【摘要】 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。更多还原

【Abstract】 Objective To investigate the effect of RPL34-AS1 on the proliferation and apoptosis of ovarian cancer cells and whether its mechanism is related to miR-575.Method SKOV3 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.Results Compared 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.Conclusion Overexpression 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.更多还原