Studies On Classical Wnt/?-catenin Signaling Pathway And Cisplatin Resistance In Cervical Cancer

ObjectiveCervical cancer is one of the most common female malignancies worldwide,which is a big threat to women's health and life safety.Chemotherapy is an important adjuvant for clinical treatment of cervical cancer,which is critical to reducing mortality rate,prolonging survival and improving quality of life.Platinum is the first-line chemotherapy for cervical cancer treatment,especially cisplatin,which has showed a good therapeutic effect in cervical cancer.However,tumor resistance is the most important cause of chemotherapy failure in cervical cancer.At present,the detailed mechanism of drug resistance in cervical cancer has not been fully understood.Exploring the causes of resistance and finding ways to solve it is one of the focuses of current cancer research.Wnt/?-catenin signaling pathways is closely related to the occurrence and development of tumors.Many studies have found that Wnt pathway overactivation in human tumors such as ovarian cancer,renal cancer,prostate cancer and non-small cell lung cancer.It isdemonstrated that the Wnt/?-catenin singaling pathway is involved in mutipletypesof tumors.Chemotherapy resistance was the most important cause of treatment failure and poor prognosis in patients with cervical cancer.It was necessary to identify the chemoresistance mechanism of cervical cancer,deepen the understanding of complex signaling pathways regulation in the process of cervical cancer resistance.It would provide a new perspective on the drug resistance.This study was to investigate the role of Wnt/?-catenin signaling pathway on cisplatin resistance in cervical cancer and its mechanism.The research results were of great value for finding potential molecular targets related to drug resistance of cervical cancer,providing a new solution to the drug resistance.Methods1.With primary cervical cancer tissues and recurrent cisplatin-resistant cervical cancer tissues as research objects,the differences in gene expression in the two groups were analyzed by RNA-seq sequencing,and further verified by immunohistochemistry,qPCR,westernblot and other molecular biological means.The relationship between differentially expressed genes and patient prognosis was analyzed by TCGA database2.Human cervix cancer SiHa,HeLa,C33A and CaSki cell lines were induced with a final concentration of 10 mg/L cisplatin to establish the cisplatin-resistant cells.We compared the morphology,main biological characteristics and resistance index of cells to cisplatin.The real-time fluorescent quantitative PCR and Western blot were used to detect the expression of ?-catenin and memebers in Wnt/?-catenin signal pathway such as Survivin?c-myc?cyclin D1?MMP-2?Frizzled.3.The ?-catenin eukaryotic expression vector and Wnt/?-catenin pathway inhibitor XAV939 were used to treat the cervical cancer resistant cells alone or in combination.After different treatments in cisplatin-resistant cells.quantitative real-time PCR and Western blot were used to detect the mRNA and protein level of?-catenin,survivin,c-myc,cyclin D1,MMP-2 and Frizzled,flow cytometry was used to detect the apoptosis rate and MTT was to assay cell proliferation of each group.4.The xenograft model of cisplatin-resistant cervical cancer cells in nude mice was induced by subcutaneous injection.The nude mice were randomly divided into control group,cisplatin group,?-catenin+cisplatin group and XAV939+cisplatin group.After each intervention,the general condition of the nude mice and the growth of transplanted tumor were observed,the tumor volume was measured to make the growth curve,and the apoptosis of the transplanted tumor was detected by Tunel assay.Immunohistochemistry was to evaluate the expression of nuclear protein Ki-67 and Western blot was to detect the expression of Survivin,c-myc,cyclin D1,MMP-2 and Frizzled in Wnt/?-catenin pathway.ResultsPart ?:RNA-seq analysis found abnormal activation of Wnt/?-catenin signaling pathway in recurrent cisplatin-resistant cervical cancer tissues(n=3).Immunohistochemical and qPCR and Western blot in found that compared with normal cervical tissue,?-catenin in primary cervical cancer tissues have a small amount of expression,and in the recurrent cisplatin resistance of ?-catenin expression in cervical cancer tissue abnormalities increased than the other two groups(P<0.05),Western blot results also show that Wnt/?-catenin signaling pathways downstream target protein Survivin in recurrent cervical cancer tissue of the expression of cisplatin resistance rise significantly(P<0.05).The TCGA database was used to analyze the relationship between the expression of ?-catenin and Survivin in cervical cancer tissues and the survival curve of patients.It was found that the expression of?-catenin and Survivin in cervical cancer tissues was negatively correlated with the survival time of patients,and the higher the expression of ?-catenin and Survivin,the shorter the survival time of patients.Part ?:1.The RI of SiHa/DDP,HeLa/DDP,C33A/DDP and CaSki/DDP cell lines were 12.34,7.92,7.13 and 17.65,respectively,which showed moderate to severe cisplatin resistance,indicating successful cell line modeling.The apoptosis rates of SiHa/DDP,HeLa/DDP,C33A/DDP and CaSki/DDP cells induced by cisplatin were significantly lower than their parent cells.MRNA and protein levels of-catenin were higher in SiHa,HeLa,C33A and CaSki cisplatin resistant cell lines than in SiHa,HeLa,C33A and CaSki cells(P<0.05).2.The apoptosis rates of SiHa/DDP,HeLa/DDP,C33A/DDP and CaSki/DDP cells induced by cisplatin and each parental cellswere(3.3±0.64)%vs.(36.9±2.56)%,(8.56±1.27)%vs.(47.55±3.78)%,(22.71±4.19)%vs.(47.44±6.97)%and(12.38±3.56)%vs.(58.44±5.63)%respectively,which were significantly lower than their parent cells(P<0.01).3 mRNA and protein levels of-catenin in SiHa,HeLa,C33A and CaSki cell lines were higher than those in normal cervical squamous cells(Ectl/E6E7)(P<0.05),and mRNA and protein levels of-catenin in SiHa,HeLa,C33A and CaSki cisplatin resistant cell lines were higher than those in SiHa,HeLa,C33A and CaSki cells(P<0.05).Part ?:1 Survivin,c-myc,cyclin D1,mmp-2,and Frizzled mRNA and protein levels in SiHa,HeLa,C33A,and CaSki cisplatin resistant cell lines overexpressed mt-catenin were significantly higher than their parent cell lines(all P<0.05)and significantly higher(all P<0.05),The mRNA and protein levels of Survivin,c-myc,cyclin D1,mmp-2 and Frizzled in the four drug-resistant cell lines showed a decreasing trend.2.The cell proliferation results showed that,with the prolongation of time,the cell proliferation capacity of group?-catenin was the strongest in the cisplatin resistant cells of the four cervical cancers,followed by group?-catenin+XAV939,and the cell proliferation of group XAV939 was the slowest,and XAV939 significantly inhibited the cell proliferation.Cell apoptosis results showed that the overexpression of ?-catenin in SiHa/DDP,HeLa/DDP,C33A/DDP and CaSki/DDP cells was not significant,and the apoptosis rate of XAV939 group was higher than that of the-catenin group and XAV939+?-catenin group(P<0.05),and XAV939 could significantly promote cell apoptosis.Part ?:1.In vivo experimental results showed that subcutaneous tumor transplantation model of cisplatin resistant cells in nude mice of cervical cancer was successfully constructed.Compared with the control group,the volume of tumor transplantation in nude mice of cisplatin group and XAV939+cisplatin group decreased significantly compared with the control group and?-catenin+cisplatin group,while the volume of tumor transplantation in XAV939+cisplatin group decreased significantly compared with that in cisplatin group.There was no significant difference between the control group and the 40-catenin+cisplatin group,and the overall change was as follows:control group/?-catenin+cisplatin group>cisplatin group>XAV939+cisplatin group.8 The apoptosis indexes of cisplatin group,?-catenin+cisplatin group,XAV939+cisplatin group and control group werc(16.26±1.34)%,(7.96±0.53)%,(27.33±1.78)%and(6.54±0.32)%,respectively.The apoptosis index of XAV939+cisplatin group was higher than that of cisplatin group(P<0.05),and both were higher than that of control group(P<0.05).There was no significant difference in apoptotic index between-catenin+cisplatin group and control group(P>0.05).3.Immunohistochemical results showed that the average optical density of ki-67 protein expression in transplanted tumor in cisplatin group was(0.038±0.0120),lower than that in control group(P<0.05);the average optical density of ki-67 protein expression in XAV939+cisplatin group was(0.024±0.008),lower than that in control group and cisplatin group(P<0.05).4.There was no significant difference in the protein expression levels of Survivin,c-myc,cyclin D1,mmp-2 and Frizzled in the control group and the cisplatin group(P>0.05),while the protein levels of Survivin,Survivin,c-myc and mmp-2 in the cisplatin+ group were significantly higher than those in the cisplatin group(P<0.05).XAV939+ cisplatin group had significantly lower levels of-catenin,Survivin and c-myc protein than cisplatin group(P<0.05).Conclusion1.Abnormal activation of Wnt/?-catenin signaling pathway was found in recurrent cisplatin tolerant cervical cancer tissues,while the expression was weak in normal cervical tissues and primary cervical cancer tissues,which may be closely related to cisplatin tolerance of cervical cancer.2.Wnt/?-catenin signaling pathway is abnormally activated in cisplatin-resistant cells of cervical cancer,and the expression levels of key factors Survivin,c-myc,cyclin D1 and MMP-2,Frizzled are up-regulated.3.Activation of Wnt/?-catenin signaling pathway can promote the proliferation of cisplatin-resistant cells in cervical cancer,inhibit apoptosis and promote the growth of xenografts in nude mice.Inhibition of Wnt/?-catenin signaling pathway can reduce the proliferative activity of the drug resistant cells,promote cell apoptosis and inhibit the growth of transplanted tumors in nude mice;4.Activation of Wnt/?-catenin signaling pathway reduces the sensitivity of cervical cancer drug-resistant cells to cisplatin;inhibition of Wnt/?-catenin signaling pathway can increase the sensitivity of cervical cancer to cisplatin and promote the anticancer effect of cisplatin.The combination of Wnt/?-catenin inhibitor and cisplatin can promote the anticancer effect of cisplatin.