差异表达的miR-20a对胃癌细胞恶性转化的影响及其机制研究
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摘要
目的探讨微小分子非编码RNA-20a (miR-20a)在胃癌中的表达及其对胃癌细胞恶性转化的影响,并对其作用机制进行研究。方法选取2014年9月至2017年9月经江苏大学附属昆山医院病理确诊的45例患者的新鲜胃癌及癌旁组织标本,提取总RNA,qRT-PCR检测胃癌及癌旁组织中miR-20a的表达,脂质体转染寡核苷酸片段NC-inhibitor及miR-20a-inhibitor至MGC-803细胞,qRT-PCR检测转染后MGC-803细胞中miR-20a的表达水平,平板克隆实验和Transwell迁移实验分别检测转染miR-20a-inhibitor后MGC-803细胞增殖能力和迁移能力的改变,Western blot法检测转染前后MGC-803细胞AKT通路活性的改变及对上皮间质转化(EMT)蛋白表达情况的影响。结果癌组织中miR-20a/U6的表达水平为1.12±0.10,显著高于癌旁组织的0.30±0.04,差异具有显著统计学意义(P<0.01);miR-20a-inhibitor转染组miR-20a/U6的表达水平为0.65±0.04,明显低于NC-inhibitor转染组的1.01±0.14,差异具有显著统计学意义(P<0.01);miR-20a-inhibitor转染组克隆细胞数量为(70±3)个,明显低于NC-inhibitor转染组的(110±4)个,差异有统计学意义(P<0.05);miR-20a-inhibitor转染组Transwell细胞迁移数量为(35±4)个,明显低于NC-inhibitor转染组的(53±6)个,差异有统计学意义(P<0.05);与miR-20a-inhibitor转染组比较,NC-inhibitor组AKT通路活化减弱,同时上皮细胞钙粘蛋白(E-cadherin)表达减弱,神经钙粘蛋白(N-cadherin)和波形蛋白(Vimentin)表达增强。结论与癌旁组织相比,癌组织中miR-20a的表达水平显著增高,miR-20a通过促进胃癌细胞增殖和迁移途径引起胃癌细胞恶性转化。
Objective To investigate the expression of micromolecular non-coding RNA-20a(miR-20a) in gastric cancer and its effect on the malignant transformation of gastric cancer cells, and to study its mechanism.Methods A total of 45 samples of fresh gastric cancer and paracancerous tissues were collected from 45 patients pathologically diagnosed in Kunshan Hospital Affiliated to Jiangsu University from September 2014 to September 2017. Total RNA was extracted and the expression of miR-20 a in gastric cancer tissues and paracancerous tissues was detected by qRT-PCR. Liposomal transfection was performed with oligonucleotide fragment NC-inhibitor and miR-20a-inhibitor to MGC-803, then the expression of miR-20 a in transfected MGC-803 cells was detected by qRT-PCR. Cell cloning assay and Transwell assay were used to detect the proliferation and migration of MGC-803 cells after transfection of miR-20a-inhibitor. Western blot was used to detect the change in cellular AKT pathway activity and the expression of epithelial to mesenchymal transition(EMT) markers in MGC-803 cells before and after transfection. Results The expression level of miR-20a/U6 in cancer tissues was 1.12±0.10, which was significantly higher than 0.30±0.04 in the paracancerous tissues, and the difference was statistically significant(P<0.01). The expression level of miR-20a/U6 in the miR-20a-inhibitor transfection group was 0.65±0.04, which was significantly lower than 1.01±0.14 in the NC-inhibitor transfection group, and the difference was statistically significant(P<0.01). The number of cloned cells in the miR-20a-inhibitor transfection group was 70±3, which was significantly lower than 110±4 in the NC-inhibitor transfection group(P<0.05). The number of Transwell cells in the miR-20a-inhibitor transfection group was 35±4, which was significantly lower than 53±6 in the NC-inhibitor transfection group(P<0.05). Compared with the miR-20a-inhibitor transfection group, the activation of the AKT pathway was weakened in the NC-inhibitor group, while the expression of E-cadherin was attenuated in the epithelial cells, and the expression of N-cadherin and Vimentin was enhanced.Conclusion The expression level of miR-20 a in cancer tissues is significantly increased compared with paracancerous tissues. miR-20 a can induce malignant transformation of gastric cancer cells by promoting the proliferation and migration of gastric cancer cells.
Objective To investigate the expression of micromolecular non-coding RNA-20a(miR-20a) in gastric cancer and its effect on the malignant transformation of gastric cancer cells, and to study its mechanism.Methods A total of 45 samples of fresh gastric cancer and paracancerous tissues were collected from 45 patients pathologically diagnosed in Kunshan Hospital Affiliated to Jiangsu University from September 2014 to September 2017. Total RNA was extracted and the expression of miR-20 a in gastric cancer tissues and paracancerous tissues was detected by qRT-PCR. Liposomal transfection was performed with oligonucleotide fragment NC-inhibitor and miR-20a-inhibitor to MGC-803, then the expression of miR-20 a in transfected MGC-803 cells was detected by qRT-PCR. Cell cloning assay and Transwell assay were used to detect the proliferation and migration of MGC-803 cells after transfection of miR-20a-inhibitor. Western blot was used to detect the change in cellular AKT pathway activity and the expression of epithelial to mesenchymal transition(EMT) markers in MGC-803 cells before and after transfection. Results The expression level of miR-20a/U6 in cancer tissues was 1.12±0.10, which was significantly higher than 0.30±0.04 in the paracancerous tissues, and the difference was statistically significant(P<0.01). The expression level of miR-20a/U6 in the miR-20a-inhibitor transfection group was 0.65±0.04, which was significantly lower than 1.01±0.14 in the NC-inhibitor transfection group, and the difference was statistically significant(P<0.01). The number of cloned cells in the miR-20a-inhibitor transfection group was 70±3, which was significantly lower than 110±4 in the NC-inhibitor transfection group(P<0.05). The number of Transwell cells in the miR-20a-inhibitor transfection group was 35±4, which was significantly lower than 53±6 in the NC-inhibitor transfection group(P<0.05). Compared with the miR-20a-inhibitor transfection group, the activation of the AKT pathway was weakened in the NC-inhibitor group, while the expression of E-cadherin was attenuated in the epithelial cells, and the expression of N-cadherin and Vimentin was enhanced.Conclusion The expression level of miR-20 a in cancer tissues is significantly increased compared with paracancerous tissues. miR-20 a can induce malignant transformation of gastric cancer cells by promoting the proliferation and migration of gastric cancer cells.
引文
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[2] BARTEL DP. MicroRNAs:genomics, biogenesis, mechanism, and function[J]. Cell, 2004, 116(2):281-297.
[3] LEE RC, FEINBAUM RL, AMBROS V, et al. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[J]. Cell, 1993, 75(5):843-854.
[4] LEE YS, DUTTA A. MicroRNAs in cancer[J]. Annu Rev Pathol,2009, 4(1):199-227.
[5] QIANG XF, ZHANG ZW, LIU Q, et al. miR-20a promotes prostate cancer invasion and migration through targeting ABL2[J]. J Cell Biochem, 2014, 115(7):1269-1276.
[6] CHANG Y, LIU C, YANG J, et al. MiR-20a triggers metastasis of gallbladder carcinoma[J]. J Hepatol, 2013, 59(3):518-527.
[7] WANG Z, WANG B, SHI Y, et al. Oncogenic miR-20a and miR-106a enhance the invasiveness of human glioma stem cells by directly targeting TIMP-2[J]. Oncogene, 2015, 34(11):1407-1419.
[8] ZHAO S, YAO D, CHEN J, et al. MiR-20a promotes cervical cancer proliferation and metastasis in vitro and in vivo[J]. PLoS One, 2015,10(3):e0120905.
[9] XU T, JING C, SHI Y, et al. microRNA-20a enhances the epithelial-to-mesenchymal transition of colorectal cancer cells by modulating matrix metalloproteinases[J]. Exp Ther Med, 2015, 10(2):683-688.
[10] DU Y, ZHU M, ZHOU X, et al. miR-20a enhances cisplatin resistance of human gastric cancer cell line by targeting NFKBIB[J]. Tumour Biol, 2016, 37(1):1261-1269.
[11] ZHOU X, ZHU W, LI H, et al. Diagnostic value of a plasma microRNA signature in gastric cancer:a microRNA expression analysis[J].Sci Rep, 2015, 5:11251.
[12] LIU R, ZHANG C, HU Z, et al. A five-microRNA signature identified from genome-wide serum microRNA expression profiling serves as a fingerprint for gastric cancer diagnosis[J]. Eur J Cancer, 2011,47(5):784-791.
[13] WANG M, GU H, WANG S, et al. Circulating mi R-17-5p and mi R-20a:molecular markers for gastric cancer[J]. Mol Med Rep, 2012, 5(6):1514-1520.
[14] ZENG X, XIANG J, WU M, et al. Circulating miR-17, miR-20a,miR-29c, and miR-223 combined as non-invasive biomarkers in nasopharyngeal carcinoma[J]. PLoS One, 2012, 7(10):e46367.
[15] MIRZAEI HR, SAHEBKAR A, MOHAMMADI M, et al. Circulating microRNAs in hepatocellular carcinoma:potential diagnostic and prognostic biomarkers[J]. Curr Pharm Des, 2016, 22(34):52575269.
[16] HUANG F, WANG M, YANG T, et al. Gastric cancer-derived MSC-secreted PDGF-DDpromotes gastric cancer progression[J]. J Cancer Res Clin Oncol, 2014, 140(11):1835-1848.
[17] SAVAGNER P. Epithelial-mesenchymal transitions:from cell plasticity to concept elasticity[J]. Curr Top Dev Biol, 2015, 112(1):273-300.
[18] GAO J, ZHOU XL, KONG RN, et al. microRNA-126 targeting PIK3R2 promotes rheumatoid arthritis synovial fibro-blasts proliferation and resistance to apoptosis by regulating PI3K/AKT pathway[J]. Ex Mol Pathol, 2016, 100(1):192-198.