Effects of biological clock gene BMAL1 and hypoxia-inducible factor HIF-1α on proliferation, migration and radiotherapy sensitivity of nasopharyngeal carcinoma cells HONE1
Authors: Tang Yaxue , Li Yuanyuan , Zhao Chaofen , Liu Lina , He Qianyong , Li Yuxin , Zhou Dingan , Jin Feng 
Published: July 2023

Abstract
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-1α protein 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-1α gene 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-1α gene knockdown cell line, and to investigate the expression of HIF-1α protein 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-1α on 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-1α gene interference lentivirus was constructed to effectively down-regulate the expression of HIF-1α protein 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-1α protein in HONE1 of nasopharyngeal carcinoma cells, while knockdown of BMAL1 gene promoted the expression of HIF-1α protein 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-1α gene 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-1α gene 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-1α knockdown 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-1α knockdown 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-1α Gene 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-1α protein while knockdown of BMAL1 gene can promote the expression of HIF-1α protein.