Du Xinming, Liu Bing, Luan Xuerong, Cui Qing, Li Leping
Department of Gastrointestinal Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China.
Department of Gastrointestinal Surgery, Zibo Central Hospital, Zibo, Shandong 250020, P.R. China.
Exp Ther Med. 2018 Jan;15(1):599-605. doi: 10.3892/etm.2017.5354. Epub 2017 Oct 23.
Chemotherapy is an important treatment modality for gastric cancer, and multidrug resistance (MDR) represents a major obstacle for successful cancer chemotherapy. There is a lack of research on whether microRNA (miR)-30a regulation affects the chemosensitivity of resistant gastric cancer cells, and mechanisms underlying the effects of miR-30a on drug resistance and cell autophagy require further investigation. In the present study, the expression of miR-30a and its effects in cisplatin (CDDP)-resistant human gastric cancer cells were investigated. A CDDP-resistant variant of the SGC-7901 cell line (SGC-7901/CDDP) was established by exposing the cells to gradually increasing drug concentrations, and miR-30a expression was detected by reverse transcription-semi quantitative polymerase chain reaction (RT-sqPCR). To examine the effect of miR-30a expression in the SGC-7901/CDDP cells, miR30a mimics or negative control miRNA were transfected into the cells, and a Cell Counting Kit-8 assay was performed to analyze the chemosensitivity of the different cell groups. RT-sqPCR and western blot analysis were also used to measure mRNA and P-glycoprotein expression, and the light chain (LC)3-II/LC3-I ratio. Furthermore, apoptosis induced by the chemotherapeutic CDDP in the different groups was assessed using flow cytometry. The results demonstrated that low expression of miR-30a was associated with chemoresistance in gastric cancer cells, and in the chemoresistant cell line SGC7901/CDDP, CDDP-induced apoptosis was weakened. Additionally, it was demonstrated that the LC3-II/LC3-I ratio was elevated in SGC7901/CDDP cells compared with chemosensitive SGC7901 cells (P<0.001), which could be attenuated by upregulating miR-30a expression (P<0.001 vs. SGC7901/CDDP control cells). These results suggested that autophagy may contribute to drug resistance in gastric cancer cells, and that the reduction of LC3-II in response to miR-30a overexpression may inhibit chemoresistance-associated autophagy in gastric cancer cells.
化疗是胃癌的一种重要治疗方式，而多药耐药（MDR）是癌症化疗成功的主要障碍。关于微小RNA（miR）-30a调控是否影响耐药胃癌细胞的化疗敏感性，目前缺乏相关研究，miR-30a对耐药性和细胞自噬影响的机制尚需进一步研究。在本研究中，对miR-30a在顺铂（CDDP）耐药的人胃癌细胞中的表达及其作用进行了研究。通过将SGC-7901细胞系暴露于逐渐增加的药物浓度中，建立了一种CDDP耐药变体（SGC-7901/CDDP），并通过逆转录-半定量聚合酶链反应（RT-sqPCR）检测miR-30a的表达。为了检测miR-30a在SGC-7901/CDDP细胞中的表达效果，将miR-30a模拟物或阴性对照miRNA转染到细胞中，并用细胞计数试剂盒-8检测法分析不同细胞组的化疗敏感性。还使用RT-sqPCR和蛋白质印迹分析来检测mRNA和P-糖蛋白的表达，以及轻链（LC）3-II/LC3-I的比值。此外，使用流式细胞术评估不同组中化疗药物CDDP诱导的细胞凋亡。结果表明，miR-30a的低表达与胃癌细胞的化疗耐药相关，在化疗耐药细胞系SGC7901/CDDP中，CDDP诱导的细胞凋亡减弱。此外，结果表明，与化疗敏感的SGC7901细胞相比，SGC7901/CDDP细胞中LC3-II/LC3-I的比值升高（P<0.001），而上调miR-30a的表达可使其降低（与SGC7901/CDDP对照细胞相比，P<0.001）。这些结果表明，自噬可能导致胃癌细胞的耐药，并且miR-30a过表达导致的LC3-II减少可能抑制胃癌细胞中与化疗耐药相关的自噬。
