抑制COL6A1基因表达对膀胱癌T24细胞增殖和侵袭能力的影响
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摘要
目的:研究小干扰RNA抑制COL6A1基因表达对膀胱癌T24细胞增殖和侵袭能力的影响。方法:设计合成靶向COL6A1基因的siRNA,采用阳离子脂质体试剂瞬时转染膀胱癌细胞株T24,利用实时荧光定量PCR和Western blot检测RNA干扰后COL6A1基因的沉默效果,评价siRNA设计的合理性及RNA干扰抑制COL6A1表达的有效性,分别在RNA干扰后第1、2、3天应用噻唑蓝(MTT)比色法检测细胞增殖状态,并绘制生长曲线,应用Transwell法检测各组膀胱癌侵袭能力。结果:靶向COL6A1的siRNA成功抑制了膀胱癌细胞株T24中COL6A1 mRNA及蛋白表达,COL6A1-siRNA组蛋白相对表达量为阴性对照组的14.1%,mRNA相对表达量为空白对照组的20.5%;MTT比色法显示COL6A1-siRNA组细胞增殖能力显著减弱(P<0.05),在第1、2、3天后重复检测,分别达到了空白对照组的75.6%、58.2%和49.4%;Transwell细胞迁移实验显示COL6A1-siRNA组细胞迁移能力明显减弱(P<0.05),穿膜细胞数为空白对照组的45.3%。结论:利用siRNA技术能有效降低COL6A1基因的表达,同时有效抑制T24细胞的体外增殖和侵袭的能力。
Objective: To examine the influence of COL6 A1 on proliferation and invasion of bladder carcinoma cell. Method: The expression of COL6 A1 gene was knocked down using RNA interference(RNAi) in the bladder carcinoma cell T24. The transcription level of COL6 A1 was tested by RT-PCR. The expression of COL6 A1 protein was examined by Western blotting. The cell proliferation was detected by MTT. The cell' s invasion ability was performed on Matrigel transwell assay. Result: The results showed that the level of COL6 A1 mRNA and protein expression were significantly reduced after RNAi with 20.5% of control group on mRNA expression and 14.1% of control group on protein expression. MTT assay showed that the proliferation of T24 cell was decreased due to RNA interference, with OD values reaching 75.6%,58.2% and 49.4% of control group at 1 d, 2 d and 3 d respectively. Transwell assay showed that invasion abilities of T24 cells were reduced obviously due to COL6 A1 RNAi(P<0.05), the transmembrane cells were 54.3% of control group. Conclusion: Our findings revealed that down-regulated COL6 A1 could inhibit abilities of proliferation and invasion in T24 cells. COL6 A1 can serve as a potential target for gene therapy of human bladder carcinoma.
Objective: To examine the influence of COL6 A1 on proliferation and invasion of bladder carcinoma cell. Method: The expression of COL6 A1 gene was knocked down using RNA interference(RNAi) in the bladder carcinoma cell T24. The transcription level of COL6 A1 was tested by RT-PCR. The expression of COL6 A1 protein was examined by Western blotting. The cell proliferation was detected by MTT. The cell' s invasion ability was performed on Matrigel transwell assay. Result: The results showed that the level of COL6 A1 mRNA and protein expression were significantly reduced after RNAi with 20.5% of control group on mRNA expression and 14.1% of control group on protein expression. MTT assay showed that the proliferation of T24 cell was decreased due to RNA interference, with OD values reaching 75.6%,58.2% and 49.4% of control group at 1 d, 2 d and 3 d respectively. Transwell assay showed that invasion abilities of T24 cells were reduced obviously due to COL6 A1 RNAi(P<0.05), the transmembrane cells were 54.3% of control group. Conclusion: Our findings revealed that down-regulated COL6 A1 could inhibit abilities of proliferation and invasion in T24 cells. COL6 A1 can serve as a potential target for gene therapy of human bladder carcinoma.
引文
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12 Chen P,Cescon M,Bonaldo P.Collagen Ⅵ in cancer and its biological mechanisms[J].Trends Mol Med,2013,19(7):410-4177.
13 Chiu K H,Chang Y H,Wu Y S,et al.Quantitative Secretome Analysis Reveals that COL6A1 is a Metastasis-Associated Protein Using Stacking Gel-Aided Purification Combined with iTRAQ Labeling[J].J Proteome Res,2011,10(3):1110-1125.
14 Wan F,Wang H,Shen Y,et al.Upregulation of COL6A1 is predictive of poor prognosis in clear cell renal cell carcinoma patients[J].Oncotarget,2015,6(29):27378-27387.
15 Turtoi A,Blomme A,Bianchi E,et al.Accessibilome of human glioblastoma:collagen-Ⅵ-alpha-1 is a new target and a marker of poor outcome[J].J Proteome Res,2014,13(12):5660-5669.
16 蔡忠林,闫雯,周川,等.膀胱相关生物分子在增强腺病毒介导基因治疗与病毒治疗膀胱癌靶向性中的应用[J].临床泌尿外科杂志,2017,32(4):310-314.
2 Lamandé S R,Bateman J F.Collagen Ⅵ disorders:Insights on form and function in the extracellular matrix and beyond[J].Matrix Biol,2018,71-72:348-367.
3 Frka K,Facchinello N,Del Vecchio C,et al.Lentiviral-mediated RNAi in vivo silencing of Col6a1,a gene with complex tissue specific expression pattern[J].J Biotechnol,2009,141(1-2):8-17.
4 Zhu Y P,Wan F N,Shen Y J,et al.Reactive stroma component COL6A1 is upregulated in castration-resistant prostate cancer and promotes tumor growth[J].Oncotarget,2015,6(16):14488-14496.
5 Park J,Scherer P E.Adipocyte-derived endotrophin promotes malignant tumor progression[J].J Clin Invest,2012,122(11):4243.
6 Wu Z,Wang S,Jiang F,et al.Mass spectrometric detection combined with bioinformatic analysis identified possible protein markers and key pathways associated with bladder cancer[J].Gene,2017,626:407-413..
7 Irwin W A,Bergamin N,Sabatelli P,et al.Mitochondrial dysfunction and apoptosis in myopathic mice with collagen Ⅵ deficiency[J].Nat Genet,2003,35(4):367-371.
8 Lu P,Weaver V M,Werb Z.The extracellular matrix:a dynamic niche in cancer progression[J].J Cell Biol,2012,196(4):395-406.
9 Fujita A,Sato J R,Festa F,et al.Identiication of COL6A1 as a differentially expressed gene in human astrocytomas[J].Gen Mol Res,2008,7(2):371-378.
10 Voiles L,Lewis D E,Han L,et al.Overexpression of type Ⅵ collagen in neoplastic lung tissues[J].Oncol Rep,2014,32(5):1897-904.
11 Hou T,Tong C,Kazobinka G,et al.Expression of COL6A1 predicts prognosis in cervical cancer patients[J].Am J Transl Res,2016,8(6):2838-2844.
12 Chen P,Cescon M,Bonaldo P.Collagen Ⅵ in cancer and its biological mechanisms[J].Trends Mol Med,2013,19(7):410-4177.
13 Chiu K H,Chang Y H,Wu Y S,et al.Quantitative Secretome Analysis Reveals that COL6A1 is a Metastasis-Associated Protein Using Stacking Gel-Aided Purification Combined with iTRAQ Labeling[J].J Proteome Res,2011,10(3):1110-1125.
14 Wan F,Wang H,Shen Y,et al.Upregulation of COL6A1 is predictive of poor prognosis in clear cell renal cell carcinoma patients[J].Oncotarget,2015,6(29):27378-27387.
15 Turtoi A,Blomme A,Bianchi E,et al.Accessibilome of human glioblastoma:collagen-Ⅵ-alpha-1 is a new target and a marker of poor outcome[J].J Proteome Res,2014,13(12):5660-5669.
16 蔡忠林,闫雯,周川,等.膀胱相关生物分子在增强腺病毒介导基因治疗与病毒治疗膀胱癌靶向性中的应用[J].临床泌尿外科杂志,2017,32(4):310-314.