The Expression Of LILRB2 In HCC And Its Function In HCC Cells

[Background]Hepatocellular carcinoma(HCC)is the malignant tumor originating from hepatocytes and is the most common primary liver malignant tumor.HCC is the sixth most prevalent malignancy and the second most common cause of cancer-related mortality in the world.About 55%of the world s new HCC cases come from China,due to high prevalence of chronic hepatitis B virus(HBV)infection.Although the effect of comprehensive treatment for HCC has been substantially improved,clinical cure rates and long-term survival rates for HCC patients remain low.In recent years,targeted treatment has brought new opportunities for the treatment of liver cancer.However,the range of targeted drugs for HCC is still limited and drug resistance is inevitable.Therefore,it is very necessary to study the pathogenesis of HCC and explore potential effective therapeutic targets for HCC.Leucocyte immunoglobulin-like receptor(LILRs),also known as immunoglobulin-like transcriptions(ILTs)or monocytes immunoglobulin-like receptors or CD85(a-h),is a group of immunoglobulin superfamily molecules,which are expressed on cell surface.The subfamily B of LILRs is a group of inhibitory receptors,which signal via intracellular immunoreceptor tyrosine-based inhibitory motifs(ITIMs)to negatively regulate immune cell activation.Previous studies showed that several members of the LILRB subfamily(such as LILR B1,LILR B2,and LILR B4)are aberrantly overexpressed in malignant tumor cells in the blood system,such as T-cell lymphoma,B-cell chronic lymphoblastic leukemia,and acute monocyte leukemia,and are closely related to tumor disease progression.In addition,LILRB1,LILRB2,and LILRB4 also have been found to be highly expressed in a variety of solid malignant tumors,such as non-small cell lung cancer(NSCLC),breast cancer,stomach cancer,pancreatic cancer,colon cancer and ovarian cancer cells.The expression of LILRB2 is positively correlated with tumor immunosuppression,tumor cell proliferation,and invasion and metastasis.These studies indicate that LILRBs represent potential targets for tumor therapy.However,at present,there are no reports on the expression of LILR B molecules in HCC and the specific functions in the progression of HCC diseases.LILRB2,also known as ILT4 or LIR-2 or MIR10 or CD85d,is a typical representative of the LILRB family,which is mainly expressed in monocytes,dendritic cells(DCs),and endothelial cells.LILRB2 can induce the tolerogenic phenotype of human DCs,consequently inducing immunosuppressive T cells,and inhibiting T cell activation and proliferation.In recent years,increasing studies have shown that LILRB2 is a potent cancer-promoting molecule,and its cancer-promoting functions depend on the binding with its ligands.LILRB2 has been found to be overexpressed in leukemia,breast cancer,non-small cell lung cancer(NSCLC),and pancreatic cancer cells and contributes to tumor progression.So far,two types of LILRB2 ligands were found.A class of ligands is the classical or non-classical histocompatibility complex(MHC)class I molecule,known in humans as human leucocyte antigen(HLA),Among them,HLA-G has the highest bingding affinity.The other class is angioalloietin-like proteins(ANGPTLs),of which the highest binding forces are ANGPTL2 and ANGPTL5.Our previous study has demonstrated that LILRB2 is highly expressed in NSCLC cell lines and tissues,and has a significant positive correlation with the poor prognosis of NSCLC patients.LILRB2 and its two class ligands,ANGPTLs and HLA-G,were co-expressed in NSCLC and their co-expression is positively related with patients'poor overall survival(OS).Mechanistically,on one hand,LILRB2 can promote the proliferation and migration invasion of NSCLC cells by activating the ERK signaling pathway;on the other hand,LILRB2 could upregulate the expression of B7H3 by activating P3K-Akt-mTOR pathway.In summary,LILRB2 plays important roles in tumor progression in a variety of tumors and is a promising tumor therapy target.However,the expression and clinical significance of LILRB2 in HCC has not yet been reported.[Objective]In this study,we aimed to investigate the expression and effect of LILRB2 in HCC cells.We explored the relationship between LILRB2 and its legend HLA-G in HCC tissues.By manipulating LILRB2 expression in HCC cells,we detected the role of LILRB2 in promoting cell proliferation,motility and apoptotic in vitro,as well as tumor growth and metastasis in vivo,and elucidated the potential mechanisms underlying the progression-promoting effect by LILRB2 in HCC.[Methods]1.The protein expression of LILRB2 in 82 human HCC tissues and 64 peritumoral tissues was evaluated by immunohistochemistry(IHC)to evaluate the difference between tumor and peritumor tissues.The correlation between LILRB2 expression and clinicopathological features of patients was also analyzed.2.The protein expression of HLA-G in HCC was evaluated by IHC to evaluate the correlation between the expression of LILRB2 and its classic ligand HLA-G.3.The survival data of 82 HCC patients were followed and survival curves were drawn using the Kaplan-Meier method and compared with log-rank test to evaluate the prognostic role of LILRB2 in HCC patients.4.The expression levels of LILRB2 in different HCC cell lines were tested by Western blot.Over-expression or interference of LILRB2 expression in HCC cells was performed by transient transfection technique.Then MTT curve was used to measure the effect of LILRB2 on the cell growth in vitro;apoptosis analysis was tested by FACS to detect the effect of LILRB2 on the cell apoptosis in vitro;transwell migration and invasion assays were used to investigate the effect of LILRB2 on the cell motility in vitro.5.Subcutaneous transplantation tumor model was applied to examine the effect of LILRB2 on the tumorigenicity and tumor growth in vivo.Stably transfected cell lines HepG2/LILRB2,SM-7721/shLILRB2 and their corresponding control cells(5×106)were injected into the left flank of the mice.Tumor size was calculated and tumor weight was measured.6.Tail intravenous injection model was applied to examine the effect of LILRB2 on the tumor metastasis in vivo.Stably transfected cell lines HepG2/LILRB2,SM-7721/shLILRB2 and their corresponding control cells(1×106)were injected into the tail vein of the mice.The number of lung metastasis tumor nodes was counted.7.The relationships of LILRB2 and the phosphorylation level of three parallel signal transduction modules of MAPK signaling,including ERK,JNK and p38 were assessed by western blot.After treated with ERK signaling pathway inhibitor,the suppressions of cell proliferation,invasion and migration ability of LILRB2 overexpressing cells were observed.[Results]1.The expression of LILRB2 in human HCC tissue tumor cells was significantly higher than that in paracancerous tissues.LILRB2 was mainly expressed in the cytoplasm of HCC cells.There was 70.73%HCC cases(58/82)showed positive expression of LILRB2,whereas the positive expression rate of LILRB2 in peritumoral tissues was only 28.13%.The expression level of LILRB2 in HCC tissues tumor cells was significantly higher than that in peritumor tissues.LILRB2 expression was positively correlated with male(P=0.02),larger tumor size(P=0.042)and poor tumor cell differentiation(P=0.018)·2.We detected the positive correlation between the expression of LILRB2 and its ligand HLA-G in human HCC tumor cells.HLA-G was mainly stained in the cancer cell cytoplasm and membrane.Positive immunoreactivity for HLA-G was found in 52.44%HCC tissue samples(43/82)?Among LILRB2-positive cases,the positive rate of HLA-G expression was 60.34%(35/58).On the other hand,among the LILRB2-negative cases,the positive rate of HLA-G expression was only 33.3%(8/24).The expression of LILRB2 was significantly correlated with HLA-G expression(P=0.026).3.Expression of LILRB2 in human HCC tumor cells is associated with poor prognosis of patients.The median survival time of LILRB2 positive group was 30 months,while that of LILRB2 negative group was 38 months.The OS of patients with LILRB2 positive expression was much lower than that of the group with LILRB2 negative expression(P=0.0438).4.LILRB2 enhanced the proliferation,migration and invasion of HCC cells in vitro.LILRB2 was expressed in all three HCC cell lines preserved in our laboratory(HepG2,Huh7,SM-7721).And then,HepG2 cells with lowest endogenous LILRB2 expression were selected to be transfected with LILRB2 vector,leading to significant up-regulation of LILRB2.The proliferation,invasion and migration ability were significantly enhanced in HepG2 cells with LILRB2 overexpression,while the anti-apoptotic ability was not significantly affected.Next,reverse validation was implemented with SM-7721,which had the highest endogenous LILRB2 expression.After interfering the expression of LILRB2,the proliferation,invasion and migration ability of SM-7721 cells were significantly inhibited.5.LILRB2 drived HCC tumor growth and metastasis in vivo.To address whether LILRB2 could promote tumor growth in vivo,subcutaneous transplantation tumor models were established with stable transfected HepG2/LILRB2,SM-7721/shLILRB2 and their corresponding control cells.The tumor volumes were much more massive in mice with injection of HepG2/LILRB2 cells and smaller in mice with injection of SM-7721/shLILRB2 cells at the designated time points,compared with the corresponding control cells respectively.Meanwhile,to evaluate whether LILRB2 could promote distant metastasis in vivo,tail intravenous injection models were established by above mentioned stable transfected cell lines.At day 42,the number of lung metastases was much more in HepG2/LILRB2 treated mice and much less in SM-7721/sh LILRB2-treated mice,compared to corresponding control groups.6.We detected the phosphorylation of MAPK signal transduction modules,including ERK,JNK and p38 and found that the phosphorylation level of ERK was significantly increased in HepG2 cells with LILRB2 overexpressed,while significantly decreased in SM-7721 cells with LILRB2 interference.In addition,after treating with ERK signaling pathway inhibitor U0126,the proliferation,invasion and migration of HepG2 cells with LILRB2 overexpressing was significantly inhibited.[Conclusions]1.LILRB2 is highly expressed in HCC tissues and the positive expression of LILRB2 is significantly correlated with poor prognosis of HCC patients.2.The expression of LILRB2 was significantly positively correlated with its ligand HLA-G expression in human primary HCC tissues.?3.LILRB2 could promote the proliferation,migration and invasion of HCC cells in vitro and in vivo.4.LILRB2 induced the malignant phenotype of HCC cells via activating ERK signaling pathway.