| Objective: The incidence rate of ovarian cancer came in third,and the fatality rate came in first in gynecological malignancies.Ovarian cancer is characterized by occult onset,lack of early typical symptoms and mature early diagnosis methods,and easy to be widely planted and spread in pelvic and abdominal cavity.It is often at an advanced stage as soon as it is diagnosed,and it is difficult to cure.The 5-year survival rate was only 35%-38%.Although ovarian cancers usually respond well to the first line chemotherapy based on platinum compounds and taxol,majority of patients develop recurrence and chemo-resistance.Ovarian cancer is a highly heterogeneous tumor,and its pathogenesis,recurrence mechanism and drug resistance mechanism are still unclear.Despite many years of studies there is still lack of reliable diagnostic markers as well as other diagnostic methods enabling early detection and suitable for screening.Thus,current studies are aimed on finding new biomarkers with diagnostic,prognostic,and predictive potential as well as on the search for the new therapeutic targets.Regenerative gene family(REG family)belongs to the calcium-dependent lectin superfamily.These proteins are functionally like lectins and have anti-apoptotic effects.REG gene encodes a secreted protein,which plays an important role in diabetes,inflammation,autoimmunity,and carcinogenesis.With the continuous in-depth research on the occurrence and development of cancer and its mechanism,there have been reports in the literature that REG4 is highly expressed in a variety of tumors and has an important impact on the development,treatment,and prognosis of tumors.However,regarding the role and drug resistance of REG4 in ovarian cancer,the mechanism is rarely reported.This project aims to study the expression and molecular mechanism of REG4 in ovarian cancer,and explore the resistance mechanism of REG4 to the chemotherapy drugs platinum and paclitaxel in ovarian cancer,to provide a scientific experimental basis for the diagnosis,screening,treatment,and prevention of ovarian cancer.Methods:1.Oncomine database and UCSC Xena database were used to analyze the expression of REG4 in ovarian cancer and normal tissues;c Bio Portal database was used to analyze the expression of REG4 related genes in ovarian cancer;Kaplan-Meier Plotter was used to analyze the relationship between REG4 expression in ovarian cancer and the prognosis of patients with ovarian cancer;The data of ovarian cancer were downloaded from TCGA database,and the related pathway of REG4 was analyzed by GSEA;2.pc DNA3.1-REG4 eukaryotic expression plasmid and pc DNA3.1 empty vector were stably transfected into ovarian cancer cells CAOV3.REG4 overexpression cell lines were screened by real-time RT-PCR and Western blot.The effects of REG4 overexpression on proliferation,apoptosis,migration,and invasion of ovarian cancer cells CAOV3 were detected by MTT,flow cytometry and Transwell.3.Western blot was used to detect the effects of REG4 overexpression and treated with cisplatin and paclitaxel on the expression of some proteins in ovarian cancer cells CAOV3.Results:Part one: Prognostic significance and mechanism of Reg4 expression in ovarian cancer.1.Oncomine database showed that REG4 was highly expressed in a variety of cancers,obviously,it was also highly expressed in ovarian cancer(P < 0.05).UCSC Xena database analysis showed that the expression level of REG4 in ovarian cancer was significantly higher than that in normal ovarian tissue,and the expression level of REG4 in recurrent ovarian cancer was significantly higher than that in primary ovarian cancer(P<0.05);c Bio Portal database showed that the expression of REG4 was significantly negatively correlated with the expression of apoptosis factors such as Bid,and positively correlated with the expression of cyclin dependent kinase CDK4,anti-apoptosis factor Bcl-2,proliferation promoting factor RAF and drug metabolism related enzymes ALDH1A1,GSTA1,GSTA2,UGT2A3(P < 0.05).2.High REG4 expression was associated with decreased overall survival(OS),progression free survival(PFS)and post progression survival(PPS)in all patients with ovarian cancer(P < 0.05).The overall survival rate of serous,clinical stage I-IV and pathological grade II-III ovarian cancer patients with high REG4 expression was lower;the progression free survival rate of serous,clinical stage I,III,IV and pathological grade III ovarian cancer patients with low REG4 expression was better;there was a negative correlation between the expression of REG4 and the survival rate of ovarian cancer patients with clinical stage III and pathological grade II(P < 0.05).3.After stable transfection of pc DNA3.1-REG4 into ovarian cancer cells CAOV3,the overexpression expression of REG4 mRNA and protein were detected by real-time RT-PCR and Western blot(P < 0.05);4.REG4 overexpression promoted the proliferation of ovarian cancer cells CAOV3,inhibited apoptosis,and enhanced cell migration and invasion(P < 0.05).5.Up regulation of REG4 protein expression promoted Ki67,Bcl2,MMP9 and GST-π protein expression,and inhibited AIF protein expression.REG4 was negatively correlated with apoptosis related genes and positively correlated with drug metabolism cytochrome P450 related genes(P < 0.05).Part two: Prognostic significance and mechanism of Reg4 expression in ovarian cancer treated with platin / paclitaxel.1.The high expression of REG4 was associated with lower overall survival rate,progression free survival rate and late progression survival rate in all ovarian cancer patients receiving platinum chemotherapy(P < 0.05).The high expression of REG4 in all ovarian cancer patients receiving paclitaxel chemotherapy had lower overall survival rate and progression free survival rate(P < 0.05).The higher expression of REG4 was associated with lower overall survival rate in ovarian cancer patients with serous,Ⅲ-Ⅳclinical stages and all histopathological grades.The serous,all clinical stages,histopathological grade III and TP53 mutation ovarian cancer patients with high REG4 expression after platinum chemotherapy had lower progression free survival rate,and the high REG4 expression after platinum chemotherapy in ovarian cancer patients with Ⅲ-Ⅳ clinical stages and all histopathological grades was associated with poor progression free survival rate.In serous,Ⅲ-Ⅳclinical stages and histopathological gradeⅢovarian cancer patients treated with paclitaxel,the high expression of REG4 was associated with lower overall survival rate;in serous,all clinical stages and histopathological grade Ⅲ ovarian cancer patients treated with paclitaxel,the high expression of REG4 had lower progression free survival rate;in addition,the overexpression of REG4 is associated with poor post progression survival in ovarian cancer patients with histopathological grade III after paclitaxel chemotherapy.2.Overexpression of REG4 promoted the proliferation,migration,and invasion of ovarian cancer cells CAOV3 treated with cisplatin and paclitaxel,and inhibited the apoptosis of ovarian cancer cells CAOV3 treated with cisplatin and paclitaxel.3.Overexpression of REG4 promoted the expression of P-Pi3 k,P-Akt,P-m TOR,survivin,Bcl2 and GST-π in ovarian cancer cells treated with cisplatin and paclitaxel.Conclusion: The up-regulation of REG4 expression in ovarian cancer is associated with poor survival and chemotherapy resistance in patients with ovarian cancer.REG4 promotes the occurrence and development of ovarian cancer and chemotherapy resistance by regulating cell proliferation,apoptosis,migration,invasion,Pi3K/AKT/m-TOR signaling pathway and up-regulating key proteins such as GST-π,Bcl2 and Survivin.It can be used as a molecular target for ovarian cancer prognosis evaluation and gene therapy. |
