The Role And Mechanism Of Key Genes In CRL E3 Ubiquitin Ligase In Renal Carcinoma

Background and purposes:Renal cell carcinoma(RCC)is responsible for 3% of human malignancies with 102,000 mortalities per year around the world.According to the American Cancer Society,RCC is expected to contribute to 8% of 65,340 incidence cases and 14,970 death cases in 2018.Despite advances in diagnostic techniques,about 30 percent of patients with kidney cancer are diagnosed at late stages,which are less sensitive to radiotherapy and chemotherapy,and the treatment effect is poor.Using immunized with interleukin2(Il-2)and interferon(IFN-type),but the treatment is effective for only 10 to 20 percent of patients.Therefore,it is urgent to find accurate and effective anti-RCC molecular therapeutic targets,and understand the pivotal molecular events which are crucial for the development of novel therapeutic strategies for the treatment of RCC.The ubiquitin-proteasome system(UPS)fulfills essential cellular functions in eukaryotes through timely degradation of a variety of regulatory proteins.As a major protein degradation pathway,the ubiquitin-protease system participates in a series of biological processes like proliferation,differentiation,apoptosis and stress response.Therefore,dysfunction of the UPS destroys the protein hemostasis and leads to many diseases including cancer.Thus the target of the UPS has been the hot point of the anti-cancer therapy and the proteasome inhibitor Bortezomib has been approved for treatment of patients with multiple myeloma by FDA in 2008.But normal cell toxicity of Bortezmib limits the treatment and a more specific target therapy of UPS is highly needed 。 The ubiquitin-proteasome system(UPS)is a major protein degradation pathway,Cullin-RING ligases(CRLs)are the largest family of E3 ubiquitin ligases that promote the ubiquitination of about 20% of cellular proteins doomed for degradation through UPS,which participates in a series of biological processes like proliferation,differentiation,apoptosis and stress response.The activation of CRLs depends on the cullin neddylation and the RING component(RBX1/ROC1 or RBX2/ROC2/SAG/RNF7).Previous studies have reported that SAG,ROC1 and NAE are overexpressed in some cancer patients and closely related to the occurrence and development of tumors.However,whether they have any effect on renal carcinoma cells(RCC)remains unexplored.Methods:We used immunohistochemistry to detect SAG,ROC1 and NAEexpression in renal tumor adjacent tissues and malignant tissues,and observed that overexpression of SAG,ROC1 and NAE in human kidney cancer tissues,which correlated with poor patient survival.Then western blot to determine the levels of SAG,RBX1 and NAE in six RCC lines,based their levels,we will choose two lines to do siRNA silencing: a)siCont;b)siSAG;and c)siRBX1,siNAE followed by IB to determine the silencing effect.Measure the effect of siSAG vs.siRBX1,along with siCont on cell growth: a)ATP-lite proliferation;b)clonogenic survival;c)FACS for apoptosis or growth arrest;d)senescence;e)autophagy.Mechanistic study: Western blot to determine the accumulation of SAG vs.RBX1 substrates such as WEE1,p21,p27,p16,NOXA and BIM related to observed biological effects,To explore the key molecules at work.Finally,Rescuing experiments that knock down the downstream target genes at the same time,observe its biological changes.Then to explore SAG,ROC1,NAE knockdown plays a causal role via a downstream gene or substrate in suppression of tumor cell growth,.thus explore the function mechanism of SAG,ROC1,NAE in the kidney cancer.Results:Our study found overexpression of SAG,ROC1 and NAE in human kidney cancer tissues,which is associated with poor patient survival。Knockdown of SAG,ROC1 or NAE inhibits growth of renal cell carcinoma cells.SAG,ROC1,NAE silencing induces apoptosisespecially in ROC1 and SAG silencing cells.Knockdown of SAG,ROC1 or NAE induced G2 arrest and senescence 。Our study also found that silencing SAG,ROC1 or NAE can cause G2 arrest and cell senescence in renal cancer cells,while silencing SAG,ROC1 and NAE leads to accumulation of NOXA,BIM and p53.The essence of SAG,ROC1 and NAE-silenced renal cancer cell survival inhibition is to induce apoptosis of the cell lines of 786-0 and SLR20,which is shown by increased cleavage of PARP and caspase-3,induction of DNA degradation and increase of G1.In addition,SAG,ROC1 or NAE silencing can induce the accumulation of G2-M phase change inhibitors Wee1,CDC25 and cell cycle inhibitors P21 and P27.Further mechanism studies found that BIM Knockdown partially rescued inhibition of proliferation and survival induced by knockdown of SAG,ROC1 or NAE.BIM Knockdown was partially rescued apoptosis and senescence triggered by knockdown of SAG,ROC1 or NAE.Conclusions:1.Overexpression of SAG,ROC1 and NAE,is directly associated with and may even be causally related to kidney cancer development,and SAG,ROC1 or NAE overexpression may serve as a biomarker for prognosis of kidney cancer patients.2.SAG,ROC1 and NAE are required for the growth and survival of renal cell carcinoma cells.3.Growth suppression upon SAG,ROC1 and NAE depletion is mediated via apoptosis induction.Thus,accumulation of BIM upon SAG,ROC1 or NAE knockdown plays at least in part a causal role in suppression of tumor cell growth.Our study showed that SAG,ROC1,and NAE may be the target for anticancer therapy in renal carcinoma cells(RCC),and BIM may be played a critical role in SAG,ROC1-induced apoptosis and SAG,ROC1 and NAE triggered senescence.