RNA干扰HMGA2基因表达对人肾癌转移细胞株增殖与侵袭影响
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摘要
目的用分子生物学研究肾癌形成和进展,可为临床治疗提供更可靠的依据,高迁移率族蛋白A2(high mobility group A2,HMGA2)是近年来研究热点之一。本研究观察HMGA2对肾癌细胞增殖和侵袭能力影响,为肾癌进一步临床研究提供依据。方法培养人肾癌细胞系786-O、769-P、人肾癌转移细胞系ACHN、人正常肾小管上皮细胞系HKC,研究分为3组,HMGA2-siRNA组、Mock-siRNA组和未转染组。利用RNA干扰技术,瞬时转染肾癌ACHN细胞,采用逆转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)和蛋白质印迹法检测HMGA2mRNA及蛋白表达;MTT法检测干扰后ACHN细胞增殖能力;Transwell法检测干扰后ACHN细胞侵袭能力。结果 786-O、769-P、ACHN和HKC细胞系HMGA2mRNA表达量分别为0.82±0.10、0.79±0.09、0.98±0.16和0.04±0.01,组内差异有统计学意义,F=63.36,P=0.002;4种细胞系中HMGA2蛋白表达量分别为0.80±0.09、0.75±0.08、0.94±0.10和0.06±0.02,组内差异有统计学意义,F=49.09,P=0.005。选择ACHN细胞作为后续研究:成功构建特异性沉默HMGA2表达的肾癌细胞株。在转染后24、48、72和96h,HMGA2-siRNA组细胞增殖速度分别为0.69±0.02、0.90±0.05、0.99±0.07和1.10±0.09,Mock-siRNA组细胞增殖速度分别为0.89±0.03、1.36±0.06、1.82±0.08和2.10±0.10,未转染组细胞增殖速度分别为0.91±0.02、1.50±0.06、1.91±0.10和2.20±0.12。HMGA2-siRNA组细胞增殖速度低于Mock-siRNA组和未转染组,F组别=630.724,P<0.001;F时间=566.968,P<0.001;F组别×时间=51.328,P=0.002。在转染后48h,HMGA2-siRNA组、Mock-siRNA组及未转染组穿过细胞数的比值分别为0.34±0.04,0.87±0.09和0.91±0.10,F=6.42,P=0.039。结论用HMGA2-siRNA干扰ACHN细胞,可抑制细胞增殖及侵袭能力,HMGA2基因在肾癌发生、发展中可能发挥"癌基因"作用,HMGA2基因及蛋白表达可能与肿瘤形成、进展和转移密切相关,有望成为肾癌治疗的一个重要靶点。
OBJECTIVE Renal carcinoma(RC)is a common malignancy of the urinary system.The molecular biological changes which would clearly result in the tumor formation and progression could provide reasonable basis for the choice of clinical treatment.The high mobility group A2(HMGA2)is one of the oncogene-research hotspots in recent years.This study aimed to observe the effect of HMGA2 on the proliferation and invasion ability of renal cell carcinoma(RCC)ACHN cells.Expected results will provide targeted gene therapy of renal carcinoma with new theoretical basis and choose direction.METHODS Human Renal proximal tubular epithelial cell lines(HKC),Human renal carcinoma cell line786-O,769-P,human renal cell carcinoma metastatic cell lines ACHN cell were cultured.HMGA2 siRNA,Mock-siRNA and their negative control group were designed and synthesized.Subsequently,ACHN cells were transiently transfected by using RNA interference technology.Finally,HMGA2 mRNA and protein expression levels were detected using reverse transcription polymerase chain reaction(RT-PCR)and western blot.ACHN cell's proliferation ability was observed by MTT,and ACHN cell's invasion ability was detected by the Transwell method.RESULTS The relative expression levels of HMGA2 mRNA in 786-O,769-P,ACHN and HKC cell lines were 0.82±0.10,0.79±0.09,0.98±0.16 and 0.04±0.01(F=63.36,P=0.002),respectively,and the HMGA2 protein relative expression levels were 0.80±0.09,0.75±0.08,0.94±0.10,0.06±0.02,with significant difference(F=49.09,P=0.005).The RCC cell strain,in which HMGA2 expression had been specifically silenced,was successfully constructed.The proliferation rate of the HMGA2-siRNA group was 0.69±0.02,0.90±0.05,0.99±0.07,1.10±0.09 at 24,48,72 and 96 hour time points after transfection,while that of the Mock-siRNA group was0.89±0.03,1.36±0.06,1.82±0.08,2.10±0.10 and the non-transfected group was 0.91±0.02,1.50±0.06,1.91±0.10,2.20±0.12,respectively.The proliferation rate of the HMGA2-siRNA group was markedly lower than the Mock-siRNA group and non-transfected group(F=18.35,P=0.012).Invasion ability in the HMGA2-siRNA group was significantly lower than in the Mock-siRNA group and non-transfected group at 48 hours after transfection(0.34±0.04,0.87±0.09,0.91±0.10,F=6.42,P=0.039).CONCLUSIONS Silence HMGA2 gene expression can obviously inhibit renal carcinoma cell proliferation and invasion ability.The HMGA2 gene may play a role of an "oncogene" in the occurrence and development of RCC.The expressions of HMGA2 gene in renal carcinoma were closely correlated with the formation,progression and metastasis of tumor and provide specific targets for the targeted therapy of RCC.Further in-depth studies on the HMGA2 gene is of great significance for gene therapy of RCC.
OBJECTIVE Renal carcinoma(RC)is a common malignancy of the urinary system.The molecular biological changes which would clearly result in the tumor formation and progression could provide reasonable basis for the choice of clinical treatment.The high mobility group A2(HMGA2)is one of the oncogene-research hotspots in recent years.This study aimed to observe the effect of HMGA2 on the proliferation and invasion ability of renal cell carcinoma(RCC)ACHN cells.Expected results will provide targeted gene therapy of renal carcinoma with new theoretical basis and choose direction.METHODS Human Renal proximal tubular epithelial cell lines(HKC),Human renal carcinoma cell line786-O,769-P,human renal cell carcinoma metastatic cell lines ACHN cell were cultured.HMGA2 siRNA,Mock-siRNA and their negative control group were designed and synthesized.Subsequently,ACHN cells were transiently transfected by using RNA interference technology.Finally,HMGA2 mRNA and protein expression levels were detected using reverse transcription polymerase chain reaction(RT-PCR)and western blot.ACHN cell's proliferation ability was observed by MTT,and ACHN cell's invasion ability was detected by the Transwell method.RESULTS The relative expression levels of HMGA2 mRNA in 786-O,769-P,ACHN and HKC cell lines were 0.82±0.10,0.79±0.09,0.98±0.16 and 0.04±0.01(F=63.36,P=0.002),respectively,and the HMGA2 protein relative expression levels were 0.80±0.09,0.75±0.08,0.94±0.10,0.06±0.02,with significant difference(F=49.09,P=0.005).The RCC cell strain,in which HMGA2 expression had been specifically silenced,was successfully constructed.The proliferation rate of the HMGA2-siRNA group was 0.69±0.02,0.90±0.05,0.99±0.07,1.10±0.09 at 24,48,72 and 96 hour time points after transfection,while that of the Mock-siRNA group was0.89±0.03,1.36±0.06,1.82±0.08,2.10±0.10 and the non-transfected group was 0.91±0.02,1.50±0.06,1.91±0.10,2.20±0.12,respectively.The proliferation rate of the HMGA2-siRNA group was markedly lower than the Mock-siRNA group and non-transfected group(F=18.35,P=0.012).Invasion ability in the HMGA2-siRNA group was significantly lower than in the Mock-siRNA group and non-transfected group at 48 hours after transfection(0.34±0.04,0.87±0.09,0.91±0.10,F=6.42,P=0.039).CONCLUSIONS Silence HMGA2 gene expression can obviously inhibit renal carcinoma cell proliferation and invasion ability.The HMGA2 gene may play a role of an "oncogene" in the occurrence and development of RCC.The expressions of HMGA2 gene in renal carcinoma were closely correlated with the formation,progression and metastasis of tumor and provide specific targets for the targeted therapy of RCC.Further in-depth studies on the HMGA2 gene is of great significance for gene therapy of RCC.
引文
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[13]Wu ZY,Wang SM,Chen ZH,et al.MiR-204regulates HMGA2expression and inhibits cell proliferation in human thyroid cancer[J].Cancer Biomark,2015,15(5):535-542.
[14]Huage ML,Chen CC,Chang LC.Gene expressions of HMGA and HMGB are associated with stage and metastasis in colorectal cancer[J].Int Colorectal Dis,2017,24(11):128l-1286.
[15]叶志华,黄耿,桂定文.抑制高迁移率组蛋白A2对肾癌细胞侵袭转移能力的影响[J].现代泌尿生殖肿瘤杂志,2018,2(10):39-42.
[16]Ljungberg B,Campbell SC,Choi HY,et al.The epidemiology of renal cell carcinoma[J].Eur Urol,2011,60(4):615-621.
[17]Wu Y,Zhou BP.New insights of epithelia-lmesenchymal transition in cancer metastasis[J].Acta Biochim Biophys Sin(Shang hai),2008,40(7):643-650.
[18]刘颖,王子明,付启忠,等.HMGA2在肾癌中的表达及临床意义[J].中华肿瘤杂志,2017,39(2),127-132.
[19]Zhao XP,Zhang H,Jiao JY,et al.Overexpression of HMGA2promotes tongue cancer metastasis through EMT pathway[J].Transl Med,2016,14(6):26-31.
[20]Wei L,Liu X,Zhang W,et al.Overexpression and oncogenic function of HMGA2in endometrial serous carcinogenesis[J].Am J Cancer Res,2016,6(2):249-259.
[21]Xia YY,Yin L,Tian H,et al.HMGA2is associated with epithelia-l mesenchymal transition and can predict poor prognosis in nasopharyngeal carcinoma[J].Onco Targets Ther,2015,8:169-176.
[2]Gao X,Dai M,Li Q,et al.HMGA2regulates lung cancer proliferation and metastasis[J].Thorac Cancer,2017,8(5):501-510.
[3]Liu Y,Fu QZ,Pu L,et al.HMGA2expression in renal carcinoma and its clinical significane[J].J Med Biochem,2015,34(3):338-343.
[4]Timmons L,Court D,Fire A.Ingestion of bacterially expressed RNAs can produce specific and potent genetic interference in Caenorhabditis elegans[J].Gene,2001,263:103-112.
[5]Motoyama K,Inoue H,Nakamura Y,et al.Clinical significance of high mobility group A2in human gastric cancer and its relationship to let-7 MicroRNA family[J].Clin Cancer Res,2017,14:2334-2340.
[6]Liu,Wang ZM,Fu QZH,et al.Effect of RNA interference in HMGA2expression on the proliferation and invasion ability of renal cell carcinoma ACHN cells[J].Molecular Medicine Reports,2017,16(4):5107-5112.
[7]Mayr C,Hemann MT,Bartel DP.Disrupting the pairing between let-7and HMGA2enhances oncogenic transformation[J].Science,2012,315:1576-1579.
[8]Sun J,Sun B,Sun R,et al.HMGA2promotes vasculogenic mimicry and tumor aggressiveness by upregulating Twist1in gastric carcinoma[J].Sci Rep,2017,7(1):2229.
[9]Borrmann L,Schwanbeck R,Heyduk T,et al.High mobility group A2protein and its derivatives bind a specific region of the promoter of DNA repair gene ERCCI and medulate its activity[J].Nucleic Acids Res,2013,3l(23):6841-6851.
[10]Eide HA,Halvorsen AR,Bjaanaes MM,et al.The MYCN-HM-GA2-CDKN2Apathway in non-small cell lung carcinoma--differences in histological subtypes[J].BMC Cancer,2016,16:71.
[11]Li W,Wang Z,Zha L,et al.HMGA2regulates epithelial mesenchymal transition and the acquisition of tumor stem cell properties through TWIST1in gastric cancer[J].Oncol Rep,2017,37(1):185-192.
[12]Shi Z,Li X,Wu D,et al.Silencing of HMGA2suppresses cellular proliferation,migration,invasion,and epithelial mesenchymal transition in bladder cancer[J].Tumour Biol,2016,37(6):7515-7523.
[13]Wu ZY,Wang SM,Chen ZH,et al.MiR-204regulates HMGA2expression and inhibits cell proliferation in human thyroid cancer[J].Cancer Biomark,2015,15(5):535-542.
[14]Huage ML,Chen CC,Chang LC.Gene expressions of HMGA and HMGB are associated with stage and metastasis in colorectal cancer[J].Int Colorectal Dis,2017,24(11):128l-1286.
[15]叶志华,黄耿,桂定文.抑制高迁移率组蛋白A2对肾癌细胞侵袭转移能力的影响[J].现代泌尿生殖肿瘤杂志,2018,2(10):39-42.
[16]Ljungberg B,Campbell SC,Choi HY,et al.The epidemiology of renal cell carcinoma[J].Eur Urol,2011,60(4):615-621.
[17]Wu Y,Zhou BP.New insights of epithelia-lmesenchymal transition in cancer metastasis[J].Acta Biochim Biophys Sin(Shang hai),2008,40(7):643-650.
[18]刘颖,王子明,付启忠,等.HMGA2在肾癌中的表达及临床意义[J].中华肿瘤杂志,2017,39(2),127-132.
[19]Zhao XP,Zhang H,Jiao JY,et al.Overexpression of HMGA2promotes tongue cancer metastasis through EMT pathway[J].Transl Med,2016,14(6):26-31.
[20]Wei L,Liu X,Zhang W,et al.Overexpression and oncogenic function of HMGA2in endometrial serous carcinogenesis[J].Am J Cancer Res,2016,6(2):249-259.
[21]Xia YY,Yin L,Tian H,et al.HMGA2is associated with epithelia-l mesenchymal transition and can predict poor prognosis in nasopharyngeal carcinoma[J].Onco Targets Ther,2015,8:169-176.