微RNA-19b-3p通过靶向原癌基因鼠双微体4影响胰腺癌的发展

MicroRNA-19b-3p affects the development of pancreatic cancer by targeting murine double microsome 4

目的探讨微RNA(miR)-19b-3p对胰腺癌PANC-1细胞原癌基因鼠双微体4(MDM4)表达和细胞增殖、凋亡活性的影响。方法采用脂质体转染法转染micrONrno-miR-19b-3p mimic(拟似组)、micrOFF mmu-miR-19b-3p inhibitor(阻遏组)和Lipofectamine 2000转染试剂(阴性组)至人胰腺癌PANC-1细胞,并以未特殊处理正常生长的PANC-1细胞作为空白组,采用荧光显微镜观察各组细胞转染是否成功,使用荧光实时定量PCR法检测微RNA-19b-3p转录水平以判断转染效率。转染24、48、72 h采用MTT法检测各组细胞增殖活性,采用荧光实时定量PCR法检测细胞内微RNA-19b-3p、MDM4含量,转染72 h采用Annexin V-FITC/PI双染法检测转染后PANC-1细胞凋亡情况。转染72 h采用免疫印迹法检测各组PANC-1细胞MDM4蛋白表达水平。结果转染后48 h 4组均有明显绿色荧光,微RNA-19b-3p转染PANC-1细胞转染效率为72.1%。转染24、48、72 h时拟似组PANC-1细胞增殖活性、微RNA-19b-3p mRNA、FL-MDM4、S-MDM4表达水平都高于空白组、阴性组和阻遏组(均P<0.05),阻遏组PANC-1细胞增殖活性和微RNA-19b-3p mRNA、FL-MDM4、S-MDM4表达水平低于空白组、阴性组和拟似组(均P<0.05),空白组和阴性组细胞增殖活性和微RNA-19b-3p mRNA、FL-MDM4、S-MDM4表达水平差异无统计学意义(均P>0.05)。空白组、阴性组、拟似组和阻遏组4组PANC-1细胞增殖活性和微RNA-19b-3p mRNA、FL-MDM4、S-MDM4表达均与时间呈现正相关(细胞增殖活性r=0.629、0.582、0.524、0.472,微RNA-19b-3p mRNA表达水平r=0.449、0.475、0.501、0.399,FL-MDM4表达水平r=0.504、0.423、0.447、0.431,S-MDM4表达水平r=0.472、0.428、0.414、0.408,均P<0.05)。转染72 h时拟似组细胞凋亡水平低于空白组、阴性组、阻遏组[(2.02±0.48)%比(3.48±0.63)%、(3.52±0.52)%、(8.23±0.82)%,均P<0.05],阻遏组细胞凋亡水平高于空白组、阴性组和拟似组,差异均有统计学意义(均P<0.05)。转染72 h拟似组MDM4蛋白表达水平高于空白组、阴性组和阻遏组[(1.162±0.114)比(0.749±0.126)、(0.663±0.137)、(0.347±0.092),均P<0.05],阻遏组MDM4蛋白表达水平低于空白组和阴性组,差异均有统计学意义(均P<0.05)。结论微RNA-19b-3p可能通过影响MDM4表达,进而影响p53基因活性,促进胰腺癌的增殖,微RNA-19b-3p可能成为治疗胰腺癌新的靶点。

Objective To investigate the effect of MicroRNA(miR)-19b-3p on expression,proliferation and apoptosis activities of pancreatic cancer PANC-1 cells via targeting murine double microsome 4(MDM4),a proto-oncogene.Methods The liposome transfection method was used to transfect the human PANC-1 cells with micrON rno-19b-3p(mimic group),micrOFF mmu-miR-19b-3p inhibitor(suppresion group)and Lipofectamine 2000(negative group).Normally growing PANC-1 cells without special treatment were used as blank groups.The real-time quantitative PCR was used to detect the miR-19b-3p transcription level to determine the effect of transfection.MTT assay was used to detect cell proliferation activity at 24,48 and 72 h after transfection.The contents of miR-19b-3p and MDM4 was detected by real-time quantitative PCR at 24,48 and 72 h after transfection.Annexin V-FITC/PI dual staining was used to detect the apoptosis of PANC-1 cells 72 h after transfection.Western blotting was used to detect the expression of MDM4 protein in each group of PANC-1 cells at 72 h after transfection.Results At 48 h after transfection,all four groups showed obvious green fluorescence,and the transfection efficiency of microRNA-19b-3p into PANC-1 cells was 72.1%.At 24,48,and 72 h after transfection,the proliferation activity of PANC-1 cells,the expression levels of microRNA-19b-3p mRNA,FL-MDM4,and S-MDM4 in the mimic group were all higher than those in the blank group,negative group and the suppression group(all P<0.05),and were the lowest in the suppression group compared with the blank group,negative group and mimic groups(all P<0.05).There were no significant differences in cell proliferation activity and expression levels of microRNA-19b-3p mRNA,FL-MDM4,and S-MDM4 between the blank and negative groups(all P>0.05).The PANC-1 cell proliferation activity(r=0.629,0.582,0.524,0.472)and expression levels of miR-19b-3p mRNA(r=0.449,0.475,0.501,0.399),FL-MDM4(r=0.504,0.423,0.447,0.431),and S-MDM4(r=0.472,0.428,0.414,0.408)in the blank,negative and mimic and suppression groups showed a time-correlated manner(all P<0.05).At 72 h after transfection,the apoptosis level in the mimic group was lower those in the blank,negative group,and suppression groups[(2.02±0.48)%vs(3.48±0.63)%,(3.52±0.52)%,and(8.23±0.82)%,all P<0.05],and that in the suppression group was higher than those in the blank,negative and mimic groups,with statistically significant differences(all P<0.05).At 72 h after transfection,the expression level of MDM4 protein in the mimic group was higher than those in the blank,negative and suppression groups[(1.162±0.114)vs(0.749±0.126),(0.663±0.137),and(0.347±0.092),all P<0.05],and that in the suppression group was lower than those in the blank and negative groups,with statistically significant differences(all P<0.05).Conclusion miR-19b-3p may affect the expression of MDM4 and then the activity of p53 gene to promote the proliferation of pancreatic cancer,and therefore can be a new target for the treatment of pancreatic cancer.