| PurposeTo investigate the different expression of circadian rhythm genes and autophagy-related genes in ovarian cancer OVCAR3 cells and OVCAR3/DDP cells,and the relationship of their expression.To explore the possible mechanism of circadian rhythm genes affecting cisplatin sensitivity in ovarian cancer OVCAR3 cells.MethodsFirst,Using whole human genome Oligo arrays(Affymetrix),circadian rhythm genes and autophagy-related genes that significantly differentially expressed between OVCAR3/DDP and OVCAR3 cell lines were identified.Selected genes were confirmed by RT-qPCR.Second,the expression of autophagy-related genes(LC3、BECN1 and P62)among the two cell lines were measured by in ovarian cancer OVCAR3 cells and OVCAR3/DDP cells,and Using immunofluorescence experiments to identify the distribution and expression differences of LC3 protein.Furthermore,OVCAR3 cell lines were treated with different concentrations(0 μg/ml、0.5 μg/ml、1 μg/ml、2 μg/ml、4 μg/ml)of cisplatin for 12 h 、 24 h and 48 h,we examined circadian rhythm genes and autophagy-related genes after treating by RT-qPCR,explored LC3,BECN1 and P62 protein by in ovarian cancer OVCAR3 cells and OVCAR3/DDP cells,,and investigated their correlation.Finally,we treated OVCAR3 cell lines with autophagy inducer rapamycin for 24 h,measured circadian rhythm and autophagy-related genes expression changes by RT-qPCR,using in ovarian cancer OVCAR3 cells and OVCAR3/DDP cells,to detect the changes of LC3 protein expression and explored the mechanism involved.Results1.The results of whole human genome Oligo arrays showed that 13 Autophagy-related genes and 6 circadian rhythm genes were significantly different in two cell lines,of autophagy-related genes,11 were up and 2 were down.All circadian rhythm differential genes were up-regulated.2.The results of RT-qPCR showed that the different expressions of circadian rhythm genes(CLOCK,PER1,CRY2),mTOR and autophagy-related genes(BECN1,P62,LC3)in OVCAR3 and OVCAR3/DDP cells,which was consistent with the human genome Oligo arrays results,and the differences were statistically significant(P<0.05).3.Immunofluorescence indicated that a large amount of LC3 protein was expressed in the cytoplasm of OVCAR3/DDP cells around the nucleus,and LC3 protein was significantly less in OVCAR3 cells.4.The results of in ovarian cancer OVCAR3 cells and OVCAR3/DDP cells,showed that compared with OVCAR3 cells,the expression of BECN1 and LC3 was enhanced with the time and concentration in OVCAR3/DDP cells.The conversion of LC3-I to LC3-II was enhanced and the expression of P62 was down-regulated,the difference was statistically significant(P<0.05).5.After treatment of cisplatin,RT-qPCR showed the expressions of CLOCK,PER1,CRY2,BECN1 and LC3 mRNA were enhanced in OVCAR3 cells,The trends of them were the same.The expressions of mTOR and P62 were weakened,and the difference was statistically significant(P<0.05).The results of in ovarian cancer OVCAR3 cells and OVCAR3/DDP cells,showed the expression of LC3 protein was enhanced,and the expression of mTOR and P62 protein was weakened,and the difference was statistically significant(P<0.05).6.After treatment of rapamycin in OVCAR3 cells,the mRNA expressions of CLOCK,PER1 and CRY2 were not changed by RT-qPCR.while level of BECN1 and P62,mTOR were significantly up-regulated and down-regulated respectively.Western blot results suggested that the expression of LC3 protein were significantly increased.Conclusions1.We found that selected genes,including circadian rhythm genes(CLOCK PER1、CRY2),autophagy-related genes(LC3、BECN1、P62)and mTOR were confirmed to be significantly differentially expressed by whole human genome Oligo arrays(Affymetrix),RT-qPCR and in ovarian cancer OVCAR3 cells and OVCAR3/DDP cells,.2.We demonstrated up-regulation of circadian rhythm genes are associated with autophagy in two cell lines.3.Circadian rhythm genes may inactivate autophagy via mTOR pathway and affect cisplatin sensitivity in ovarian cancer OVCAR3 cells. |
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