LncRNA SNHG16 Promotes The Survival Of Bladder Cancer Cells By Binding YBX1 To Inhibit P53 Signaling Pathway

Objective: Bladder cancer is the most common malignant tumor in urinary system.which is characterized by easy recurrence,progression and metastasis.Bladder cancer can generally be divided into muscular invasive type and non-muscular invasive type.At present,surgery is the main treatment for bladder cancer,but more than 50% of the patients have recurrence after operation,and more than 30% of the recurrence cases are accompanied by increased malignancy and invasive ability.The overall 5-year survival rate is between 23% and 48%.Cisplatin-based chemotherapy is the main treatment for inoperable and metastatic myometrial invasive bladder cancer.However,due to the occurrence of chemical resistance,a large number of patients with tumor recurrence.Therefore,it is of great significance to study the mechanism of the occurrence and treatment.Methods: 1、The subcellular localization of SNHG16 in bladder cancer cells was proved by nuclear-cytoplasmic separation test and FISH test;The expression of SNHG16 in bladder cancer and adjacent tissues and its relationship with clinical prognosis were analyzed by TCGA and GEO database;Collect surgical excised bladder cancer tissues and adjacent tissues and cultured bladder cancer cells to detect the expression level of SNHG16 in tissues and cells by q RT-PCR;Prognostic information of 35 patients with clinical tissue specimens was followed up after operation to validate the results of public database and establish a clinical prognostic model to evaluate the role of SNHG16 as a prognostic predictor.2、CCK8 and colony formation assays were used to detect the effect of SNHG16 on the increase of bladder cells;Annexin V-FITC/PI double staining flow cytometry was used to investigate the effect of silencing SNHG16 induced by cisplatin on the apoptosis of bladder cancer cells.The effects of SNHG16 on the migration and invasion of bladder cancer cells were determined by scratch assay and Transwell assay.The effect of SNHG16 on chemical resistance of bladder cancer cells after cisplatin intervention was detected by Western Blot assay.The promotion effect of SNHG16/YBX1/p53 on the growth of bladder cancer was verified by subcutaneous tumor formation in nude mice.3、Searching for SNHG16 Direct Binding Protein by RNA pull down Test;The sense band was analyzed and identified by mass spectrometry.YBX1,which is related to the malignant progression of bladder cancer,was identified as a research protein by literature search and bioinformatics analysis for subsequent experiments.Reverse verification of YBX1 and SNHG16 as direct binding proteins by RNA co-immunoprecipitation experiments;The possible signal pathway mediated by SNHG16 was analyzed by GSEA.The relationship between SNHG16 and p53 protein expression was studied by Western Blot;The interaction between YBX1 and p53 protein in bladder cancer cells overexpressing SNHG16 was determined by Co IP assay;The possible signal pathways mediated by YBX1 were analyzed by GSEA,and the intersection of YBX1 and SNHG16 was selected as the key research pathway by comparing the signal pathways mediated by SNHG16.Recovery experiment verified the regulatory relationship between SNHG16 and YBX1 binding through p53 signaling pathway;Subcutaneous tumorigenesis test in nude mice verified the promoting effect of SNHG16 and YBX1 on the growth of bladder cancer.The promotion effect of SNHG16/YBX1/p53 on the growth of bladder cancer was verified by subcutaneous tumor formation in nude mice.Results:1、SNHG16 was expressed in both nucleus and cytoplasm of bladder cancer cells;The results of public database and clinical samples showed that the expression of SNHG16 in bladder cancer tissues was significantly up-regulated compared with normal adjacent tissues(P<0.05),and the expression level of SNHG16 was negatively correlated with the survival time of bladder cancer patients(P<0.001).The expression of SNHG16 in UMUC3、T24、J82 cells was up-regulated compared with sv-huc-1 cells(P<0.05);The results of clinical prognosis model based on self-built clinical case database showed that SNHG16 as a predictor of clinical prognosis could significantly improve the predictive efficiency of prognosis model(P<0.05);2、The proliferation,migration and invasion of UMUC3 and T24 cells decreased significantly(P<0.05)after knock-down of SNHG16,and the level of apoptosis increased significantly(P<0.05)after overexpression of SNHG16;In addition,SNHG16 increased cisplatin resistance in UMUC3 and T24 cells.In vivo experiments,SNHG16 significantly promoted the growth of bladder cancer tumors.3 、 RNA pull down combined with mass spectrometry analysis found that SNHG16 can directly bind to YBX1 protein.RIP experiments reversely verified the direct binding relationship between the two;SNHG16 and YBX1 protein have a positive regulatory relationship in T24 and UMUC3 cells;GSEA analysis showed that the high expression SNHG16 group can significantly enrich the p53 pathway;SNHG16 has a negative regulation on p53 protein relation.Co IP experiments showed that YBX1 interacted with p53 in T24 cells and UMUC3 cells overexpressing SNHG16;GSEA analysis results showed that the p53 signaling pathway is a common regulatory pathway of SNHG16 and YBX1;Recovery experiments demonstrated that the role of SNHG16 in regulating p53 pathway was at least partly through YBX1;The vivo experiments demonstrated the promoting effects of SNHG16/YBX1/p53 on the growth and survival of bladder cancer cells.Conclusion: 1、SNHG16 is highly expressed in bladder cancer cells and tissues,which is associated with the occurrence and development of bladder cancer and poor prognosis of patients.SNHG16 can be used as a prognostic predictor for patients with bladder cancer,significantly improving the predictive efficacy of the prognostic prediction model.2 、SNHG16 can promote the proliferation,migration and invasion of bladder cancer cells,inhibit apoptosis,enhance the chemical resistance of bladder cells to cisplatin,and promote the growth of tumor in nude mice.3、SNHG16 can promote bladder cancer cell survival by directly binding to YBX1 protein and reducing p53 protein level.