miR-181c-5p调控BIRC5对前列腺癌细胞生物学行为的影响

Effects of miR-181c-5p on biological behaviors of prostate cancer cells by regulating BIRC5

目的探讨miR-181c-5p对前列腺癌细胞生物学行为的影响及其作用机制。方法通过TCGA数据库中前列腺癌数据分析BIRC5、miR-181c-5p与前列腺癌病理关系,采用miRNA靶基因预测数据库分析miR-181c-5p与BIRC5靶结合位点并使用双色荧光素酶活性实验验证,Western blot检测miR-181c-5p过表达细胞中BIRC5蛋白表达。以前列腺癌细胞PC3、DU145为研究背景,构建miR-181c-5p过表达细胞系(miR-181c-5p组)及其阴性对照(miR-NC组)并用qRT-PCR验证,CCK-8法检测细胞增殖情况[450 nm处光密度值(OD_(450 nm)值)],流式细胞术检测细胞周期分布和细胞凋亡率,Western blot检测细胞增殖、凋亡相关蛋白表达。建立miR-181c-5p/BIRC5双过表达调细胞系(miR-181c-5p+BIRC5组),用上述相同方法检测细胞生长、细胞周期分布、细胞凋亡率及相关蛋白表达。结果BIRC5在前列腺癌组织表达升高且在肿瘤高侵犯程度、淋巴结转移和复发患者呈现更高表达趋势,BIRC5高表达患者生存情况较差;miR-181c-5p在前列腺癌癌组织表达降低,miR-181c-5p水平与BIRC5水平呈负相关,miR-181c-5p靶向抑制BIRC5表达。在PC3、DU145细胞中,miR-181c-5p组细胞miR-181c-5p水平高于miR-NC组(P<0.05),OD_(450 nm)值和S期细胞百分比低于miR-NC组(P<0.05),G_(0)/G_(1)期细胞百分比、细胞凋亡率和BAX、caspase-3、PARP蛋白表达高于miR-NC组(P<0.05),CDK2、CCNB1、BCL-2蛋白表达弱于miR-NC组(P<0.05)。miR-181c-5p+BIRC5组细胞的BIRC5蛋白表达和OD_(450 nm)值高于miR-181c-5p组(P<0.05),G_(0)/G_(1)期细胞百分比低于miR-181c-5p组(P<0.05),S期细胞百分比高于miR-181c-5p组(P<0.05),细胞凋亡率低于miR-181c-5p组(P<0.05),CDK2、CCNB1和BCL-2蛋白表达高于miR-181c-5p组,BAX、caspase-3、PARP蛋白表达低于miR-181c-5p组。结论miR-181-5p可以靶作用BIRC5抑制人前列腺癌细胞增殖,使细胞周期在G_(0)/G_(1)期阻滞,促进细胞凋亡。

Objective To explore the effects and action mechanism of miR-181c-5p on biological behaviors of prostate cancer cells.Methods The pathological relationship between BIRC5,miR-181c-5p and prostate cancer was analyzed based on prostate cancer data in TCGA database.The target binding site of miR-181c-5p and BIRC5 was analyzed by miRNA target gene prediction database,and was verified by double luciferase activity assay.The expression of BIRC5 protein in miR-181c-5p overexpression cells was detected by Western blot.The prostate cancer cells PC3 and DU145 were selected to construct cell line with miR-181c-5p overexpression(miR-181c-5p group)and its negative control(miR-NC group),and qRT-PCR verification was conducted.The cells proliferation[optical density at 450 nm site(OD_(450 nm))]was detected by CCK-8.Distribution of cell cycles and apoptosis rate were detected by flow cytometry.Expressions of proliferation and apoptosis related proteins were detected by Western blot.The cell line with miR-181c-5p/BIRC5 overexpression was constructed(miR-181c-5p+BIRC5 group).Cells growth,distribution of cell cycles,apoptosis rate and expressions of related proteins were detected by the above methods.Results The expression of BIRC5 was up-regulated in prostate cancer tissues,and it was higher in patients with high tumor invasion,lymph node metastasis and recurrence.Patients exhibiting high expression of BIRC5 demonstrated poor survival rates.The expression of miR-181c-5p was down-regulated in prostate cancer tissues.The level of miR-181c-5p was negatively correlated with BIRC5 level,and miR-181c-5p could inhibit BIRC5 expression.In PC3 and DU145,miR-181c-5p level in miR-181c-5p group was higher than that in miR-NC group(P<0.05);OD_(450 nm) and percentage of S-phase cells were lower than those in miR-NC group(P<0.05),percentage of cells in G_(0)/G_(1) phase;apoptosis rate and expressions of BAX,caspase-3 and PARP proteins were higher than those in miR-NC group(P<0.05);expressions of CDK2,CCNB1 and BCL-2 proteins were lower than those in miR-NC group(P<0.05).The expression of BIRC5 protein and OD_(450 nm) in miR-181c-5p+BIRC5 group were higher than those in miR-181c-5p group(P<0.05),percentage of cells in G_(0)/G_(1) phase was lower than that in miR-181c-5p group(P<0.05);percentage of S-phase cells was higher than that in miR-181c-5p group(P<0.05);apoptosis rate was lower than that in miR-181c-5p group(P<0.05);expressions of CDK2,CCNB1 and BCL-2 proteins were higher than those in miR-181c-5p group;expressions of BAX,caspase-3 and PARP proteins were lower than those in miR-181c-5p group.Conclusion miR-181-5p can inhibit the proliferation of human prostate cancer cells by targeting BIRC5,block cells in G_(0)/G_(1) phase and promote cells apoptosis.