干扰lncRNA RHPN1-AS1表达通过靶向miR-339-5p对肝癌细胞增殖和凋亡的影响

目的:探讨长链非编码RNA PCNA1(lncRNA RHPN1)反义AS1(RHPN1-AS1)对肝癌细胞增殖和凋亡的影响及其作用机制。方法:将si-NC(lncRNA RHPN1-AS1阴性对照组)、si-RHPN1-AS1(沉默lncRNA RHPN1-AS1组)、pcDNA(真核表达载体组)、pcDNA-RHPN1-AS1(沉默lncRNA RHPN1-AS1真核表达载体组)、miR-NC(微小RNA-339-5p阴性对照组)及miR-339-5p(微小RNA-339-5p组)分别转染至肝癌细胞Huh7中,记为si-NC组、si-RHPN1-AS1组、pcDNA组、pcDNA-RHPN1-AS1组、miR-NC组和miR-339-5p组;将si-RHPN1-AS1质粒分别与anti-miRNC(微小RNA-339-5p抗结剂阴性对照)、anti-miR-339-5p(微小RNA-339-5p抗结剂)共转染至Huh7细胞中,记为si-RHPN1-AS1+anti-miR-NC组(沉默lncRNA RHPN1-AS1+微小RNA-339-5p抗结剂阴性对照组)、si-RHPN1-AS1+anti-miR-339-5p组(沉默lncRNA RHPN1-AS1+微小RNA-339-5p抗结剂组);转染均采用脂质体法。采用实时荧光定量聚合酶链反应(PCR)(RT-qPCR)检测正常肝细胞(THLE-2)、肝癌细胞株(Huh7、MHCCLM3、MHCC97H)中miR-339-5p和RHPN1-AS1表达水平;双荧光素酶报告实验检测RHPN1-AS1和miR-339-5p的靶向关系;用蛋白质印迹法检测细胞周期素D1(Cyclin D1)蛋白、P21蛋白、B细胞淋巴瘤/白血病-2(Bcl-2)及Bcl-2相关X蛋白(Bax)的表达水平;用流式细胞术检测细胞凋亡情况;用四甲基偶氮唑(MTT)比色法检测细胞活性。结果:与正常肝细胞THLE-2相比,肝癌细胞株Huh7、MHCCLM3、MHCC97H中RHPN1-AS1高表达,miR-339-5p低表达。RHPN1-AS1靶向调控miR-339-5p的表达。干扰RHPN1-AS1表达和miR-339-5p过表达的肝癌细胞Huh7中P21蛋白、Bax蛋白表达水平显著升高,Cyclin D1、Bcl-2水平显著降低,细胞活性显著降低,细胞凋亡率显著升高。下调miR-339-5p表达逆转了干扰RHPN1-AS1表达对肝癌细胞Huh7的增殖抑制和凋亡促进作用。结论:干扰RHPN1-AS1表达可抑制肝癌细胞的增殖,促进细胞凋亡,其机制可能与miR-339-5p表达有关,将可为肝癌的靶向治疗提供新靶点。

Study on the effect of interfering the expression of lncRNA RHPN1-AS1 on proliferation and apoptosis of hepatoma cells by targeting miR-339-5p

Objective:To explore the effect of long non-coding RNA PCNA1(lncRNA RHPN1)antisense AS1(RHPN1-AS1)on the proliferation and apoptosis of hepatoma cells and its mechanism.Methods:si-NC(lncRNA RHPN1-AS1 negative control group),si-RHPN1-AS1(silencing lncRNA RHPN1-AS1 group),pcDNA(eukaryotic expression vector group),pcDNA-RHPN1-AS1(silencing lncRNA RHPN1-AS1 eukaryotic expression vector group),miR-NC(microRNA-339-5p negative control group),miR-339-5p(microRNA-339-5p group)were transfected into liver cancer cell Huh7,and they were denoted as si-NC group,si-RHPN1-AS1 group,pcDNA group,pcDNA-RHPN1-AS1 group,miR-NC group and miR-339-5p group,respectively.And the si-RHPN1-AS1 plasmid was combined respectively with anti-miR-NC(microRNA-339-5p anti-tackiness agent negative control)and anti-miR-339-5p(microRNA-339-5p anti-tackiness agent)to co-transfect into Huh7 cells,and they were denoted as si-RHPN1-AS1+anti-miR-NC group(silencing lncRNA RHPN1-AS1+microRNA-339-5p anti-tackiness agent negative control group),si-RHPN1-AS1+antimiR-339-5p group(silencing lncRNA RHPN1-AS1+microRNA-339-5p anti-tackiness agent group),respectively.All of transfection adopted liposome method.Real-time fluorescent quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression levels of miR-339-5p and RHPN1-AS1 in normal liver cells(THLE-2)and hepatoma cell lines(Huh7,MHCCLM3,MHCC97H).And dual luciferase report experiment was used to detect targeting relationship between RHPN1-AS1 and miR-339-5p.Western blotting was used to detect the expression levels of Cyclin D1 protein,P21 protein,B-cell lymphoma/leukemia-2(Bcl-2)and Bcl-2 related X Protein(Bax).Flow cytometry was used to detect cell apoptosis,and methyl thiazolyl tetrazolium(MTT)colorimetric method was used to detect cell viability.Results:Compared with normal liver cell THLE-2,RHPN1-AS1 was highly expressed in hepatoma cell lines Huh7,MHCCLM3 and MHCC97H,and miR-339-5p was lowly expressed in these cells.RHPN1-AS1 could targeting regulate the expression of miR-339-5p.And the expression levels of P21 protein and Bax protein in hepatoma cell Huh7,that interfered the expression of RHPN1-AS1 and has overexpression of miR-339-5p,were significantly enhanced.And the expression levels of Cyclin D1 and Bcl-2 were significantly reduced,and the cell activity was significantly reduced,while the apoptotic rate was significantly increased in hepatoma cells Huh7.The down-regulation of miR-339-5p expression could reverse the effects that interfered RHPN1-AS1 expression on proliferation inhibition and apoptosis promotion of hepatoma Huh7 cells.Conclusion:Interfering with the expression of RHPN1-AS1 can inhibit the proliferation of hepatoma cells and promote cell apoptosis.The mechanism may be related to the expression of miR-339-5p,which can provide new target spot for targeted therapy of liver cancer.