The Involvement And Mechanisms Of TLR4in The Regulation Of Proliferation In Hepatocellular Carcinoma

IntroductionHepatocelluar carcinoma(HCC) is one of the common maliganancies that threaten human lives, and patients have bad prognosis and high mortality rate. HCC is closely related to chronic inflammation, and usually originates from steatosis, alcoholic liver disease and cirrhrosis.TLR4is a membrane protein which expresses on immune cells and epithelial cells, and its activation by exogenous and endogenous ligands can initiate MyD88-dependent and MyD88-dependent signaling, lead to downstream NF-κB pathway and MAPK pathway activation, and inflammatory gene transcription. TLR4not only plays a critical role in cell confrontation with exogenous infections and wound healing, but also promotes the initiation and progression of tumors when aberrantly activated.TLR4expressed by hepatocytes can be activated by the elevated endotoxin in chronic liver disease, and promotes alcoholic steatosis, alcoholic hepatitis, hepatic fibrosis and hepatic cirrhosis. So, we speculate that TLR4is involved in the initiation and progression of HCC.This study investigated the expression of TLR4in HCC cell lines and HCC specimens and explored the effects and mechanisms involved in cell modulation of TLR4activation. Part Ⅰ The expression of TLR4in hepatocellular carcinoma cell lines and its effect on cell proliferation when activatedObjectives To investigate TLR4expression in cell lines and its effects on cell proliferation.Methods Western Blot was adopted to measure TLR4protein expression, cell proliferation rate was investigated by MTT, and flow cytometry was utilized to evaluate apoptosis rate and cell cycle。Relults Compared to immortalized hepatocyte L02, the expression of TLR4was elevated in both hepatocellular carcinoma cell lines HepG2and PLC. LPS stimulation led to increased apoptosis, increased G0-G1phase cell percentage, decreased G2-M phase cell percentage, and weak cell proliferation before24hours in L02and PLC. While LPS stimulation promoted cell rapid proliferation after24hours in all three cell lines..Conclusion The expression of TLR4was elevated in hepatocellular carcinoma and could promote cell proliferation. Part Ⅱ the effects of TLR4activation by LPS on signaling in hepatocellular carcinomaObjectives To clarify the mechanisms underlying the promotion of proliferation by TLR4activation in HCC.Methods Western Blot was used to measure the activation of MAPK pathway and NF-κB pathway.Relults After30and90minutes’ administration of LPS, phosph-IκB-α in both L02and PLC were significantly elevated compared to control, while down-regualted in HepG2. Phosph-JNK and phosph-ERK were also fast elevated after LPS administration compared to control in there cell lines. Phosph-p38was increased in L02, while down-regulated in HepG2and PLC.Conclusion The activation by LPS of TLR4in hepatocellular carcinoma cells can save cells from apoptosis, promote cell survival and proliferation by elevating activities of JNK MAPK pathway、ERK MAPK pathway and NF-kB pathway Part Ⅲ The expression of TLR4and its correlation with clinicopathological indications in hepatocellular carcinomaObjectives To clarify the expression of TLR4and its correlation with clinicopathological indications in61hepatocellular carcinomas.Methods Immunohistochemistry was performed in61hepatocellular carcinomas and the correlation between TLR4expression and clinicopathological indications was statistically analyzed.Relults TLR4was detected in the cytoplasm. Hepatocytes were weakly stained, and in some cases had much stronger stain in cancer cells and paracacerous cells.29.5%cases were scored2and9.8%cases were scored3. We didn’t observe significant correlation between TLR4expression and tumor grades, tumor size, p53expression, HBsAg and HBcAg. While, TLR4expression positively correlated with age (r=0.287,P<0.05)Conclusion According to the increased expression of TLR4in cancer cells compared to hepatocytes, we speculated that TLR4may participate in the progression of hepatocellular carcinoma.