miR-204过表达抑制成视网膜细胞瘤细胞的增殖与侵袭及其可能的机制
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摘要
目的:观察miR-204对成视网膜细胞瘤(retinoblastoma,RB)细胞增殖与侵袭的影响,探讨其可能的调控机制。方法:应用实时荧光定量PCR(q RT-PCR)检测RB细胞系Y79、SO-RB50、HXO-Rb44和人正常视网膜色素上皮细胞系h TERT RPE-1中miR-204的表达水平。将Y79细胞系分成阴性对照组和miR-204组,分别应用脂质体转染法转染NC-mimics和miR-204 mimics,CCK-8增殖实验检测miR-204表达对Y79细胞增殖的影响,细胞划痕实验和Transwell小室法检测miR-204对Y79细胞迁移和侵袭的影响,应用生物学信息法预测miR-204的可能作用靶基因,应用q RT-PCR和Western blotting检测miR-204对靶基因高迁移率族蛋白A2(high mobility group AT-hook 2,HMGA2)m RNA和蛋白表达的影响。结果:miR-204在RB细胞系Y79、SO-RB50、HXO-Rb44中的表达较人正常视网膜色素上皮细胞系h TERT RPE-1明显降低(P<0.01)。转染miR-204 mimics后,Y79细胞中miR-204表达明显升高(P<0.01)、细胞增殖能力明显下降(P<0.01)、迁移及侵袭能力明显降低(P<0.01),miR-204的靶基因HMGA2 m RNA和蛋白的表达明显下降(P<0.01)。结论:miR-204在RB细胞系中低表达,过表达miR-204能够抑制RB细胞的增殖、迁移及侵袭能力,其机制可能与下调HMGA2基因的表达有关。
Objective: To observe the effects of miR-204 on the proliferation and invasion of retinoblastoma(RB) cells and to explore the potential regulatory mechanism. Methods: The expression level of miR-204 in RB cell lines(Y79, SO-RB50, and HXO-Rb44) as well as in normal human retinal pigment epithelial cell line h TERT RPE-1 was detected using q RT-PCR. The Y79 cells were divided into two groups(negative control group and miR-204 group) by respectively transfecting Y79 cells with NC-mimics and miR-204 mimics using liposome transfection method. The effects of miR-204 on Y79 cell proliferation was detected with CCK-8 assay; while the effect of miR-204 on migration and invasion of Y79 cells were determined by cell scratch assay and Transwell assay, respectively. Besides,the potential target gene of miR-204 was predicted by bioinformatics; and the influence of miR-204 on the expression of high mobility group AT-hook 2 gene(HMGA2) at both m RNA and protein levels was detected using q RT-PCR and Western blotting, respectively.Results: miR-204 expression in RB cell lines Y79, SO-RB50 and HXO-Rb44 was remarkably lower than that in normal human retinal pigment epithelial cell line h TERT RPE-1(P<0.01). miR-204 expression in Y79 cells was markedly up-regulated after transfection with miR-204 mimics(P<0.01) along with significantly reduced cell proliferation, migration and invasion capacities(all P<0.01), and m RNA and protein expressions of HMGA2 were also outstandingly reduced(P<0.01). Conclusion: miR-204 is lowly expressed in RB cell lines; in addition, miR-204 over-expression can suppress RB cell proliferation, migration and invasion, the mechanism of which might be related to down-regulation of the expression of HMGA2.
Objective: To observe the effects of miR-204 on the proliferation and invasion of retinoblastoma(RB) cells and to explore the potential regulatory mechanism. Methods: The expression level of miR-204 in RB cell lines(Y79, SO-RB50, and HXO-Rb44) as well as in normal human retinal pigment epithelial cell line h TERT RPE-1 was detected using q RT-PCR. The Y79 cells were divided into two groups(negative control group and miR-204 group) by respectively transfecting Y79 cells with NC-mimics and miR-204 mimics using liposome transfection method. The effects of miR-204 on Y79 cell proliferation was detected with CCK-8 assay; while the effect of miR-204 on migration and invasion of Y79 cells were determined by cell scratch assay and Transwell assay, respectively. Besides,the potential target gene of miR-204 was predicted by bioinformatics; and the influence of miR-204 on the expression of high mobility group AT-hook 2 gene(HMGA2) at both m RNA and protein levels was detected using q RT-PCR and Western blotting, respectively.Results: miR-204 expression in RB cell lines Y79, SO-RB50 and HXO-Rb44 was remarkably lower than that in normal human retinal pigment epithelial cell line h TERT RPE-1(P<0.01). miR-204 expression in Y79 cells was markedly up-regulated after transfection with miR-204 mimics(P<0.01) along with significantly reduced cell proliferation, migration and invasion capacities(all P<0.01), and m RNA and protein expressions of HMGA2 were also outstandingly reduced(P<0.01). Conclusion: miR-204 is lowly expressed in RB cell lines; in addition, miR-204 over-expression can suppress RB cell proliferation, migration and invasion, the mechanism of which might be related to down-regulation of the expression of HMGA2.
引文
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[33]NALINI V,DEEPA P R,RAGURAMAN R,et al.Targeting HMGA2 in retinoblastoma cells in vitro using the aptamer strategy[J].Ocul Oncol Pathol,2016,2(4):262-269.DOI:10.1159/000447300.
[34]SINGH M K,SINGH L,SEN S,et al.Role of high-mobility group protein A isoforms and their clinicopathologic significance in primary retinoblastoma[J].Appl Immunohistochem Mol Morphol,2017,25(4):244-250.DOI:10.1097/PAI.0000000000000295.
[2]GRUBER A R,MARTIN G,MULLER P,et al.Global 3'UTR shortening has a limited effect on protein abundance in proliferating T cells[J/OL].Nat Commun,2014,5:5465[2018-03-17].https://www.nature.com/articles/ncomms6465.DOI:10.1038/ncomms6465.
[3]ZHOU P,XU W,PENG X,et al.Large-scale screens of mi RNAm RNA interactions unveiled that the 3'UTR of a gene is targeted by multiple mi RNAs[J/OL].PLo S One,2013,8(7):e68204[2018-03-17].http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0068204.DOI:10.1371/journal.pone.0068204.
[4]DI LEVA G,GAROFALO M,CROCE C M.Micro RNAs in cancer[J].Annu Rev Pathol,2014,9:287-314.DOI:10.1146/annurevpathol-012513-104715.
[5]李佳桐,常莉.micro RNAs在视网膜母细胞瘤的研究进展[J].解剖科学进展,2016,22(4):439-441.
[6]WANG F E,ZHANG C,MAMINISHKIS A,et al.Micro RNA-204/211 alters epithelial physiology[J].FASEB J,2010,24(5):1552-1571.DOI:10.1096/fj.08-125856.
[7]OHANA R,WEIMAN-KELMAN B,RAVIV S,et al.Micro RNAs are essential for differentiation of the retinal pigmented epithelium and maturation of adjacent photoreceptors[J].Development,2015,142(14):2487-2498.DOI:10.1242/dev.121533.
[8]LI T,PAN H,LI R.The dual regulatory role of mi R-204 in cancer[J].Tumour Biol,2016,37(9):11667-11677.DOI:10.1007/s13277-016-5144-5.
[9]TSAI S C,HUANG S F,CHIANG J H,et al.The differential regulation of micro RNAs is associated with oral cancer[J].Oncol Rep,2017,38(3):1613-1620.DOI:10.3892/or.2017.5811.
[10]WU H,LIANG Y,SHEN L,et al.Micro RNA-204 modulates colorectal cancer cell sensitivity in response to 5-fluorouracil-based treatment by targeting high mobility group protein A2[J].Biol Open,2016,5(5):563-570.DOI:10.1242/bio.015008.
[11]WU Z Y,WANG S M,CHEN Z H,et al.Mi R-204 regulates HMGA2 expression and inhibits cell proliferation in human thyroid cancer[J].Cancer Biomark,2015,15(5):535-542.DOI:10.3233/CBM-150492.
[12]SINGH R P,MASSACHI L,MANICKAVEL S,et al.The role of mi RNA in inflammation and autoimmunity[J].Autoimmun Rev,2013,12(12):1160-1165.DOI:10.1016/j.autrev.2013.07.003.
[13]WANG Z,YAO Y J,ZHENG F,et al.Mir-138-5p acts as a tumor suppressor by targeting pyruvate dehydrogenase kinase 1 in human retinoblastoma[J].Eur Rev Med Pharmacol Sci,2017,21(24):5624-5629.DOI:10.26355/eurrev_201712_14005.
[14]SHAO X L,CHEN Y,GAO L.Mi R-200c suppresses the migration of retinoblastoma cells by reversing epithelial mesenchymal transition[J].Int J Ophthalmol,2017,10(8):1195-1202.DOI:10.18240/ijo.2017.08.02.
[15]ZHAO Y,ZHANG S,ZHANG Y.Micro RNA-320 inhibits cell proliferation,migration and invasion in retinoblastoma by targeting specificity protein 1[J].Mol Med Rep,2017,16(2):2191-2198.DOI:10.3892/mmr.2017.6767.
[16]YANG L,WEI N,WANG L,et al.mi R-498 promotes cell proliferation and inhibits cell apoptosis in retinoblastoma by directly targeting CCPG1[J].Childs Nerv Syst,2018,34(3):417-422.DOI:10.1007/s00381-017-3622-8.
[17]ZHANG M,LI Q,PAN Y,et al.Micro RNA-655 attenuates the malignant biological behaviours of retinoblastoma cells by directly targeting PAX6 and suppressing the ERK and p38 MAPK signalling pathways[J].Oncol Rep,2018,39(4):2040-2050.DOI:10.3892/or.2018.6264.
[18]YANG G,FU Y,ZHANG L,et al.mi R106b regulates retinoblastoma Y79 cells through Runx3[J].Oncol Rep,2017,38(5):3039-3043.DOI:10.3892/or.2017.5931.
[19]SHU L,ZHANG Z,CAI Y.Micro RNA-204 inhibits cell migration and invasion in human cervical cancer by regulating transcription factor 12[J].Oncol Lett,2018,15(1):161-166.DOI:10.3892/ol.2017.7343.
[20]SONG S,FAJOL A,TU X,et al.mi R-204 suppresses the development and progression of human glioblastoma by targeting ATF2[J].Oncotarget,2016,7(43):70058-70065.DOI:10.18632/oncotarget.11732.
[21]JIANG G,WEN L,ZHENG H,et al.mi R-204-5p targeting SIRT1regulates hepatocellular carcinoma progression[J].Cell Biochem Funct,2016,34(7):505-510.DOI:10.1002/cbf.3223.
[22]SHRESTHA S,YANG C D,HONG H C,et al.Integrated micro RNAm RNA analysis reveals mir-204 inhibits cell proliferation in gastric cancer by targeting CKS1B,CXCL1 and GPRC5A[J/OL].Int J Mol Sci,2017,19(1).pii:E87[2018-03-17].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5496037/.DOI:10.3390/ijms19010087.
[23]WU Z Y,WANG S M,CHEN Z H,et al.Mi R-204 regulates HMGA2 expression and inhibits cell proliferation in human thyroid cancer[J].Cancer Biomark,2015,15(5):535-542.DOI:10.3233/CBM-150492.
[24]TSAI S C,HUANG S F,CHIANG J H,et al.The differential regulation of micro RNAs is associated with oral cancer[J].Oncol Rep,2017,38(3):1613-1620.DOI:10.3892/or.2017.5811.
[25]WU H,LIANG Y,SHEN L,et al.Micro RNA-204 modulates colorectal cancer cell sensitivity in response to 5-fluorouracil-based treatment by targeting high mobility group protein A2[J].Biol Open,2016,5(5):563-570.DOI:10.1242/bio.015008.
[26]PALLANTE P,SEPE R,PUCA F,et al.High mobility group a proteins as tumor markers[J].Front Med(Lausanne),2015,2:15.DOI:10.3389/fmed.2015.00015.
[27]FUSCO A,FEDELE M.Roles of HMGA proteins in cancer[J].Nat Rev Cancer,2007,7(12):899-910.DOI:10.1038/nrc2271.
[28]JIN L,LLOYD R V,HENRY M R,et al.The diagnostic utility of combination of HMGA2 and IMP3 q RT-PCR testing in thyroid neoplasms[J].Appl Immunohistochem Mol Morphol,2015,23(1):36-43.DOI:10.1097/PAI.0000000000000031.
[29]ZHU C,LI J,CHENG G,et al.mi R-154 inhibits EMT by targeting HMGA2 in prostate cancer cells[J].Mol Cell Biochem,2013,379(1/2):69-75.DOI:10.1007/s11010-013-1628-4.
[30]DING X,WANG Y,MA X,et al.Expression of HMGA2 in bladder cancer and its association with epithelial-to-mesenchymal transition[J].Cell Prolif,2014,47(2):146-151.DOI:10.1111/cpr.12096.
[31]KONG D,SU G,ZHA L,et al.Coexpression of HMGA2 and Oct4predicts an unfavorable prognosis in human gastric cancer[J].Med Oncol,2014,31(8):130.DOI:10.1007/s12032-014-0130-5.
[32]VENKATESAN N,KRISHNAKUMAR S,DEEPA P R,et al.Molecular deregulation induced by silencing of the high mobility group protein A2 gene in retinoblastoma cells[J].Mol Vis,2012,18:2420-2437.
[33]NALINI V,DEEPA P R,RAGURAMAN R,et al.Targeting HMGA2 in retinoblastoma cells in vitro using the aptamer strategy[J].Ocul Oncol Pathol,2016,2(4):262-269.DOI:10.1159/000447300.
[34]SINGH M K,SINGH L,SEN S,et al.Role of high-mobility group protein A isoforms and their clinicopathologic significance in primary retinoblastoma[J].Appl Immunohistochem Mol Morphol,2017,25(4):244-250.DOI:10.1097/PAI.0000000000000295.