Function And Molecular Mechanism Of Cullin1 Gene In The Development Of Human Renal Cell Carcinoma

Objective The purpose of our study is to evaluate the expression of Cullin1 in renal cell carcinoma and normal renal tissue and to detect the effects of Cullin1 gene on renal cancer cell proliferation, migration, invasion and the mechanisms underlying these effects.Methods In vitro: First,Immunohistochemistry was used to assess the expression of Cullin1 in 307 renal cancer tissues and 34 normal renal tissues, and its correlation with clinicopathological features and prognosis of renal cancer were also analyzed; Using Cullin1 small interfering RNA(si RNA) which was synthesized chemically in vitro to transfect 786-O and ACHN renal cancer cells and to investigate the effects of Cullin1 si RNA on Cullin1 expression in 786-O and ACHN cells by Western Blot. Flow cytometry was used to detect the cell cycle and CCK-8 assay was used to measure the proliferation abilities of 786-O and ACHN cells after Cullin1 knockdown. Cell migration assay was used to detect the migration abilities of 786-O and ACHN cells after Cullin1 knockdown. Cell invasion assay was used to detect the migration abilities of 786-O and ACHN cells after Cullin1 knockdown. After knockdown of Cullin1 gene in renal cancer 786-O and ACHN cells, we detect the p21,p27,Cyclin D1, Cyclin E2, MMP-2, MMP-9, TIMP-1 and TIMP-2 activities of the two renal cancer cells by Western Blot; 786-O-Luc was used to establish a xenograft model in immunodeficient BALB/c nude mice. Prior to implantation, The Cullin1 knockdown 786-O-Luc cell lines(Cullin1KD-786-O-Luc) and control 786-O-Luc cell lines(Control-786-O-Luc) were established by infecting with lentivirus packing Cullin1 sh RNA expression vector and control vector respectively. Target cells were infected with lentivirus for 48 hours then selected with puromycin for 4 weeks..In vitro:Female BALB/c nude mice(4 weeks of age) were used. Subconfluent 786-O-Luc-lenti-Ctrl and 786-O-Luc-lenti-sh Cullin1 cells were harvested and resuspended in matrigel which was dilution with RPMI 1640 medium in 1:1 ratio to a density of 5×107 cells/m L. 200μl cell suspension of 786-O-Luc-lenti-Control were injected subcutaneously into right flank of each animal, and 200μl cell suspension of 786-O-Luc-lenti-sh Cullin1 were injected subcutaneously into left flank of each animal. One month after injection, the pictures were captured using bioluminescence imaging with a Night OWL Imaging System.Results In vitro,(1) IHC showed that the expression of Cullin1 was increased significantly in RCC tissue compared with NRT(P<0.05) and it was correlated with renal carcinoma p T status(P<0.05),Cullin1 expression has no correlation with the survival of patients.(2) Western Blot showed that the Cullin1 expression is decreased after Cullin1 knockdown in both 786-O and ACHN renal carcinoma cells.(3) The CCK-8 assay shows that Cullin1 interference could drastically decreased the ability of cell proliferation in both renal cancer cells and arrest cell cycle at G1 phase after Cullin1 knockdown.(4) In cell migration assay, our data revealed that Cullin1 knockdown decreased cell migration ability of 786-O and ACHN by 53% and 79% when compared with the corresponding control. In cell invasion assay, cell invasion ability of 786-O and ACHN decreased95% and 44%.(5)Western Blot analysis showed that the expression of Cyclin D1、Cyclin E2、MMP-9 is significantly inhibited while p21、p27 and TIMP-1 is increased after the silencing of Cullin1 in 786-O and ACHN cells and no significant changes in MMP-2 and TIMP-2 protein expression were found.In vivo:Thirty days after implantation, in vivo imaging analysis of the mice revealed that the growth of tumors was significantly inhibited by decreased expression of Cullin1.Conclusion We founded that Cullin1 staining was increased significantly in RCC tissue compared with NRT. Knockdown of Cullin1 leads to inhibition of renal cancer cell proliferation in both vivo and vitro and arrested cell cycle at G1 phase by upregulating p21 and p27 levels which could down regulating Cyclin D1 and Cyclin E2 protein levels. Knockdown of Cullin1 inhibits the RCC cells migration and invasion abilities by up-regulating the expression of TIMP-1 that inhibit the expression of the MMP-9 which caused the ability of renal cancer cells for degrading components of ECM and basement membrane was decreased.