| 张海生,黄河,徐震.微小 RNA-203a-3p下调 Mink相关肽 1基因表达抑制非小细胞肺癌细胞转移潜能的实验研究[J].安徽医药,2021,25(10):2080-2085. |
| 微小 RNA-203a-3p下调 Mink相关肽 1基因表达抑制非小细胞肺癌细胞转移潜能的实验研究 |
| MicroRNA-203a-3p (miR-203a-3p) down-regulates the expression of Mink-related peptide 1 (KCNE2) gene to inhibit the metastasis potential of non-small cell lung cancer cells |
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| DOI：10.3969/j.issn.1009-6469.2021.10.040 |
| 中文关键词: 癌，非小细胞肺 微小 RNA-203a-3p Mink相关肽 1 细胞运动 肿瘤侵润 细胞增殖 |
| 英文关键词: Carcinoma,non-small-celllung miR-203a-3p KCNE2 Cellmovement Neoplasminvasiveness Cellproliferation |
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| 中文摘要: |
| 目的探讨微小 RNA-203a-3p(miR-203a-3p)对非小细胞肺癌恶性生物学行为的影响及机制。方法本研究于 2018年 7月至 2019年 6月进行，正常肺上皮细胞 BEAS-2B以及肺癌细胞 H1299、SPC-A-1、NC-H446购自中国科学院上海细胞库；实时荧光定量 PCR(qRT-PCR)检测 miR-203a-3p和 Mink相关肽 1(KCNE2)mRNA表达水平；蛋白质印迹法检测 KCNE2蛋白表达；将 SPC-A-1细胞分为 miR-203a-3p(转染 miR-203a-3p模拟物)组、 miR-con组(转染 miR-203a-3p模拟物阴性对照)、 si-KCNE2组(转染 KCNE2干扰表达载体)、 si-con组(转染 KCNE2干扰表达载体阴性对照)、 miR-203a-3p+pcDNA组(转染 miR-203a-3p模拟物和 KCNE2过表达载体阴性对照)、 miR-203a-3p+pcDNA-KCNE2组(转染 miR-203a-3p模拟物和 KCNE2过表达载体)；四甲基偶氮唑盐比色法( MTT)法检测细胞活性； Transwell检测细胞迁移和侵袭；蛋白质印迹法检测细胞周期蛋白 D1(Cyclin D1)、细胞周期素依赖蛋白激酶 6(CDK6)、基质金属蛋白酶( MMP)-2、MMP-9蛋白表达；双荧光素酶报告实验检测 miR-203a-3p和 KCNE2的靶向关系。结果与 BEAS-2B细胞相比，肺癌细胞 H1299、SPC-A-1、NC-H446中 miR-203a-3p表达水平显著降低［( 0.27±0.05)、(0.18±0.07)、(0.25±0.04)比( 1.00±0.24)］KCNE2 mRNA表达水平显著升高［( 4.37±0.43)、(5.24±0.34)、(6.39± 0.35)比( 1.00±0.08)］，KCNE2蛋白表达水平显著升高［(，0.73±0.07)、(0.75±0.06)、(0.59±0.05)比( 0.38±0.04)］(P<0.05)。与 miR-NC组相比， miR-203a-3p组 SPC-A-1细胞活性降低［48 h为( 0.42±0.05)比( 0.55±0.05)72 h为( 0.63±0.07)比( 0.84±0.09)］迁移细胞数减少［(79.90±5.21)个比(172.13±8.57)个］，侵袭细胞数减少［(45.89±3.50)个，比( 101.83±9.55)个］； Cyclin D1、，CDK6、MMP-2、MMP-9蛋白表达水平降低( P<0.05)。与 si-NC组相比， si-KCNE2组 SPC-A-1细胞活性降低［48 h为( 0.44±0.05)比( 0.58±0.05)72 h为( 0.63±0.08)比( 0.94±0.09)］，迁移细胞数减少［( 71.71±4.52)个比( 174.56±13.67)个］侵袭细胞数减少［( 35.68±3.27)个，比( 81.37±5.46)个］； Cyclin D1、CDK6、MMP-2、MMP-9蛋白表达水平降低( P<0.05)。 miR-20，3a-3p靶向调控 KCNE2的表达； KCNE2过表达部分逆转 miR-203a-3p对肺癌细胞 SPC-A-1的作用。结论 miR-203a-3p可通过下调 KCNE2抑制肺癌细胞的增殖、迁移和侵袭。 |
| 英文摘要: |
| Objective To explore the effect and mechanism of microRNA-203a-3p (miR-203a-3p) on the malignant biological behavior of non-small cell lung cancer.Methods This research was conducted from July 2018 to June 2019. Normal lung epithelial cells BEAS-2B and lung cancer cells H1299, SPC-A-1, NC-H446 were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences; real-time fluorescent quantitative PCR (qRT-PCR) was used to detect expression levels of miR-203a-3p and Mink-related peptide 1 (KCNE2) mRNA; Western blotting method to detect KCNE2 protein expression; SPC-A-1 cells were assigned into miR-203a3p (transfection miR-203a-3p mimic) group, miR-con group (transfection miR-203a-3p mimic negative control), si-KCNE2 group (transfected with KCNE2 interference expression vector), si-con group (transfected KCNE2 interference expression vector negative control), miR-203a-3p+pcDNA group (transfected with miR-203a-3p mimic and KCNE2 overexpression vector negative control), miR-203a-3p+ pcDNA-KCNE2 group (transfected with miR-203a-3p mimic and KCNE2 overexpression vector); tetramethylazolium salt colorimetricmethod (MTT) method to detect cell activity; Transwell method to detect cell migration and invasion; Western blot method to detect Cyclin D1, Cyclin-dependent protein kinase 6 (CDK6), matrix metalloproteinase (MMP)-2, MMP-9 protein expressions; dual luciferase reporter experiment to detect the targeting relationship between miR-203a-3p and KCNE2.Results Compared with BEAS-2B cells, the expression level of miR-203a-3p in lung cancer cells H1299, SPC-A-1, NC-H446 was significantly reduced [(0.27±0.05), (0.18±0.07), (0.25±0.04) vs. (1.00±0.24)], KCNE2 mRNA expression level was increased significantly [(4.37±0.43), (5.24±0.34), (6.39±0.35) vs. (1.00±0.08)], KCNE2 protein expression level was significantly increased [(0.73 ±0.07), (0.75±0.06), (0.59±0.05) vs. (0.38±0.04)] (P< 0.05). Compared with the miR-NC group, the cell viability of SPC-A-1 in the miR-203a-3p group was reduced [48h: (0.42±0.05) vs. (0.55±0.05), 72h: (0.63±0.07) vs. (0.84±0.09) ], the number of migrating cells was decreased [(79.90±5.21) vs. (172.13±8.57)], the number of invasive cells was decreased [(45.89±3.50) vs. (101.83±9.55)]; the expression levels of Cyclin D1, CDK6, MMP-2, MMP-9 protein were decreased (P<0.05). Compared with the si-NC group, the cell viability of SPC-A-1 in the si-KCNE2 group was reduced [48h: (0.44±0.05) vs. (0.58±0.05), 72h: (0.63±0.08) vs. (0.94±0.09)], the number of migrating cells was decreased [(71.71±4.52) vs. (174.56± 13.67)], the number of invasive cells was decreased [(35.68±3.27) vs. (81.37±5.46)]; Cyclin D1, CDK6, MMP-2, MMP -9 protein expression levels were decreased (P<0.05). miR-203a-3p targeted and regulated the expression of KCNE2; KCNE2 overexpression partially reversed the effect of miR-203a-3p on lung cancer cells SPC-A-1.Conclusion miR-203a-3p can inhibit the proliferation, migration and invasion of lung cancer cells by down-regulating KCNE2. |
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