Mechanism Of M2-TAMs Induced Chemotherapy-Resistant In Hepatocellular Carcinoma Cells Through IL-17/IL-17R-CMA Pathway

Objectives To investigate M2 tumor associated macrophages(M2-TAMs)activated the molecular chaperone-mediated autophagy(CMA)pathway through IL-17/IL-17 R and down-regulated the apoptosis sensitivity of oxaliplatin(OXA)chemotherapy in hepatocellular carcinoma cells(HCC),then further enriching the mechanisms of tumor drug resistance,so as to broaden the idea of establishing anti-tumor resistance measures.Methods Human mononuclear macrophages(THP1)were cultured in vitro and induced to differentiate into M2-TAMs,after OXA treatment,and detected the expression of IL-17 by Western Blot and ELISA.Co-cultured M2-TAMs with HepG2 and SMMC7721 cells for 24 hours in Transwell chambers and treated with OXA for 24 h,then removed the culture medium,continuous cultured with DMEM and extracted cell proteins of 0h,6h,12 h,24h and 48 h,then used Western Blot to detect the expression of IL-17 R,the expression of apoptosis-related proteins Bax,Caspase-3,Cleaved-caspase-3 and antiapoptosis-related protein Bcl-2,as well as the expression of CMA signaling pathway key protein Lamp-2A and HSC70.Co-cultured M2-TAMs with HepG2 cells for 24 h,and the cells were harvested after 24 h of OXA treatment,then used flow cytometry to detect cell apoptosis.Knocked down the expression of IL-17 R in Hep G2 cells by siRNA technology,and co-cultured with M2-TAMs for 24 h,after 24 h treatment of OXA,the expression of apoptosis-related proteins Bax,Caspase-3,Cleaved-caspase-3,and anti-apoptosis-related protein Bcl-2,as well as the expression of Lamp-2A and HSC70 were detected by Western Blot.Interfered Lamp-2A expression in HepG2 cells with siRNA technique,and co-cultured with M2-TAMs for 24 h,after treated with OXA for 24 h,the expression of Bax,Caspase-3,Cleaved-caspase-3 and Bcl-2 were detected by Western Blot.Using independent sample t-test and single-factor ANOVA as statistical method,and using SPSS17.0 Software and Excel forms to process data,with P < 0.05 showed the difference was statistically significant.Results 1 The THP1 cells were successfully induced to differentiate into M2-TAMs.Under the treatment of OXA,M2-TAMs produced a large amount of IL-17,and the expression of IL-17 R in the hepatoma cells co-cultured with M2-TAMs also gradually increased.2 The expression of apoptosis-related proteins Bax,Caspase-3,and Cleaved-caspase-3 in hepatoma cells co-cultured with M2-TAMs was first increased and then decreased,and the expression level of anti-apoptotic related protein Bcl-2 was increased.At the same time,flow cytometry results showed that the apoptosis rate of HepG2 cells co-cultured with M2-TAMs(42.47%)was significantly lower than that of HepG2 alone(60.51%).3 The expression of Lamp-2A and HSC70 in hepatoma cells co-cultured with M2-TAMs increased gradually under the action of OXA,and the signal pathway of CMA was activated.After interfering with the expression of IL-17 R in HepG2 cells,the expression of Lamp-2A and HSC70 decreased and CMA signaling pathway activation was inhibited,while the expression of apoptosis-related proteins Bax,Caspase-3,Cleaved-caspase-3 was increased,the expression anti-apoptotic related protein Bcl-2 was increased and the sensitivity of apoptosis was increased.4 Interfering with the expression of Lamp-2A in HepG2 cells and co-cultured with M2-TAMs,the expression of apoptosis-related proteins Bax,Caspase-3,and Cleaved-caspase-3 was increased,the expression of anti-apoptosis related protein Bcl-2 was increased,and the apoptosis sensitivity was increased under the treatment of OXA.Conclusions M2-TAMs produced a large amount of IL-17 under the treatment of OXA,by binding to IL-17 R on the surface of hepatoma cells,activated CMA signaling pathway,then down-regulated the apoptosis sensitivity to OXA and lead to the chemo-resistance of HCC.