| Objective:Acquired radioresistance is thought to be the main cause of recurrent metastasis after failed radiotherapy for nasopharyngeal carcinoma(NPC),possibly related to X-ray-induced activation of epithelial-mesenchymal transformation(EMT).The circadian clock gene BMAL1 has been shown to correlate with radiotherapy sensitivity for NPC,but the specific mechanism has not been reported.This study aims to explore the effect and molecular mechanism of the circadian clock gene BMAL1 on acquired radioresistance and EMT of NPC through in vitro and in vivo experiments,so as to provide a theoretical basis and laboratory basis for improving the efficiency of clinical radiotherapy.Methods:1.To explore the expression of BMAL1 gene in NPC cell lines,the establishment and biological behavior of radiation-resistant cell lines,and EMT verification:(1)The expression of BMAL1 protein in human nasopharyngeal immortalized epithelial cells NP69 compared with NPC cell lines CNE1,HONE1 and 5-8F was detected by western blotting;(2)NPC radiation-resistant cells 5-8FR(70Gy)were constructed by irradiating 5-8F cells in fractional 2Gy cycles,and the construction of radiation-resistant cell lines was verified by cloning formation assays;(3)The changes in EMT-related indexes,proliferation,migration and invasion ability in5-8FR were verified by western blotting,cell counting kit-8 assays,wound-healing tests and transwell assays.2.To explore the effect of BMAL1 gene on NPC acquired radioresistance:(1)the lentivirus NPC radio-resistant cell line 5-8FR of BMAL1 gene overexpression(BMAL1-OE)and its blank control group(OENC)was used to verify the transfection efficiency by western blotting;(2)The correlation between radiation dose and BMAL1 protein expression was explored by western blotting;(3)Cloning formation assays were used to explore the effect of BMAL1 gene on NPC acquired radioresistance in radiation-resistant cell lines 5-8FR.3.To explore the effects of BMAL1 gene on TGF-β1/Smads pathway:(1)The overexpression(BMAL1-OE)and knockout(sh-BMAL1)of BMAL1 gene and its blank control group(OENC/sh NC)were used to infect lentivirus with NPC cell lines5-8F and HONE1,respectively,and verified the transfection efficiency by western blotting;(2)The expression of TGF-β1/Smads pathway-related proteins in NPC cells was detected by western blotting when overexpressing and interfering with BMAL1 gene in NPC cells;4.To explore the mechanism of BMAL1 gene affecting NPC-acquired radioresistance:(1)In vitro,BMAL1 gene was explored by western blotting,cell counting kit-8 assays,wound-healing tests,transwell assays,flow cytometry,EDU methods and nuclear plasma separation tests to explore the EMT,proliferation,migration,invasion ability,apoptosis after radiotherapy,DNA synthesis ability,effects of nuclear translocation of BMAL1 gene in 5-8FR cell lines when recombinant human TGF-β1 stimulatory factor was added.(2)In vivo,the growth curve of nude mice transplanted tumors before and after radiotherapy was drawn by the subcutaneous tumorigenic model of nude mice,and after the sacrifice of nude mice,the transplanted tumors were weighed and subjected to HE staining,immunohistochemical staining and TUNEL staining,and the influence mechanism of BMAL1 gene on EMT and NPC radiosensitivity was explored.Results:1.The expression of BMAL1 gene in NPC cell lines,the establishment and biological behavior of radiation-resistant cell lines,and the EMT verification results suggested:(1)Compared with normal nasopharyngeal epithelial cells,BMAL1 expression in NPC cell lines was significantly downregulated(p < 0.05);(2)The cloning formation assays showed that the colony formation rate of radiation-resistant cell lines 5-8FR was higher than that of 5-8F at different radiation doses(p < 0.05),and the radiosensitization ratio of radiation-resistant cell lines 5-8FR was 1.256(> 1)by fitting the dose-survival curve and calculating,indicating that the radiation-resistant cell line 5-8FR was successfully constructed;(3)EMT activation existed in radiation-resistant cell lines 5-8FR,accompanied by significant enhancement of proliferation,migration and invasion(p < 0.05).2.The results of the study on the effect of BMAL1 gene on NPC acquired radioresistance showed that:(1)the overexpression of BMAL1 gene(BMAL1-OE)lentivirus effectively upregulated the expression of BMAL1 protein(p < 0.05)in radiation-resistant cell lines 5-8FR,and successfully screened out stable cell lines;(2)The cloning formation assays showed that the colony formation rate(p < 0.05)was lower in the 5-8FR BMAL1-OE group than in the control group,and the radiosensitization ratio of the cells in the 5-8FR BMAL1-OE group was 0.801(< 1)by fitting the dose-survival curve and calculating the radiosensitivity ratio of the5-8FR BMAL1-OE group,suggesting that the BMAL1 gene could significantly improve the radiosensitivity of the radiation-resistant cell line 5-8FR and reverse the acquired radioresistance of NPCs.3.The effects of BMAL1 gene on TGF-β1/Smads pathway showed that:(1)BMAL1 gene overexpression(BMAL1-OE)and interference lentivirus(sh-BMAL1)effectively upregulated 5-8F and downregulated the expression of BMAL1 protein(p< 0.05)in HONE1 cell lines,and successfully screened stable cell lines;(2)Overexpression of BMAL1 gene significantly inhibited the expression of TGF-β1/Smads pathway-related protein in NPC cells compared with its control group,while interference with BMAL1 gene had opposite results(p < 0.05).4.The results of the mechanism study of BMAL1 gene and NPC acquired radioresistance suggest that:(1)under the induction of exogenous recombinant human TGF-β1 stimulating factor,the activity of TGF-β1/Smads signaling pathway is enhanced,and EMT is further activated in radiation-resistant cell lines 5-8FR,accompanied by further improvement of proliferation,migration and invasion ability;At the same time,after radiotherapy,anti-apoptosis and DNA synthesis ability were improved,p-Smad2 was promoted to intranuclear migration,and the expression of intranuclear transcription factor Snail1 was upregulated(p < 0.05).However,overexpression of BMAL1 gene can reverse the above induction effect of TGF-β1,manifested as inactivated EMT,significantly inhibiting proliferation,migration and invasion ability,significantly increasing apoptosis after radiotherapy of radiation-resistant cell lines 5-8FR,inhibiting DNA synthesis ability after radiotherapy,reducing p-Smad2 nuclear translocation,and significantly decreasing the expression of intranuclear transcription factor Snail1(p < 0.05).(2)In vivo tests showed that exogenous addition of TGF-β1 stimulating factor could promote the growth of transplanted tumors,promote EMT,and increase radioresistance(p < 0.05);Overexpression of BMAL1 gene can reverse the above induced effects of TGF-β1,which is manifested as significantly inhibiting the growth of nude mouse transplant tumors,inactivating EMT,and promoting apoptosis after radiotherapy,thereby improving the radiosensitivity of NPC(p < 0.05).Conclusions:1.The circadian clock gene BMAL1 inhibits the expression of TGF-β1/Smads signaling pathway protein in NPC.2.The circadian clock gene BMAL1 inhibits radiation-induced EMT through inactivation of TGF-β1/Smads pathway,and inhibits the proliferation,migration and invasion ability of radiation-resistant cell lines of NPC.3.The circadian clock gene BMAL1 is related to the acquired radioresistance of NPC,inhibits radiation-induced EMT by inactivating TGF-β1/Smads/Snail1 axis,and induces radiosensitization of NPC. |
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