Function And Mechanism Study Of LPS Regulates Immune Escape By Affecting M6A Modification Of LncRNA MIR155HG In Hepatocellular Carcinoma

Background:The incidence of liver cancer in China is among the highest in the world.The number of cases of morbidity and deaths account for more than half of the global cases.Attributed to the late detection and late staging diagnosis of most patients with hepatocellular carcinoma(HCC)in China,about 20% of patients are surgically resectable when diagnosed.Regrettably for most patients with HCC,the available treatments are very limited.Although sorafenib has always been the gold standard for treatment of advanced,unresectable HCC,the improvement of patient survival time is not satisfactory.Immunotherapy arises at the historic moment,which brings dawn for patients with HCC.However,the low response rate of immunotherapy,together with the expensive treatment costs,prohibited a large proportion of patients from undergoing treatment.In order to improve the efficacy of immunotherapy,it is urgent to gain insight into the immune microenvironment and uncover the mechanisms by which shapes the immune escape in HCC.Intestinal bacteria have been reported to be involved in the occurrence and development of many diseases,including tumors.In particular,the special anatomical structure of the liver determines that it has a closer relationship with gut bacteria.In-depth study of the influence of intestinal products on the immune microenvironment is beneficial to unveil the complex immune microenvironment of HCC.Studies have demonstrated that LPS can stimulate target cells through TLR4 binding to the adaptor protein myeloid differentiation marker 88(My D88),exerting an important influence on the immune response,either mediating antitumor immunity or promoting immune escape.While the role of LPS in the regulation of HCC immune microenvironment is not clear.Therefore,exploring the impact of LPS on the immune microenvironment and its molecular mechanism may provide a new direction for improving the efficacy of immunotherapy for HCC.Long non-coding RNAs(lnc RNAs)are a class of non-coding protein transcripts with nucleotides more than 200,which participated in the regulation various stages of growth and development as well as physiological and pathological processes.In recent years,continuous research has found that lnc RNAs participated in the immune regulation process in a variety of ways,including anti-tumor immune and immune escape.LncRNA MIR155 HG,also known as B cell integration cluster(BIC),is considered to play an important function in tumor development and immune regulation.But the role of MIR155 HG in HCC has not been reported.Therefore,this study focused on exploring the role and mechanism of MIR155 HG in LPS mediated immune escape of HCC.Methods:Analyze the expression and prognostic values of MIR155 HG and the correlation between MIR155 HG and immune cells and immune molecules in various tumors of TCGA(The cancer genome atlas)database by GEPIA(The gene expression profiling interactive analysis)and TIMER(Tumor immune estimation resource)online database.Detecting the relationship between MIR155 HG and immune checkpoint molecules PD-L1 and CTLA4 in clinical tissues of cholangiocarcinoma and HCC by q RT-PCR.Plasma samples were collected from patients with HCC who had not received preoperative treatment.Detected and comparison the plasma LPS levels by limulus assay in HCC patients with cirrhosis and patients without cirrhosis.The influence of LPS on the expression of immune checkpoint molecules PD-1 and PD-L1 was examined in mice.The influence of LPS on PD-L1 was examined in HCC cells.T cell-mediated tumor killing experiment were used to analyzes the cell killing effect of activated T cells on tumor cells stimulated by LPS.In order to understanding the mechanism of PD-L1 regulated by LPS,the GEPIA2 online tool was utilized to analyze the correlation between TLR4 and My D88,the major molecules activated by LPS,with MIR155 HG and PD-L1.The regulatory effect of LPS on MIR155 HG as well as MIR155 HG on PD-L1 was verified in HCC cells.To analyze the role of METTL14 on the process of MIR155 HG expression induced by LPS,RNA pull-down,RIP(RNA binding protein immunoprecipitation)and dual luciferase reporter assays were performed.The subcellular localization of MIR155 HG in HCC cells were detected using FISH(fluorescence in situ hybridization)experiments as well as the lnc Locator database.Mi RNAs containing the binding sites of MIR155 HG and the downstream target genes of mi RNAs were predicted by star Base 3.0 database.Detecting the interaction of MIR155 HG with mi RNA,mi RNA with downstream target genes using dual luciferase reporter assay.Then validation the regulation of PD-L1 by MIR155 HG in vivo.Furthermore,we investigate the effect of MIR155 HG on the growth of HCC in vitro and vivo.Finally,the regulatory axis of LPS-METTL14-MIR155HG-PD-L1 was verified by TCGA database,clinical tissue samples and mouse in vivo.Results:1.In various types of tumors,the expression of MIR155 HG is different between tumor and normal tissues,and is related to tumor stage and prognosis.MIR155 HG can be used as a prognostic indicator in cholangiocarcinoma,glioblastoma multiforme and skin cutaneous melanoma and so on.2.MIR155 HG is closely related to the infiltration of immune cells,the expression of immune molecules,especially the expression of PD-1,PD-L1 and CTLA4 in most types of tumors including HCC.3.The TCGA HCC data shown that LPS activated pathway molecules(TLR4 and My D88)are positive correlated with MIR155 HG and PD-L1.Clinical specimen results showed that the plasma LPS levels and the PD-L1 expression in tumor were higher in HCC patients with cirrhosis than without cirrhosis.4.Intraperitoneal injection of LPS in vivo upregulated the expression of PD-1 and PD-L1 in HCC tissues,promoted the growth of tumor,and knockdown of MIR155 HG in HCC cells inhibited the expression of PD-L1.5.Both LPS stimulation and overexpression of MIR155 HG in HCC cells up-regulated the expression of PD-L1,and the PD-L1 inhibited T cell-mediated killing.Rescue experiments shown that MIR155 HG played a critical regulatory role in the process of PD-L1 induced by LPS.6.LPS promotes the m6 A modification of MIR155 HG by upregulating RNA methyltransferase METTL14,stabilizing MIR155 HG through an m6A-ELAVL1 dependent manner.7.MIR155 HG competitively inhibits mi R-223-3p binding to the STAT1 3’UTR to upregulate PD-L1 expression through a ce RNA mechanism in HCC cell.Further experiment confirming that MIR155 HG regulated the expression of PD-L1 through mi R-223-3p/STAT1 pathway in vivo.8.MIR155 HG had no significant effect on HCC cell growth and migration.9.The expression of PD-L1 in tumor tissues of HCC patients is associated with LPS pathway and METTL14-MIR155HG-STAT1 signaling.10.The METTL14-MIR155HG-PD-L1 axis is upregulated in tumor tissues of HCC patients with cirrhosis.Conclusions:The present study revealed that LPS caused immune escape of HCC by affecting the level of m6 A modification of MIR155 HG to upregulate PD-L1.The formation of immune escape microenvironment in HCC patients with cirrhosis may be closely related to high levels of LPS.The current results not only provide novel insights into the molecular mechanisms underlying HCC induced by LPS,but also paves the way for the development of more effective immunotherapeutic strategies for HCC.