RESUMEN
Objective:To investigate the effect of BMAL1 gene on the proliferation, migration and invasion ability of radiation-resistant nasopharyngeal carcinoma cell line (5-8FR) and the molecular mechanism. Methods:A multi-target click model was constructed for radiation-resistant nasopharyngeal carcinoma cell line 5-8FR by low-dose fractionated irradiation, and the results of clone formation assay were used to fit the multi-target click model and calculate the sensitization ratio of radiotherapy. The expression levels of PI3K/Akt/MMP-2/9 signaling pathway-related proteins in 5-8FR and control 5-8F cell lines were detected by Western blot. The overexpression and knockdown vectors of BMAL1 gene were constructed and transfected with 5-8F and 5-8F cell lines, respectively. The BMAL1 gene overexpression (pcDNA-BMAL1) and its control (pcDNA) and interference (BMAL1-shRNA) and control (con-shRNA) cell lines were stably transfected with nasopharyngeal carcinoma cell line 5-8F and radiation-resistant cell line 5-8FR, respectively. Western blot was performed to verify the infection efficiency and detect the changes of PI3K/Akt/MMP-2/9 signaling pathway-related proteins after overexpression or interference of BMAL1 gene in both groups of cells. CCK-8 assay, cell scratch test and Transwell chamber test were conducted to investigate the proliferation, migration and invasion capabilities of 5-8FR cell line after overexpression or interference of BMAL1 gene. Results:BMAL1 gene expression was down-regulated, and those of PI3K/Akt pathway proteins and downstream related molecules of MMP-2 and MMP-9 were up-regulated, and TIMP-2 and TIMP-1 expression was down-regulated in nasopharyngeal carcinoma radiation-resistant cell lines. Overexpression of BMAL1 gene inhibited the expression of PI3K/Akt pathway proteins and downstream related molecules of MMP-2 and MMP-9, promoted the expression of TIMP-2 and TIMP-1, and inhibited the proliferation, migration and invasion capabilities of radiation-resistant nasopharyngeal carcinoma cells, while interference with BMAL1 gene yielded the opposite results. Conclusions:BMAL1 gene can reverse the expression of PI3K/Akt/MMP-2/9 signaling pathway-related proteins in radiation-resistant nasopharyngeal carcinoma cell lines and inhibit the proliferation, migration and invasion capabilities of radiation-resistant nasopharyngeal carcinoma cell lines.
RESUMEN
Objective: To investigate the molecular change of extracellular signal-regulated protein kinase (ERK) in Ras-MAPK signaling pathway after K 562 cells in nude mice were treated with simvastatin to elucidate whether ERK is involved in the regulation of simvastatin-induced apoptosis of K 562 cells. Methods: K 562 cells, the chronic myeloid leukemia (CML) cell line, were cultured in vitro and used to establish the K 562 xenografted model in BALB/c-nu/nu mice. The cell cycle of K 562 cells of control and two treatment groups was detected by flow cytometry (FCM). The advanced stage apoptotic change of K 562 cells induced by simvastatin was detected by TdT-mediated dUTP nick end labeling (TUNEL). The differential expressions of N-Ras and ERK 1 mRNA in Ras-MAPK signaling pathway were detected by RT-PCR. The changes of p-ERK protein were measured by immunohistochemical LDP method. Results: Simvastatin markedly inhibited the growth of xenografted K 562 tumor in nude mice. The volume and weight of tumors were significantly reduced with the increase in the dosage of simvastatin (P < 0.05, P < 0.01). Injection of 0.05 mg simvastatin per time induced significant G0/G1 arrest of K 562 cells in nude mice. Apoptosis of K 562 cells of nude mice could be induced by simvastatin at different dosages and the apoptotic rate was elevated by increasing the dosage of simvastatin (P < 0.01). The mRNA expressions of N-Ras and ERK 1 mRNA in K 562 cells were down-regulated by simvastatin at different dosages (P < 0.01). Compared with the control group, the p-ERK protein expression was down-regulated in 2 simvastatin-treated groups (P = 0.01, P < 0.01), respectively. Conclusion: Simvastatin depends on the Ras-MAPK signaling pathway. Downregulation of ERK mRNA and protein is involved in the induction of apoptosis of K 562 by simvastatin.