| Part One:Mucin MUC5AC as a novel molecular marker for the diagnosis of cervical adenocarcinomaBackgroundCervical cancer is one of the most common gynecologic malignant tumor in the world.In recent years,the incidence of squamous cell carcinoma has declined due to the promotion of cervical cancer screening and HPV testing in women.However,as cervical adenocarcinoma is difficult to find in the early stage of carcinogenesis,and the existing screening methods such as cytology screening sensitivity is poor,the incidence of cervical adenocarcinoma has increased relatively,and gradually showing a younger trend.Although the treatment of cervical adenocarcinoma and adeno-squamous cell carcinoma is similar to that of cervical squamous cell carcinoma,the etiology,histological type and biological behavior of adenocarcinoma are different from that of squamous cell carcinoma.Adenocarcinoma shows more invasive,easy to relapse after surgery,more biological characteristics of metastasis,relatively low sensitivity to chemotherapy and radiotherapy,and worse prognosis than that of the same period of squamous cell carcinoma.Therefore,different treatment plans for different pathological types are gradually put on the agenda.Its foundation is to make clear pathological diagnosis and accurate classification of cervical cancer.At present,CK7,P63,P40 and CK5/6 are often used in combination for the differential diagnosis of poorly differentiated cervical squamous cell carcinoma and adenocarcinoma,but CK7 has poor specificity,so the search for new adenocarcinoma markers is the objective of this study.In this study,immunohistochemistry was used to detect the expression of MUC5AC,CK7,CK5/6,P40 and P63 proteins in 101 cases of poorly differentiated cervical squamous cell carcinoma and 108 cases of adenocarcinoma,in order to explore whether MUC5AC can be used as a new molecular marker for the diagnosis of cervical adenocarcinoma,to distinguish cervical adenocarcinoma from squamous cell carcinoma and to investigate its expression in different types of cervical adenocarcinoma.Method1.Selection of study casesCases diagnosed and classified as cervical poorly differentiated squamous cell carcinoma and adenocarcinoma were collected from Qi Lu Hospital of Shandong University in the past 13 years from January 2005 to February 2018,and the relevant clinical data were improved.The selected cases were reviewed and interpreted by two pathologists.2.Immunohistochemistry and correlation analysisImmunohistochemical staining of MUC5AC,CK7,CK5/6,P40 and P63 were performed on selected clinical cervical squamous cell carcinoma and adenocarcinoma specimens to analyze the expression of MUC5AC in different pathological types and adenocarcinoma subtypes.Conclusion1.The expressions of MUC5AC,CK7,CK5/6,P40 and P63 in cervical adenocarcinoma and squamous cell carcinoma were significantly different.2.The specificity of MUC5AC in the diagnosis of cervical adenocarcinoma was better than that of CK7,and there was no significant difference in the expression of MUC5AC in different cervical adenocarcinoma subtypes.3.The expression of CK7 had no significant effect on the differentiation degree of cervical adenocarcinoma,but the lower the degree of cervical differentiation,the higher the expression rate of MUC5AC was.4.MUC5AC combined with P40 and P63 can be used for the diagnosis and differential diagnosis of cervical adenocarcinoma and squamous cell carcinoma in clinical pathological work.Part Two:the regulatory effects and related mechanisms of?-arrestin2 on multidrug resistance gene 1(MDR1)in cervical adenocarcinomaBackgroundCervical cancer is one of the most common gynecological malignant tumors.The incidence of cervical adenocarcinoma has increased significantly in recent years.At present,for the treatment of cervical adenocarcinoma,chemotherapy is a very effective treatment,however,in the clinical chemotherapy process,the emergence of multidrug resistance has seriously affected its treatment effect,which is the main problem facing effective tumor treatment.Multidrug resistance(MDR)refers to the fact that once tumor cells develop resistance to a chemotherapy drug during chemotherapy,they will also have the same effect on other drugs with the same or different structures or targets.The P-glycoprotein encoded by the MDR1 gene is one of the ABC family of proteins,is set in the ATP binding protein on the cell membrane.We found that in clinical specimens of cervical adenocarcinoma,?-arrestin2 is closely related to the expression of MDR 1.Therefore,our research mainly intend to study the regulatory role and related mechanisms of ?-arrestin2 in the multidrug resistance gene 1(MDR1)of cervical adenocarcinoma,and looked for therapeutic targets for the regulation of P-gp expression,thus bringing new hope for the reversal of multidrug resistance of tumors.Method1.Construct overexpression vectorsThe full-length expression vector pcDNA3.1-?-arrestin2-GFP was presented by the Professor Deling Yin of Eastern Tennessee University.2.Cell culture and transfectionThe above plasmid vectors were transfected into two types of cervical adenocarcinoma cell lines HeLa and C33A,respectively.After further culture for 48-72h,the mRNA and protein expressions of ?-arrestin2 and MDR1/P-gp in each group were detected by RT-qPCR and Western blot,respectively.3.Detection of changes in the function of cervical adenocarcinoma cells after overexpression of ?-arrestin2 gene.MTT method was used to detect the changes of cell lethal dose to half cells of adriamycin,5-fluorouracil and cisplatin in each group.CCK8 was used to reflect the proliferation of tumor cells after overexpression of ?-arrestin2 gene.After over-expression of ?-arrestin2 gene,Western blot and RT-qPCR were used to detect the interaction between the expression of ?-arrestin2 gene and relative drug-resistant proteins and downstream signaling pathways.Conclusion1.The expression of ?-arrestin2 in cervical adenocarcinoma was related to the degree of differentiation.The higher the degree of tumor differentiation,the higher the positive rate of ?-arrestin2 was.2.The mRNA level of MDR1 gene increased after the up-regulation of ?-arrestin2 expression in cervical adenocarcinoma cells,and the difference was statistically significant.However,the expression of P-gp protein was significantly higher than that of mRNA.The up-regulation of P-gp expression may be regulated by both the transcription level and the post-transcription level.3.After overexpression of ?-arrestin2 gene,the proliferation rate of cervical adenocarcinoma cells was slowed down,but the resistance to adriamycin,cisplatin,5.fluorouracil and other chemotherapy drugs was demonstrated.4.The expression of NF-?B protein and mTOR pathway related proteins in cervical adenocarcinoma cells were increased after overexpression of ?-arrestin2,while the expression of PRMT7 was decreased.It is suggested that ?-arrestin2 may regulate the expression of P-gp through NF-?B pathway and mTOR pathway.On the one hand,the transcription of MDR1 gene could be promoted by the activation of NF-?B pathway;On the other hand,?-arrestin2 could regulate the up-regulation of mTOR pathway to inhibit the expression of PRMT7,and then induce the demmethylation of MDR1 gene,so as to up-regulate P-gp expression to mediate the occurrence of multidrug resistance in cervical adenocarcinoma. |
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