Objective: To explore the effects of K-ras gene on the expressions of oncogenes and cancer suppressor genes in human bronchial epithelial (HBE) cells which were exposed to PM(2.5). Methods: According to the mRNA sequence of K-ras gene provided by GenBank in September 2019, interference sequences were designed and synthesized, and the recombinant lentiviral vector was transfected into HBE cell to construct the K-ras gene-silenced cells. HBE cells and K-ras gene-silenced cells were exposed to 10 μg/ml, 50 μg/ml PM(2.5) suspension and 10 μmol/L Cr(6+). Real-time fluorescent quantitative PCR was used to detect the mRNA expression levels of c-myc, c-fos, N-ras, cyclin-D1, p16 and p53 genes, the expression levels of p53 and c-myc proteins were detected by Western blot. Results: In K-ras silenced cell group, K-ras mRNA expression level decreased (80.5%±3.6%) and K-ras protein level decreased (58.9%±4.7%) when compared with the control group (P<0.01) . Compared with the correspoding cell control group without exposure, the mRNA expression levels of c-myc, c-fos, N-ras and cyclin-D1 genes in HBE cell group exposed to different concentrations of PM(2.5), K-ras silenced cell group exposed to different concentrations of PM(2.5), HBE cell group exposed to 10 μmol/L Cr(6+) and K-ras silenced cell group exposed to 10 μmol/L Cr(6+) were increased, the mRNA expressions of p16 and p53 genes were decreased (P<0.01) . Compared with HBE cell group exposed to 10 μg/ml PM(2.5), the mRNA expressions of c-myc, c-fos and p16 genes in K-ras silenced cells exposed to 10 μg/ml PM(2.5) were decreased, and the p53 mRNA level was increased (P<0.01) . Compared with HBE cell group exposed to 50 μg/ml PM(2.5), the mRNA expression levels of c-fos, N-ras, cyclin-D1, p16 and p53 genes in K-ras silenced cell group exposed to 50 μg/ml PM(2.5) were decreased (P<0.01) . Compared with the HBE cell group without exposure, c-myc protein increased and p53 protein decreased in HBE cells exposed to 50 μg/ml PM(2.5) (P<0.05) . Compared with the K-ras silenced cell group without exposure, c-myc protein increased in K-ras silenced cells exposed to 50 μg/ml PM(2.5) (P<0.05) . Conclusion: PM(2.5) can increase the expression levels of oncogenes in HBE cells, and K-ras gene silencing can inhibit the expression levels of oncogenes in HBE cells treated with PM(2.5).
目的： 探讨K-ras基因对PM(2.5)染毒人支气管上皮（HBE）细胞部分癌基因和抑癌基因表达的影响。 方法： 于2019年9月，根据K-ras基因mRNA序列，设计合成干扰序列，转染HBE细胞构建K-ras基因沉默细胞。用10、50 μg/ml PM(2.5)混悬液及10 μmol/L Cr(6+)分别染毒HBE细胞和K-ras基因沉默细胞，实时荧光定量PCR检测c-myc、c-fos、N-ras、cyclin-D1、p16、p53基因的mRNA表达水平，Western blot检测c-myc和p53蛋白表达水平。 结果： K-ras基因沉默细胞中K-ras基因mRNA表达水平下降（80.5%±3.6%），K-ras蛋白表达水平下降（58.9%±4.7%）（P<0.01）。各浓度PM(2.5)染毒HBE细胞组、K-ras沉默细胞组及10 μmol/L Cr(6+)染毒HBE细胞组、K-ras沉默细胞组c-myc、c-fos、N-ras、cyclin-D1基因mRNA表达水平均高于未染毒相应细胞对照组，p16和p53基因mRNA表达水平低于未染毒相应细胞对照组（P<0.01）；10 μg/ml PM(2.5)染毒K-ras沉默细胞组c-myc、c-fos、p16基因mRNA表达水平低于10 μg/ml PM(2.5)染毒HBE细胞组，p53基因mRNA表达水平高于10 μg/ml PM(2.5)染毒HBE细胞组（P<0.01）；50 μg/ml PM(2.5)染毒K-ras沉默细胞组c-fos、N-ras、cyclin-D1、p16和p53基因mRNA表达水平均低于50 μg/ml PM(2.5)染毒HBE细胞组（P<0.01）。50 μg/ml PM(2.5)染毒HBE细胞组c-myc蛋白表达水平高于未染毒HBE细胞组，p53蛋白表达水平低于未染毒HBE细胞组（P<0.05）；50 μg/ml PM(2.5)染毒K-ras沉默细胞组c-myc蛋白表达水平高于未染毒K-ras沉默细胞组（P<0.05）。 结论： PM(2.5)可引起HBE细胞癌基因表达升高，K-ras基因沉默能抑制PM(2.5)诱导HBE细胞癌基因的表达。.
Keywords: Gene silencing; Human bronchial epithelial cells; K-ras; Oncogenes; Particulate matter 2.5, PM(2.5).