| Objective: Bladder Cancer occurs in the bladder mucosa.In terms of pathological type,urothelial carcinoma is the most common pathological type of bladder cancer.Compared with those that did not penetrate into the muscular layer,tumors that infiltrated into the muscular layer were more malignant and more likely to develop distant metastasis.At present,there are many measures to treat bladder cancer.Surgical treatment is suitable for patients with no metastasis in the early stage,while chemotherapy and immunotherapy are suitable for patients with metastasis in the late stage.Although the prognosis of early tumors without metastasis is good after surgical treatment,tumors that have invaded the muscular layer often recur or metastasize after surgery.In recent years,the death rate of bladder cancer has steadily increased.In order to improve the outcome of patients with bladder cancer,it is urgent to find new therapeutic targets.VGLL(vestigial like family member)are mammalian homologues of degenerative genes in Drosophila,including VGLL1-4..VGLL family members can bind to TEA domain transcription factor TEAD(TEA domain transcription factor)by their own TDU motif,thus playing the role of transcription cofactors.It is well known that YAP/TAZ can act as a transcription cofactor and bind to transcription factor TEAD4 to regulate the transcription of downstream target genes of TEAD4,thus playing a biological function.Studies have reported that although VGLL1 and YAP/TAZ have different motifs,VGLL1 can still compete with YAP/TAZ for TEAD4 binding sites.VGLL1 can both promote and inhibit the development of tumors.Depending on the tumor type,VGLL1 plays a different role in different tumors.VGLL1 is significantly highly expressed in bladder cancer,but there are few reports of VGLL1 in bladder cancer.Therefore,we chose VGLL1 as the research object and tried to explore the mechanism of its role in bladder cancer.The key molecules of PI3K/AKT signaling pathway include PI3 K and AKT.PI3 K and AKT are both silk/threonine protein kinases with silk/threonine kinase activity,while PI3 K also has phosphatidyl inositol activity.After the extracellular signal binds to the cell membrane surface receptor,the receptor is self-phosphorylated to activate PI3 K and AKT successively,thus regulating proliferation,apoptosis and other cell phenotypes.According to relevant literature reports,PI3K/AKT pathway is involved in promoting various phenotypes of bladder cancer,but it has not been reported that PI3K/AKT can regulate VGLL1 in bladder cancer.In this study,on the basis of confirming that VGLL1 can promote the proliferation of bladder cancer cells and epithelial mesenchymal transformation through cell experiments,further explore the regulatory mechanism of the PI3K/AKT pathway on VGLL1,and construct the PI3K/Akt-VGLL1-Tead4 signaling axis.To provide a theoretical basis for VGLL1 as a biomarker and therapeutic target for bladder cancer.Meanwhile,in this study,flubanserin targeted at VGLL1-TEAD4 complex was screened by network pharmacological means,and its ability to inhibit the migration and invasion of bladder cancer cells was successfully verified,providing a new means for the treatment of bladder cancer.Methods:1.q-PCR or Western Blot was used to detect the m RNA and protein expression levels of VGLL1 and TEAD4 in SV-HUC-1 and 6 kinds of bladder cancer cells,the efficiency of overexpression or knockdown of VGLL1 in BIU-87 or TCCSUP cells,and the transcriptional changes of MMP9 after knockdown of VGLL1.2.The effect of knockdown or overexpression of VGLL1 on proliferation of BCa cells was investigated by CCK-8 and clonal formation experiments.3.Scratch callus and Transwell experiments were conducted to investigate the effect of knockdown or overexpression of VGLL1 on the invasion and metastasis of bladder cancer cells,to detect the effect of addition of PI3 K activator 740-YP on the migration and invasion ability of bladder cancer cells,to detect the effect of TEAD4 knockdown on the migration and invasion ability of bladder cancer cells,and todetect the effect of flubanserin treatment on the bladder The influence of cancer cell invasion and metastasis.4.q PCR and Western Blot analysis was performed to explore the effect of VGLL1 on key EMT molecules,the effect of knockdown or overexpression of VGLL1 on PI3K/AKT pathway star protein,the changes of VGLL1 after addition of PI3K/AKT pathway activator 740-YP and PI3K/AKT pathwayinhibitor LY294002,and the key score of EMT after TEAD4 knockdown The change of the son.5.KEGG enrichment analysis screened highly correlated signal pathways.6.The changes in VGLL1 localization after addition of PI3 K activator 740-YP and PI3 K inhibitor LY294002 and the changes in VGLL1 and TEAD4 nuclear localization after TEAD4 knockdown were detected by immunofluorescence and nuclear plasma separation experiments.7.Co-IP assay was used to detect the interaction between VGLL1 and TEAD4 after addition of PI3 K pathway activator 740-YP,TEAD4 knockdown and flubanserin.8.Luciferase report experiment detected changes in the recruitment of MMP9 promoter by VGLL1 after TEAD4 knockdown.9.The effect of overexpression of VGLL1 on bladder cancer lung metastasis in nude mice was detected.10.Network pharmacology screened drugs targeting VGLL1-TEAD4 complex.Results:1.Compared with normal uroepithelial SV-HUC-1,VGLL1 was highly expressed in bladder cancer cell lines T24,RT4 and TCCSUP,and was low expressed in BIU-87,UMUC3 and J82.2.VGLL1 regulates the proliferation,invasion and metastasis of bladder cancer cells.3.KEGG enrichment analysis found that VGLL1 was associated with PI3K/AKT pathway in BCa.4.VGLL1 is located downstream of the PI3K/AKT pathway and regulated at the protein level.5.PI3K/AKT promotes the entry of VGLL1 into the nucleus.6.VGLL1,as a cotranscription factor,regulates bladder cancer EMT after binding with TEAD4.7.As a cotranscription factor,VGLL1 regulates the transcription of target gene MMP9 after binding with TEAD4.8.VGLL1 promotes lung metastasis of bladder cancer.9.Flubanserin inhibited bladder cancer cell migration and invasion by targeting VGLL1-TEAD4.Conclusion:1.VGLL1 affects the migration and invasion of bladder cancer cells by regulating epithelial interstitial transformation.2.The PI3K/AKT pathway regulates the expression of VGLL1 at the protein level and promotes its entry into the nucleus.3.VGLL1,as a cotranscription factor,regulates bladder cancer EMT after binding with TEAD4.4.As a cotranscription factor,VGLL1 regulates the transcription of target gene MMP9 after binding with TEAD4.5.VGLL1 promotes lung metastasis of bladder cancer.6.Flubanserin inhibited bladder cancer cell migration and invasion by targeting VGLL1-TEAD4... |
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