The Role Of Oct4 In Promoting Radioresistance And EMT Induced By Irradiation In Cervical Cancer Cells

Part ? Establishment and characteristics of radioresistant cells of human cervical cancer cell lines Objective To establish radioresistant human cervical cancer lines,to determine some of their characteristics.Methods(1)Cervical cancer cell lines Hela and Siha(the parental cells)were selected to culture in the medium and exposure to X-rays ionizing radiation with a cumulative dose of 70 Gy(2 Gy per fraction,twice a week).And then survived cells were cultured for 5 generations and named as Hela-R and Siha-R.(2)In contrast to the parental cells,some biological characteristics of Hela-R and Siha-R were determined using the following methods.The changes of cellular morphology were observed by microscope.The radioresistance were measured by survival fraction analysis after exposure to gradual dose ranging from 0 to 8 Gy.The number of colony formation after exposure to 4 Gy and 6 Gy were determined by plating colony formation assay.The cell apoptosis rates were analyzed by flow cytometry at 48 hours after exposure to 0,6 and 12 Gy.The nuclear foci-formation of ?-H2 AX were detected after exposure to a single dose of 5 Gy at 24 hours.The expression of RAD21,TGF-?1,Oct4 and E-cadherin,N-cadherin and Vimentin were detected by PCR and Western Blot.The capacity of migration and invasion were separately tested by Transwell with or whithout Matrigel assay.Results The Hela-R and Siha-R cells lost their original cellular morphology and presented spindle-like and loss of cellular polarity compared to the parental cells.The Hela-R and Siha-R cells exhibited markedly increased resistance to irradiation.The apoptosis rates of Hela-R and Siha-R cells were much lower than their parental cells at the same dose of irradiation.The number of ?-H2 AX foci in Hela-R and Siha-R cells were lower at 24 hours than the parental cells.PCR and Western Blot analysis showed that the expression of RAD21,TGF-?1,Oct4,N-cadherin and Vimentin were significantly increased while E-cadherin decreased.Moreover,the Hela-R and Siha-R cells enhanced migration and invasion ability.Conclusion Hela-R and Siha-R cells had a higher resistance to irradiation.The expression of RAD21,TGF-?1 and Oct4 increased in radioresistant cells.Besides,the radioresistant cells exhibited EMT and enhanced migration and invasion.Part ? The effects of knockdown of Oct4 by siRNA on radioresistance and EMT of radioresistant cellsObjective To clarify the relationship between Oct4 and irradiation-induced radioresistance and EMT.Methods(1)Radioresistant cells were transfected with Oct4-siRNA and then transfection efficiency were detected by Western Blot.(2)Compared with the empty vector transfection group,after knockdown of Oct4 the following methods were used to detect the relevant changes.Survival fraction were analyzed after exposure to gradual dose ranging from 0 to 8 Gy.The number of residual ?-H2 AX forci in the nucleus at 24 hours after 5 Gy irradiation.The cell apoptosis rates were analyzed by flow cytometry at 48 hours after exposure to 0,6 and 12 Gy.The expression of E-cadherin,N-cadherin and Vimentin were detected by Western Blot.The migration and invasion ability were tested by Transwell with or without Matrigel assay.Results The expression of Oct4 in the radioresistant cells were markedly decreased after transfected with Oct4-siRNA.Compared with the empty vector transfection group,the cell survival curve presented that the radioresistance of radioresistant cells decreased significantly.The number of residual ?-H2 AX forci in the nucleus were significantly increased at 24 hours after 5 Gy irradiation.The apoptosis rates of the radioresistant cells at 48 hours after exposure to 6 Gy and 12 Gy were much higher after knockdown of Oct4.Transwell with or without Matrigel assay exhibited attenuated migration and invasion ability of the radioresistant cells.Conclusion Oct4 might be one of the important factors which could promote radioresistance and EMT induced by irradiation in radioresistant cells.Moreover,Oct4 could enhance migration and invasion.Knockdown of Oct4 expression could decrease radioresistance and inhibit EMT as well as migration and invasion.Part ? The regulation of Oct4 expression by RAD21 and TGF-?1 in radioresistant cellsObjective To investigate whether RAD21 and TGF-?1 could regulate Oct4 expression in radioresistant cells.Methods Constructed the siRNA against RAD21,TGF-?1 and Oct4 and transfected the radioresistant cells with these siRNAs respectively.The changes of these protein expressions were detected by Western Blot.The capacity of RAD21 binding to TGF-?1 promoter regions was detected by Ch IP assay.Constructed RAD21 overexpression plasmids and transfected Hela and Siha with RAD21 overexpression plasmids.And then detected the expression of RAD21,TGF-?1 and Oct4.Using TGF-? receptor inhibitor LY2109761(10 umol/L)to treat radioresistant cells and then detected following changes.The change of radioresistance were detected by survial frction analysis.The number of ?-H2 AX forci in the nucleus at 24 hours after 5 Gy irradiation.The expression of Oct4 after 48 hours were detected by Western Blot.The migration and invasion ability were tested by Transwell with or without Matrigel assay.Results The expression of TGF-?1 and Oct4 were decreased after knockdown of RAD21.The expression of Oct4 markedly decreased after downregulation of TGF-?1.However,there was no significant change on the expression of RAD21 and TGF-?1 after knockdown of Oct4.Ch IP assay showed that RAD21 could directly bind to the promoter regions of TGF-?1.Overexpression of RAD21 could increase expression of TGF-?1 and Oct4.Treated radioresistant cells with LY2109761 could decrease its radioresistance and inhibit the expression of Oct4.Moreover,LY2109761 could reverse EMT and inhibit migration and invasion.Conclusion RAD21 and TGF-?1 were regulators of Oct4 and they could promote Oct4 expression.RAD21 could directly bind to the promoter regions of TGF-?1 and facilitate TGF-?1 expression.Knockdown of RAD21 or TGF-? receptor inhibitor could promote radiosensitivity and reverse EMT as well as decrease the capacity of migration and invasion.