expression levels of PTIP. PTIP levels were

analyzed by immunoblot. n=3. (D) Overexpression of Gm10451

increased PTIP levels. PTIP levels were analyzed by immunoblot.

n=3. (E) Gm10451 rescued the inhibitory effect of miR-338-3p on

PTIP expression. PTIP expression levels were analyzed by

immunoblot. n=3. (F) Flow cytometry analyses of Insulin+ PTIP

siRNA / NC LV-CMV cells and PTIP siRNA/Gm10451LV-CMV and

NC siRNA/NC LV-CMV at day 6. Data are presented as mean ±

s.e.m. n=3. ***P<0.001, Student’s t test. (G) qPCR analysis of

pancreatic islet functional genes and key transcription factors of all

groups. Data are presented as mean ± s.e.m. n=3. **P<0.01,

***P<0.001, Student’s t test.

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Figure 7: Reduced genome-wide H3K4me3 after PTIP

knockdown in pancreatic endocrine progenitors. (A)

Knockdown of PTIP reduced the expression levels of H3K4me3.

H3K4me3 levels were analyzed by immunoblot. n=3. (B) Pie charts

show the distribution of different peaks in genes between PTIP

siRNA group and NC group (including Gene Up2k, Gene Down2k,

Exon, Intron, Intergenic). (C) Volcano plot of the genes with

different H3K4me3 binding levels. Scatter points represent genes,

the x axis is the log-transformed fold change (logFC) of the genes

with different H3K4me3 binding levels in NC siRNA versus PTIP

siRNA cells, and the y axis represents the possibility that a gene

has statistical significance in its differential H3K4me3 binding levels

(genes with downregulated H3K4me3 binding levels in PTIP siRNA

group are highlighted as green dots; genes involved in stem cell

differentiation and pancreas development and function are marked).

(D) Gene ontology analysis of genes with differential H3K4me3

binding levels by bubble charts. The top 20 biological processes

are listed. (E) qPCR analysis of genes marked in the Volcano plot.

Data are presented as mean ± s.e.m. n=3. *P<0.05, **P<0.01,

***P<0.001, Student’s t test.

Figure 8: Gm10451-silenced β-like cells cannot effectively

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reverse hyperglycemia in streptozotocin (STZ)-induced

diabetic mice. (A) Workflow of the experimental protocol used for

decellularized rat pancreatic scaffold-based iPSC-derived β-like

cell transplantation. (B) Fasting blood glucose levels

post-transplantation. Data are presented as mean ± s.e.m. n=7.

*P<0.05, Student’s t test. (C) Insulin/Nkx6.1/Mafa immunostaining

of grafts of Gm10451 siRNA group, comparing with NC group.

Scale bar represents 100µm. n=7. (D) The hypothetical model

illustrating how Gm10451may regulate PTIP transcription by

targeting miR-338-3p.

Supplementary Figure 1: The mRNA-lncRNA networks in

profile 22. Blue dots are protein-coding RNAs. Red blocks are

lncRNAs.

Supplementary Figure2: The genomic and epigenomic

landscape and copy number of lncRNA Gm10451. (A) Data is

viewed by IGV. (B) The UCSC custom track mode and Jaspar

database are used to indicate the TF binding region together with

the 10ways species conserved region track. (C) The standard

curves with limit dilution approaches using Gm10451 expressing

vector pMD@19-T as standard templates. (D) qPCR analysis of the

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exact copy numbers of Gm10451.Data are presented as mean ±

s.e.m. n=3.

Supplementary Figure3: Structure of Lentivirus-CMVGm10451.

Supplementary Figure 4: Predicted ceRNA network of

Gm10451-miRNA-mRNA. Square colored in red is lncRNA

Gm10451.Triangles colored in green are miRNAs and circles

colored in red are protein-coding RNAs.

Supplementary Figure5: The binding sites to miR-338-3p is

necessary for Gm10451 to reverse the negative influence of

PTIP knockdown. Flow cytometry analyses of Insulin+ cells of

PTIP siRNA / NC LV-CMV mut group and PTIP siRNA/Gm10451

LV-CMV mut group and NC siRNA/NC LV-CMV mut group at day 6.

Data are presented as mean ± s.e.m. n=3. ***P<0.001,

****P<1×10-4, Student’s t test.

Supplementary Figure 6: Preparation of decellularized rat

pancreatic scaffolds and dorsal subcutaneous transplantation.

(A) Harvested rat pancreas and spleen. (B) The decellularized

scaffold pancreas. (C) The decellularized scaffold was cut into

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1×1cm2 slices and cultured with 1×106 cells in 6-well plates. (D)

Scaffolds were then transplanted into STZ-induced diabetic mice

(n=7).

Supplementary data 1: The list of differentially expressed

lncRNAs and mRNAs detected by RNA-seq.

Supplementary data 2: The list of H3K4me3 differentially

enriched genes detected by CHIP-seq.

Supplementary table 1: The list of primers used for reverse

transcription and real-time PCR. The list of sequence for

miR-338-3p agomir/antagomir and Gm10451 siRNA and

PTIP siRNA.

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