Cancer-associated Fibroblasts Infiltration Related Factor RUNX2 Promotes The Progression Of Bladder Cancer

Objective: Bladder cancer is one of the top ten malignant tumors in the world,causing approximately 150,000 deaths each year.The prognosis of advanced bladder cancer is poor,and the 5-year survival rate is less than 5%.There is still no effective treatment for advanced bladder cancer,and the mechanism of bladder cancer progression is not clear.Tumor stroma is an important part of the tumor microenvironment(TME).Cancerassociated fibroblasts(CAFs),as the main cellular components in the tumor stroma,contribute to the occurrence and development of cancer,immune escape,and treatment resistance.However,the research of CAFs in bladder cancer is still in its infancy,and the related molecular mechanism is still unclear.Methods: Using the identified bladder cancer-specific CAFs gene signature from our previous work,we calculated CAFs infiltration scores of multiple bladder cancer transcriptome data samples in The Cancer Genome Atlas(TCGA)and Gene Expression Omnibus(GEO)based on the single sample gene set enrichment analysis(ss GSEA).Combining with relevant clinical data information,we showed the relationship between CAFs and bladder cancer progression and prognosis.We used multiple data in TCGA and GEO to verify the expression of RUNX2 in different clinical stages,and performed western blot assay to compare the protein expression of RUNX2 in bladder cancer and adjacent normal tissues.To ensure the prognosis significance,we used Cox proportionalhazards model to evaluate the relationship between survival and RUNX2.Combining the relevant data of bladder cancer tissues and cell lines in TCGA and GEO,we used gene set enrichment analysis(GSEA)to predict the function and mechanism of RUNX2.After silencing RUNX2 in bladder cancer cells,we verified the effect of RUNX2 on migration and invasion through Transwell and scratch experiments,and verified the effect of RUNX2 on proliferation through real-time cell analysis,clone formation,and EDU proliferation assays.In the tail vein metastasis model,we verified the effect of RUNX2 on the metastatic function of bladder cancer cells.RNA-Seq was used to identify potential downstream genes of RUNX2.Real-time quantitative PCR was used to detect the RNA expression changes of downstream genes after intervention of RUNX2 expression.The protein expression of downstream genes were verified by western blot.According to the predicted transcription binding site,chromatin Immunoprecipitation(Ch IP)assay was used to prove the specific binding ability of RUNX2 with the corresponding promoter DNA fragment.Finally,we overexpressed RUNX2 and silenced downstream genes to perform rescue experiments of migration function.Results: 1.Compared with previous CAFs evaluation algorithms,bladder cancer-specific CAFs gene signature had a high correlation in calculating the level of CAFs infiltration in bladder cancer samples.The high degree of CAFs infiltration in bladder cancer was related to tumor progression and poor prognosis.2.Overexpressed RUNX2 in bladder cancer was significantly related to the level of infiltration of CAFs,tumor progression and recurrence after treatment.RUNX2 was also an independent prognostic factor in bladder cancer.3.According to the RNA-Seq of bladder cancer tissues and bladder cancer cell lines,we predicted that RUNX2 is closely related to the extracellular matrix(ECM)remodeling function in bladder cancer.4.In vitro assays,knocking down RUNX2 significantly inhibited the migration,invasion and proliferation of bladder cancer cells.In vivo assays,silencing RUNX2 significantly inhibited the metastasis of bladder cancer cells.RUNX2 may activated the Wnt/?-catenin pathway through phosphorylation of GSK3? to promote the progression of bladder cancer.5.After knocking down and overexpressing RUNX2,the RNA and protein expression levels of potential downstream genes FN1 and THBS1 decreased and increased.Ch IP assay proved that RUNX2 regulates the expression of FN1 through transcriptional regulation.6.Knockdown of FN1 and THBS1 can significantly inhibit the migration and invasion of bladder cancer.The decreased expression of FN1 and THBS1 can down-regulate the expression of RUNX2,suggesting that RUNX2 and its downstream genes FN1 and THBS1 may have a positive feedback loop regulation relationship.FN1 and THBS1 are associated with CAFs infiltration,progression and poor prognosis of bladder cancer.In rescue assays,separately knocking down FN1 and THBS1 could rescue the enhanced migration effect caused by overexpression of RUNX2.Conclusions: 1.High CAFs infiltration in bladder cancer was related to cancer progression and poor prognosis.RUNX2 was a transcription factor associated with CAFs infiltration of bladder cancer.RUNX2 was overexpressed in bladder cancer and was associated with tumor progression.RUNX2 was also an independent prognostic factor for bladder cancer.2.RUNX2 was closely correlated to ECM remodeling in biological function prediction,which CAFs play a role in remodeling ECM in TME.RUNX2 promoted the proliferation and metastasis of bladder cancer cells,and promoted the progress of bladder cancer through the activation of Wnt / ?-catenin pathway.3.CAFs infiltration related factor RUNX2 promoted the metastasis of bladder cancer through FN1 and THBS1.Both the high expression of FN1 and THBS1 were related to high infiltration of CAFs,advanced stage and poor prognosis of bladder cancer.RUNX2 may directly regulate the expression of FN1 through transcriptional regulation,and FN1 and THBS1 also regulated the expression of RUNX2.There may be a positive feedback regulation relationship between them,which can interfere with CAFs infiltration to promote the progress of bladder cancer.