Hepatic Stellate Cell-derived Galectin-1’s Promotion Of Hepatocellular Carcinoma Through Immunosuppression Can Be Down-regulated By MiRNA-22

Background:Hepatic stellate cells(HSCs)are known to induce immune privilege and to play a key role in hepatocellular carcinoma(HCC)development.Galectin-1 and mi RNA-22(mi R-22),which have been shown to be deregulated in HCC,are regarded as prognostic indicators for patients with HCC.However,whether galectin-1 and mi R-22 are involved in the immunosuppressive capacity of HSCs in HCC remains unclear.Methods and Results:Part 1 Investigation into gaelctin-1’s expression and function in HSCHSCs isolated from normal liver tissues(N-HSCs)were identified with α-smooth muscle actin expression.After T cells and HSCs were co-cultured(with different ratio),the apoptosis rate of T cells and production of cytokines(IFN-γ and IL-10)were investigated and the results verified HSC’ immunosuppressive capacity.Real-time quantitative polymerase chain reaction(RT-q PCR),Western blotting(WB)analysis,Immunohistochemistry(IHC)test and ELISA were used and the results confirmed the expression of galectin-1 in HSCs.Pre-treatment with interference or overexpression of galectin-1 in HSCs could influence the HSC’ immunosuppressive capacity.The above results indicated that(a)human HSCs can express and release galectin-1 and(b)HSC-derived galectin-1 plays a critical role in HSC-induced T cell apoptosis and Th cytokine balance skewing.Part 2 Investigation into the correlation between HSC-derived galectin-1 expression and the progression of HCCHSCs isolated from HCC tissues were identified as Ca-HSCs.The comparison between N-HSCs and Ca-HSCs about the galectin-1 expression and immunosuppressive capacity showed that Ca-HSCs exhibited significantly higher galectin-1 expression and possessed more powerful immunosuppressive capacity.The tissue specimens and clinical data of 162 HCC patients were collected.The immunofluorescence assay was performed to confirm the co-expression of α-SMA and galectin-1 in liver,and the IHC test with consecutive paraffin-embedded sections was performed to verify the co-expression of α-SMA and galectin-1 in liver stromal cells.Moreover,the correlation between galectin-1 expression,CD3 expression and clinicopathological features in 162 patients with HCC was examined,and the results showed that high galectin-1 and low CD3 expression levels were associated with poor prognosis of patients with HCC.The above results indicated that HSC-derived galectin-1 can promote the progression of HCC by improving immune privilege.Part 3 Investigation into the correlation between mi RNA-22 expression and HSC-derived galectin-1 expression and the immunosuppressive capacity of HSCRT-PCR was performed to test the mi R-22 and galectin-1 expression of Ca-HSCs and N-HSCs,and the results showed that the expression of galectin-1 and mi R-22 in HSCs exhibited a negative correlation.Besides,compared with N-HSCs,the mi R-22 expression of Ca-HSCs was significantly lower and the galectin-1 expression was much higher.Luciferase assay was performed to verify the directly regulatory relationship between galectin-1 and mi R-22 expression in HSCs.The changes in mi R-22 expression could markedly affect HSC-derived galectin-1 expression and HSC-induced T cell apoptosis and cytokine production(IFN-γ and IL-10).The above results indicated that the galectin-1 expression of HSCs and those HSC-derived galectin-1-involved immune effects can be inhibited by mi R-22.Conclusions:The immunosuppressive microenvironment in HCC promoted by HSC-derived galectin-1 can be down-regulated by miR-22.