| Endometrial carcinoma is one of the common tumors in female genital tract.Along with the development of economy and the extension of average life span,the incidence of endometrial carcinoma is increasing year by year,and the patients is getting younger.Especially for patients with advanced,recurrent or metastatic endometrial carcinoma,there is still a lack of high efficiency and low toxicity treatment.With the proposal and in-depth research of molecular classification of endometrial carcinoma,molecular targeted therapy may become an important strategy for the treatment of endometrial carcinoma.Nuclear receptor interacting protein 1(NRIP1)is a class of transcription cofactors.It can interact with many nuclear receptors and transcription factors,has important biological functions.At the same time,NRIP1 has been found to regulate key steps in the occurrence and progression of a variety of tumors.The mutation rate of NRIP1 in endometrial carcinoma has been reported as high as 7.07-12.50%,but no study has revealed the biological function and the mechanism of NRIP1 mutation in endometrial carcinoma.Objective: To study the biological function and the molecular mechanism of NRIP1 mutationsdriven endometrial tumorigenesis.Methods:(1)The mutations of NRIP1 in endometrial carcinoma was analyzed by TCGA database,and the expression of NRIP1 in endometrial carcinoma tissue samples was investigated by HE staining and immunohistochemical staining.(2)NRIP1 knockout endometrial carcinoma cell lines was constructed by CRISPR/Cas9 technology,and the biological function of NRIP1 in endometrial carcinoma cells was studied through CCK-8 cell proliferation assay,plate clone formation assay,Ed U cell proliferation assay,Transwell assay and soft AGAR clone formation assay.(3)The gene expression differences between NRIP1 knockout cell lines and wild-type cell lines were analyzed by RNA-seq,and the signaling pathways affected by NRIP1 deletion were analyzed by KEGG.(4)q RT-PCR verified the downstream target genes of NRIP1 regulating HIF signaling pathway.(5)Through CCK-8 cell proliferation assay,plate clone formation assay,Transwell assay and soft AGAR clone formation assay,as well as q RT-PCR,NRIP1 deletion was verified to promote the malignant transformation of endometrial carcinoma cells by regulating HIF signaling pathway.(6)The molecular mechanism of NRIP1 deletion regulating HIF signaling pathway was studied by q RT-PCR,Western blot and CO-IP technique.Results:(1)NRIP1 is a high frequency mutant gene of endometrial carcinoma.The expression level of NRIP1 in endometrial carcinoma tissues was significantly lower than that in normal endometrial tissues.(2)The Ishikawa cell line with NRIP1 knockout was successfully constructed.Cell phenotypic experiments showed that NRIP1 knockout cells had enhanced biological functions such as proliferation,migration,invasion and anchoring independent growth.(3)Compared with wild-type Ishikawa cells,there were 653 differentially expressed protein-coding genes in NRIP1 knockout cells,which were enriched in multiple signaling pathways,including hypoxia-inducible factor(HIF)pathway.(4)The m RNA expression levels of target genes of HIF pathway including SERPINE,DDIT4,BNIP3,PGK1,ALDOC,ENO1,BNIP3 L,CA9 and PKM2 were significantly increased in NRIP1 knockout cells,indicating that HIF signaling pathway was activated after the inactivation of NRIP1.(5)Malignant phenotypes of NRIP1 knockout cells,such as proliferation,migration and anchorage independent growth,were enhanced under hypoxia.The m RNA expression levels of target genes of HIF pathway,such as SERPINE,DDIT4,BNIP3,PGK1,ALDOC,ENO1,BNIP3 L,CA9 and PKM2 were significantly increased under hypoxia.(6)Absence of NRIP1 did not affect the expression of HIF-1α and HIF-2α,but activated the HIF pathway by directly binding to HIF-1 α and HIF-2α.Conclusion: This study confirmed that NRIP1 is a high frequency mutant gene of endometrial carcinoma.The deletion of NRIP1 promotes malignant transformation such as tumor growth,migration and invasion,suggesting that NRIP1 plays a role as a tumor suppressor gene in endometrial carcinoma and plays a carcinogenic role after mutation.One of the molecular mechanisms of NRIP1’s antitumor effect is that it interacts directly with HIF-1α and HIF-2α to inhibit the activation of HIF-1 α and HIF-2α downstream target genes,thereby inhibiting the HIF signaling pathway.In other words,NRIP1 inactivation mutations can activate the HIF signaling pathway. |
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