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RESEARCH COMMUNICATION Induction of MiR-17-3p and MiR-160a by TNFa and LPS Xin Jiang 1 * and Nancy Li 2 1 Signosis, Inc., Sunnyvale, CA, USA 2 University of Southern California, Los Angeles, CA, USA MicroRNAs (miRNAs), small non-coding molecules, regulate gene expression in response to stimuli. Lipopolysaccharide (LPS) was reported to induce the expression of miR-146 and miR-155 in HL-60. The effects of LPS and the related stimulus, tumour necrosis factor alpha (TNFa), on miRNA expression required to be further studied. Using T7-oligo ligation assay (OLA)-based miRNA array, we profiled the expression of 132 miRNAs and identified a number of TNFa-regulated miRNAs in HeLa cells, including miR-17-3p and miR-106a. TNFa induction of miR-17-3p and miR-106a was verified by Northern blot analysis with RNU48 normalization. Northern blot analysis also showed that LPS was able to induce the expression of both miR-17-3p and miR-106a in HeLa cells. Furthermore, both array assay and Northern blot analysis showed that the expression levels of miR-146 and miR-155 were either low or undetectable in HeLa cells and TNFa- and LPS- mediated induction of these two miRNAs was not found. Luciferase reporter analysis confirmed the induction of miR-17-3p and miR-106a in response to TNFa and LPS treatment in HeLa cells. These results suggested that the expression of miR-17-3p and miR-106a is regulated by TNFa and LPS in HeLa cells. Copyright # 2011 John Wiley & Sons, Ltd. key words — TNFa; LPS; miR-17-3p; miR-106a; HeLa; array; miRNA INTRODUCTION Tumour necrosis factor alpha (TNFa) is a multi-function molecule that can induce cell proliferation and differen- tiation in many types of cells. It is capable of inducing apoptotic or necrotic cell death of certain tumour cell lines. 1,2 The protein is also responsible for the induction of insulin resistance and the development of obesity or diabetes in adipose tissues. 3 TNFa executes its functions by binding to TNF receptor (TNFR) superfamily, mainly TNFR1, the primary receptor for soluble TNFa , and TNFR2, the predominant receptor for membrane-associated TNFa . These receptors activate several intracellular signalling pathways. Two of them are most important, the IkB kinase (IKK) and mitogen-activated protein kinase (MAPK) pathways, which result in the activation of NFkB and AP-1 transcription factors, respectively. 4 The activated transcription factors regulate the expression of target genes by binding to specific DNA-binding elements present in promoters. Like TNFa , lipopolysaccharide (LPS) is also capable of activating IKK and MAPK cascades. 5 The serial biochemical events from initial extra-cellular stimuli, distinct intracellular signalling pathways, activated transcription factors to bound unique promoters lead to expression of target genes. The expression of every single gene is under tight control. MicroRNAs (miRNAs), recently discovered molecules, provide additional controls to the complicated regulation of gene expression. Therefore, studies of the expression and function of miRNAs will help us further understand gene expression and regulation. miRNAs control gene expression at the post-transcrip- tional level through selectively binding to complementary messenger RNA sequences. 6,7 Computational and exper- imental approaches predict that the human genome encodes as many as 1000 miRNAs, regulating 30% of protein- coding genes. 8,9 miRNAs have shown to be critical to varieties of biological functions, including development, cell differentiation, proliferation, apoptosis and maintenance of stemness and imprinting. 10 miRNAs also play roles in major diseases, such as cancer and viral infections. 11,12 Altered expression of miRNAs has been revealed in a large number of cancers. Microarray profiling has revealed differential expression of miRNAs in different tissues and in various human tumours, such as miR-21, miR-155, miR-10b, miR-125b and miR-145 in breast cancers. 13 cell biochemistry and function Cell Biochem Funct 2011; 29: 164–170. Published online 2 February 2011 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cbf.1728 *Correspondence to: X. Jiang, Signosis, Inc., 528 Weddell Drive, #5, Sunnyvale, CA 94089, USA. E-mail: [email protected] Copyright # 2011 John Wiley & Sons, Ltd. Received 14 October 2010 Revised 7 December 2010 Accepted 18 December 2010

Induction of MiR-17-3p and MiR-160a by TNFα and LPS

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Page 1: Induction of MiR-17-3p and MiR-160a by TNFα and LPS

cell biochemistry and function

Cell Biochem Funct 2011; 29: 164–170.

Published online 2 February 2011 in Wiley Online Library

(wileyonlinelibrary.com) DOI: 10.1002/cbf.1728

RESEARCH COMMUNICATION

Induction of MiR-17-3p and MiR-160a by TNFa and LPS

Xin Jiang 1* and Nancy Li 2

1Signosis, Inc., Sunnyvale, CA, USA2University of Southern California, Los Angeles, CA, USA

MicroRNAs (miRNAs), small non-coding molecules, regulate gene expression in response to stimuli. Lipopolysaccharide (LPS) was reportedto induce the expression of miR-146 and miR-155 in HL-60. The effects of LPS and the related stimulus, tumour necrosis factor alpha(TNFa), on miRNA expression required to be further studied. Using T7-oligo ligation assay (OLA)-based miRNA array, we profiled theexpression of 132 miRNAs and identified a number of TNFa-regulated miRNAs in HeLa cells, including miR-17-3p and miR-106a. TNFainduction of miR-17-3p and miR-106a was verified by Northern blot analysis with RNU48 normalization. Northern blot analysis also showedthat LPS was able to induce the expression of both miR-17-3p and miR-106a in HeLa cells. Furthermore, both array assay and Northern blotanalysis showed that the expression levels of miR-146 and miR-155 were either low or undetectable in HeLa cells and TNFa- and LPS-mediated induction of these two miRNAs was not found. Luciferase reporter analysis confirmed the induction of miR-17-3p and miR-106a inresponse to TNFa and LPS treatment in HeLa cells. These results suggested that the expression of miR-17-3p and miR-106a is regulated byTNFa and LPS in HeLa cells. Copyright # 2011 John Wiley & Sons, Ltd.

key words — TNFa; LPS; miR-17-3p; miR-106a; HeLa; array; miRNA

INTRODUCTION

Tumour necrosis factor alpha (TNFa) is a multi-functionmolecule that can induce cell proliferation and differen-tiation in many types of cells. It is capable of inducingapoptotic or necrotic cell death of certain tumour celllines.1,2 The protein is also responsible for the inductionof insulin resistance and the development of obesity ordiabetes in adipose tissues.3 TNFa executes its functions bybinding to TNF receptor (TNFR) superfamily, mainlyTNFR1, the primary receptor for soluble TNFa, and TNFR2,the predominant receptor for membrane-associated TNFa.These receptors activate several intracellular signallingpathways. Two of them are most important, the IkB kinase(IKK) and mitogen-activated protein kinase (MAPK)pathways, which result in the activation of NFkB andAP-1 transcription factors, respectively.4 The activatedtranscription factors regulate the expression of target genesby binding to specific DNA-binding elements present inpromoters. Like TNFa, lipopolysaccharide (LPS) is alsocapable of activating IKK and MAPK cascades.5 The serial

* Correspondence to: X. Jiang, Signosis, Inc., 528 Weddell Drive, #5,Sunnyvale, CA 94089, USA. E-mail: [email protected]

Copyright # 2011 John Wiley & Sons, Ltd.

biochemical events from initial extra-cellular stimuli,distinct intracellular signalling pathways, activatedtranscription factors to bound unique promoters lead toexpression of target genes. The expression of every singlegene is under tight control. MicroRNAs (miRNAs), recentlydiscovered molecules, provide additional controls to thecomplicated regulation of gene expression. Therefore,studies of the expression and function of miRNAs willhelp us further understand gene expression and regulation.

miRNAs control gene expression at the post-transcrip-tional level through selectively binding to complementarymessenger RNA sequences.6,7 Computational and exper-imental approaches predict that the human genome encodesas many as 1000 miRNAs, regulating �30% of protein-coding genes.8,9 miRNAs have shown to be critical tovarieties of biological functions, including development,cell differentiation, proliferation, apoptosis and maintenanceof stemness and imprinting.10 miRNAs also play roles inmajor diseases, such as cancer and viral infections.11,12

Altered expression of miRNAs has been revealed in alarge number of cancers. Microarray profiling has revealeddifferential expression of miRNAs in different tissues andin various human tumours, such as miR-21, miR-155,miR-10b, miR-125b and miR-145 in breast cancers.13

Received 14 October 2010Revised 7 December 2010

Accepted 18 December 2010

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INDUCTION OF mir-17-3p AND mir-160a 165

Identification of differentially expressed miRNAs is criticalto uncovering the biological functions of miRNAs thatattribute to or associate with human diseases.

The expression of miRNAs is recently linked to innateimmune response. Microarray profiling of 200 miRNAs hasshown that miR-146, miR-132 and miRNA-155 are inducedin LPS-treated THP-1 (a human acute monocytic leukaemiacell line).14 The study indicates that LPS induction of miR-146a is also found in human U937, Mono-Mac-6, andmouse WEHI-3, but not in human B-cell lines Ramos,Bjab and Namalwa. IRAK1 and TRAF6 are on the list ofalgorithm-predicted miR-146 targets, which are key mole-cules in LPS-mediated signalling pathways.

To profile the expression of miRNAs, we recentlydeveloped a novel array technology with a high discrimi-nation power in differentiating miRNAs and isoforms.From more than 500þ human miRNAs listed in the miRNAdatabase, we selected 132 miRNAs that have been reportedto be associated with cancers, proliferation, and apoptosis.In the present study, we employed the array to analyse theexpression of these miRNAs in HeLa cells and the regulationof the miRNAs upon the treatment of TNFa. A number ofmiRNAs have been identified to be regulated by TNFa.Two of them, miR-17-3p and miR-106a, were verified withNorthern blot analysis. Induction of miR-17-3p andmiR-106a was also found in LPS-treated HeLa cells. BothTNFa- and LPS-mediated inductions of miR-17-3p andmiR-106a expression were confirmed in vivo with luciferasereporters.

MATERIALS AND METHODS

HeLa cells were cultured in 100 cm culture plates inDulbecco’s Minimal Essential Eagle’s medium (Invitrogen)supplemented with 10% FBS, 1% non-essential minimalamino acids, 100 U/ml penicillin and 0.1 mg/ml streptomy-cin. Cells (about 80% confluent) were treated with 20 ng/mlTNFa and 1 mg/ml LPS for 16 h in serum-free medium,respectively. For transfection, HeLa cells (4� 105) wereseeded on a six-well plate the day before transfection in themedium without antibiotics. The cells were transfected with3 mg of pmiR-106a-Luc and pmiR17-3p-Luc using Fugene 6(Roche) for 16 h, respectively. TNF (20 ng/ml) and 100 ngLPS were added to the cells during the transfection andincubated for 16 h.

miRNA array assays were performed following theprocedure as described in miRNA array kit user manual(Signosis, Inc., Sunnyvale, CA, USA). Five microgramstotal RNA first incubated with an oligo mix. A pair of theoligos hybridized and aligned with a specific miRNA to formDNA/RNA duplex. The duplexes were then isolated fromfree oligos with magnetic streptavidin conjugated beads.The aligned oligos were ligated with T4 DNA ligase, andused as templates to convert into DNA double strands.They were then transcribed into RNA in the presence ofbiotin-UTP with T7 RNA polymerase for linear amplifica-tion and generation of biotin labelled probes. The productswere used as probes for array hybridization. A unique

Copyright # 2011 John Wiley & Sons, Ltd.

tag sequence assigned to each miRNA was introduced inthe oligo sequence, which were characterized with amembrane array spotted with complementary tag sequence.After overnight hybridization, the array was washed twotimes with Hybridization Wash Buffer, each for 20 min.The membrane was blocked with Block Buffer at roomtemperature for 30 min. The biotin-labelled probe was thendetected with Streptavidin-HRP. After washing three timeswith Detection Wash Buffer, the membrane was overlaidwith lumino/enhancer and substrate for 5 min. The imagewas acquired using a FluorChem imager (Alpha InnotechCorp).

Total RNA was prepared with Trizol procedure. Approxi-mately 5 mg of total RNA was loaded onto a 15%polyacrylamide urea denature gel and run in 0.5� TBE at70 V for about 45 min. Molecular markers (20 and 60 ntbiotin-labelled oligonucleotides) were loaded with RNAsamples side-by-side. After transferred in 0.5� TBE ontoa nylon membrane at 70 V for 60 min, RNA samples andthe molecular marker were immobilized on the membranethrough UV crosslinking. The membrane was hybridizedwith biotin labelled miRNA probe at 428C for overnightand washed twice with Hybridization Wash Buffer. Themembrane was then blocked at room temperature for 15 minand detected with streptavidin-HRP. After washing withdetection wash buffer, the membrane was overlaid withlumino/enhancer and substrate for 5 min. The image wasacquired using a FluorChem imager (Alpha Innotech Corp).

RESULTS AND DISCUSSION

Among 500þ human miRNAs listed in the miRNAdatabase, <200 miRNAs were found to be associatedwith cancers, proliferation or apoptosis. From literatures, wecollected 132 miRNAs that belong to the category and usedthem for array development. We took the followingthree aspects into consideration during array developmentas miRNAs are different from large messenger RNAs:(i) miRNAs are small sized molecules with quite a bigdifference in abundance, (ii) mature miRNAs co-exist withtheir precursor pre-miRNA and pri-miRNA, only differingin length and (iii) many miRNAs are very closely related insequences, such as isoforms, differing by only one or afew nucleotides. Therefore, we cannot simply employ theconventional mircoarray technologies. A number of miRNAmicroarray products are commercially available, but someof them either need pre-isolation of the microRNA, or lackthe discriminative power to differentiate between isoforms.

We developed T7-oligo ligation assay (OLA) miRNAarray. In the array assay, each miRNA molecule was targetedby two oligos. As shown in Figure 1, each of the oligoshybridized with a half molecule of the target miRNA to forma RNA/DNA duplex. When the sequences were perfectlymatched, they were hybridized with the miRNA and the jointwas ligated by DNA ligase. A single nucleotide differencebetween miRNA and the oligos will block either thehybridization or the ligation, by which miRNA isoform canbe differentially analysed. Due to the small size of miRNA,

Cell Biochem Funct 2011; 29: 164–170.

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Figure 1. The diagram of miRNA array of miRNA array. Six steps of miRNA array assay were included, the formation of DNA/RNA duplexes, DNA ligationof two aligned oligos, conversion into DNA double strands, T7 transcription, probe hybridization and detection.

166 x. jiang and n. li

half of the size, only 10–12 nucleotides, hybridized witha oligo. In order to stabilize the RNA/DNA duplex, weintroduced stacking sequences to each side of the molecule.The ligated molecules were subjected to linear amplificationvia T7 transcription into RNA in the presence of biotin-UTP,which were used as probes for array hybridization.To differentiate each isoform, we assigned unique tagsequences to the ligation oligos. The tag sequences werethen differentiated by the complementary sequences spottedon a membrane. Because tag sequences were introducedto represent single nucleotide differences, those sequence-closely related miRNAs can be easily distinguished by array

Copyright # 2011 John Wiley & Sons, Ltd.

hybridization. We applied the array to analyse the membersof let7 family and found that the let7 family members can bedifferentially detected by the array (data not shown).

To profile the expression of these 132 miRNAs (Table 1)and examine how many of the miRNAs were regulated byTNFa, we treated HeLa cells with TNFa prior to preparationof the total RNA. Cells without treatment were used ascontrol. Total RNAs from both treated and untreated cellswere subjected to T7-OLA miRNA array assay. The amountof RNA from each sample used for array analysis wasnormalized with a more stable small nucleolar house-keeping RNA, RNU48. As shown in the Figure 2A and

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Table 1. Schematic diagram of miRNA array; 132 miRNAs were collected and spotted on the array

Let-7a Let-7b Let-7c Let-7d Let-7aLe Let-7f Let-7g Let-7i miR-1 miR-7miR-9 miR-10a miR-15a miR-15b miR-16 miR-17-5p miR-18a miR-18b miR-19a miR-19bmiR-20a miR-21 miR-25 miR-28 miR-34a miR-124a miR-34a miR-34a miR-3 4a miR-125bmiR-126 miR-131 miR-133a miR-133b miR-143 miR-145 miR-146a miR-146b miR-148a miR-155miR-181a miR-181b miR-181c miR-182 miR-192 miR-194 miR-195 miR-199a miR-199b miR-199a

miR-200a miR-200c miR-204 miR-206 miR-216 miR-223 miR-204 miR-342 miR-368 miR-375miR-216 miR-223 miR-224 miR-342 miR-368 miR-375 miR-26a miR-26b miR-26a miR-26bmiR-29a miR-29b miR-29c miR-30a-3p miR-30a-5p miR-30b miR-30c miR-92 miR-29b miR-93miR-95 miR-101-1 miR-103 miR-106a miR-106b miR-107 miR-128a miR-128b miR-132 miR-134miR-135b miR-136 miR-137 miR-140 miR-140miR-1 miR-142-3p miR-149 miR-140 miR-135bmiR-1 miR-153miR-154 miR-181d miR-183 miR-185 miR-186 miR-18 miR-190 miR-190miR-1 miR-196a miR-196bmiR-197 miR-198 miR-200b miR-200bmiR-2 miR-203 miR-205 miR-210 miR-214 miR-215 miR-218miR-219 miR-221 miR-222 miR-296 miR-372 miR-373 miR-488 miR-100 miR-127 miR-142-5pmiR-31 miR-213 RNU48

INDUCTION OF mir-17-3p AND mir-160a 167

Table 2, total of 8 miRNAs were found to be up- or down-regulated by TNFa, each with a minimal change of twofolds.Six of them were up-regulated and two down-regulated.Two previously identified miRNAs that are differentiallyexpressed in HL-60, miR-146 and miR-155, showedundetectable in HeLa cells. The result indicates thatexpression of miR-146 and miR-155 is different in HeLaand HL-60.

We chose miR-17-3p and miR-106a for further validationwith Northern blot analysis and in vivo luciferase reporterassay. For Northern blot analysis, total RNAs were preparedfrom both TNFa-treated HeLa and untreated HeLa cellsand subject to electrophoresis on 15% urea denature gel. Todeterminate the size of the detection band, we introducedtwo oligocleotides of 20 and 60 nt pre-labelled with biotinas molecular markers. After separation and transfer, the

Figure 2. miRNA array analysis of RNAs from HeLa and TNFa-treated HeLa celcells and TNFa-treated HeLa cells. miR-17-3p and miR-106a are labelled in the

Copyright # 2011 John Wiley & Sons, Ltd.

expression of miR-17-3p and miR-106a was detectedwith biotin-labelled oligonucleotides complementary tothe targets. Hybridized probes and the molecular markerswere then detected with streptavidin-HRP and a chemilu-minescent substrate. As shown in the Figure 3, the sizes ofboth miR-17-3p and miR-106a were expected to be around20 nt. Their expression levels were low in HeLa cells andTNFa-treatment induced the expression of both miRNAs.To normalize the two RNA samples with and without TNFatreatment, the blot was also detected with RNU48 probe.After normalization, the difference in intensity was observedto be more than twofolds by densitometry analysis.

LPS, similar to TNFa, activates both IKK and MAPKand consequently NFkB and AP1. To examine whether LPScould induce miR-17-3p and miR-106a, we treated HeLacells with 1 mg/ml of LPS for 16 h. Total RNAs were

ls. The array procedure was performed using total RNA from untreated HeLaboxes.

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Table 2. Data analysis of miRNA array analyses of untreated HeLa and TNFa-treated HeLa cells; the hybridization signals on the array were converted intodigits with softwares built in FluoChem image system

Let-7a 2.64 2.325 Let-7b 7.21 3.605 Let-7c 3.005 2.185 Let-7d 2.795 2.265 Let-7e 3.22 2.58miR-9 7.915 8.865 miR-10a 3.31 2.805 miR-15a 3.97 2.365 miR-15b 3.3 2.6 miR-16 36.51 42.675miR-20a 3.445 2.79 miR-21 6.06 7.53 miR-25 3.35 2.41 miR-28 3.465 2.315 miR-34a 3.815 2.6miR-126 3.285 2.75 miR-131 3.51 2.79 miR-133a 3.29 2.46 miR-133b 3.485 2.445 miR-143 3.27 2.435miR-181a 4.49 4.41 miR-181b 3.69 3.925 miR-181c 9.34 11.71 miR-182 7.845 5.855 miR-192 3.61 2.62miR-200a 3.77 3.395 miR-200c 3.76 2.8 miR-204 3.385 2.87 miR-206 4.82 3.455 miR-216 5.045 3.02miR-9-1 95.03 87.89 miR-10b 5.025 4.45 miR-17-3p 8.75 22.13 miR-22 15.59 32.56 miR-23a 8.34 11.53miR-29a 2.93 2.865 miR-29b 3.97 3.185 miR-29c 3.155 2.84 miR-30a-3p 3.535 4.54 miR-30a-5p 20.485 44.365miR-95 2.745 2.75 miR-101-1 3.24 3.315 miR-103 2.755 3.4 miR-106a 8.38 30.245 miR-106b 33.72 50.415miR-135b 5.27 5.185 miR-136 5.775 7.265 miR-137 69.86 84.34 miR-140 3.78 5.3 miR-141 7.55 12.255miR-154 6.585 4.245 miR-181d 9.545 9.44 miR-183 17.345 29.33 miR-185 3.245 3.35 miR-186 4.05 3.525miR-197 64.185 69.8 miR-198 3.055 3.275 miR-200b 10.475 16.255 miR-202 7.71 25.1 miR-203 4.075 3.775miR-219 4.08 3.795 miR-221 4.66 4.655 miR-222 5.025 4.02 miR-296 4.42 5.035 miR-372 4.4 4.25miR-31 10.755 5.84 miR-213 7.925 5.755 RNU48 96.93 85.73Let-7f 3.315 2.505 Let-7g 3.315 2.375 Let-7i 3.13 2.46 miR-1 3.26 2.84 miR-7 8.62 15.295miR-17-5p 13.145 12.42 miR-18a 16.97 6.765 miR-18b 3.315 2.64 miR-19a 6.93 3.835 miR-19b 96.895 84.905miR-99a 34.995 33.545 miR-122a 83.95 75.46 miR-124a 4.815 3.6 miR-125a 16.845 7.74 miR-125b 5.01 4.285miR-145 9.925 3.67 miR-146a 3.36 2.74 miR-146b 3.345 2.81 miR-148a 3.635 2.975 miR-155 4.82 3.64miR-194 3.545 2.8 miR-195 3.345 2.755 miR-199a 66.405 42.26 miR-199b 3.725 3.75 miR-199a

�3.895 3.375

miR-223 4.525 2.865 miR-224 10.52 5.705 miR-342 77.975 69.43 miR-368 3.645 3.895 miR-375 9.55 10.555miR-24 76.07 59.125 miR-26a 33.44 35.55 miR-26b 3.85 3.35 miR-27a 3.935 3.67 miR-27b 3.86 3.29miR-30b 35.305 42.28 miR-30c 62.065 86.835 miR-92 4.455 6.785 miR-92b 3.645 3.135 miR-93 28.665 38.27miR-107 3.99 5.115 miR-128a 4.205 3.575 miR-128b 3.1 3 miR-132 3.88 4.23 miR-134 4.525 4.08miR-142-3p 3.385 3.145 miR-149 7.57 4.63 miR-150 44.56 28 miR-151 31.29 41.295 miR-153 4.525 4.12miR-188 56.185 79.065 miR-190 8.04 9.495 miR-191 11.815 11.425 miR-196a 96.465 85.22 miR-196b 4.9 5.45miR-205 15.085 29.485 miR-210 4.54 3.96 miR-214 5.705 5.57 miR-215 4.045 3.48 miR-218 4.32 4miR-373 32.52 46.51 miR-488 89.185 83.84 miR-100 52.145 78.86 miR-127 85.485 81.415 miR-142-5p 4.385 4.1

168 x. jiang and n. li

extracted from LPS-treated and untreated cells andsubjected to Northern blot analysis. Both miR-17-3p andmiR-106a were shown to be induced in LPS-treated cellscompared to untreated cells (Figure 3). The expression ofRNU48 in LPS-treated cells was similar to untreated cells.

We also performed Northern blot analyses of TNFa-treated and untreated RNA samples with miR-146 and miR-

Figure 3. Northern blot analysis of miR-17-3p and miR-106a in HeLa and TNFatreated cells, separated with 15% urea denature gel. After transfer, miRNAs wer

Copyright # 2011 John Wiley & Sons, Ltd.

155 probes, respectively, which demonstrated LPS inductionin HL60 in a previous study.14 To examine whether these twomiRNA were induced in HeLa cells, we used miR-146 andmiR-155 probes to hybridize the Northern blot of total RNAsprepared from TNFa- and LPS-treated and untreated cells.We were unable to detect the expression of both miRNAs inHeLa, which was agreeable to our array analysis (data not

- or LPS-treated HeLa cells. Total RNA was prepared from TNFa-and LPS-e detected with complementary oligo probes.

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Figure 4. Analysis of miRNA-mediated repression of luciferase geneexpression with miRNA luciferase reporter vectors. (A) The diagram ofmiRNA luciferase reporter vectors. (B) HeLa cells were transfected withmiR-17-3p and miR-106a luciferase reporter, respectively treated with orwithout TNFa or LPS. Cells were subject to luciferase analysis. Grey bar isfor HeLa cells without treatment, the shade bar is for HeLa treated with TNFor LPS.

INDUCTION OF mir-17-3p AND mir-160a 169

shown). In addition, the induction of miR-146 and miR-155expressions was undetectable in TNFa- and LPS-treatedcells (data not shown). These results suggested that miR-146and miR-155 were not induced by TNFa and LPS inHeLa cells. As a previous study done at Baltimore lab,14

LPS-mediated induction of miR-146a is quite differentamong different cells. The induction was found in HL-60and THP-1 as well as other myeloid cell lines, while not in Bcell lines.14 The induction of miR-146a was not observedin 293 cells, a human embryonic kidney cell line.14 Ourresults provide additional evidence to support the differentexpression of miR-146a in different cell lines. Althoughwe do not know the molecular mechanism underlying theexpression and induction, the difference of miR-146 andmiR-155 between HeLa and HL-60 as well as other celllines could provide a good model system for the study of theexpression and induction of these two miRNAs.

Luciferase reporter assay is a common approach tomonitor the expression of induced miRNAs in cells. Thevector contains a unique miRNA target site within 30UTR atthe downstream of luciferase gene. The target site is asequence perfectly complementary to a specific miRNA.When the miRNA is expressed, it binds to the sequenceand results in repression of luciferase gene expression.Therefore, luciferase activity represents the expression andactivity of a miRNA. In the study, we used miR-17-3pand miR-106a luciferase reporters to monitor TNFa- andLPS-mediated induction of miR-17-3p and miR-106a. Avector without any miRNA target site was used as a control.The vectors were used to transfect to HeLa cells with Fugene6 overnight while TNFa and LPS treatments were applied,respectively. After the cells were collected, cell lysates wereprepared for the measurement of luciferase activities. Asshown in the Figure 4, luciferase activities were significantlyreduced in TNFa- and LPS-treated cells compared tocontrol untreated cells. The control vector without anymiRNA target site showed no difference between TNFa- orLPS-treated and -untreated cells. This result suggested thatTNFa- and LPS-induced miR-17-3p and miR-106a boundto the target sites of the luciferase reporter vectors andrepressed the expression of luciferase gene.

As demonstrated in the Northern blot analysis andluciferase reporter assay, miR-17-3p and miR-106a ident-ified by miRNA array were indeed induced by TNFa andLPS, although a few more miRNAs that were identified inthe study need further validation. miR-17-3p and miR-106abelong to two different clusters, miR-17-92 and miR-106a-363, respectively. The miR-17-92 and miR-106-363 clustersshow a high homology. Comparison between these twoclusters indicates miR-92-2 and miR-19b-2 of the miR-106a-363 cluster are identical to miR-92-1 and miR19b-1 ofthe miR-17-92 cluster. In addition, miR-106a, miR-20b andmiR-18b share high homology with miR-17-5p, miR-20aand miR-18a of the miR-17-92 cluster, respectively.15 Moreimportantly, both clusters have shown to be involvedin cancers.15,16 Searching the targets of miR-17-3p andmiR-106a will be an important question to answer. There aretwo approaches to answer the question. First, the potential

Copyright # 2011 John Wiley & Sons, Ltd. Cell Biochem Funct 2011; 29: 164–170.

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170 x. jiang and n. li

targets can be identified by silicon methods. RNA-inducedsilencing complex-mediated interaction between a maturemiRNA and its binding site depends on sequence comple-mentarities between miRNA and its targets. For an animalmiRNA, the sequence complementarities with its targetsare usually restricted to the 50 region of a miRNA, termed‘seed region’.17–19 The absence of perfect complementa-rities between animal miRNAs and their targets leads totranslational repression. A number of software areavailable to identify potential gene targets, from which aspecific group of genes that associate with proliferation,differentiation, apoptosis and immune response. Experimentalsupport of the association of the miRNAs with the targetgenes will uncover their roles in the regulation of proli-feration, differentiation, apoptosis and immune responses.Second approach is experimental tests of those identifiedtargets of miR-106a and miR-17-3p. Via literature search,we found a couple of genes, E2F1 and RB, are regulatedby miR-106a20,21 and TSP1 by miR-17-92 cluster.22 Thefunctions of these genes are shown to be associated with cellgrowth and proliferation. Further studies to link miR-106aand miR-17-3p with their target genes will facilitate theelucidation of regulation of these miRNAs in TNFa andLPS induction.

ACKNOWLEDGEMENTS

This work was supported by Signosis Inc.

CONFLICT OF INTEREST

No conflict of interest.

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