Effect Of NLRC5 On Vascular Endothelial Growth Factor-A (VEGF-A) Expression In Hepatocellular Carcinoma And Its Molecular Mechanism

Hepatocellular carcinoma(HCC)is frequent malignancy and most lethal cancer in the world and its incidence and mortality aggrandized rapidly.With the high malignant degree and low 5-year-survival rate,HCC has become one of the most concerning topics.Chinese patients with HCC accounts for about more than 50% of the total number of patients around the world.The prognosis of HCC after conventional treatments including surgery,chemotherapy,radiation still remains generally poor.Thus,elucidating the molecular mechanism of HCC is crucial for the prevention and treatment of the disease.As the sensors for innate immunity,pattern recognition receptors(PRRs)are dual in the regulation of tumor immunity.They can inhibit the progression of tumor through keeping the host-microflora homeostasis and eliminating the death or mutant cells and recognize abnormal signals to induce immunogenic tumor cell death on one hand,they can also contribute to tumorigenesis in accelerating chronic inflammation and induction of regulatory cell populations and immunosuppressive cytokines to facilitate tumor growth on the other hand.Nucleotide-binding oligomerization domain-like receptors(NLRs)as a member of PRRs have been implicated in tumorigenesis including NOD1,NOD2,NLRP1,NLRP3,NLRP7 and NLRP12,especially there is interactive between the occurrence and development of gastrointestinal cancer.NLR protein family contains NLRC5(NLR family,CARD domain containing 5).NLRC5 is verified the role in innate immune response and inflammation.However,the function and mechanism of NLRC5 has not been completely explained.In addition,there is a report representing the expression and oncogenic role of NLRC5 in HCC.It has also shown that NLRC5 takes part in cell proliferation,migration and invasion in HCC by the Wnt/b-catenin signaling pathway.Vascular endothelial growth factor-A(VEGF-A)has been relevant with the tumorigenesis including HCC.Increased expression of VEGF-A was found in human HCC tissue and it was frequently related to clinical stages and prognosis.However,its relationship with NLRC5 is still unknown.Therefore,our studies research the effect and potential mechanism of NLRC5 in tumorigenesis,elucidate whether NLRC5 modulated VEGF-A expression.So this study mainly divided into the following several parts:1.The expression of NLRC5 and VEGF-A in human HCC tissues and human hepatoma carcinoma cell lines The immunohistochemical analysis and western blot found that NLRC5 and VEGF-A was positive for HCC tissues.We detected HCC cell lines including Hep G2,SMMC-7721 and BEL-7402 for investigation.And L02 cell was included as the normal control.Results shown that protein expressions of NLRC5 and VEGF-A in HCC cell lines were much higher compared with that in control L02 cells.2.The expression of NLRC5 and VEGF-A in nude mice6 × 106 cells / 100?L(sh NLRC5 or control)in PBS were inoculated subcutaneously into the left armpit of each mouse.After 43 days,tumors were entirely stripped and photographed.The result indicated that NLRC5 knock-down had antitumoractivity in vivo as witnessed by the decreased tumor volume and tumor weight.Western blot displayed that the expression of NLRC5 and VEGF-A reduced in sh NLRC5 compared with the control.3.The effect of NLRC5 up-regulation on the expression of VEGF-A and cell proliferation of Hep G2 cells NLRC5 up-regulation increased the level of VEGF-A,respectively,by Western blot,q RT-PCR and Elisa.What's more,the result of MTT assay,cell cycle analysis and plate-colony formation assay shown that NLRC5 overexpression promoted proliferation of Hep G2 cell.4.The effect of NLRC5 drown-regulation on the expression of VEGF-A and cell proliferation of Hep G2 cells In order to study the influence of drown-regulation NLRC5 to VEGF-A expression,Western blot,q RT-PCR and Elisa were used to measure the level of VEGF-A.It was found that NLRC5-si RNA decreased the expression and secretion of VEGF-A.A slower proliferation rate was detected in NLRC5-si RNA cells compared with control-si RNA cells by MTT assay.Silencing NLRC5 decreased the ability of Hep G2 cells to form colonies by plate-colony formation assay.There were increased NLRC5-si RNA Hep G2 cells arrested in G1 phase compared with control-si RNA cells by cell cycle analysis.5.The portion mechanisms of NLRC5 regulating VEGF-A expression To investigate whether NLRC5-regulated VEGF-A expression was adjusted through the PI3K/AKT signaling pathway,we texted the protein levels of p AKT(s473)when NLRC5 expression levels were modulated in Hep G2 cells and HCC tissue.The research found that the expression of p AKT(s473)was remarkably increased in human HCC tissues.NLRC5 up-regulation significantly increased the protein levels of p AKT(s473)compared with that control cells.It turned out the opposite result of NLRC5down-regulated express cell.In addition,we stimulated p EGFP-C2 transfected Hep G2 cells or NLRC5-overexpressing cells with LY294002(25 m M)which was an AKT inhibitor.At 48 h after LY294002(25 m M)treatment,the level of p AKT(s473)decreased significantly in p EGFP-C2 transfected Hep G2 cells or NLRC5-overexpressing cells by Western blot and q RT-PCR,the same as VEGF-A m RNA.Result of ELISA assay also indicated LY294002(25 m M)could inhibited the secretion of VEGF-A in p EGFP-C2 transfected Hep G2 cells or NLRC5-overexpressing cells.These data indicate that NLRC5 regulates VEGF-A activity through the AKT signaling pathway in Hep G2 cells.