lncRNA RP11-635L1.2在膀胱癌组织中的表达及对膀胱癌细胞增殖和侵袭的影响

Expression of lncRNA RP11-635L1.2 in Bladder Cancer Tissue and Its Effect on the Proliferation and Invasion of Bladder Cancer Cells

目的探讨在膀胱癌组织中长链非编码RNA(lncRNA)RP11-635L1.2的表达变化及对膀胱癌细胞增殖和侵袭的影响。方法通过GEPIA数据库分析膀胱癌组织中RP11-635L1.2的表达变化。行qRT-PCR检测RP11-635L1.2在膀胱癌细胞系MGH-U3、253J、J82、T24中的相对表达量。选择RP11-635L1.2表达最低的膀胱癌细胞(J82细胞),分为NC组和RP11-635L1.2组,分别转染空载质粒和RP11-635L1.2过表达质粒。qRT-PCR检测验证转染效率。采用CCK-8法和Transwell实验分别观察RP11-635L1.2对膀胱癌细胞增殖和侵袭的影响。利用LncCeRBase数据库预测RP11-635L1.2靶基因,采用双荧光素酶报告实验验证RP11-635L1.2和miR-373-3p的靶向性。qRT-PCR检测2组细胞中miR-373-3p的相对表达量。行Western blot实验检测2组细胞中EGFR/AKT信号通路蛋白的表达情况。结果RP11-635L1.2在膀胱癌组织中表达水平明显低于癌旁正常组织(P<0.01)。RP11-635L1.2在膀胱癌细胞系MGH-U3、253J、J82、T24中的表达水平明显低于在永生化膀胱上皮细胞SV-HUC-1中的表达水平(P<0.05,P<0.01),且在J82细胞中的表达水平最低。相比NC组,上调RP11-635L1.2后,J82细胞增殖能力及侵袭能力显著下降(P<0.05,P<0.01)。RP11-635L1.2和miR-373-3p具有靶向性(P<0.01)。相比NC组,RP11-635L1.2组miR-373-3p表达被明显抑制(P<0.01),EGFR/AKT信号通路蛋白p-EGFR、p-AKT、p-ERK、p-JAK2、p-STAT3表达量明显下降(P<0.01)。结论RP11-635L1.2在膀胱癌组织和细胞系中呈现低表达,上调RP11-635L1.2通过与miR-373-3p结合抑制膀胱癌细胞的增殖、侵袭,其可能成为膀胱癌新的诊断标志物和治疗靶点。

Objective To investigate changes in the expression of long non-coding RNA(lncRNA)RP11-635 L1-2 in bladder cancer tissues and its effects on the proliferation and invasion of bladder cancer cells.Methods The changes in the expression of RP11-635 L1-2 in bladder cancer was analyzed by GEPIA database.The relative expression levels of RP11-635 L1-2 in bladder cancer cell lines MGH-U3,253 J,J82 and T24 were detected by qRT-PCR.Bladder cancer cells(J82 cells)with the lowest expression of RP11-635 L1-2 were selected and divided into NC group and RP11-635 L1-2 group,and transfected with no-load plasmid and RP11-635 L1-2 overexpression plasmid,respectively.The transfection efficiency was detected by qRT-PCR.CCK-8 assay and Transwell assay were used to observe the effects of RP11-635 L1-2 on the proliferation and invasion of bladder cancer cells.LncCeRBase database was used to predict RP11-635 L1-2 target genes,and dual luciferase reporting assay was used to verify the targeting of RP11-635 L1-2 and miR-373-3 p.The relative expression of miR-373-3 p in the two groups of cells was detected by qRT-PCR.Western blot assay was performed to detect the expression of EGFR/AKT signaling pathway protein in the two groups.Results The expression level of RP11-635 L1-2 in bladder cancer tissues was significantly lower than that in adjacent tissues(P<0.01).The expression level of RP11-635 L1-2 in bladder cancer cell lines MGH-U3,253 J,J82 and T24 was significantly lower than that in immortalized bladder epithelial cell SV-HUC-1(P<0.05,P<0.01),and the expression level of RP11-635 L1-2 was the lowest in J82 cells.Compared with NC group,the proliferation ability and invasion ability of J82 cells were significantly decreased after up-regulation of RP11-635 L1-2(P<0.05,P<0.01).Rp11-635 l1-2 and miR-373-3 p were targeted(P<0.01).Compared with the NC group,the expression of miR-373-3 p in RP11-635 L1-2 group was significantly inhibited(P<0.01),and the expression levels of EGFR/AKT signaling pathway proteins P-EGFR,p-Akt,p-ERK,p-JAK2 and P-Stat3 were significantly decreased(P<0.01).Conclusion RP11-635 L1-2 is low expressed in bladder cancer tissues and cell lines,and up-regulated RP11-635 L1-2 inhibits the proliferation and invasion of bladder cancer cells through binding with miR-373-3 p,which may become a new diagnostic marker and therapeutic target for bladder cancer.