miR-29a靶向调控PDGFB促进非小细胞肺癌细胞凋亡

miR-29a Promotes Apoptosis in Non-small Cell Lung Cancer Cells by Targeting PDGFB

为了探究miR-29a对非小细胞肺癌细胞增殖和凋亡的影响及分子机制,本研究通过荧光定量PCR检测肺癌组织、癌旁组织、肺癌细胞以及人正常肺支气管上皮细胞BEAS-2B中miR-29a的表达,在肺癌A549转染miR-29a mimics后,使用荧光定量PCR和CCK-8法分别检测miR-29a的表达以及各组细胞的活力,使用流式细胞术检测A549细胞凋亡;通过荧光定量PCR检测肺癌组织、癌旁组织PDGFB m RNA的表达,采用Western blot检测PDGFB蛋白的表达;使用双荧光素酶报告基因检测miR-29a可能的靶基因;在肺癌A549细胞转染miR-29a mimics后继续转染PDGFB过表达质粒,通过qPCR和Western blotting分别检测PDGFB mRNA和蛋白的表达。结果表明,与癌旁组织相比,miR-29a在肺癌组织的表达显著下调(p<0.01),PDGFB在肺癌组织的表达显著增加(p<0.01);转染miR-29a mimics后,肺癌A549细胞中miR-29a表达显著增加(p<0.01);CCK-8法结果显示miR-29a mimics组A549肺癌细胞在24 h和48 h后细胞增值率较miR-NC对照组显著降低(p<0.01);流式细胞术结果显示miR-29a mimics组的细胞凋亡率较miR-NC对照组显著增加(p<0.01);与miR-NC+PDGFB 3’UTR WT组相比,miR-29a mimics+PDGFB 3’UTR WT组的荧光强度显著降低(p<0.01);荧光定量PCR和Western blotting显示miR-29a mimics+PDGFB组PDGFB m RNA和蛋白表达量与miR-29a mimics+vector组相比显著增加(p<0.01)。本研究结果表明miR-29a在肺癌组织和肺癌细胞株中低表达,及抑制PDGFB的表达并且促进肺癌细胞凋亡。

To explore the effect and molecular mechanism of miR-29a on proliferation and apoptosis of non-small cell lung cancer cells, Real-time PCR was used to detect the expression of miR-29a in lung cancer tissues and paracancerous tissues, lung cancer cells and human normal lung bronchial epithelial BEAS-2 B cells. After transfected with miR-29 a mimics, the expression of miR-29a and the viability of each group cells were detected by real-time PCR and CCK-8. Apoptosis of A549 cells was detected by flow cytometry. The expression of PDGFB mRNA in lung cancer tissues and paracancerous tissues was detected by real-time PCR, and the expression of PDGFB protein was detected by Western blotting. The possible target genes of miR-29a were detected by the dual luciferase reporter gene assay system. After transfection of miR-29 a mimics into lung cancer A549 cells, the PDGFB overexpression plasmid was further transfected, and PDGFB mRNA and protein expression was detected by fluorescence quantitative PCR and Western blotting. The results showed that compared with adjacent tissues, the expression of miR-29a was significantly down regulated in lung cancer(p<0.01) and the expression of PDGFB was significantly increased(p<0.01). After transfection of miR-29a mimics, the expression of miR-29a increased significantly in lung cancer A549 cells(p<0.01). CCK-8 assay showed that the cell proliferation rate of A549 lung cancer cells in mi R-29a mimics group after 24 h and 48 h was significantly lower than that in mi R-NC control group(p<0.01).The results of flow cytometry showed that the apoptosis rate of miR-29a mimics group was significantly higher than that of miR-NC control group. Compared with miR-NC+PDGFB 3’UTR WT group, the fluorescence intensity of miR-29a mimics+PDGFB 3’UTR WT group decreased significantly(p<0.01). Fluorescence quantitative PCR and Western blotting showed that the expression of PDGFB mRNA and protein in miR-29 a mimics +PDGFB group increased significantly compared with miR-29a mimics+vector group(p<0.01). The results of this study showed that the expression of miR-29 a was decreased in lung cancer tissues and lung cancer cell lines, which inhibited the expression of PDGFB and promotes apoptosis in lung cancer cells.