HnRNP F Promotes The Proliferation Of Bladder Cancer Cell Via Regulating TPX2 Expression

BACKGROUD&OBJECTIVE:Bladder cancer(BC)is one of the most common malignant tumors of the genitourinary system worldwide,it ranks second place among the malignant tumors of urinary system with the characteristics of high recurrence rate and high mortality,and its incidence is increasing.The development of BC,involving a multi-gene,multi-factor and multi-step process,is complex,among which,the aberrant proliferation of BC cell plays a crucial role.At present,although rapid progress of the research and treatment therapy have been made,the recurrence and poor prognosis of BC caused by various factors after treatment are still great challenges.Therefore,the clarification of cell proliferation mechanism in BC is of great importance for our understanding on BC,and for promoting clinical application.Previous studies have shown that Heterogeneous nuclear ribonucleoprotein F(hnRNP F)is significant highly expressed in multiple malignant tumors with the role of promoting the progression of them.In our previous study,we concluded that hnRNP F was significantly increased in human BC tissues and was closely associated with poor prognosis of patients with BC.HnRNP F could promote the invasion,migration and EMT processes of BC cells.HnRNP F could bind to the 3’UTR region of snail mRNA to promote its stablility,thereby promotes the EMT in BC cells.In this present research,based on previous studies,we intended to detect the expression level of hnRNP F in human BC,to explore its effect on the proliferation of BC cells and its molecular mechanism affecting the proliferation of BC cells.Chapter 1:HnRNP F is increased in BC tissues and could promote the proliferation of BC cellsMethods:The expression of hnRNP F in BC tissues was detected by western blot.HnRNP F shRNA lentivirus was constructed and then transfected into BC cells to inhibit the expression of hnNRP F protein.The effect of hnRNP F on the proli feration of BC cells was detected by MTT proliferation assay and cloning formation assay.The alteration of the cell cycle distribution by hnRNP F of BC cells was determined by flow cytometry.The effect of hnRNP F on the KI67 proliferation index of BC cells was detected by immunocytochemistryRESULTS:The expression of hnRNP F was increased in BC tissues,and was higher in EJ and UMUC-3 cells than T24 and 5637 cells.HnRNP F could promote the proliferation of BC cells in vitro.HnRNP F could promote the Gl/S transition and accelerate cell cycle progression of BC cells.Chapter 2:Mechanism of HnRNP F on promoting the proliferation of BC cellsMethods:The protein molecules binded to hnRNP F was explored by mass spectrometry and co-immunoprecipitation.The distribution of hnRNP F and TPX2 proteins in BC cells was detected by dual-fluorescence immunolocalization method.The relationship between hnRNP F and TPX2 proteins in BC tissues was detected by western blotting.The TPX2 overexpression or interference plasmid were constructed and infected to alter the level of TPX2 in BC cells.The interference and overexpression efficiency of TPX2 plasmids,the altered expression of hnRNP F,cyclin DI and p21 upon TPX2 knockdown or overexpression were detected by western blotting.The effect of TPX2 on cell proliferation regulated by hnRNP F was detected by MTT proliferation assay and cloning formation assay.Flow cytometry was used to detect the effect of TPX2 on cell cycle influented by hnRNP F.RESULTS:HnRNP F was binded to TPX2 protein in EJ and UMUC-3 cells,and a positive correlation expression existed between the two proteins.HnRNP F could change the expression of cyclin D1 and p21 by regulating TPX2 protein,thereby drove the cell cycle progression and promoted proliferation of BC cells.The interference with TPX2 could interdict the promoting role of hnRNP F on cell cycle progression and cell proliferation in BC.CONCLUSION:HnRNP F promotes the proliferation of BC cell via regulating TPX2.