lncRNA RPL34-AS1通过靶向调控miR-575表达对卵巢癌SKOV3细胞增殖和凋亡的影响

目的探究RPL34-AS1对卵巢癌细胞增殖、迁移的影响及其作用机制。 方法取对数生长期SKOV3细胞用无血清培养基同步化12 h，将pcDNA、pcDNA-RPL34-AS1、si-NC、si-RPL34-AS1、anti-miR-NC、anti-miR-575转染至SKOV3细胞中，分别记为pcDNA组、pcDNA-RPL34-AS1组、si-NC组、si-RPL34-AS1组、anti-miR-NC组、anti-miR-575组；将pcDNA-RPL34-AS1与miR-NC、miR-575分别共转染至SKOV3细胞中，记为pcDNA-RPL34-AS1+miR-NC组、pcDNA-RPL34-AS1+miR-575组。实时荧光定量PCR （RT-qPCR）检测临床组织标本及转染后各组细胞中RPL34-AS1和miR-575的表达水平；双荧光素酶报告实验检测RPL34-AS1和miR-575的靶向关系；四甲基偶氮唑盐比色法（MTT）检测细胞存活率；流式细胞术检测细胞凋亡；蛋白质印迹（Western blot）法检测细胞周期蛋白D1（Cyclin D1）、细胞周期蛋白依赖性激酶抑制剂1A （p21）、B细胞淋巴瘤/白血病-2 （Bcl-2）、Bcl-2相关X蛋白（Bax）蛋白表达水平。两组间比较采用独立样本t检验，多组间比较采用单因素方差分析，组间两两比较采用LSD-t检验。 结果与癌旁组织相比，卵巢癌组织中RPL34-AS1表达水平降低（1.00±0.08比0.47±0.05），miR-575表达水平升高（1.01±0.07比3.12±0.28）（P < 0.05）。转染si-RPL34-AS1后，细胞活性升高（48 h：0.68±0.06比0.55±0.05；72 h：0.99±0.08比0.71±0.06），G1期细胞所占比例降低（13.42±1.38比32.15±2.11），S期细胞所占比例升高（53.75±5.22比34.69±3.41），细胞凋亡率降低（4.31±0.42比9.25±0.91），CyclinD1、Bcl-2表达水平升高（0.92±0.08比0.71±0.07；0.86±0.07比0.61±0.06），p21、Bax表达水平降低（0.13±0.02比0.29±0.03；0.19±0.02比0.31±0.03） （P均< 0.05）。RPL34-AS1靶向调控miR-575，过表达RPL34-AS1或抑制miR-575后可抑制细胞活性，阻滞细胞周期和促进细胞凋亡。miR-575过表达逆转了RPL34-AS1过表达对卵巢癌SKOV3细胞增殖抑制和凋亡促进的作用。 结论过表达RPL34-AS1可抑制卵巢癌SKOV3细胞增殖，促进细胞凋亡，其机制可能与下调miR-575有关。

Effects of lncRNA RPL34-AS1 on the proliferation and apoptosis of ovarian cancer cells by targeted regulation of miR-575 expression

ObjectiveTo investigate the effect of RPL34-AS1 on the proliferation and apoptosis of ovarian cancer cells and whether its mechanism is related to miR-575. MethodSKOV3 cells in logarithmic growth phase were synchronized with serum-free mediumfor 12 h and pcDNA, pcDNA-RPL34-AS1, si-NC, si-NC, si-RPL34-AS1, si-RPL34-AS1, anti-miR-NC, anti-anti-miR-NC, anti-miR-575 were transfected into SKOV3 cells, respectively labeled as pcDNA group, pcDNA-RPL34-AS1 group, si-NC group, si-NC group, si-RPL34-AS1 group, si-RPL34-AS1 group, anti-miR-NC group, anti-miR-NC group, anti-miR-575 group; PcDNA-RPL34-AS1 was co-transfected into SKOV3 cells with miR-NC and miR-575, respectively, labeled as pcDNA-RPL34-AS1+miR-NC group, pcDNA-RPL34-AS1+miR-575 group. Real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the expression level of RPL34-AS1 and miR-575 in clinical tissue specimens and cells of each group after transfection; dual luciferase reporter assay was used to detect the targeting relationship between RPL34-AS1 and miR-575; tetramethylazozolium salt colorimetric method (MTT) was used to detect cell viability; flow cytometry was used to detect apoptosis; Western Blot was used to detect expression of Cyclin D1, Cyclin-dependent kinase inhibitor 1A (p21) , B-cell lymphoma/leukemia-2 (Bcl-2) , Bcl-2 related X protein (Bax) proteins.The comparison between the two groups was performed using the independent sample t test, and the comparison between multiple groups was performed using the one-way analysis of variance; LSD-t test was used for pairwise comparison between groups. ResultsCompared with adjacent tissues, the expression level of RPL34-AS1 was decreased in ovarian cancer tissues, the expression level ofmiR-575 was increased (1.01±0.07 vs 3.12±0.28) (P < 0.05) . After transfection with si-RPL34-AS1, cell viability was increased (48 h: 0.68±0.06 vs 0.55±0.05; 72 h: 0.99±0.08 vs 0.71±0.06) , and the proportion of G1 phase cells was decreased (13.42±1.38 vs 32.15±2.11) , the proportion of S-phase cells was increased (53.75±5.22 vs 34.69±3.41) , the apoptosis rate was decreased (4.31±0.42 vs 9.25±0.91) , and the expression level of CyclinD1 and Bcl-2 were increased (0.92±0.08 vs 0.71±0.07; 0.86±0.07 vs 0.61±0.06) , the expression level of p21 and Bax were decreased (0.13±0.02 vs 0.29±0.03; 0.19±0.02 vs 0.31±0.03) (all P < 0.05) . RPL34-AS1 targets and regulates miR-575. Overexpression of RPL34-AS1 or inhibition of miR-575 can inhibit cell activity, block the cell cycle and promote apoptosis. MiR-575 overexpression reversed RPL34-AS1 overexpression effect on ovarian cancer SKOV3 cell proliferation inhibition as well as its apoptosis promotion. ConclusionOverexpression of RPL34-AS1 can inhibit the proliferation of ovarian cancer SKOV3 cells and promote cell apoptosis, which may be related to the down-regulation of miR-575.