摘要
目的 探讨微小RNA-1291（miR-1291）对肾癌细胞中锌指蛋白8（PHF8）基因表达的调控作用和对肾癌细胞周期及增殖的影响。方法 实时荧光定量聚合酶链反应（qRT-PCR）检测肾癌细胞株OSRC-2、ACHN、A498、786-O和人类近端肾小管上皮细胞HK-2中miR-1291表达水平，以表达量最低的肾癌细胞株为实验对象分别转染miR-1291（miR-1291组）和miR-NC（miR-NC组）。qRT-PCR检测转染后细胞中miR-1291和PHF8 mRNA的表达水平。Western blotting检测PHF8、细胞周期蛋白依赖性激酶6（CDK6）和Cyclin D1蛋白的表达水平。双荧光素酶报告基因系统检测miR-1291对PHF8转录活性的影响。流式细胞术检测细胞周期分布。四甲基偶氮唑蓝（MTT）实验和集落形成实验分别检测细胞活力和增殖能力。结果肾癌细胞株OS-RC-2、ACHN、A498、786-O和人类近端肾小管上皮细胞HK-2中miR-1291的表达量分别为0.64±0.17、0.60±0.15、0.29±0.08、0.63±0.08、1.01±0.17，差异有统计学意义（F=13.790，P〈0.001）。与肾癌细胞株OS-RC2、ACHN、786-O相比，A498细胞株中的miR-1291表达量最低（P=0.002，P=0.006，P=0.003）。转染后的miR-NC组和miR1291组A498细胞中miR-1291表达量分别为1.00±0.03和775.25±329.91，差异有统计学意义（t=4.694，P=0.003）；PHF8 mRNA表达量分别为1.00±0.11和0.57±0.18，差异有统计学意义（t=4.122，P=0.006）。Western blotting实验结果与qRT-PCR结果一致，且CDK6和Cyclin D1蛋白表达明显降低。双荧光素酶报告基因显示miR-1291可以直接作用于靶基因PHF8的3′-非翻译区,抑制荧光素酶活性。与miR-NC组比较，转染miR-1291后肾癌细胞在S期（23.40±4.29∶32.19±2.64，t=3.491，P=0.013）和G2M期（14.38±4.05∶25.59±6.01，t=3.095，P=0.021）的细胞比例下降，在G0-G1期的细胞比例升高（62.22±7.56∶42.22±5.23，t=4.351，P=0.005）。MTT实验显示，转染miR-1291的细胞活力明显下降。集落形成实验显示，A498细胞在miR-NC组和miR-1291组形成的集落数分别为246.64±39.94和87.34±21.93，差异有统计学意义（t=6.993，P〈0.001）。结论miR-1291在肾癌细胞株中表达显著下降，miR-1291可通过靶向干扰PHF8基因的表达显著抑制肾癌细胞的增殖，可能有助于开发新的肾癌治疗靶点。
Objective To investigate the effects of microRNA-1291 （miR-1291） on the expression of Zinc finger protein 8 （PHF8） gene in renal cell carcinoma and its effect on cell cycle and proliferation of renal cell carcinoma. MethodsRealtime fluorescence quantitative polymerase chain reaction （qRT-PCR） was used to detect the expression of miR1291 in renal cell carcinoma cell lines OS-RC-2, ACHN, A498, 786-O and human proximal tubular epithelial cells HK-2. miR-1291 （miR-1291 group） and miR-NC （miR-NC group） were transfected into the renal cell lines with the lowest expression of miR1291. qRTPCR was used to detect the expression of miR-1291 and PHF8 mRNA in the transfected cells. The expression levels of PHF8, Cyclindependent kinase 6 （CDK6） and Cyclin D1 were detected by Western blotting. The effect of miR-1291 on the transcriptional activity of PHF8 was detected by double luciferase reporter gene system. Flow cytometry was used to detect cell cycle distribution. Methyl thiazolyl tetrazolium （MTT） assay and colony formation assay were used to detect cell viability and proliferation. ResultsThe expressions of miR1291 in renal carcinoma cell lines OS-RC-2, ACHN, A498, 786-O and human proximal renal tubular epithelial cell HK2 were 0.64±0.17, 0.60±0.15, 0.29±0.08, 0.63±0.08 and 1.01±0.17 respectively, with a significant difference （F=13.790, P〈0.001）. Compared with renal carcinoma cell lines OS-RC-2, ACHN and 786-O, the expression level of miR-1291 in A498 cell line was the lowest （P=0.002, P=0.006, P=0.003）. The expression levels of miR1291 in A498 cell lines of miRNC group and miR1291 group were 1.00±0.03 and 775.25±329.91 respectively, with a significant difference （t=4.694, P=0.003）; and the expression levels of PHF8 mRNA were 1.00±0.11 and 0.57±0.18 respectively, with a significant difference （t=4.122, P=0.006）. The results of Western blotting were consistent with the results of qRTPCR, and the expressions of CDK6 and Cyclin D1 were significantly decreased. The double luciferase reporter gene showed that miR1291 could directly inhibit the activity of luciferase in the 3′ untranslated region of target gene PHF8. Compared with miR-NC group, the proportion of renal carcinoma cells in S phase （23.40±4.29 vs. 32.19±2.64; t=3.491, P=0.013） and G2-M phase （14.38±4.05 vs. 25.59±6.01; t=3.095, P=0.021） decreased; and the proportion of cells in G0-G1 phase increased （62.22±7.56 vs. 42.22±5.23, t=4.351, P=0.005）. MTT assay showed that the cell viability of miR1291 was significantly decreased. Colony formation experiments showed that the numbers of colonies formed by A498 cells in miR-NC group and miR1291 group were 246.64±39.94 and 87.34±21.93 respectively, with a significant difference （t=6.993, P〈0.001）. ConclusionThe expression of miR1291 is significantly decreased in renal cancer cell lines. miR1291 can significantly inhibit the proliferation of renal cell carcinoma cells by targeting interfering PHF8 gene expression, which may contribute to the development of new renal cancer target.
作者
叶志华
黄耿
付金伦
桂定文
Ye Zhihua;Huang Geng;Fu Jinlun;Gui Dingwen(Department of Urological Surgery, Huangshi Central Hospital of Edong Healthcare Group （ Affiliated Hospital of Hubei Polytechnic Univer- sity;Hubei Key Laboratory of Kidney Disease Pathogenesis and Intervention, Huangshi 435000, Chin)
出处
《国际肿瘤学杂志》
CAS
2018年第3期129-133,共5页
Journal of International Oncology
关键词
肾肿瘤
微RNAS
细胞周期
细胞增殖
锌指蛋白8
Kidney neoplasms
MicroRNAs
Cell cycle
Cell proliferation
Zinc finger protein 8
