| Background&AimsLiver cancer,the sixth most common cancer globally and the third leading cause of cancer death,is one of the malignant tumors that seriously endanger human health.In China,liver cancer is the fifth most common cancer,with the second mortality rate only to lung cancer.At present,liver resection is still the first choice for the treatment of liver cancer,but five-year recurrence rates approach 70%postresection,which is an important risk factor for the prognosis of liver cancer.However,the mechanism underlying hepatocarcinogenesis is not clearly illustrated.More and more studies have demonstrated that liver regeneration is closely related to the occurrence of liver cancer.The liver possesses powerful regeneration capacity.Following viral or alcoholism-induced liver injury and partial surgical resection,the remaining hepatocytes replenish the lost tissue,restoring the liver to its full size and functionality.In chronic liver injury,in which hepatocytes exhibit severe necrosis,apoptosis,and aging,hepatic progenitor cells and biliary epithelial cells will be activated and participate in liver regeneration.Some studies have indicated that hepatocytes harbor a degree of plasticity,and can dedifferentiate into hepatic progenitor cells or transdifferentiate into biliary epithelial cells following injury.Recent studies have shown that the chronic inflammatory microenvironment leads to gene mutation and epigenetic changes of cells involved in liver regeneration,which finally contribute to malignant transformation and conversion into tumor-initiating cells.On the background of chronic liver injury,partial hepatectomy promoted inflammation-induced DNA damaged cell proliferation and tumorigenesis.PHx surgery triggers the increased expression of different cytokines and growth factors to initiate hepatocyte proliferation and liver regeneration,so enriched cytokines,and growth factors potentially promote tumor progression.Thus,there is a close relationship among the inflammation environment,liver regeneration,and hepatocarcinogenesis,however,the effect and mechanism of the inflammation environment and liver regeneration on hepatocarcinogenesis need further to be investigated.LPS/TLR4/NF-κB signaling pathway is an important pathway that mediates inflammatory immune signals and plays an important role in maintaining physiological homeostasis and the development of diseases.Under normal physiological conditions,blood in the portal vein from the gastrointestinal tract contains a large number of lipopolysaccharide（LPS）produced by intestinal bacteria,which are absorbed and cleared by hepatocytes.Previous reports have demonstrated that LPS plays an important role in liver regeneration and maintaining cell stemness.In the initial phase of liver regeneration after PHx,LPS can activate the NF-κB signaling pathway by TLR4 and promote hepatocytes to enter the cell cycle and proliferate.Also,LPS can regulate the self-renewal of stem cells and inhibit differentiation by upregulating stem cell markers.In chronic liver injury,the function of the intestinal barrier is destroyed,leading to ectopic intestinal bacteria and the increase of LPS in the liver,which further aggravates liver injury and hepatocarcinogenesis.In the present study,we aimed to investigate the role and mechanism of the LPS/TLR4signaling pathway in maintaining cell stemness in the portal vein area in vivo and in vitro;then analyze whether LPS can participate in liver regeneration after partial hepatectomy by regulating cell stemness,and the mechanism underlying the phenomenon;finally,explore the relationship between liver regeneration and hepatocarcinogenesis,as well as the role and mechanism of LPS downstream molecules in hepatocarcinogenesis.We hope to reveal the internal relationship between liver regeneration and hepatocarcinogenesis,and provide a new theoretical basis for the prevention and treatment of liver cancer.PartⅠThe role and mechanism of lipopolysaccharide in the maintaining of hepatocyte stemness in normal physiological stateObjective:The liver possesses a powerful regeneration ability,which is correlated with the stemness of hepatocytes in the portal vein（PV）.Present studies have shown that the cell stemness in the portal vein area is much higher,and hepatocytes in the portal vein area are exposed to a large amount of portal blood containing lipopolysaccharide.Many studies have shown that LPS is closely related to the maintenance of cell stemness.This part aims to explore the effect of LPS on hepatocyte stemness and its regulatory mechanism.Methods:1.IHC and IF staining were used to examine the location of stem cell markers;the concentration of LPS in the portal vein and inferior vena cava were detected;analysis of the correlation between them;2.Then,we assessed the effect of LPS on stemness maintenance in mice by using antibiotics to eliminate LPS and knocking out the LPS receptor,TLR4.3.In vitro,the effect of LPS on the stemness of hepatocytes was investigated by colony and sphere formation assays and assessment of pluripotent and stem cell marker expression by q RT-PCR,western blot,IF staining.q RT-PCR,western blot,IF staining were used to analyze the effect of LPS on hepatocyte reprogramming;4.The effect of LPS on the reprogramming ability of hepatocytes was analyzed by real-time PCR,Western blot,and immunofluorescence;5.Differentiation assay and cell transplantation assay were used to explore the role of LPS on hepatocyte differentiation;6.After silencing TLR4,colony and sphere formation assays,western blot was administrated to study whether LPS regulated cell stemness by TLR4;7.Western blot was used to detect the expression of YAP1 in LPS treated hepatocytes;after silencing YAP1,colony and sphere formation assays,western blot was administrated to study whether YAP1 mediates the regulation of LPS on cell stemness;8.The model of portal vein branch ligation in WT and TLR4-/-mice was used to examine the correlation among LPS,YAP1,and Sox9.Results:1.Sox9+cells were located in the PV area,where the concentration of LPS was much higher than that in the CV area;elimination of LPS by antibiotics inhibited the expression of the stem cell marker Sox9.2.LPS promoted colony and sphere formation and induced the upregulation of pluripotent and stem cell markers in AML12 cells.3.After silencing of TLR4 or YAP1,the promotion of LPS on cell stemness in hepatocytes was inhibited.4.The concentration of LPS was positively correlated with the expression of YAP1 and Sox9 in the PV area in the model of portal vein branch ligation.Conclusions:Our study implies a correlation between LPS/TLR4/YAP1 signaling and cell stemness,and LPS was shown to play an essential role in maintaining the homeostasis of hepatocyte stemness in the PV area.However,the mechanism by which LPS induces the activation of YAP1 needs to be further investigated.PartⅡThe effect and mechanism of lipopolysaccharide on hepatocyte stemness in liver regeneration after PHxObjective:Liver regeneration is a fundamental pathophysiological process that occurs after the loss of hepatic tissue,and it is critical for promoting functional recovery and maintaining homeostasis.Hepatocytes have powerful phenotypic plasticity and can dedifferentiate into Sox9+HNF4α+hepatocytes with the characteristics of hepatic progenitor cells.LPS has been reported to be correlated with cell stemness and liver regeneration.In this part,we aimed to analyze whether LPS can promote the conversion of hepatocytes into Sox9+HNF4α+hepatocytes to participate in liver regeneration by regulating cell stemness,and the mechanism underlying this process.Methods:1.Western blot,IF,IHC were used to analyze the expression of stemness molecules in liver regeneration after PHx.2.After silencing of Sox9,IHC staining and the recovery ratio of liver-to-body weight were used to study the role of Sox9+HNF4α+hepatocytes in liver regeneration.3.Hepatocyte transplantation and chimeric Fah-/-mice were used to evaluate the cell origin of Sox9+HNF4α+hepatocytes.4.Analysis of the concentration of LPS in the portal vein after PHx and the expression of Sox9 in hepatocytes after administration with LPS to evaluate whether LPS/TLR4 signaling participates in the activation of Sox9+HNF4α+hepatocytes.5.The NF-κB pathway PCR array was used to screen key molecules that participated in liver regeneration,western blot and IF assay were used to verify the role of key molecules.6.Construct transgenic mice of key molecules and analysis the effect of key molecules on the activation of Sox9+HNF4α+hepatocytes and liver regeneration.7.Overexpression of key molecule in TLR4-/-mice and analysis whether LPS/TLR4signaling regulated the activation of Sox9+HNF4α+hepatocytes and liver regeneration by key molecules.8.Using qRT-PCR、western blot、IF、Chip-PCR、immunoprecipitation to analyze the effect and mechanism of key molecules on the expression of Sox9.Results:1.The number of Sox9+HNF4α+hepatocytes was highly increased in the initial phase of liver regeneration after PHx;Knocking down the expression of Sox9 inhibited hepatocyte proliferation and the recovery of liver-to-body-weight ratio;Sox9+HNF4α+hepatocytes derived from the dedifferentiation of mature hepatocytes.2.Activated LPS/TLR4 signaling promoted the dedifferentiation of mature hepatocytes,hepatocyte proliferation,and the recovery of liver-to-body-weight ratio after PHx.3.Bcl3,as a master downstream molecule of the LPS/TLR4 signaling,promoted the activation of Sox9+HNF4α+hepatocytes and liver regeneration after PHx.4.Bcl3 bound to and deubiquitinated YAP1,which contributed to the accumulation of YAP1 in hepatocytes and upregulated Sox9 expression.Conclusions:In liver regeneration after partial hepatectomy,mature hepatocytes could dedifferentiate into Sox9+HNF4α+hepatocytes,which possessed high proliferation and stemness.Mechanistically,in the initial phase,LPS could upregulate the expression of Bcl3 by TLR4.And then,Bcl3 formed a complex with and deubiquitinated YAP1 and further induced YAP1 to translocate into the nucleus,resulting in Sox9 upregulation and mature hepatocyte conversion.Our findings demonstrated that Bcl3 promoted Sox9+HNF4α+hepatocytes to participate in liver regeneration,which might be a potential target for promoting regeneration after liver injury.PartⅢThe role and mechanism of liver regeneration in the occurrence of liver cancerObjective:Hepatic resection is the preferred treatment of choice for liver cancer,however,postoperative recurrence is an important risk factor for the prognosis.Recent studies have indicated that liver regeneration is closely correlated with hepatocarcinogenesis,but the mechanism underlying the phenomenon is not elucidated.Herein,we aimed to investigate the relationship between liver regeneration and hepatocarcinogenesis,and the role and mechanism of Bcl3 in tumorigenesis.Methods:1.In the model of DEN-induced HCC,we performed PHx surgery and analyzed the effect of liver regeneration after PHx on hepatocarcinogenesis.2.IHC staining was used to analyze the activation of hepatic progenitor cells after PHx in the model of DEN-induced HCC.3.Western blot was used to investigate the expression of Bcl3 in the process of tumorigenesis;Bcl3 deficient mice were used to examine the role of Bcl3 in hepatocarcinogenesis and the activation of hepatic progenitor cells.4.In the process of DEN-induced HCC,IHC,q RT-PCR,and western blot assay were used to investigate the activation of hepatic progenitor cells and the expression of genes associated with chromosomal instability.5.Xenograft animal studies were used to analyze the effect of Bcl3 on xenografted Hepa1-6 cells growth in BALB/c-nu mice.6.Western blot,immunoprecipitation,ubiquitination assays were used to investigate the effect of USP7 on the expression of Bcl3.7.In DEN+PHx induced HCC in mice and Hepa1-6 xenografts established in BALB/c-nu mice,P5091 was used to investigate whether target Bcl3 could inhibit tumorigenesis and tumor progression.8.Analyze the expression of Bcl3 and Sox9 in liver cancer tissues and adjacent tissues,and its relationship with the prognosis of patients.Results:1.Liver regeneration after PHx accelerated hepatocarcinogenesis induced by DEN.2.In the process of DEN+PHx induced HCC,Bcl3 expression and the activation of hepatic progenitor cells were increased.3.Bcl3 deficiency inhibited the activation of hepatic progenitor cells and the promotion of liver regeneration on hepatocarcinogenesis.4.On the chronic liver injury induced by DEN,PHx surgery promoted the upregulation of genes associated with chromosomal instability in hepatic progenitor cells.5.USP7 bound to and stabilized Bcl3,and contributed to the accumulation of Bcl3;6.The inhibitor of USP7 P5091 induced the ubiquitination of Bcl3 and inhibited the expression of Bcl3.7.P5091 inhibited hepatocarcinogenesis in the model of DEN+PHx induced HCC and repressed the growth of tumor cells in tumor-bearing BALB/c-nu mice.7.Bcl3 was positively correlated with Sox9 expression and poor prognosis in human HCC.Conclusions:Our study demonstrated that PHx surgery accelerated hepatocarcinogenesis by inducing hepatic progenitor cell activation and chromosome instability on the background of chronic liver injury.Furthermore,we found that Bcl3 participated in the activation of hepatic progenitor cells and the promotion of liver regeneration on hepatocarcinogenesis.We revealed that hepatic progenitor cells were the potential cell origin of tumor occurrence after surgical resection,and genomic instability of hepatic progenitor cells contributed to malignant transformation and tumor progression under the regenerative proliferative stress. |
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