The Mechanism Of SNORA9 Targeted MiR-451a Regulated HIPPO Pathway In Endometrial Cancer

Objective: Endometrial carcinoma(EC)is the fourth most common female tumor and one of the most common epithelial malignant tumors in the reproductive system.Although it seems that surgery and chemoradiotherapy are effective for stage I/II of EC,there are still a large number of patients with advanced stage or patients who have undergone surgery and chemoradiotherapy will have uncontrollable recurrence and multiple metastases.Emerging evidences suggest that dysregulation of small nucleolar RNA(Sno RNA)is involved in the development and progression of various tumors.Previous studies have shown that SNORA9 is abnormally expressed in estrogen-associated breast cancer,but there are no studies on SNORA9 in EC,which is also an estrogen-associated tumor of the reproductive system.The purpose of this study was to investigate the effect of SNORA9 on the malignant biological behavior of EC cells and its related mechanism.Methods: 1.Real-time PCR was used to detect the expression level of SNORA9 in EC tissues and normal endometrial tissues;Transfect EC line cells,Ishikawa and HEC-1A cells with overexpression and silencing SNORA9 lentivirus,and verify the transfection efficiency by Real-time PCR.The changes of cell proliferation were detected by CCK-8assay,and cell cycle and cell apoptosis were detected by flow cytometry.2.Bioinformatics software starbase2.0 was used to screen out miRNA interacting with SNORA9;Real-time PCR was used to detect the expression level of miR-451 a in EC tissues and normal tissues.The wild type and mutant SNORA9 expression vectors were established,and the wildtype and mutant SNORA9 expression vectors were co-transfected with miR-451 a,respectively.The binding effect and binding site of the wild-type and mutant SNORA9 expression vectors were detected by double luciferase reporter gene assay.Ishikawa and HEC-1A cell lines with miR-451 a knockdown were established,and the transfection efficiency was verified by real-time PCR.EC cell lines co-transfected with overexpression and knockdown of SNORA9 and overexpression and knockdown of miR-451 a were constructed,respectively.Cell proliferation,cell cycle and apoptosis after co-transfection were detected by CCK-8 assay and flow cytometry.3.EC cell lines with overexpression and knockdown of SNORA9 and co-transfection of overexpression and knockdown of miR-451 a were constructed.The protein levels of HIPPO signaling pathway-related proteins LATS1,P-LATS1,YAP and PYAP were detected by Westen blot assay.Results: 1.The expression of SNORA9 in EC tissues was significantly higher than that in normal endometrial tissues;With SNORA9 overexpression,the cell proliferation and the proportion of cells in G2-M phase increased in Ishikawa and HEC-1A cell lines,and the cell apoptosis decreased.On the contrary,with SNORA9 knockdown,the proliferation of cells in Ishikawa and HEC-1A cell lines decreased,the proportion of cells in G2-M phase decreased,and the cells apoptosis increased.(All P < 0.05).2.The bioinformatics software(Starbasev2.0)was used to predict whether SNORA9 had binding sites with miR-451 a,and the interaction sites between SNORA9 and miR-451 a were verified by the dual luciferin reporter gene assay.Different expression of miR-451 a was detected in EC and normal tissues by Real-time PCR.In the Ishikawa cells and HEC-1A cells with miR-451 a knockdown,the cell proliferation increased,the proportion of cells in G2-M phase increased,the proportion of cells in G0-G1 phase decreased,and the cell apoptosis decreased.EC cell lines with co-transfection of SNORA9 and miR-451 a were constructed.The results showed that when SNORA9 was overexpressed and miR-451 a was knocked down at the same time,cell proliferation and proportion of G2-M phase cells increased most significantly,while the proportion of G0-G1 phase cells and cell apoptosis decreased most significantly.On the contrary,when SNORA9 was knocked down and miR-451 a was overexpressed at the same time,the proliferation and proportion of cells in G2-M phase decreased most significantly,and the proportion of cells in G0-G1 phase and cell apoptosis increased most significantly.(All P < 0.05).3.EC cell lines co-transfected with SNORA9 and miR-451 a were constructed.Protein levels of HIPPO signaling pathway related proteins were detected,and it was found that overexpression of SNORA9 and knockdown of miR-451 a reduced the protein expression levels of P-LATS1 and P-YAP.The protein expression levels of LATS1 and YAP were increased.Conversely,knockdown of SNORA9 and overexpression of miR-451 a increased the protein expression levels of P-LATS1 and P-YAP,and decreased the protein expression levels of LATS1 and YAP.(All P < 0.05).Conclusions: 1.SNORA9 is highly expressed in EC tissues.Overexpression of SNORA9 promotes the malignant biological behavior of EC cells,while knockdown of SNORA9 inhibits the malignant biological behavior of EC cells.2.There is a binding site between SNORA9 and miR-451 a,and miR-451 a is underexpressed in EC tissues.Knockdown of miR-451 a can promote the malignant biological behavior of EC cells.By targeting miR-451 a,SNORA9 can further influence the malignant biological behavior of EC cells.3.Overexpression of SNORA9 and knockdown of miR-451 a can inhibit the phosphorylation of LATS1 and YAP,promote the protein expression of LATS1 and YAP,and activate HIPPO pathway,thus promoting the malignant biological behavior of EC cells.