Biological Functions Of HnRNP F And Its Mechanism Of Promoting EMT In Bladder Cancer

BACKGROUD&OBJECTIVE:Bladder cancer(BC)ranks second place in the disease with malignant growth of urinary and its incidence is increasing gradually.At diagnosis,approximately 70%of new bladder cancers are superficial non-muscle invasive tumors.Moreover,up to 50-70%of those cases will recur and 10%～20%of recurrent tumors will progress into deeper layers,despite treatment by transurethral resection combined with intravesical chemotherapy.The extent of tumor invasion is closely correlated with clinical prognosis of bladder cancer,which muscle invasive bladder cancer can progress to life threatening metastases and result in bladder cancer-related deaths.Although the methods of diagnosis and treatment have improved greatly in recent years,it is still unsatisfactory to prevent the recurrence and progression of bladder cancer.Therefore,elucidating pathologic mechanisms underlying bladder cancer invasion and metastasis is of considerable basic scientific significance and clinical application value.In our previous studies,comparative proteomic strategy,combined with high flux two-dimensional electrophoresis(2-DE)and protein mass spectrum identification,were used to screen out and identify 15 BC differently expressed proteins between bladder cancer tissues and adjacent normal tissues,in which nuclear heterogeneous ribonucleoprotein F(HnRNP F)is up-regulated in bladder cancer tissues in comparison with adjacent normal tissues.It’s reported that HnRNP play a vital role on tumor progressions in multiple cancers such as liver cancer,colorectal cancer and cervical cancer.But its specific biological functions of bladder cancer occurrence and progression and its pathologic molecular mechanisms still need to be clarified.This study intends to measure HnRNP F expression in bladder cancer tissues and explore the relationship between its expression and clinical stage,pathological grade and survival prognosis of patients with BC.Then exploring the biological functions of HnRNP F in BC cell lines with a series of experiments in vitro and vivo.Finally exploring the potential pathologic molecular mechanism of HnRNP F in bladder cancer.Part 1:HnRNP F expression in BC tissues and its clinical significanceMethods:HnRNP F expression were examined by qRT-PCR,Western blot and IHC in BC tissues,and exploring its clinical significance.Results:HnRNP F expression in bladder cancer tissues were obviously higher than adjacent non-carcinoma tissues,as an oncogene,the expression of HnRNP F level were closely related to muscular invasion,clinical stage and survival prognosis;HnRNP F is an independent risk factor influencing the survival prognosis of patients with bladder cancer.Part 2.Effect of HnRNP F on the biological functions in BCMethods:Establishment of stable knockdown and overexpression HnRNP F cell lines.CCK8 assay,plate clone formation assay,flow cytometry technique(cell cycle distribution),and nude mice subcutaneous tumor model were used to detect the effects of HnRNP F on BC cell proliferation in vitro and vivo.The effects of HnRNP F on invasion and migration of bladder cancer cells were detected by Transwell invasion assay,Wound healing experiment and nude mice lung metastasis tumor model of BC cells in vitro and vivo.Western blot and qRT-PCR were used to detect the alterations of the biomarkers and related transcriptional factors(TFs)of EMT.Results:Depletion of HnRNP F inhibits proliferation,invasion and migration ability of bladder cancer cell lines,and delays the cell cycle;up-regulation of HnRNP F promotes proliferation,invasion and migration ability of bladder cancer cell lines,in addition,accelerates the cell cycle;HnRNP F can promote the EMT progress in BC.3.The related mechanisms of HnRNP F in EMT progress in BCMethods:Rescue experiments,RNA stability,RNA immunoprecipitation(RIP),qRT-PCR and RNA domain detection were used to detect the related mechanisms of HnRNP F in the EMT in bladder cancer.Results:HnRNP F can participate in the EMT process through promoting the stability of mRNA and binding to its special regions containing ARE in 3’untranslated region of the Snail mRNA.CONCLUSION:HnRNP F promotes the EMT involved in bladder cancer evolution through enhancing Snail mRNA stability and binding to its special regions containing ARE in 3’ untranslated region of the Snail mRNA.