miR-130b在人视网膜母细胞瘤中的表达及促癌机制研究

Study on the expression and cancer-promoting mechanism of miR-130b in human retinoblastoma

目的:检测miR-130b在人视网膜母细胞瘤(RB)中的表达并初步探究其促癌机制。。方法:采用qRT-PCR检测miR-130b在人RB癌组织及癌旁组织、人RB细胞系(HXO-Rb44与Y79)中的表达;采用qRT-PCR、Western Blot及免疫荧光实验检测miR-130b过表达及干扰前后HXO-Rb44与Y79细胞中PTEN的表达水平;采用双荧光素酶报告基因试验验证miR-130b与PTEN的靶向关系;采用共转染实验考察PTEN与miR-130b影响RB细胞系PI3K/Akt信号通路表达的关系。结果:miR-130b在RB癌组织中的表达水平显著高于癌旁组织(P<0. 05)。与ACBRI-181细胞比较,miR-130b在HXO-Rb44与Y79细胞中的表达水平显著增高(P <0. 05)。与RB癌组织比较,PTEN在其癌旁组织中的表达水平显著增高(P<0. 05); miR-130b表达水平与PTEN表达水平呈负相关性(P<0. 001)。过表达miR-130b后的HXO-Rb44细胞PTEN mRNA与蛋白表达水平均显著降低,而干扰miR-130b后的Y79细胞PTEN mRNA与蛋白表达水平均显著升高(P<0. 05)。与miR-130b mimics+PTEN-NC组比较,miR-130b mimics+wt-PTEN组荧光素酶活性明显降低(P<0. 05)。共转染miR-130b mimics+PTEN-NC HXO-Rb44细胞p-Akt 308与p-Akt 473蛋白表达水平显著增高(P<0. 05),PTEN蛋白表达水平显著降低(P<0. 05);共转染miR-130b mimics+PTEN HXO-Rb44细胞中,以上三种蛋白表达水平均未发生明显改变。结论:miR-130b在RB组织及细胞系中呈高水平表达,PTEN为miR-130b的靶基因,miR-130b可能是经负向调控PTEN对PI3K/Akt信号通路的表达产生影响,最终发挥促癌作用。

AIM:To study the expression and cancer-promoting mechanism of miR-130b in human retinoblastoma(RB).METHODS:Detected the expression levels of miR-130b in human RB carcinoma tissues,adjacent tissues and human RB cell lines(HXO-Rb44 and Y79)by qRT-PCR;detected the expression levels of PTEN in HXO-Rb44 and Y79 cells by qRT-PCR,Western Blot and immunofluorescence;verified the target relationship between miR-130b and PTEN by dual-luciferase reporter gene test;the co-transfection test was used to investigate the relationship between PTEN and miR-130b on the expression of PI3K/Akt signaling pathway in RB cell line.RESULTS:The expression level of miR-130b in cancer tissue of RB was significantly higher than that of paracancerous tissue(P<0.05).Compared with axben-181 cells,the expression level of miR-130b in HXO-Rb44 and Y79 cells was significantly increased(P<0.05).Compared with RB cancer tissue,the expression level of PTEN in its paracancerous tissue was significantly increased(P<0.05).The expression level of miR-130b was negatively correlated with the expression level of PTEN(P<0.001).The mRNA and protein expression levels of PTEN in HXO-Rb44 cells overexpressing miR-130b were significantly reduced,while the mRNA and protein expression levels of PTEN in Y79 cells after miR-130b interference were significantly increased(P<0.05).Compared with miR-130b mimics+PTEN-NC group,the luciferase activity of miR-130b mimics+wt-PTEN group was significantly reduced(P<0.05).In the HXO-Rb44 cells co-transfected with miR-130b mimics+PTEN-NC,the expression levels of p-Akt 308 and p-Akt 473 protein were significantly increased(P<0.05),while the expression levels of PTEN protein were significantly decreased(P<0.05).In the HXO-Rb44 cells co-transfected with miR-130b mimics+PTEN,no significant changes were observed in the above three proteins.CONCLUSION:miR-130b is highly expressed in RB tissues and cell lines.PTEN is the target gene of miR-130b,and miR-130b may negatively regulate PTEN to affect the expression of PI3K/Akt signaling pathway and ultimately play a role in promoting cancer.