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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front. Med.    2015, Vol. 9 Issue (3) : 331-343     DOI: 10.1007/s11684-015-0409-8
RESEARCH ARTICLE |
MicroRNA-142-3p and microRNA-142-5p are downregulated in hepatocellular carcinoma and exhibit synergistic effects on cell motility
Felice Ho-Ching Tsang,Sandy Leung-Kuen Au,Lai Wei,Dorothy Ngo-Yin Fan,Joyce Man-Fong Lee,Carmen Chak-Lui Wong,Irene Oi-Lin Ng(),Chun-Ming Wong()
State Key Laboratory for Liver Research and Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
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Abstract  

MicroRNAs (miRNAs), an important class of small non-coding RNAs, regulate gene expression at the post-transcriptional level. miRNAs are involved in a wide range of biological processes and implicated in different diseases, including cancers. In this study, miRNA profiling and qRT-PCR validation revealed that miR-142-3p and miR-142-5p were significantly downregulated in hepatocellular carcinoma (HCC) and their expression levels decreased as the disease progressed. The ectopic expression of miR-142 significantly reduced HCC cell migration and invasion. Overexpression of either miR-142-3p or miR-142-5p suppressed HCC cell migration, and overexpression of both synergistically inhibited cell migration, which indicated that miR-142-3p and miR-142-5p may cooperatively regulate cell movement. miR-142-3p and miR-142-5p, which are mature miRNAs derived from the 3′- and 5′-strands of the precursor miR-142, target distinct pools of genes because of their different seed sequences. Pathway enrichment analysis showed a strong association of the putative gene targets of miR-142-3p and miR-142-5p with several cell motility-associated pathways, including those regulating actin cytoskeleton, adherens junctions, and focal adhesion. Importantly, a number of the putative gene targets were also significantly upregulated in human HCC cells. Moreover, overexpression of miR-142 significantly abrogated stress fiber formation in HCC cells and led to cell shrinkage. This study shows that mature miR-142 pairs collaboratively regulate different components of distinct signaling cascades and therefore affects the motility of HCC cells.

Keywords hepatocellular carcinoma      microRNA      metastasis      cytoskeletal reorganization     
Corresponding Authors: Irene Oi-Lin Ng,Chun-Ming Wong   
Just Accepted Date: 14 July 2015   Online First Date: 21 August 2015    Issue Date: 26 August 2015
URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-015-0409-8     OR     http://academic.hep.com.cn/fmd/EN/Y2015/V9/I3/331
Fig.1  Expression levels of miR-142-5p and miR-142-3p in HCC samples. (A) Expression levels of both miR-142-3p and miR-142-5p were significantly downregulated in primary HCC samples compared with their corresponding NT liver samples. Expression data were obtained through qRT-PCR. Statistical comparison was performed using paired t-test. (B) In the paired primary HCC cohort (N = 70), underexpression of miR-142-3p and miR-142-5p was observed in 71.4% (50/70) of the cases. Data are presented as log2(ratio) of miR-142 expression in primary HCC samples compared with their corresponding NT liver samples. miR-142 underexpression (red bars) cases were defined as log2(ratio)<1, whereas miR-142 overexpression (blue bars) cases were defined as log2(ratio)>1. (C) Significant and positive correlation between miR-142-3p and miR-142-5p was shown in both primary HCC tumor and NT liver samples, indicating that both miR-142-3p and miR-142-5p were concurrently expressed in the clinical samples. Correlations were determined by linear regression model, with R2 and P values indicated. (D) Expression levels of both miR-142-3p and miR-142-5p gradually decreased from normal liver, chronic hepatitis, cirrhotic liver, early HCC, and advanced HCC, indicating the involvement of both strands in multistep HCC progression. (NL, normal liver; CH, chronic hepatitis; CL, cirrhotic liver; Early HCC refers to pTNM stage I and II; and Advanced HCC refers to pTNM stage III and IV.)
Fig.2  Overexpression of miR-142 reduced HCC cell migration. Overexpression of miR-142 in BEL-7402 and SMMC-7721 cell lines significantly attenuated HCC cell migration compared with EV control as demonstrated by the transwell cell migration assay. Statistical comparison was performed using t-test (*** P<0.0001).
Fig.3  miR-142-3p and miR-142-5p exhibited an additive inhibitory effect on HCC cell migration. Overexpression of either miR-142-3p or miR-142-5p significantly reduced cell migration in BEL-7402 cells, whereas overexpression of both further inhibited cell migration, suggesting that miR-142-3p and miR-142-5p coordinately regulate cell movement. Statistical comparison was performed using t-test (** P<0.001, *** P<0.0001).
KEGG pathway name KEGG pathway number Number of genes -ln (P value)
Colorectal cancer hsa05210 18 13.04
Phosphatidylinositol signaling system hsa04070 16 13.04
Regulation of actin cytoskeleton hsa04810 31 11.25
Adherens junction hsa04520 15 10.59
Pancreatic cancer hsa05212 15 10.31
Inositol phosphate metabolism hsa00562 11 9.34
Ubiquitin mediated proteolysis hsa04120 21 9.03
Chronic myeloid leukemia hsa05220 14 7.77
Renal cell carcinoma hsa05211 13 7.58
mTOR signaling pathway hsa04150 10 7.16
TGF-β signaling pathway hsa04350 15 6.85
Insulin signaling pathway hsa04910 19 5.48
Amyotrophic lateral sclerosis (ALS) hsa05030 5 4.99
MAPK signaling pathway hsa04010 29 4.93
Tight junction hsa04530 18 4.87
Wnt signaling pathway hsa04310 19 4.75
Long-term potentiation hsa04720 10 4.08
Glioma hsa05214 10 3.93
Acute myeloid leukemia hsa05221 9 3.80
Focal adhesion hsa04510 22 3.65
Oxidative phosphorylation hsa00190 2 3.48
Cell communication hsa01430 3 3.40
Calcium signaling pathway hsa04020 19 3.24
Axon guidance hsa04360 15 3.03
Melanoma hsa05218 10 2.92
Ribosome hsa03010 1 2.88
Jak-STAT signaling pathways hsa04630 17 2.61
Prostate cancer hsa05215 11 2.35
T cell receptor signaling pathway hsa04660 11 2.27
Circadian rhythm hsa04710 3 2.26
Tab.1  Pathway enrichment analysis of miR-142-3p and miR-142-5p
Fig.4  Putative targets of miR-142-3p and miR-142-5p were involved in the regulation of cytoskeleton. (A) Putative target genes of miR-142-3p (highlighted in red), miR-142-5p (highlighted in blue) and both miR-142-3p and miR-142-5p (highlighted in purple) are indicated in the pathway map adopted from the KEGG pathway. (B) Expression levels of various components of pathways regulating actin cytoskeleton. ACTN4, DIAPH3, FGF13, ITGAV, MYH9, NCKAP1, PIP4K2C, RAC1, RHOA, and SSH2 were significantly upregulated in HCCs compared with the corresponding NT liver samples. Data on these expression levels were obtained from the RNA sequencing data in the TCGA database. Statistical comparison was performed using t-test.
Fig.5  Overexpression of miR-142 disrupted stress fiber network and changed the morphology of HCC cells. (A) Stress fibers were demonstrated by phalloidin staining (red, indicated by the arrow). SMMC-7721 cells overexpressing miR-142 exhibited a loss of stress fibers compared with the vector control cells. (B) Overexpression of miR-142 in BEL-7402 cells reduced the cell cytoplasm. (C) Effect of overexpressing miR-142 on cell morphology was investigated using SEM under a magnification of 12 000×. A significant shrinkage was observed in miR-142 overexpressing HCC cells compared with the EV control.
1 Esquela-Kerscher A, Slack FJ. Oncomirs—microRNAs with a role in cancer. Nat Rev Cancer 2006; 6(4): 259–269
doi: 10.1038/nrc1840 pmid: 16557279
2 Landgraf P, Rusu M, Sheridan R, Sewer A, Iovino N, Aravin A, Pfeffer S, Rice A, Kamphorst AO, Landthaler M, Lin C, Socci ND, Hermida L, Fulci V, Chiaretti S, Foà R, Schliwka J, Fuchs U, Novosel A, Müller RU, Schermer B, Bissels U, Inman J, Phan Q, Chien M, Weir DB, Choksi R, De Vita G, Frezzetti D, Trompeter HI, Hornung V, Teng G, Hartmann G, Palkovits M, Di Lauro R, Wernet P, Macino G, Rogler CE, Nagle JW, Ju J, Papavasiliou FN, Benzing T, Lichter P, Tam W, Brownstein MJ, Bosio A, Borkhardt A, Russo JJ, Sander C, Zavolan M, Tuschl T. A mammalian microRNA expression atlas based on small RNA library sequencing. Cell 2007; 129(7): 1401–1414
doi: 10.1016/j.cell.2007.04.040 pmid: 17604727
3 Zhang X, Yan Z, Zhang J, Gong L, Li W, Cui J, Liu Y, Gao Z, Li J, Shen L, Lu Y. Combination of hsa-miR-375 and hsa-miR-142-5p as a predictor for recurrence risk in gastric cancer patients following surgical resection. Ann Oncol 2011; 22(10): 2257–2266
doi: 10.1093/annonc/mdq758 pmid: 21343377
4 Wu L, Cai C, Wang X, Liu M, Li X, Tang H. MicroRNA-142-3p, a new regulator of RAC1, suppresses the migration and invasion of hepatocellular carcinoma cells. FEBS Lett 2011; 585(9): 1322–1330
doi: 10.1016/j.febslet.2011.03.067 pmid: 21482222
5 Shen WW, Zeng Z, Zhu WX, Fu GH. miR-142-3p functions as a tumor suppressor by targeting CD133, ABCG2, and Lgr5 in colon cancer cells. J Mol Med (Berl)2013; 91(8): 989–1000
doi: 10.1007/s00109-013-1037-x pmid: 23619912
6 MacKenzie TN, Mujumdar N, Banerjee S, Sangwan V, Sarver A, Vickers S, Subramanian S, Saluja AK. Triptolide induces the expression of miR-142-3p: a negative regulator of heat shock protein 70 and pancreatic cancer cell proliferation. Mol Cancer Ther 2013; 12(7): 1266–1275
doi: 10.1158/1535-7163.MCT-12-1231 pmid: 23635652
7 Wong CM, Kai AK, Tsang FH, Ng IO. Regulation of hepatocarcinogenesis by microRNAs. Front Biosci (Elite Ed)2013; 5: 49–60
pmid: 23276969
8 Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 2005; 120(1): 15–20
doi: 10.1016/j.cell.2004.12.035 pmid: 15652477
9 Chen K, Rajewsky N. Natural selection on human microRNA binding sites inferred from SNP data. Nat Genet 2006; 38(12): 1452–1456
doi: 10.1038/ng1910 pmid: 17072316
10 Maragkakis M, Alexiou P, Papadopoulos GL, Reczko M, Dalamagas T, Giannopoulos G, Goumas G, Koukis E, Kourtis K, Simossis VA, Sethupathy P, Vergoulis T, Koziris N, Sellis T, Tsanakas P, Hatzigeorgiou AG. Accurate microRNA target prediction correlates with protein repression levels. BMC Bioinformatics 2009; 10(1): 295
doi: 10.1186/1471-2105-10-295 pmid: 19765283
11 Papadopoulos GL, Alexiou P, Maragkakis M, Reczko M, Hatzigeorgiou AG. DIANA-mirPath: integrating human and mouse microRNAs in pathways. Bioinformatics 2009; 25(15): 1991–1993
doi: 10.1093/bioinformatics/btp299 pmid: 19435746
12 Kanehisa M, Goto S. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res 2000; 28(1): 27–30
doi: 10.1093/nar/28.1.27 pmid: 10592173
13 Wong CM, Wong CC, Lee JM, Fan DN, Au SL, Ng IO. Sequential alterations of microRNA expression in hepatocellular carcinoma development and venous metastasis. Hepatology 2012; 55(5): 1453–1461
doi: 10.1002/hep.25512 pmid: 22135159
14 Wong CC, Wong CM, Tung EK, Man K, Ng IO. Rho-kinase 2 is frequently overexpressed in hepatocellular carcinoma and involved in tumor invasion. Hepatology 2009; 49(5): 1583–1594
doi: 10.1002/hep.22836 pmid: 19205033
15 Fukui K, Tamura S, Wada A, Kamada Y, Sawai Y, Imanaka K, Kudara T, Shimomura I, Hayashi N. Expression and prognostic role of RhoA GTPases in hepatocellular carcinoma. J Cancer Res Clin Oncol 2006; 132(10): 627–633
doi: 10.1007/s00432-006-0107-7 pmid: 16810502
16 Khvorova A, Reynolds A, Jayasena SD. Functional siRNAs and miRNAs exhibit strand bias. Cell 2003; 115(2): 209–216
doi: 10.1016/S0092-8674(03)00801-8 pmid: 14567918
17 Schwarz DS, Hutvágner G, Du T, Xu Z, Aronin N, Zamore PD. Asymmetry in the assembly of the RNAi enzyme complex. Cell 2003; 115(2): 199–208
doi: 10.1016/S0092-8674(03)00759-1 pmid: 14567917
18 Ro S, Park C, Young D, Sanders KM, Yan W. Tissue-dependent paired expression of miRNAs. Nucleic Acids Res 2007; 35(17): 5944–5953
doi: 10.1093/nar/gkm641 pmid: 17726050
19 Kozomara A, Griffiths-Jones S. miRBase: integrating microRNA annotation and deep-sequencing data. Nucleic Acids Res 2011; 39(Database issue): D152–D157
doi: 10.1093/nar/gkq1027 pmid: 21037258
20 Shan SW, Fang L, Shatseva T, Rutnam ZJ, Yang X, Du W, Lu WY, Xuan JW, Deng Z, Yang BB. Mature miR-17-5p and passenger miR-17-3p induce hepatocellular carcinoma by targeting PTEN, GalNT7 and vimentin in different signal pathways. J Cell Sci 2013; 126(Pt 6): 1517–1530
doi: 10.1242/jcs.122895 pmid: 23418359
21 Uchino K, Takeshita F, Takahashi RU, Kosaka N, Fujiwara K, Naruoka H, Sonoke S, Yano J, Sasaki H, Nozawa S, Yoshiike M, Kitajima K, Chikaraishi T, Ochiya T. Therapeutic effects of microRNA-582-5p and <?Pub Caret?>-3p on the inhibition of bladder cancer progression. Mol Ther 2013; 21(3): 610–619
doi: 10.1038/mt.2012.269 pmid: 23295946
22 Wang F, Wang XS, Yang GH, Zhai PF, Xiao Z, Xia LY, Chen LR, Wang Y, Wang XZ, Bi LX, Liu N, Yu Y, Gao D, Huang BT, Wang J, Zhou DB, Gong JN, Zhao HL, Bi XH, Yu J, Zhang JW. miR-29a and miR-142-3p downregulation and diagnostic implication in human acute myeloid leukemia. Mol Biol Rep 2012; 39(3): 2713–2722
doi: 10.1007/s11033-011-1026-5 pmid: 21678057
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