| Objective:This study was aimed to investigate the expression and effects of RPS15A gene in glioblastoma cells in vivo and in vitro, and investigate whether the effects of RPS15A were associated with cell cycle regulatory proteins and signaling pathways, in order to found a new biological gene target in diagnosis and prognosis of glioblastoma and a new idea in glioblastoma therapy.Methods:In this study, we used quantitative real-time PCR and Western Blot to detect expression of RPS15A in tumor tissues and normal tissues, as well as the expression in parafin sections of patients by HE assay. In vitro, we established RPS15A knockdown cell line by transduced glioblastoma U87 cells with shRPS15A-containing lentivirus. Real-time quantitative PCR and Western Blot showed that Lv-shRPS15A U87 cells successfully established, MTT assay also showed that the viability of Lv-shRPS15A U87 cells was higher than that of U87 cells transduced with PLV-Ctr. We analyzed the cell-cycle progression of U87 cells transduced with Lv-shRPS15A and PLV-Ctr by flow cytometry. To determine whether RPS 15A knockdown inhibits migration of glioblastoma cells, we examined its effect on U87 cells transduced with Lv-shRPS15A and PLV-Ctr by cell scratch and transwell assay. Furthermore, the growth of glioblastoma cell-transplanted tumors in nude mice was measured by transduced with Lv-shRPS15A and PLV-Ctr. Besides, expressions of phosphorylated AKT, P18INK4C and CDK4were also determined by Western blot.Results:In this study, we investigate the mRNA and protein expression of RPS15A in clinical samples by real-time quantitative PCR and Western Blot experiments, we found that both of them were highly expressed in glioblastoma tissues than that in normal tissues. The results show that, compared to normal brain tissues, the expression of RPS15A in glioblastoma is increased. Similarly, we analyzed the expression of RPS15A in 123 parafin sections of glioma by HE assay. Factors such as age, gender, histological type and degree of the World Health Organization were analyzed respectively. We found that the number of high expression of RPS15A was 43 cases in patients older than 50 years,71.7% of the total samples, the number of low expression of RPS15A is 17 cases,28.3% of the total samples; and the number of high expression of RPS15A was 47 cases in patients younger than 50 years,74.6% of the total samples, the number of low expression of RPS15A is 16 cases,25.4% of the total samples. In male patients, the number of high expression of RPS15A was 58 cases,79.5% of the total samples, the number of low expression of RPS15A is 15 cases,20.5% of the total samples; in female patients, the number of high expression of RPS15A was 32 cases,64% of the total samples, the number of low expression of RPS15A is 18 cases,36% of the total samples. In astrocytic glioma, the number of high expression of RPS15A was 9 cases,64.3% of the total samples, the number of low expression of RPS15A is 5 cases,35.7% of the total samples; in oligodendroglioma, the number of high expression of RPS15A was 6 cases,66.6% of the total samples, the number of low expression of RPS15A is 3 cases,33.3% of the total samples; in anaplastic astrocytoma, the number of high expression of RPS15A was 47 cases,78.3% of the total samples, the number of low expression of RPS15A is 13 cases,21.7% of the total samples; in glioblastoma, the number of high expression of RPS15A was 30 cases,75% of the total samples, the number of low expression of RPS15A is 10 cases,25% of the total samples. The number of high expression of RPS15A was 8 cases in WHO stage Ⅰ and Ⅱ,34.8% of the total samples, the number of low expression of RPS15A is 15 cases,65.2% of the total samples; the number of high expression of RPS15A was 84 cases in WHO stage Ⅲ and Ⅳ,84% of the total samples, the number of low expression of RPS15A is 16 cases,16% of the total samples. In summary, compared with normal brain tissues, RPS15A was specifically overexpressed in human glioblastoma tumor tissues, and the expression of RPS15A was significantly increased in parafin sections of patients in WHO stage Ⅲ and IV by HE assay. All results indicted that RPS15A may play an important role in the development of glioblastoma. In addition, we investigate the possible relationship between survival time and RPS15A expression in glioma patients, and found that RPS15A expression could be a prognostic marker of glioma. Analysis of patients’survival time showed that patients with higher level of RPS15A expression in tumors suffered a worse survival than those with lower level of RPS15A expression. We successfully established RPS15A knockdown U87 cells by transduced with shRPS15A-containing lentivirus to investigate the biological functions of RPS15A in vitro. Real-time quantitative PCR and Western Blot proved the knockdown. When U87 cells transduced with Lv-shRPS15A, proliferation inhibition was measured by MTT colorimetric assay. Similarly, the fraction of RPS15A knockdown U87 cells in the G0/G1 phase was greatly increased. Moreover, the increase of cell number in G0/G1 phase was accompanied with a decrease of cell number in both S and G2/M phases. These results indicate that RPS15A knockdown-induced inhibition of U87 cell proliferation was likely due to G0/G1 phase arrest of the cell cycle. Besides, cells migration was also observed to be inhibited compared with U87 cells transduced with PLV-Ctr in cell scratch and transwell experiments. To provide direct evidence that RPS15A knockdown really inhibits tumor development, we injected U87 cells transduced with Lv-shRPS15A to the flank of nude mice. The RPS15A knockdown-induced reduction of tumor volumes was significant during the period of observation (three weeks). It has been shown that MAPKs and PI3K-AKT-mTOR are major signalling pathways that have been identified as important in cancer, we thus examined whether RPS15A had effect on activation of MAPKs and PI3K-AKT-mTOR. phosphorylated ERK and AKT were analyzed by Western blot-ting using the antibodies against the phosphorylated form of these kinases. The level of phosphorylated AKT was greatly increased while the level of phosphorylated ERK was not affected. These results suggest that RPS15A might affect AKT pathway to inhibit proliferation of U87 cells. In addition, RPS15A knockdown also reduced expression of cell cycle regulation protein CDK4 while enhanced expression of p18 INK4C.Conclusion:RPS15A was specifically overexpressed in human glioblastoma tumor tissues, and RPS15A knockdown inhibited proliferation and migration of glioblastoma cells in vitro. Transduced with shRPS15A-containing lentivirus, led to depression of phosphorylated AKT, cell cycle arrest in G0/G1 phase and inhibition of proliferation in U87 cells. In addition, also reduced expression of cell cycle regulation protein CDK4 while enhanced expression of p18 INK4C. Furthermore, the growth of glioblastoma cell-transplanted tumors in nude mice was inhibited by transduced with Lv-shRPS15A. In conclusion, our findings indicate that RPS15A promotes cell proliferation and migration in glioblastoma for the first time. RPS15A might play a distinct role in glioblastoma and serve as a potential target for therapy. |
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