The Mechanism Of Lipopolysaccharide Promote Abberant Differentiation Of Hepatic Progenitor Cells Contributes To The Formation Of Hepatocarcinogenesis Microenvironment

【Background and Object】Hepatocellular carcinoma(HCC)is one of the common malignant tumors,especially in China.Its mortality rate ranks third in malignant tumors.HCC is often formed on the basis of chronic liver damage such as liver fibrosis and cirrhosis.Abnormal differentiation of hepatic progenitor cells(HPCs)is considered to be one of the origins of liver cancer.In physiological conditions,HPCs can participate in the repair process of chronic liver injury through hepatic and biliary differentiation.HPCs are also considered as an important source of fibroblasts and hepatocellular carcinoma initiation cells under pathological conditions,which participate in liver fibrosis and hepatocarcinogenesis.However,the potential mechanism by which HPCs are activated to form liver cancer is still unclear.More and more studies have shown that chronic liver injury is an important pathological process leading to liver cancer.During chronic liver injury,intestinal permeability increases due to abnormal portal hypertension and abnormal bile metabolism,leading to disorder of intestinal flora,so that lipopolysaccharide(LPS)in the intestine enters the liver through the portal vein and the concentration of LPS continues to rise.LPS is a product of self-dissolving and releasing from the cell wall of dead Gram-negative bacteria and has various biological activities.Under physiological conditions,part of the LPS produced by intestinal bacteria enters the capillaries through the intestinal mucosa and enters the liver through the portal vein and is then detoxified.LPS is always present in the so-called "hepatocarcinoma trilogy" of "hepatitis-hepatic cirrhosis-hepatocarcinogenesis".LPS is an important pro-inflammatory factor that promotes inflammatory factors such as interleukin-1-beta(IL-1β),interleukin-6(IL-6),interleukin-8(IL-8),interleukin-12(IL-12),type I interferons(IFNs),tranforming growth factor-beta(TGF-β)and tumor necrosis factor-alpha(TNF-α).Therefore,LPS might play an important role in liver fibrosis and liver cancer.However,the effect of LPS on the function of HPCs is still less studied.Exploring the effects of LPS and HPCs on hepatocarcinogenesis and studying the effects and mechanisms of LPS on HPCs function can not only deeply understand the effects of the interaction of inflammation and HPCs on tumorigenesis in tumor microenvironment,but also provide theoretical guidance for further intervention.We first used the liver cancer clinical specimens and the rat primary liver cancer animal model to observe the correlation between the activation of liver precursor cells and the degree of liver fibrosis and liver cancer,and found that the activation of liver precursor cells and the formation of liver fibrosis It is closely related to the occurrence of liver cancer.We investigated the effects of HPCs on hepatocarcinogenesis by infusion of HPCs in a rat model of primary liver cancer induced by DEN.It was found that infusion of exogenous HPCs promoted the formation of liver fibrosis.And the occurrence of liver cancer,lipopolysaccharides(LPS)play an important role in the above process.Interestingly,LPS does not directly induce HPCs to form tumors,but induces the transformation of HPCs into myofibroblasts,which have the ability to induce HPCs to form tumors.Further studies have found that myofibroblasts induce the inactivation of Ras and p53 signaling pathways in HPCs by secreting IL-6 and TNF-α and ultimately promote the proliferation and abnormal transformation of HPCs.The TLR4 signaling pathway mediates LPS-induced differentiation of HPCs into myofibroblasts and ultimately promotes abnormal differentiation of liver precursor cells.In addition,by correlating the expression levels of activated HPCs,liver fibrosis,IL-6 and TNF-α with liver cancer recurrence in clinical samples of liver cancer,it was found that the above indicators were closely related to postoperative recurrence of liver cancer.Our results suggest that myofibroblasts and liver cancer cells in the liver cancer microenvironment are derived from liver precursor cells.Hepatic progenitor cells-derived myofibroblasts constitute the tumor microenvironment and promote the proliferation and abnormal transformation of liver precursor cells.Lipopolysaccharide not only participates in the induction of chronic inflammation microenvironment in the liver,but also plays an important role in regulating the plasticity of liver precursor cells to promote liver cancer.Section 1 The correlation between hepatic progenitor cells activation、fibrosis level and hepatocarcinogenesisIn order to study the role of HPCs in the hepatocarcinogenesis,we used DEN induced rat primary liver cancer model and liver cancer clinical tissue specimens to detect fibrosis levels and HPCs activation in different stages of HCC and adjacent tissues of HCC tissues and analyzed the relationship.The results showed that with the progress of HCC,the level of fibrosis and the activation of HPCs gradually increased and the activation of HPCs had a certain correlation with the level of hepatic fibrosis.In addition,this phenomenon also exists in tissues adjacent to liver cancer.The level of fibrosis and the number of HPCs activation are related to the survival of patients.In order to further clarify the role of HPCs in the occurence of hepatocarcinoma,we used the DEN-induced rat primary liver cancer model to observe the effect of HPCs on tumorigenesis,liver function and survival.The results showed that HPCs infusion promoted the tumorigenesis,increased liver damage and shortened survival time.Further pathological examination revealed that HPCs infusion promoted the level of liver fibrosis,which showed as increasement in fibrous tissue structure and the expression of α-SMA.To investigate the distribution of exogenous HPCs in the liver after infusion,we transfected HPCs with GFP-tagged lentiviral vecto and found that most of the GFP-positive HPCs located in the liver interstitial site and expressed α-SMA.The results mentioned above suggest that the number of HPCs has a correlation with the level of liver fibrosis.In the process of hepatocarcinogenesis,HPCs can promote liver fibrosis and tumorigenesis,and may differentiate into fibroblasts.Section 2 The effect of LPS promoted abberant differentiation of hepatic progenitor cells contributed to the formation of hepatocarcinogenesis microenvironmentLPS concentration continues to increase during the occurrence of liver cancer.Then,does LPS participate in the role of HPCs in liver fibrosis and liver cancer? To confirm this hypothesis,we used the DEN-induced rat primary liver cancer model to remove LPS by antibiotics and observed the effect of exogenous HPCs infusion on liver fibrosis,liver cancer,liver function and survival time.It was found that after LPS clearance,HCC was inhibited in rats,liver function improved and the survival time of HCC rats was prolonged.Further histopathological examination revealed that the level of liver fibrosis was also reduced after LPS inhibition.In order to further clarify the effect of LPS on the function of HPCs,we used LPS to treat HPCs cell lines.Using transcriptome sequencing technology to analyze the effect of LPS on HPCs gene expression,we found that the expression of fibrosis-related genes was up-regulated,but had no effect on the expression of tumor-associated genes.To further confirm this phenomenon,we isolated the GFP-positive HPCs that have moved to the liver of DEN-induced HCC model by primary separation technique and examined the expression of fibroblast-related genes in GFP+ cells by RT-PCR and Western Blot.The result demonstrated that the expression of fibroblast-related genes increased significantly.This result confirms the hypothesis that HPCs can differentiate into fibroblasts.These results suggest that HPCs are affected by LPS in the microenvironment of liver cancer,differentiate into fibroblasts,further induce the malignant transformation of HPCs,and promote the occurrence of HCC.Section 3 The mechanism of LPS promoted abberant differentiation of hepatic progenitor cells contributed to the formation of hepatocarcinogenesis microenvironmentIn the previous section,we confirmed that LPS induced differentiation of hepatic precursor cells into fibroblasts.Then subcutaneous tumor formation experiments in nude mice showed that LPS treatment of HPCs could not form tumors.However,tumor emerged when LPS-treated HPCs were mixed with normal HPCs.Next,we explored the potential mechanism.We used conditioned medium collected from normal HPCs and LPS treated HPCs to treat HPCs.Gene chip technology showed that conditioned medium from LPS treated HPCs activated the expression of Ras,which is tumor-associated signaling pathways in HPCs and inhibited the expression of p53,which is tumor suppressor signaling pathway.Bioplex cytokine chip was used to detect the cytokine profile of myofibroblast differentiated from LPS treated HPCs.The result demonstrated that LPS induces HPCs to differentiate into fibroblasts and secrete a large amount of IL-6 and TNF-α,which suggested that IL-6 and TNF-a might lead to the abnormal expression of Ras and p53 signaling pathways in HPCs and promote their abnormal differentiation which eventally caused the tumorigenesis.Toll-like receptor 4(TLR4)is an important receptor for LPS.In order to detect the role of TLR4 in the differentiation of HPCs induced by LPS and its role in liver cancer,we used TLR4 sh RNA to inhibit the expression of TLR4 in HPCs and found that the levels of IL-6 and TNF-α were decreased.Subcutaneous tumor formation in nude mice showed that the effect of LPS treated HPCs on the tumorigenesis potential of HPCs was also inhibited after knocking out TLR4.At the same time,knockdown of TLR4 in HPCs reversed the promotion of tumorigenesis by HPCs infusion.This result suggests that the TLR4 signaling pathway mediates the differentiation of HPCs into fibroblasts induced by LPS and ultimately promotes the abnormal differentiation of HPCs.Finally,the relationship among the expression levels of IL-6 and TNF-α,the activation of HPCs,the level of liver fibrosis in HCC clinical samples and the recurrence time of HCC patients were comprehensively analysised.It was found that IL-6 and TNF-α were closely related to the indicators mentioned above.The results of this study confirmed that LPS induced differentiation of HPCs into fibroblasts by binding to TLR4,secreting IL-6 and TNF-α,and leading to the abnormal expression of Ras and P53 signaling pathways in HPCs,which promoted the malignant transformation of HPCs and ultimately led to hepatocarcinogenesis.