Circadian Clock Gene BMAL1 Inhibits Tumorigenesis And Increases Paclitaxel Sensitivity In Tongue Squamous Cell Carcinoma

Part 1 Expression of circadian clock gene BMAL1 in tongue squamous cell carcinoma and their relationship with clinical significanceObjective:To investigate the expression levels of BMAL1 in tongue squamous cell carcinoma(TSCC)tissues and cells,and assess their relationship with clinical significance.Methods:Immunofluorescence,western blot analysis and qRT-PCR analysis were used to analyze the levels of BMAL1 at each time point as specified in advance in 53 TSCC tissue samples from TSCC patients and 50 non-tumorous tissue samples from other patients;immunofluorescence and Western blot analysis were used to analyze the levels of BMAL1 at CT 0 and CT 8 in SCC9,SCC25,CAL27 cells and human tongue keratinocytes(KCs)after synchronization;qRT-PCR analysis was used to analyze the circadian rhythm of BMAL1 in SCC9,SCC25,CAL27 cells and KCs after synchronization.Results:(1)Compared with the normal tongue epithelial tissues collected at 3 different periods of time,the average mRNA levels of BMAL1 were significantly reduced in both TSCC tissues and paired adjacent noncancerous tissues;(2)Compared with KCs,the average mRNA.levels of BMAL1 were significantly reduced in human tongue cancerous cells SCC9,SCC25 and CAL27;(3)Compared with KCs,we also noticed that human tongue cancerous cells SCC9,SCC25 and CAL27 displayed different BMAL1 expression rhythmic pattern characterized by shorter cycle and weaker amplitude.Conclusion:Our data indicated that the levels of circadian clock gene BMAL1 were reduced in both TSCC tissues and cells,and displayed different expression rhythmic pattern.It is suggested that the abnormal expression of circadian clock gene and the circadian rhythm disorders were closely correlated with TSCC,which have clinical significance in the prevention and treatment of TSCC.Part 2 Effects of circadian clock gene BMAL1 on the biological behavior of TSCCObjective:To investigate the effects of BMAL1 on the biological behavior of TSCC and the correlative mechanisms in vivo and in vitro.Methods:We designed and constructed BMAL1-overexpression plasmids and CRISP/Cas9 specific BMAL1-knockout plasmids,transfected them into human tongue cancerous cells SCC9,SCC25 and CAL27 by lipo2000 to upregulate or down-regulate the expression levels of BMAL1,and used the cells transfected with empty vector plasmids or wild type cells as control.First,CCK-8 assays,scratch wound-healing assays and matrigel invasion assays were used to analyze the effects of upregulating or downregulating BMAL1 on the proliferation,migration and invasion abilities of TSCC cells.Then,subcutaneous xenografted tumor mice models of SCC9 cell lines were established to evaluate the effects of upregulating or downregulating BMAL1 on the growth of TSCC cells.Finally,CCK-8 assays,Annexin V FITC/PI double staining flow cytometry,PI single staining flow cytometry and western blot analysis were performed to evaluate the effects of upregulating or downregulating BMAL1 on paclitaxel sensitivity in TSCC.Results:(1)We designed and constructed BMAL1-overexpression plasmids and CRISP/Cas9 specific BMAL1-knockout plasmids,up-regulated or down-regulated the expression levels of BMAL1 effectively,and screened stable transfected cell lines successfully;(2)Our results of CCK-8 assays,scratch wound-healing assays and matrigel invasion assays showed that the proliferation,migration and invasion abilities of BMAL1-overexpession TSCC cells all declined significantly;(3)The results of CCK-8 assays,scratch wound-healing assays and matrigel invasion assays showed that the proliferation,migration and invasion abilities of BMAL1-knockout TSCC cells all increased significantly;(4)The data of subcutaneous xenografted tumor mice models confirmed that BMAL1-overexpression could inhibit the growth rate of TSCC cells and BMAL1-knockout could accelerate the growth rate of TSCC cells in vivo;(5)The data of CCK-8 assays,Annexin V FITC/PI double staining flow cytometry,PI single staining flow cytometry and western blot analysis displayed the paclitaxel sensitivity of BMAL1-overexpression TSCC cells was enhanced,and the.proliferation inhibition and apoptosis phenomena were more obvious;(6)The data of CCK-8 assays,Annexin V FITC/PI double staining flow cytometry,PI single staining flow cytometry and western blot analysis displayed the paclitaxel sensitivity of BMAL1-knockout TSCC cells was reduced,and the abilities of paclitaxel in cell proliferation inhibition and apoptosis were weakened.Conclusion:BMAL1-overexpression or BMAL1-knockout in TSCC can inhibit or activate proliferation,migration and invasion respectively;subcutaneous xenografted tumor mice model of BMAL1-overexpression or BMAL1-knockout SCC9 cells showed the growth rate of tumors slowed down or speeded up obviously;BMAL1-overexpression TSCC cells can enhance the paclitaxel sensitivity and BMAL1-knockout TSCC cells can reduce the paclitaxel sensitivity.Part 3 Mechanism study of circadian clock gene BMAL1 on improving paclitaxel sensitivityObjective:To preliminarily investigate the regulatory mechanism of circadian clock gene BMAL1 improving paclitaxel sensitivity.Methods:First,we used transcriptome sequencing(RNA-seq)assay to compare the differences in gene transcription between BMAL1-overexpression SCC9 and WT SCC9 and preliminarily screened TERT,which is the target of BMAL1 improving paclitaxel sensitivity,by combining the results of qRT-PCR analysis.Then,we used qRT-PCR analysis and western blot analysis to explore the negative correlation between the expression of BMAL1 and TERT in TSCC tissues and cells.Next,we constructed and transfected TERT-overexpression plasmids or TERT-shRNA plasmids,using Annexin V FITC/PI double staining flow cytometry,PI single staining flow cytometry and western blot analysis to explore whether TERT-overexpression could reverse the effect of BMAL1 improving paclitaxel sensitivity and whether downregulation of TERT could cut down the effect of paclitaxel directly.Finally,we used Chromatin immunoprecipitation(ChIP)assay,coimmunoprecipitation(Co-IP)assay,double immunofluorescence staining and luciferase reporter assay to confirm the mechanism how BMAL1 regulates TERT.Results:(1)Our data of RNA-seq assay showed that the expression levels of TERT declined significantly in BAAL1-overexpression TSCC cells;(2)We used qRT-PCR analysis and western blot analysis,and discovered the significant negative correlation between the expression of BMAL1 and TERT in TSCC tissues and cells;(3)We discovered that co-transfection with BMAL1-overexpression plasmids and TTERT-overexpression plasmids could reverse the effect of BMAL1 improving paclitaxel sensitivity,and particular downregulation of TERT could improve the effect of paclitaxel directly in TSCC cells;(4)The results of Co-IP assay and double immunofluorescence staining confirmed that BMAL1 and EZH2 could combine with each other in nucleus.ChIP assay indicated that BMAL1 recruited EZH2 and bound to the promoter of TERT together.And luciferase reporter assay discovered that BMAL1 could inhibit TERT transcription after recruiting EZH2 and could promote TERT transcription after EZH2 ablated in TSCC cells.Conclusion:In TSCC cells,we confirmed that TERT is the target of BMAL1 improving paclitaxel sensitivity and BMAL1 could improve the paclitaxel sensitivity by downregulating TERT.Soon afterwards,the mechanism study confirmed that BMAL1 could inhibit TERT transcription by recruiting EZH2 and binding to the promoter of TERT.Part 4 Cooperativity study between circadian clock gene BMAL1 and Paclitaxel chronotherapyObjective:To find the best administration timing of synergistic effect between circadian clock gene BMAL1 and paclitaxel chronotherapyMethods:We transplanted BMAL1-overexpression SCC9 cells,WT SCC9 cells and BMAL1-knockout SCC9 cells into the subcutaneous tissues in nude mice.After 5 weeks,the tumors grew up and were collected at ZT2,ZT6,ZT10,ZT14,ZT18 and ZT22 respectively.qRT-PCR analysis was used to detect the circadian rhyth:m of BMAL1 and TERT in tumors.Then,we transplanted BMAL1-overexpression SCC9 cells,WT SCC9 cells and BM4L1-knockout SCC9 cells into subcutaneous tissues in nude mice again.After two weeks,we started to inject paclitaxel solution intraperitoneally at six different time points(ZT2,ZT6,ZT10,ZT14,ZT18 and ZT22)twice a week.Samples were taken after 4 weeks of drug injection.And then,we compared the differences of paclitaxel efficacy on the tumors between different administration timing.Results:(1)In SCC9/BMAL1 group and SCC9/wild type group,the BMAL1 expression levels showed significant rhythmic changes,while in SCC9/BMAL1?KO group,the BMAL1 levels did not change significantly;(2)In SCC9/BMAL1 group,the peak and valley of BMAL1 expression were at ZT10 and ZT22,while in SCC9/wild type group,the peak and valley of BMAL1 expression were at ZT6 and ZT18;(3)In SCC9/BMAL1 group and SCC9/wild type group,the fluctuation in the efficacy kinetics of paclitaxel was basically consistent with the expression fluctuation of BMAL1,while in SCC9/BMAL1-KO group,there was no significant fluctuation in the efficacy kinetics among all the experiment time points.Conclusion:The therapeutic efficacy of paclitaxel on TSCC was positively correlated with the expression of circadian clock gene BMAL1.BMAL1 is a direct molecular target for determining the best administration timing of paclitaxel in TSCC treatment.