微小核糖核酸-24-3p抑制肝细胞癌增殖、迁移及侵袭机制

Mechanism of microRNA-24-3p inhibiting proliferation,migration and invasion of hepatocellular carcinoma

目的探讨微小核糖核酸-24-3p(miR-24-3p)对肝癌细胞增殖、迁移及侵袭的影响及其机制。方法收集2019年6月至2021年8月长江大学附属荆州医院诊治的肝癌患者肝癌组织及癌旁组织。分别将miR-NC组、miR-24-3p inhibitor组、pcDNA3.1组、pcDNA3.1-WNK2组、miR-24-3p inhibitor+pcDNA3.1-NC组、miR-24-3p inhibitor+pcDNA3.1-WNK2组、miR-24-3p inhibitor+shRNA-NC组、miR-24-3p inhibitor+sh-WNK2组转染至HepG2细胞。实时荧光定量聚合酶链反应(RT-qPCR)检测miR-24-3p表达;蛋白质印迹法(Western blot)检测WNK赖氨酸缺陷型蛋白激酶2(WNK2)、细胞周期蛋白D1(Cyclin D1)、金属基质蛋白2(MMP-2)蛋白表达;噻唑蓝(MTT)法检测细胞增殖;Transwell法检测细胞侵袭、迁移情况;荧光素酶报告基因实验验证miR-24-3p与WNK2的靶向关系;体内成瘤实验检测肿瘤体积及重量。两组间均数比较采用t检验;多组间均数比较采用单因素方差分析(组间比较采用SNK检验)。结果肝癌组织miR-24-3p表达显著高于癌旁组织(1.13±0.04比2.16±0.28),WNK2蛋白表达显著低于癌旁组织(1.56±0.23比0.54±0.11),差异有统计学意义(t=14.104、15.495,P<0.05)。HepG2、Hep3B、MHCC97H及Huh7细胞中miR-24-3p表达显著高于L02细胞(分别1.68±0.20、1.26±0.13、1.31±0.21、1.43±0.24比1.01±0.10),差异有统计学意义(F=8.892,P<0.05)。miR-24-3p inhibitor组miR-24-3p表达、细胞存活率、Cyclin D1及MMP-2、迁移及侵袭数显著低于对照组及miR-NC组[0.51±0.04比1.01±0.08、(53.61±6.30)%比(97.08±9.26)%、0.49±0.14比1.26±0.22、0.71±0.10比1.35±0.35、92.47±12.18比154.01±25.30、70.10±8.08比130.10±22.30],而WNK2表达显著高于对照组及miR-NC组(1.61±0.29比0.98±0.12),差异有统计学意义(t=12.500、8.769、6.603、3.931、4.901、5.656、4.489,P<0.05)。pcDNA3.1-WNK2组WNK2表达显著高于对照组及pcDNA3.1-NC组(1.58±0.36比0.99±0.10),细胞存活率、Cyclin D1及MMP-2表达、迁移及侵袭数显著低于对照组及pcDNA3.1-NC组[(58.22±21.13)%比(101.07±13.20)%、0.62±0.20比1.29±0.23、0.71±0.26比1.37±0.25、79.40±8.45比155.31±22.35、64.89±9.02比131.62±27.23],差异有统计学意义(t=3.531、3.846、4.915、4.092、7.104、5.186,P<0.05)。pmirGLO-WNK2-wt+miR-24-3p mimics组荧光素酶活性显著低于pmirGLO-WNK2-wt+miR-NC组(0.57±0.09比0.94±0.12),差异有统计学意义(t=5.516,P<0.05)。miR-24-3p inhibitor+sh-WNK2组细胞存活率、Cyclin D1及MMP-2表达、迁移及侵袭数显著高于miR-24-3p inhibitor+shRNA-NC组与miR-24-3p inhibitor组,WNK2表达显著低于miR-24-3p inhibitor+shRNA-NC组与miR-24-3p inhibitor组,差异有统计学意义(F=17.215、28.073、4.839、19.136、53.869、6.565,P<0.05)。结论miR-24-3p可抑制肝癌增殖、迁移及侵袭,其机制可能与调控WNK2表达有关。

Objective To explore the mechanism of microRNA-24-3p inhibiting proliferation,migration and invasion of hepatocellular carcinoma(HCC).Methods Liver cancer tissues and paracancerous tissues of liver cancer patients diagnosed and treated from June 2019 to August 2021 were collected.The miR-NC group,miR-24-3p inhibitor group,pcDNA3.1 group,pcDNA3.1-WNK lysine deficient protein kinase 2(WNK2)group,miR-24-3p inhibitor+pcDNA3.1-NC group,miR-24-3p inhibitor+pcDNA3.1-WNK2 group,miR-24-3p inhibitor+shRNA-NC group,miR-24-3p inhibitor+sh-WNK2 group were set up in HepG2 cells.Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression of miR-24-3p.Western blotting was used to detect the protein expression of WNK2,Cyclin D1,and matrix metalloproteinase(MMP)-2.The methyl thiazolyl tetrazolium(MTT)method was used to detect cell proliferation.The Transwell method was used to detect cell invasion and migration.The luciferase reporter gene experiment was done to verify the targeting relationship between miR-24-3p and WNK2;The in vivo tumor formation experiment was carried out to detect tumor volume and weight.Results The expression of miR-24-3p in liver cancer tissue was significantly higher than that in para-cancerous tissue(1.13±0.04 vs.2.16±0.28),and the expression of WNK2 protein was significantly lower than that in para-cancerous tissue(1.56±0.23 vs.0.54±0.11),and the difference was statistically significant(t=14.104,15.495,P<0.05).The expression of miR-24-3p in HepG2,Hep3B,MHCC97H and Huh7 cells was significantly higher than that in L02 cells(respectively 1.68±0.20,1.26±0.13,1.31±0.21,1.43±0.24 vs.1.01±0.10),and the difference was statistically significant(F=8.892,P<0.05).In miR-24-3p inhibitor group,miR-24-3p expression,cell survival rate,expression of Cyclin D1 and MMP-2,the number of migrating and invasive cells were significantly reduced as compared with those in the control group and miR-NC group[0.51±0.04 vs.1.01±0.08,(53.61±6.30)%vs.(97.08±9.26)%,0.49±0.14 vs.1.26±0.22,0.71±0.10 vs.1.35±0.35,92.47±12.18 vs.154.01±25.30,70.10±8.08 vs.130.10±22.30],and the expression of WNK2 was significantly higher than that of the control group and miR-NC group(1.61±0.29 vs.0.98±0.12),the difference was statistically significant(t=12.500,8.769,6.603,3.931,4.901,5.656,4.489,P<0.05).The expression of WNK2 in the pcDNA3.1-WNK2 group was significantly higher than that in the control group and the pcDNA3.1-NC group(1.58±0.36 vs.0.99±0.10),and the cell survival rate,the expression of Cyclin D1 and MMP-2,the number of migrating and invasive cells were significantly reduced as compared with those in the control group and pcDNA3.1-NC group[(58.22±21.13)%vs.(101.07±13.20)%,0.62±0.20 vs.1.29±0.23,0.71±0.26 vs.1.37±0.25,79.40±8.45 vs.155.31±22.35,64.89±9.02 vs.131.62±27.23],the difference was statistically significant(t=3.531,3.846,4.915,4.092,7.104,5.186,P<0.05).The luciferase activity of the pmirGLO-WNK2-wt+miR-24-3p mimics group was significantly lower than that of the pmirGLO-WNK2-wt+miR-NC group(0.57±0.09 vs.0.94±0.12),and the difference was statistically significant(t=5.516,P<0.05).The cell survival rate,the expression of Cyclin D1 and MMP-2,the number of migrating and invasive cells in the miR-24-3p inhibitor+sh-WNK2 group were significantly increased as compared with those in the miR-24-3p inhibitor+shRNA-NC group and the miR-24-3p inhibitor group,and the expression of WNK2 was significantly higher than miR-24-3p inhibitor+shRNA-NC group and miR-24-3p inhibitor group,the difference was statistically significant(F=17.215,28.073,4.839,19.136,53.869,6.565,P<0.05).Conclusion MiR-24-3p can inhibit the proliferation,migration and invasion of liver cancer probably by the regulation of WNK2 expression.