Objective To understand the effects of clock gene BMAL1 and HIF-1α(Hypoxia inducible factor-1α)on proliferation,migration and sensitivity to radiotherapy of nasopharyngeal carcinoma cells HONE1.At the same time,whet...Objective To understand the effects of clock gene BMAL1 and HIF-1α(Hypoxia inducible factor-1α)on proliferation,migration and sensitivity to radiotherapy of nasopharyngeal carcinoma cells HONE1.At the same time,whether the biological clock gene BMAL1 can affect the expression of HIF-1αprotein was investigated.It will lay the foundation for further study on the correlation between clock gene BMAL1 and HIF pathway.Methods BMAL1 gene overexpression and interference lentivirus and HIF-1αgene interference lentivirus were constructed respectively,and were transfected into nasopharyngeal carcinoma cells HONE1.Western blot was used to verify the establishment of overexpressed and knockdown BMAL1 cell lines and HIF-1αgene knockdown cell line,and to investigate the expression of HIF-1αprotein in overexpressed and knockdown BMAL1 cell lines.CCK-8 cell proliferation test and scratch test were used to analyze the proliferation and migration ability of cells.Cell apoptosis after radiotherapy was analyzed by flow cytometry.The effects of BMAL1 and HIF-1αon the sensitivity of HONE1 radiotherapy in nasopharyngeal carcinoma cells after X-ray irradiation at different doses(0Gy,2Gy,4Gy,6Gy)were detected by clone formation assay.Results The overexpression of BMAL1 gene and lentivirus interference were constructed to effectively up regulate and down regulate the expression of BMAL1 protein in nasopharyngeal carcinoma cells HONE1.Meanwhile,HIF-1αgene interference lentivirus was constructed to effectively down-regulate the expression of HIF-1αprotein in nasopharyngeal carcinoma cell line HONE1,and successfully screen out stable nasopharyngeal carcinoma cell lines.Western blot results showed that overexpression of BMAL1 gene could inhibit the expression of HIF-1αprotein in HONE1 of nasopharyngeal carcinoma cells,while knockdown of BMAL1 gene promoted the expression of HIF-1αprotein in HONE1 of nasopharyngeal carcinoma cells(P<0.05).CCK-8 cell proliferation and scratch test showed that overexpression of BMAL1 gene or knockdown of HIF-1αgene could inhibit the proliferation and migration of HONE1 cells(P<0.05).Flow cytometry results showed that after 8Gy irradiation for 72 h,the apoptosis rate of BMALl gene overexpression group was higher than that of the overexpression control group,similarly,the apoptosis rate of HIF-1αgene knockdown group was higher than that of the knockdown control group(P<0.05).After X-ray irradiation at different doses(0Gy,2Gy,4Gy,6Gy),clon-formation experiment showed that the clon-formation rate and cell survival fraction of BMALl overexpression group or HIF-1αknockdown group were lower than those of negative control group(P<0.05).Sigmaplot analysis showed that the D0,Dq and SF2 of the BMAL1 overexpression group or HIF-1αknockdown group were lower than those of the negative control group,and the radiosensitization ratios were 1.381 and 1.063,respectively.Conclusion Overexpression of BMAL1 gene can inhibit the proliferation and migration of nasopharyngeal carcinoma cell line HONE1,increase apoptosis after radiotherapy and improve radiosensitivity.Knock down HIF-1αGene can inhibit the proliferation and migration of nasopharyngeal carcinoma cell line HONE1,increase apoptosis after radiotherapy and improve radiosensitivity.In nasopharyngeal carcinoma cells HONE1,overexpression of BMAL1 gene can inhibit the expression of HIF-1αprotein while knockdown of BMAL1 gene can promote the expression of HIF-1αprotein.展开更多
基金supported in part by grants from the National Natural Science Foundation of China under grant number 82060556,81560437the Department of Science and Technology,Guizhou Province,under grant number[2018]2755+3 种基金the Ordinary Colleges and Universities Youth Science and Technology Talent Growth Project,Guizhou Province,under grant number[2021]187The Health Commission Science and Technology Fund,Guizhou Provincial under grant number gzwkj2021-050Guizhou Medical University 2021 National Foundation Cultivation Project[20NSP041]the Hospital-level Science and Technology Project of Guizhou Cancer Hospital under grant number YJ2019-33.
文摘Objective To understand the effects of clock gene BMAL1 and HIF-1α(Hypoxia inducible factor-1α)on proliferation,migration and sensitivity to radiotherapy of nasopharyngeal carcinoma cells HONE1.At the same time,whether the biological clock gene BMAL1 can affect the expression of HIF-1αprotein was investigated.It will lay the foundation for further study on the correlation between clock gene BMAL1 and HIF pathway.Methods BMAL1 gene overexpression and interference lentivirus and HIF-1αgene interference lentivirus were constructed respectively,and were transfected into nasopharyngeal carcinoma cells HONE1.Western blot was used to verify the establishment of overexpressed and knockdown BMAL1 cell lines and HIF-1αgene knockdown cell line,and to investigate the expression of HIF-1αprotein in overexpressed and knockdown BMAL1 cell lines.CCK-8 cell proliferation test and scratch test were used to analyze the proliferation and migration ability of cells.Cell apoptosis after radiotherapy was analyzed by flow cytometry.The effects of BMAL1 and HIF-1αon the sensitivity of HONE1 radiotherapy in nasopharyngeal carcinoma cells after X-ray irradiation at different doses(0Gy,2Gy,4Gy,6Gy)were detected by clone formation assay.Results The overexpression of BMAL1 gene and lentivirus interference were constructed to effectively up regulate and down regulate the expression of BMAL1 protein in nasopharyngeal carcinoma cells HONE1.Meanwhile,HIF-1αgene interference lentivirus was constructed to effectively down-regulate the expression of HIF-1αprotein in nasopharyngeal carcinoma cell line HONE1,and successfully screen out stable nasopharyngeal carcinoma cell lines.Western blot results showed that overexpression of BMAL1 gene could inhibit the expression of HIF-1αprotein in HONE1 of nasopharyngeal carcinoma cells,while knockdown of BMAL1 gene promoted the expression of HIF-1αprotein in HONE1 of nasopharyngeal carcinoma cells(P<0.05).CCK-8 cell proliferation and scratch test showed that overexpression of BMAL1 gene or knockdown of HIF-1αgene could inhibit the proliferation and migration of HONE1 cells(P<0.05).Flow cytometry results showed that after 8Gy irradiation for 72 h,the apoptosis rate of BMALl gene overexpression group was higher than that of the overexpression control group,similarly,the apoptosis rate of HIF-1αgene knockdown group was higher than that of the knockdown control group(P<0.05).After X-ray irradiation at different doses(0Gy,2Gy,4Gy,6Gy),clon-formation experiment showed that the clon-formation rate and cell survival fraction of BMALl overexpression group or HIF-1αknockdown group were lower than those of negative control group(P<0.05).Sigmaplot analysis showed that the D0,Dq and SF2 of the BMAL1 overexpression group or HIF-1αknockdown group were lower than those of the negative control group,and the radiosensitization ratios were 1.381 and 1.063,respectively.Conclusion Overexpression of BMAL1 gene can inhibit the proliferation and migration of nasopharyngeal carcinoma cell line HONE1,increase apoptosis after radiotherapy and improve radiosensitivity.Knock down HIF-1αGene can inhibit the proliferation and migration of nasopharyngeal carcinoma cell line HONE1,increase apoptosis after radiotherapy and improve radiosensitivity.In nasopharyngeal carcinoma cells HONE1,overexpression of BMAL1 gene can inhibit the expression of HIF-1αprotein while knockdown of BMAL1 gene can promote the expression of HIF-1αprotein.
