Effects Of MAGE-A3 Gene On Proliferation And Apoptosis Of Human Cervical Cancer Cells And Its Possible Mechanism

BackgroundAs a common malignant tumour,cervical cancer ranks fourth in the cause of cancer-relative death all over the world,and it remains one of the major causes of cancer-related death in women worldwide.Although cervical cancer treatment options,including radiotherapy,chemotherapy,and surgery,have made significant progress,the overall 5-year survival rate of patients with cervical cancer remains unfavourable because of the recurrence and metastasis.Therefore,the development of new diagnosis and treatment strategies is required to reduce recurrence and improve the survival rate.Melanoma-associated antigen A3(MAGE-A3)gene is a cancer-testis antigen(CTAs)gene whose expression has been demonstrated in a wide array of malignancies,including melanoma,breast,colorectal,gastric,lung and pancreatic cancer.Emerging data have reported that aberrant expression of MAGEA3 in many tumour types has been shown to correlate with poor clinical outcome.To investigate whether MAGEA3 is involved in the tumorigenesis and progression of human cervical cancer,our group had previously detected the expression of MAGEA3 by quantitative RT-PCR,Western blot and immunohistochemical methods in cervical lesions tissues compared with normal tissues.The results showed that the expression of MAGEA3 in CC tissues was significantly higher than that of the normal group.Moreover,The expression level of MAGEA3 was positively correlated with the clinical stage,pathological grade,lymphatic metastasis of cervical cancer.The expression of MAGE-A3 was further detected in cervical cancer cell line and immortalized normal cervical cell line(End1/E6E7),the results showed that the protein level of MAGE-A3 m RNA was significantly higher in cervical cancer cell line compared with End/E6E7.Base on this,the present study was performed to determine the effects of MAGE-A3 on cervical cancer cells,and to investigate the underlying mechanism of MAGEA3 in carcinogenesis.Chapter I Construction and identification of cervical cancer cell lines with overexpression and silencing of MAGE-A3 genesObjective: To detect the expression of MAGE-A3 in cervical cancer cell line and construct overexpression and silencing MAGE-A3 cervical cancer cell line,which provides the basis for exploring the role of MAGE-A3 in cervical cancer.Methods:1.The cervical cancer cell line(Hela、Siha、C33A、Caski)was selected as the study object.Real-time fluorescence quantitative PCR(q RT-PCR)and western blotting(WB)were used to detect the m RNA and protein expression in the cervical cancer cell lines.2.Cervical cancer cells were infected with MAGE-A3-gene-overexpressed lentivirus(LV-MAGEA3)to construct the MAGE-A3 gene overexpressed cells.The infection or transfection efficiency was detected by q RT-PCR and WB.3.Using transient transfection,cervical cancer cells were infected with the MAGEA3-specific si RNA(si RNA-MAGEA3)to establish the MAGE-A3 gene silenced cells.The infection or transfection efficiency was detected by q RT-PCR and WB,and the effective si RNA interference chain was screened.Results:1.qRT-PCR and WB test results showed that among the four cervical cancer cell lines,including Caski,Hela,C33 A,and Siha,the expression of MAGE-A3 was relatively high in Hela and relatively low in Siha.2.Siha cells were selected as the object of overexpression of MAGE-A3.The empty vector lentivirus(negative control,Lv-NC)and overexpression lentivirus(Lv-MAGEA3)were transfected into Siha cells,respectively.The results of q RT-PCR and WB showed that compared with Lv-NC,the expression of MAGE-A3 in Lv-MAGEA3 was significantly up-regulated.3.Hela cells were selected as the object of silence MAGE-A3.The negative control si RNA(si RNA-NC)and MAGEA3-specific si RNA(si MAGEA3-1,si MAGEA3-2,si MAGEA3-3)were transfected into Hela cells,respectively.The results of q RT-PCR and WB showed that the expression of MAGE-A3 in the si RNA-NC group was not significantly different from that in the Blank control.The gene knockdown efficiency of si MAGEA3-1,si MAGEA3-2 and si MAGEA3-3 were(88.31±1.28)%,(77.64±4.33)%and(16.27±3.41)%,respectively.Among them,si MAGEA3-1 and si MAGEA3-2 were the most significant efficiency,and they were applied for further experiments.Conclusion:Hela cells had the endogenous highest level of MAGEA3 expression,and Siha had the lowest expression level.The MAGE-A3 overexpression lentivirus successfully constructed the MAGE-A3 overexpression cervical cancer cell line,and the MAGE-A3 silencing cervical cancer cell line was successively constructed using MAGE-A3-specific si RNA.Chapter II Effects of MAGE-A3 on proliferation and apoptosis of cervical cancer cellsObjective: To evaluate the effects of MAGE-A3 on cell proliferation,cell cycle and apoptosis of cervical cancer cells.Methods:1.Using CCK-8 、 EdU cell proliferation experiments to evaluate the effects of overexpression or silencing MAGE-A3 on the proliferation of cervical cancer cells.2.Using flow cytometry to evaluate the effects of overexpression or silencing MAGE-A3 on cell cycle and apoptosis of cervical cancer cells.Results:1.The results of CCK-8 and Ed U experimental showed that overexpression MAGE-A3 promote the proliferation ability of cervical cancer cells while silencing MAGE-A3 inhibit the proliferation ability of cervical cancer cells.2.The results of flow cytometry showed that overexpression MAGE-A3 promotes cell cycle progression in cervical cancer(S phase cell ratio increased),inhibit cell apoptosis(decreased apoptosis rate)(P<0.05).Silencing MAGE-A3 arrest the cell cycle in the G1 phase(the G1 phase cell increased,and the S phase cell decreased)and induce apoptosis(increased apoptosis rate)(P<0.05).Conclusion:MAGE-A3 promotes cervical cancer cell proliferation and cell cycle progression,inhibit apoptosis,suggesting that MAGE-A3 play a critical role in the occurrence and development of cervical cancer.Chapter III Relationship between MAGE-A3 and KAP1/P53 signaling pathway in cervical cancerObjective: To observe the effects of MAGE-A3 on KAP1/P53 signaling pathway in cervical cancer,and to explore the possible mechanism of MAGE-A3 in promoting cervical cancer.Methods:1.qRT-PCR and WB were used to detect changes in KAP1、p53 m RNA and protein expression after overexpression or silencing of MAGE-A3 genes in cervical cancer cells.2.Using WB to detect protein expression in downstream of the p53 pathway involved in the cell cycle(P21,Cyclin D1)and apoptosis(Bax,Bcl-2).3.The Nutlin3(p53 activator)was added to the medium of overexpression MAGE-A3 cells and cultured for 48 h.The changes in cervical cancer cell cycle and apoptosis were analyzed by flow cytometry.Using WB to detect the protein expression changes of p53 and its downstream genes p21 and Bax.Results:1.The results of qRT-PCR and WB showed that the KAP1 m RNA and protein expression was up-regulated and the p53 expression was down-regulated.2.WB detection results showed that in the Siha-Lv-MAGEA3 group,the expression of p21 and Bax in the downstream of p53 pathway were down-regulated,the expression of Cyclin D1 and Bcl-2 were up-regulated,and the apoptosis protein cleaved-caspase3 expression was decreased(P < 0.05).The detection results of Hela-si MAGEA3-1 and si MAGEA3-2 groups were contrary to the above,and the difference was statistically significant.3.The results of flow cytometry showed that compared with Lv-NC group,the S phase cells were increased and the apoptosis rate decreased in the Lv-MAGEA3 group,and the expression of p53、p21、Bax protein was down-regulated(P<0.05).Nevertheless,after cell cultured with Nutlin3 48 h,the S phase cells decreased,the G1 phase cells increased,and the apoptosis rate increased.WB detection showed that the expression of p53、p21 and Bax protein was up-regulated compared with the Lv-MAGEA3 group(P<0.05).Conclusion:MAGE-A3 promotes proliferation and inhibit apoptosis of cervical cancer cells through KAP1/p53 signaling pathway.