沉默LncRNA OIP5-AS1通过上调miR-34c-5p表达增加A549R细胞放射敏感性

目的 探讨LncRNA OIP5-AS1对非小细胞肺癌放射抗拒细胞A549R的放射敏感性影响及作用机制。方法 X射线6Gy照射5次A549细胞建立放射抗拒细胞A549R。qRT-PCR检测A549、A549R细胞OIP5-AS1和miR-34c-5p表达水平。转染OIP5-AS1抑制剂或miR-34c-5p模拟物至A549R细胞,OIP5-AS1过表达质粒至A549细胞。流式细胞仪检测细胞凋亡,克隆形成实验检测细胞放射敏感性,蛋白印记检测p-Chk2和p-ATM蛋白表达水平。双荧光素酶实验验证OIP5-AS1与miR-34c-5p之间关系。结果 与A549细胞比,A549R细胞中OIP5-AS1表达上调(1.97±0.11︰1.01±0.05,P<0.05),miR-34c-5p表达下调(0.43±0.02︰1.02±0.06,P<0.05)。沉默OIP5-AS1+6Gy组A549R细胞中p-Chk2和p-ATM蛋白水平低于沉默对照+6Gy组(0.43±0.03︰1.39±0.15和0.51±0.05︰1.21±0.11,P<0.05),但凋亡率增加(13.29±1.25)%︰(28.47±2.31)%,P<0.05]。过表达OIP5-AS1+6Gy组A549细胞中p-Chk2和p-ATM蛋白水平高于过表达对照+6Gy组(1.23±0.13︰0.75±0.06和1.08±0.11︰0.59±0.04,P<0.05)。抑制miR-34c-5p表达逆转了沉默OIP5-AS1对A549R细胞存活分数影响,增敏比为1.42。OIP5-AS1负调控miR-34c-5p表达。结论 沉默OIP5-AS1通过上调miR-34c-5p表达增强A549R细胞放射敏感性,为放疗提供潜在的靶点。

Silencing LncRNA OIP5-AS1 increases radiosensitivity of non-small cell lung cancer A549 cell line by up-regulating miR-34c-5p expression

Objective To investigate the effect of LncRNA OIP5-AS1 on radiosensitivity of non-small cell lung cancer (NSCLC) cells and its mechanism. Methods The radiation-resistant cell A549R was established by using A549 cells irradiated by X-ray 6Gy in 5 fractions. The expression levels of OIP5-AS1 and miR-34c-5p in A549 and A549R cells were detected by qRT-PCR. OIP5-AS1 inhibitor or miR-34c-5p mimetic was transfected into A549R cells, or OIP5-AS1 overexpression plasmid was transfected into A549 cells. Cell apoptosis was detected by flow cytometry. Cell radiosensitivity was analyzed by colony formation assay. The expression levels of p-Chk2 and p-ATM proteins were measured by Western blot. Dual luciferase assay was adopted to verify the relationship between OIP5-AS1 and miR-34c-5p. Results Compared with A549 cells, the expression of OIP5-AS1 was significantly up-regulated in A549R cells (1.97±0.11 vs.1.01±0.05,P<0.05), whereas the expression of miR-34c-5p was remarkably down-regulated (0.43±0.02 vs.1.02±0.06,P<0.05). The expression levels of p-Chk2 and p-ATM proteins in A549R cells in the silencing OIP5-AS1+6Gy group were significantly lower (0.43±0.03 vs.1.39±0.15,0.51±0.0 5 vs.1.21± 0.11,both P<0.05), whereas the apoptotic rate was significantly higher than those in the silencing control+6Gy group (13.29±1.25)% vs. (28.47±2.31)%,P<0.05)]. The expression levels of p-Chk2 and p-ATM proteins in A549 cells in overexpressing OIP5-AS1+6Gy group were significantly higher than those in overexpression control+6Gy group (1.23±0.13 vs.0.75±0.06,1.08±0.11 vs.0.59±0.04,both P<0.05). Inhibiting miR-34c-5p expression reversed the effect of silencing OIP5-AS1 on survival fraction of A549R cells (SER=1.42). OIP5-AS1 negatively regulated the expression of miR-34c-5p. Conclusion Silencing OIP5-AS1 enhances the radiosensitivity of radiation-resistant A549 cells by up-regulating the expression of miR-34c-5p, providing a potential target for radiotherapy of NSCLC cells.