Upregulation Of SOCS3by Liver X Receptor And5-Aza-2’-deoxycytidine Respectively Suppresses The Proliferation Of HCC And RCC Cells

Background：SOCS3(suppressor of cytokine signaling3) is an important suppressor of tumorgrowth,it can be induced in many different kinds of tissue cells such as hepatocyte.SOSC3protein is characterized structurally by an N-terminal domain of variable length andsequence,a central SH2domain,and a C-terminal SOCS box.As a negative regulator ofJAK-STAT signaling pathway,SOCS3play a very important role in suppression of tumorgrowth and induction of tumor apoptosis.On the one hand,SOCS3can suppresse theproliferation of cancer cells through p21induction;On the other hand,overexpression ofSOCS3lead to apoptosis of cancer cells in a way of suppression of Bcl-2and induction ofcasepase3.However,it is unclear for the regulatory mechanism of SOCS3geneexpression.LXR are ligand-activated transcriptional factors and belongs to nuclear receptorsuperfamily.As a muti-functional transcriptional factor,LXRs are extensively involved in theregulation of glucose,fatty acid,and cholesterol metabolism,inflammation,andneurodegenerative disease such as Alzheimer＇s disease.In recent years,some study indicatethat LXRs has a new function in anti-tumor.LXRs can exhibit it＇s potential of suppressionof cell grow in many different kinds of human tumors including hepatocellularcarcinoma， Prostate cancer，breast cancer，Colon cancer, cervical cancer, non-hodgkin＇slymphoma, and ovarian cancer, etc.But the mechanism of LXRs in anti-tumor is not thatclear now. LXR and SOCS3have the potential of anti-tumor.furthermore,SOCS3is in avery low level in most of cancer cells. there is no final conclusion about whether thereexists a link between the anti-tumor action of LXR agonists and the induction of SOCS3.Asan inhibitor of DNA methyltrsansferase,5-Aza-2＇-deoxycytidine has a wide range ofanti-cancer activities.5-Aza-2＇-deoxycytidine can lead to cell cycle arrest at G2phase inhuman cancer cells,but the molecular mechanisms of this cel cycle are poorly understood. Objective：To explore the molecular mechanism of LXR-mediated SOCS3induction and the roleof SOCS3in suppressing hepatocellular carcinoma cells proliferation; To explore themolecular mechanism of5-Aza-2＇-deoxycytidine-mediated cell cycle arrest in RCC cells.Methods：(1) We use two agonists of LXR to treat hepatocellular carcinoma cells(HepG2andHep3B) for24h,then extracted the total mRNA and protein of the cells,at last tested theexpression of SOCS3,cyclin D1,p21and p27by RT-PCR,QRT-PCR and Western Blot.(2) We predict whether there is a LXR binding site in Socs3promoter region using anuclear website,subsequently,construct recombinant plasmids of SOCS3promoter region.Inthe end,dual luciferase reporter assay was used to measure the influence of LXR agonists onthe transcriptional activity of Socs3gene promoter.(3) The influence of LXR agonists on proliferation and cell cycle of HCC cells wasevaluated by CCK-8and flow cytometry.(4) After knockdown of SOCS3with siRNA for SOCS3,we treated HepG2cells withTO901317for48h,and then CCK-8,flow cytometry and WB were performed to measure it＇s affect on cell proliferation,cell cycle,and the level of cylcin D1,p21and p27.(5) We implanted HepG2cells into axillae of nude mice.When palpable tumors wereperformed,we injected TO901317(25mg/kg/d) to peritoneum.Two weeks later,tumors wereharvested for analysis of the tumor size,the levels of SOCS3mRNA and protein byQRT-PCR,WB and Immunological Histological Chemistry.(6) CCK-8and flow cytometry were used to measure the proliferarion of RCC cellsafter being treated by5-Aza-2＇-deoxycytidine.(7) We used RT-PCR to detect the mRNA level of SOCS3and DNAmethyltransferases in769-Ps after exposure to5-Aza-2＇-deoxycytidine and used WB todetect the level of SOCS3and p21protein in the same condition.(8)769-P cells were transiently transfected with SOCS3-specific siRNA.Five-hourslater, the cells were treated with5μM5-Aza-2＇-deoxycytidine.Aftern48h,the cells werecollected to analyses the levels of SOCS3and p21.Results：(1) two agonists of LXR can upregulate the mRNA and protein level of SOCS3in a dose dependent manner.(2) LXRs agonists can’t increase transcriptional activity of SOSC3gene promoter.Thereason for SOCS3mRNA upregulation is mainly because LXR agonists increase the mRNAstability of SOCS3.(3) TO901317can suppress the proliferation of HCC cells and induce cell cycle arrestat G1phase in a dose dependent manner.(4) Knockdown of SOCS3in HCC cells remarkably attenuated TO901317-mediatedantitumor effect.(5)LXRs agonist TO901317can repress HCC xenograft tumore growth and increasemRNA and protein level of SOCS3.(6)5-Aza-2＇-deoxycytidine can lead to cell cycle arrest and inhibit proliferation ofRCC cells.(7)5-Aza-2＇-deoxycytidine decrease the mRNA levels of DNMT3a an DNMT3b andincrease SOCS3mRNA level,in addition,it also increase the protein level of SOCS3andp21.(8) The induction of p21were weakened after knockdown of SOCS3.Conclusion：(1) LXRs agonists upregulate mRNA and protein levels of SOCS3through increasingSOCS3mRNA stability in HCC cells.(2) LXRs agonists lead to cell cycle arrest and suppression of cell proliferate throughthe pathway:LXR-SCOS3-cyclin D1/p21/p27in HCC cells.5-Aza-2＇-deoxycytidine caninduce cell cycle arrest at G2phase through SOCS3meidiated p21induction in RCC cells.