A Study On Function And Mechanism Of PRMT5 In Endometrial Carcinoma

[Background]Endometrial carcinoma is the second largest gynecological cancer next to cervical cancer,and the incidence of which is increasing year by year.Protein arginine methyltransferase 5(PRMT5)is an emerging epigenetic enzyme.It regulates the transcription of target genes mainly by symmetrically dimethylating histone H4 arginine 3,histone H3 arginine 8 or histone H2A arginine 3.Our research intend to study the biological effect and mechanism of PRMT5 in the progression of type 1 endometrial carcinoma,and to explore the relationship between the expression and the progression and poor prognosis of the disease,finding new diagnostic marker and therapeutic target for endometrial carcinoma.[Method]1.case and specimen selectionCases were selected from Qilu hospital of shandong university,including samples of endometrial adenocarcinoma and mucinous adenocarcinoma of endometrium,simple endometrial hyperplasia and complex hyperplasia tissues.Complete all the related clinical pathologic data and send all sections for re-diagnosis and judgement.2.Immunohistochemistry and correlation analysis.Comparing the expression discrepancy of PRMT5 in different types of EC and hyperplasia endometrium tissue through immunohistochemical techniques.Analyze the correlation between the expression of PRMT5 and clinicopathological characteristics of EC(age,histological grade,lymph node metastasis,etc),and that of PRMT5 expression and the expression of ERa.3.Expression of PRMT5 in two cell lines.Through Western Blot and rt-qPCR,the expression differences of PRMT5 in the endometrial cancer cell lines were compared at mRNA level and protein level.4.Cell culture and transfection.The PRMT5 shRNA was designed to transfect Ishikawa cells with ERapositive.The transfection efficiency was Evaluated by rt-pcr and Western blot.5.Changes in endometrial cancer cells after PRMT5 silencingTranswell experiment was used to detect changes in tumor cell invasion and migration ability after the silence of PRMT5 gene.Cck-8 method was used to reflect the proliferation of the cells after transfection.Clonal and population analyses help us study the ability of tumor cells extensive self-renew.We also use Annexin-PE 7-AAD Apoptosis Detection Kit to test cell apoptosis by flow cytometer.Western Blot and rt-qPCR were used to find the interaction between the expression of PRMT5 and the proteins associated with proliferation and apoptosis.6.Explore the influence of PRMT5 silencing on estrogen receptor ER expression.The expression of estrogen receptor ERawas compared with the NC group,and the downstream target gene of PRMT5 was predicted and verified by Western blot.7.Test the combination of PRMT5 and steroid receptor assisted activation protein-1(SRC-1)and estrogen receptor ER.The combination of PRMT5 and steroid receptor assisted activation protein-1(SRC-1)and estrogen receptor ERa can be verified by co-ip experiments.8.Explore the influence of E2 on PRMT5.The effect of E2 on the PRMT5 expression was observed through cell line starvation and gradient dosing experiment of E2.[Conclusion]1.High expression of PRMT5 can be seen in endometrial adenocarcinoma tissue,mainly in cytoplasm and some nuclei,which is lost in endometrial mucinous adenocarcinoma.The staining-positive rate of simple hyperplasia endometrium,complexhyperplasia endometrium,endometrial atypical hyperplasia andendometrial adenocarcinoma tissue was 39%,77%,90%and 95%respectively,which is statistically significant(P<0.001).The expression was positively correlated with the differentiation of tumor.Moreover,there was a statistical association between PRMT5 expression and histological classification,ki-67 and ERaexpression of endometrial adenocarcinoma.2.The silencing of PRMT5 gene in endometrial cancer cells significantly increased apoptosis,and reduced the proliferation and clone ability of tumor cells,.The migration and invasion ability of tumor cellsalso decreased significantly,indicating that PRMT5 can regulate the proliferation and apoptosis of tumor cells,thereby affecting its occurrence and development.PRMT5 can promote cell proliferation byup regulating c-Myc,and inhibit cell apoptosisby up-regulating Bcl-2 and down-regulating P53 expression.Therefore,the development of gene therapy drugs for PRMT5 may become a new target for therapy of endometrial cancer.3.Through the analysis of signaling pathways,we found that the amount of estrogen receptor expression was inhibited after PRMT5 silence.ERa may contribute to the proliferation of tumor cells under the regulation of PRMT5.What’s more,PRMT5 can form a complex with SRC1 and ERa,,which verified the former conclusion.4.It was found that E2 could inhibit the expression of PRMT5 through gradient dosing experiment of E2,possibly because ERahas an Adverse effect on PRMT5.