姜黄素调控LINC00491/miR-532-3p轴抑制前列腺癌发生发展的机制研究

Curcumin inhibits occurrence and development of prostate cancer via regulating LINC00491/miR-532-3p axis

目的探讨姜黄素调控LINC00491/miR-532-3p轴抑制前列腺癌发生发展的机制。方法取人前列腺癌细胞株22RV1体外培养后分为正常对照组、姜黄素组、姜黄素+si-LINC00491组、姜黄素+si-LINC00491+miR-532-3p inhibitor组。RT-PCR检测LINC00491、miR-532-3p表达水平;双荧光素酶实验检测LINC00491、miR-532-3p的靶向关系;细胞计数试剂盒8(CCK8)、流式细胞术、Transwell实验分别检测细胞增殖、凋亡、迁移及侵袭等肿瘤恶性行为;Western blot检测细胞增殖相关KI67、凋亡相关半胱氨酸蛋白酶3(Caspase 3)、迁移侵袭相关基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)蛋白表达。取60只BALB/c裸小鼠建立前列腺癌皮下移植瘤模型,将建模成功的裸小鼠随机分为空白对照组、姜黄素组、姜黄素+oe-LINC00491组、oe-LINC00491组、姜黄素+sh-LINC00491组和sh-LINC00491组。观察瘤块大小、侵袭、转移和腹腔播散情况,记录动物生存期;RNA FISH检测LINC00491在原发癌灶和转移癌灶中的定位和表达水平的变化;TdT介导的dUTP缺口末端标记技术(TUNEL)染色检测细胞凋亡;RT-PCR检测LINC00491、miR-532-3p表达水平;Western blot检测KI67、Caspase 3、MMP-2、MMP-9蛋白表达。结果姜黄素可抑制前列癌细胞及前列腺癌荷瘤裸小鼠癌组织中LINC00491的表达,促进miR-532-3p的表达,抑制LINC00491表达可加强姜黄素的作用,而下调miR-532-3p的表达可逆转抑制LINC00491表达对姜黄素作用的影响。荧光素酶实验结果显示,LINC00491可靶向调控miR-532-3p的表达。体外细胞实验结果显示,姜黄素可提高人前列腺癌细胞凋亡率,降低细胞迁移和侵袭能力,抑制细胞增殖相关KI67、迁移侵袭相关MMP-2、MMP-9蛋白表达,促进凋亡相关Caspase 3蛋白表达,且下调LINC00491表达可加强姜黄素对前列癌细胞恶性行为及上述蛋白表达的作用,下调miR-532-3p表达可逆转下调LINC00491表达对前列癌细胞恶性行为及上述蛋白表达的作用。动物体内实验结果同样显示,姜黄素可提高前列腺癌荷瘤裸小鼠生存期,抑制肿瘤生长和转移,促进癌组织细胞凋亡,抑制KI67、MMP-2、MMP-9蛋白表达,促进Caspase 3蛋白表达,且下调LINC00491表达可加强姜黄素对前列腺癌荷瘤裸小鼠癌组织生长、转移及上述蛋白表达的作用。结论动物实验表明,姜黄素可通过靶向调控LINC00491/miR-532-3p轴,抑制KI67、MMP-2、MMP-9表达,促进Caspase 3表达,发挥抑制前列腺癌发生发展的作用。

Objective To explore the underlying mechanism by which curcumin regulates LINCO0491/microRNA-532-3p(miR-532-3p)axis to inhibit the malignant behavior of prostate cancer.Methods Human prostate cancer cells 22RV1 were cultured and subdivided into the control group,the curcumin group,the curcumin+si-LINCO0491 group,the curcumin+si-LINC00491+miR-532-3p inhibitor group.The expressions of LINC00491 and miR-532-3p in four groups were measured by RT-PCR;Dual luciferase assay was used to determine the targeting relationship of LINCO0491 and miR-532-3p;Cell Counting Kit-8(CCK8),flow cytometry,and Transwell assays were used to analyze tumor malignant behaviors such as cell proliferation,apoptosis,migration and invasion,respectively;Western blot was used to detect the protein expressions of cell proliferation-related KI67,apoptosis-related cysteine protease 3(Caspase 3),migration and invasion-related matrix metalloproteinase-2(MMP-2),and matrix metalloproteinase-9(MMP-9).60 BALB/c nude mice were selected to establish the subcutaneous transplanted tumor model of prostate cancer,and the modeled nude mice were randomly divided into the control group,the curcumin group,the curcumin+oe-LINC00491 group,the oe-LINC00491 group,the curcumin+sh-LINC00491 group and the sh-LINC00491 group.Tumor size,invasion,metastasis,and intraperitoneal dissemination were observed,and the survival time of the animals was recorded;RNA FISH was used to detect the localization and expression changes of LINCO0491 in primary and metastatic tumors;Cell apoptosis was analyzed by TdT-mediated dUTP nick-end labeling(TUNEL)staining;The expressions of LINCO0491 and miR-532-3p were detected by RT-PCR;The protein expressions of KI67,Caspase 3,MMP-2 and MMP-9 were detected by Western blot.Results Curcumin obviously inhibited the expression of LINC00491 and promote the expression of miR-532-3p in prostate cancer cells and prostate cancer-bearing nude mice.Suppression of LINC00491 enhanced the effect of curcumin,but downregulation of miR-532-3p partially reversed the above effect.Luciferase assay showed that LINC00491 targeted and regulated the expression of miR-532-3p.Cell experiments in vitro demonstrated that curcumin increased the apoptosis rate of human prostate cancer cells,reduced the ability of cell migration and invasion.Meanwhile,it inhibited the protein expressions of KI67,MMP-2,and MMP-9,and promoted Caspase 3 protein expression.Downregulation of LINCO0491 enhanced the effect of curcumin on the malignant behavior of prostate cancer cells and the expression of the above proteins,and down-regulation of miR-532-3p expression can reverse the above effect.Analysis of animal experiments also revealed that curcumin significant improved the survival of prostate cancer-bearing nude mice,inhibited tumor growth and metastasis,additionally,it promoted cancer cell apoptosis and Caspase 3 protein expression,inhibited the protein expressions of KI67,MMP-2,and MMP-9.Downregulation of LINC00491 expression enhanced the effect of curcumin on the growth and metastasis of prostate cancer-bearing nude mice and the expression of the above-mentioned proteins.Conclusion The in utuo and experiment showed that the curcumin can inhibit the expressions of KI67,MMP-2,MMP-9,and promote the expression of Caspase 3 by targeting the LINC00491/miR-532-3p axis.It plays a role in inhibiting the malignant behavior of prostate cancer.