Effect Of PNN On Proliferation Of Renal Cancer Cells And Its Molecular Mechanism

Renal cell carcinoma(RCC)is one of the common malignant tumors in the urinary system.The most common type is renal tubular epithelial cell carcinoma,also known as renal cell carcinoma.It is believed that reducing the expression of cell adhesion molecules not only induces changes in cell morphology,but also reduces the ability of cells adhere between cells and extracellular matrices,thereby promoting cell migration.In addition to the adhesion function similar to desmoglein,PNN also has the alternative splicing function of m RNA.Recent studies have found that PNN also plays an important role in tumor development.However,the roles of PNN in the development of RCC carcinogenesis and its molecular mechanism are still unclear.First,we tested the expression of PNN in RCC tissues and their pair adjacent non-tumor tissues by QT-PCR,and the relationship between PNN expression and clinicolpathlogic characteristics of RCC patients was statistically analyzed.Second,QRT-PCR and Western Blot experiments were performed to investigate the expression of PNN in renal epithelial cells(HK-2)and RCC cell lines(ACHN,OS-RC-2,786-O,Caki-1),construct sh PNN recombinant plasmids and si PNN,and transfect RCC cells.Third,MTS experiments study the relationship between PNN and RCC cell proliferation.Fourth,flow cytometry analysis of the effects of PNN on the RCC cell cycle、cell apoptotic effects.Fifth,in vivo studies of the effect of reducing PNN on RCC tumor formation.Finally,through bioinformatics analysis,Western blot and CHIP experiments,the molecular mechanism of PNN conversion to RCC replication was analyzed in depth.We found that the expression level of PNN in renal cancer tissues was markedly higher than that in normal adjacent tissues.The clinical data analysis found that the expression of PNN was related to clinical stage,grade,tumor size and lymph node metastasis in patients with RCC(P < 0.05),and had nothing to do with the patient’s age,gender,and histological grade.In addition,survival analysis found that the overall survival rate of patients with metastatic clear cell renal cell carcinoma(metastatic clear-cell renal cell carcinoma,m-cc RCC)with low expression of PNN was significantly higher than that of patients with high expression.Further,we found that the expression of PNN in RCC cells was also higher than that of HK-2 cells.After decreasing the level of PNN in RCC cells,the proliferation of RCC cells was significantly suppressed,and the cells apoptosis were significantly increased,with cell cycle arrest in G0/G1.Subsequently,we studied the effect of PNN on tumor formation in vivo.Through subcutaneous tumor formation experiments in nude mice,we found that after inhibiting PNN expression,the volume and weight of renal tumors were markedly lower than those in the uninhibited group.More importantly,bioinformatics analysis and gene chip found E2F1 may be a regulation gene of PNN.When we decrease the expression of E2F1 protein,the expression of PNN also decreases.Conversely,increasing the expression of E2F1 can reverse the attenuation of cell proliferation and increase of apoptosis caused by inhibition of PNN.Meanwhile,CHIP experiments confirmed the direct regulation relationship between E2F1 and PNN,and immunohistochemical experiments also found that the expression of E2F1 and PNN was positively correlated.Therefore,inhibiting PNN expression can inhibit RCC proliferation through positive E2F1 levels,promote RCC transformation,and induce cell cycle arrest in the G0/G1 phase.In addition,RCC patients with high expression of PNN have poorer prognosis and shorter survival of metastatic patients.Therefore,it is believed that PNN is a key gene for the development of cell carcinoma,and these theoretical basis provide a new idea for regulating PNN expression may become a method for preventing and treating renal cell carcinoma.