Correlation Between Twist Expression In Epithelial Ovarian Cancer And Chemotherapy Resistance And Prognosis

Epithelial ovarian cancer（EOC）is the highest mortality among gynecological tumors.Due to the hidden anatomic position of the ovary,the absence of early symptoms and the lack of effective early detection and diagnosis methods,about 70%⁸0%of the patients were found to be in the middle and late stage,and there were extensive abdominal metastasis or distant metastasis,which seriously threatened women’s health.According to the latest NCCN guidelines,the treatment principle of EOC is still a combination of surgery and chemotherapy.With the improvement of chemotherapy regimens,althoμgh the effective rate of platinum-based chemotherapy is up to 70%⁸0%,about 70%of these patients will eventually relapse within 2 years after standardized treatment,even if complete clinical remission is achieved.The 5-year survival rate of epithelial ovarian cancer patients is always hovering around 30-40%.The recurrence of ovarian cancer patients often accompanied by platinum drug resistance,research shows that chemotherapy resistance is the main cause of ovarian cancer treatment failure.Therefore,in-depth study on the molecular mechanism of EOC resistance is the key to improve the therapeutic effect,evaluate drug resistance and predict prognosis.Twist is（basic helix-loop-helix.BHLH）transcription factor.Now studies show that Twist,as a highly conserved oncogene in the evolutionary process,is involved in the whole process of epithelial-mesenchymal transition（EMT）.Literature shows that Twist protein can be highly expressed in various malignant tumors,and it is associated with the occurrence,development,drug resistance and prognosis of cancer.There have been relevant reports about Twist gene in ovarian tumors at home and abroad,but few studies have been done on epithelial ovarian cancer.The mechanism of Twist and epithelial ovarian cancer’s occurrence,development,recurrence and drug resistance is still unclear.Since the mechanism of drug resistance of ovarian cancer and how to solve the problem of chemotherapy tolerance of clinical patients have been the focus and difficulty of basic research and clinical research of ovarian cancer,we chose the topic of the relationship between Twist gene expression and drug resistance and prognosis of epithelial ovarian cancer to study.Starting from the histological and cytological levels,this project hopes to understand the relationship between the expression of Twist in EOC and its clinicopathological characteristics,chemotherapy resistance and prognosis,and to preliminarily explore its molecular biological mechanism.This study was divided into two parts:1.Immunohistochemistry was used to detect the relationship between the expression of Twist in epithelial ovarian cancer and chemotherapy resistance and prognosis;2.Relationship between Twist expression in epithelial ovarian cancer cell line A2780 and cisplatin chemotherapy resistance.Research purpose1.Twist protein expression in epithelial carcinoma of ovary was detected2.The correlation between Twist expression and clinicopathological features,drug resistance and prognosis was analyzed based on clinical data3.si-RNA interference technique was used to study the effect of Twist protein expression changes in ovarian cell line on tumor cell proliferation and apoptosis,and to explore the possible molecular biological mechanism of Twist and platinum drug sensitivity and resistanceResearch methods1.A total of 4115 cases of patients with gynecological ovarian diseases in xijing hospital from 2011 to 2016 were reviewed,and 590 cases of patients with epithelial ovarian cancer were screened and collected.According to the inclusion criteria of primary EOC patients,initial diagnosis and treatment in our hospital,and standard post-operation platinum based chemotherapy≥4 times,101 patients with epithelial ovarian cancer were selected by the method of random sampling.Among the patients who underwent total hysterectomy and bilateral adnexectomy due to cervical intraepithelial neoplasia,20patients with normal ovarian tissue were selected by simple random method.According to WHO histological classification of ovarian cancer in 2014^[1],FIGO staging of international federation of obstetrics and gynecology in 2015^[2]and 2018 NCCN guidelines^[3],patients with ovarian cancer were divided into clinical features groups,and chemotherapy-sensitive group and chemotherapy-resistant group according to the effect of chemotherapy.Complete follow-up data were collected.2.Immunohistochemical staining was used to detect the expression status of Twist in normal ovarian tissues and epithelial ovarian cancer tissues in paraffin tissue sections.3.The relationship between Twist expression and different ages,pathological stages,pathological grades,pathological types,FIGO stages,ascites,lymph node metastasis and platinum reaction was analyzed based on clinical data.4.When different expressions of Twist were plotted in combination with follow-up data,patients’survival curves were analyzed by COX univariate analysis of risk factors affecting prognosis of patients,and multivariate COX model analysis was conducted for those with statistical differences.5.According to previous preliminary experiments,human ovarian cancer cell line A2780 was cultured,small interfering RNA was designed,and Twist-siRNA was screened for expression in A2780 cell line by reverse transcription.6.The expression of Twist in A2780 was down-regulated,and the relationship between Twist and cisplatin was investigated by CCK-8 cell proliferation experiment and flow cytometry apoptosis experiment.7.The expression of EOC cell line A2780 transfected with Twist-siRNA was detected,and the relationship between Twist and cisplatin was investigated by RT-PCR and Western-blot results.Results1.Compared with normal ovarian tissues,Twist in ovarian epithelial carcinoma tissues is highly expressed.2.The expression level of Twist in ovarian epithelial carcinoma tissues was correlated with patients’FIGO stage（P=0.006）,whether accompanied by lymph node metastasis（P=0.046）,and platinum resistance（P=0.026）.The difference of Twist expression level was not correlated with patients’age,pathological grade,pathological type and presence or absence of ascites（P>0.050）.3.The survival curve showed that patients with high expression of Twist had shorter progression free survival（PFS）and total survival（OS）periods.4.COX univariate analysis of COX regression model one by one showed that age,pathological grade,pathological classification and ascites were not prognostic risk factors（P≥0.05）.Tumor recurrence,FIGO stage,lymph node metastasis,platinum drμg reaction and high expression of Twist are risk factors for prognosis（P<0.05）.Multi-factor analysis of COX model was established for the above risk factors,which showed that drug-resistant recurrence was an independent risk factor for EOC patients（P<0.05）.5.Twist-siRNA was successfully constructed,and Twist-siRNA-771 was used for subsequent experimental studies.6.After transfection of Twist-siRNA into ovarian epithelial carcinoma A2780 cells,experiments showed that the proliferation rate of the cells was reduced,the apoptosis rate was increased,and the sensitivity to cisplatin drugs was increased.Conclusion1.Overexpression of Twist is associated with epithelial ovarian cancer.2.EOC with high expression of Twist may be more invasive.3.Patients with high expression of Twist are prone to platinum resistance.4.Patients with high expression of Twist have poor prognosis.5.Down-regulated Twist inhibited the proliferation of ovarian cancer cell A2780 and promoted its apoptosis.6.Down-regulation of Twist increases the sensitivity of proliferation of ovarian cancer cells A2780 to cisplatin.