Investigation Of The Roles Of STK32C In Bladder Cancer And Relevant Mechanism

Objective: Bladder cancer(BC)is a common tumor of the urinary system,second only to prostate cancer.Although the diagnosis and treatment of BC continue to improve,the high rate of recurrence and progression is still an important obstacle affecting the clinical treatment of BC.In recent years,multiple biomarkers have been proved to play a role in the progression of bladder cancer and related to the prognosis of patients.However,the therapeutic effects of biological targets and prognostic values of these biomarkers are unsatisfied.Our previous experiments found that STK32 C gene was highly expressed in BC tissues from TCGA database,and it was speculated that STK32 C might play a role in BC.Therefore,this study focused on the expression of STK32 C protein in tumor and adjacent tissues of patients.The effects of STK32 C on the proliferation,apoptosis,invasion and metastasis of T24 and 5637 cells were studied in vitro.Further,the possible mechanism of STK32 C in BC was preliminarily explored.Method:(1)Firstly,m RNA expression data and clinical information of 414 patients with BC were downloaded from the TCGA database,and STK32 C gene expression in BC and adjacent tissues were compared through integrated analysis of TCGA data.(2)Sixteen paired cases of BC and adjacent tissues were obtained from clinical patients after surgery,and the expression level of STK32 C protein in tissue samples was evaluated by immunohistochemistry(IHC).The expression of STK32 C in tumor and adjacent tissues was compared.(3)Lentivirus-mediated stable knockdown of STK32 C was applies to T24 and 5637 BC cell lines,and negative transfection was used as the control.Knocking down efficiency of STK32 C was detected by q RT-PCR and western blot at the gene expression and protein levels.The effects of STK32 C knockdown on the proliferation,colony formation,migration and invasion of BC cells were analyzed separately by cell MTT assay,plate cloning assay,scratching-wounding assay and Transwell assay.(4)The online database of String was used to search for the protein molecules that might be correlated with STK32 C protein.Western blot was used to detect the effect of STK32C-knockdown on the expression levels of m TOR and p-mTOR,which were key proteins in the m TOR pathway.Result:(1)According to the analysis of 414 bladder cancer cases in the TCGA database,STK32 C m RNA expression in 414 BC tissues was significantly higher than that in adjacent normal tissues(P<0.01).STK32 C m RNA was still highly expressed in tumor tissues(P<0.01)in 19 patients with paired tissues.Among the 12 samples,STK32 C m RNA was highly expressed,1 was decreased,and 6 showed no significant change.(2)IHC analysis of clinical BC cases showed that STK32 C protein was significantly highly expressed in BC tumor compared to normal adjacent tissues(P<0.01).The expression of STK32 C increased with the progression of tumor pathology.(3)The in vitro biological functions of STK32 C were analysed by constructing stable STK32 Cknockdown and negative transfection control in T24 and 5637 cells.The results showed that,compared with the control group,knockdown of STK32 C significantly inhibited the proliferation,colony formation,migration and invasion ability of tumor cells(P<0.05).(4)Using the String(protein interaction online prediction tool),we found 10 protein molecules that may be related to STK32 C protein.By analyzing these proteins,we found that m TOR had the highest correlation with STK32 C.Using western blot,we found that the expression levels of STK32 C,m TOR and p-m TOR proteins were significantly decreased after knockdown.Conclusion: STK32 C is highly expressed in BC and may be associated with clinical features.In vitro,knockdown of STK32 C significantly inhibited the proliferation,colony formation,migration and invasion of tumor cells.Meanwhile,knockdown of STK32 C protein can reduce the expression of m TOR and p-m TOR protein.Therefore,we concluded that STK32 C regulates the activity of m TOR pathway by affecting the expression and phosphorylation of m TOR protein,thereby changing the biological function of tumor cells and promoting the progress of BC.This may provide a new idea for the targeted treatment and biomarkers of clinical BC patients.