| Objective: An investigation of the potential mechanism of mir-375’s action in the treatment of sorafenib-resistant hepatocellular carcinoma.To provide a theoretical foundation for enhancing hepatocellular carcinoma treatment with sorafenib and PD1.Method:Part I: Overload of mir-375 downregulates PD-L1 expression1.In order to predict the expression of mir-375 in liver cancer,we first used the database,identified mir-375 expression in patients with hepatocellular carcinoma(HCC)and evaluated the correlation between mir-375 expression and PFS.2.Clinical samples are used to verify the conviction analysis results,a PCR analysis of 14 HCC patients showed that MIR-375 was expressed in tumor and paratumor tissues,and used immunohistochemistry to recognize the expression of PD-L1.3.We made use of RT-q PCR to test the expression of mir-375 in Hepatoma cell(Hep G2,HUH-7,SMMC-7721)and hepatocytes(LO2),and studied the potential role of mir-375 in human and mouse(Hepa1-6)hepatoma cells by transient transfection.4.The regulatory effect of mir-375 on PD-L1 was verified by Westernblot.Part II: to explore the mechanism of sorafenib resistance in HCC.1.Based on the determination of IC50 of HCC cell line by MTT,the HCC resistant strain(named SR)was constructed by increasing the concentration of IC50(initial concentration was 1/10 of IC50,final concentration was IC50).2.The characteristics of proliferation and apoptosis were detected by MTT and Flow cytometry assay.3.Western blot was used to detect the expression of AKT/Pakt,ERK1/2/PERK1/2 and PD-L1 in HCC cell lines.Part III: to investigate the effect of mi R-375 combined with sorafenib on drug-resistant cell lines.1.The recovery experiment is the same as the 2、3 steps in Part II.Part IV: to investigate the effect of mi R-375 on sorafenib in combination with immune checkpoint inhibitors.1.The baseline characteristics of 69 patients with liver cancer treated with sorafenib monotherapy and combined immune checkpoint inhibitors were systematically reviewed.2.Mice spleen lymphocyte were incubated for 48 h with sr-Hepa1-6treated with sorafenib and immune checkpoint inhibitor,respectively.The expression of CD8 + T cells was measured by Flow cytometry.Results:Part I.1.Database analysis showed that mi R-375 presented a lower pfs advantage in HCC patients with low expression levels and low expression levels in HCC tissues,but the OS results were not statistically significant.2.The expression of mi R-375 in cancer tissues was lower than that in para-cancer inhibition,and the detection results of clinical samples were consistent with the results of database analysis;the HCC patients with low expression of mi R-375 were negatively correlated with PD-L1 expression in their cancer tissues.3.The expression of mi R-375 in Hepg2,Huh-7 and SMMC-7721 was lower than that in Lo2.Mir-375 could inhibit the proliferation,induce apoptosis and down-regulate migration of human-derived and murine HCC cell lines.4.Western blot showed that mi R-375 could down-regulate the expression of PD-L1 in Hepg2,Huh-7,SMMC-7721 and Hepa1-6.Part II.1.The IC50 of Hepg2,Huh-7,SMMC-7721 and Hepa1-6were respectively7.25±0.63、10.0±1.37、11.5±1.39、9.8±1.2(u M).2.Under the microscope,the fibrosis of Hepg2,Huh-7 and Hepa1-6was aggravated,while the cytoplasm of SMMC-7721 was round.2.1 The model of sorafenib resistant cell line was established successfully.Sorafenib inhibited the proliferation and induced apoptosis of HEPG2,Huh-7,SMMC-7721 and Hepa1-6 in a concentration-dependent manner.2.2 the proliferation and apoptosis of drug-resistant hepatoma cell lines were obviously inhibited under the same intervention conditions.2.3 Western blot results: Sorafenib acquired resistance by down-regulating the expression of p-akt/p-erk1/2/pd-l1.Part III: 3.1 transient transfection showed that mi R-375 could inhibit the migration of drug-resistant hepatocellular carcinoma cell lines and enhance the effect of sorafenib on the proliferation and apoptosis of drug-resistant hepatocellular carcinoma cell lines.3.2 Western blot results suggested that mi R-375 reversed sorafenib resistance by enhancing the inactivation of akerk12pd-l1 signaling pathway.Part IV:1.In a systematic review,sorafenib in combination with the immune checkpoint inhibitor sorafenib in combination with PD1 Vs Sorafenib(median OS 13.6 months Vs 7.1 months)demonstrated a synergistic effect of the combination.1.Flow cytometry results: MIR-375 activated CD8 + T cell proliferation in combination with ICI.Conclusion : Mir-375 reversed sorafenib acquired resistance by inactivating the AKERK12PD-L1 signaling pathway.These results suggest that mi R-375 can be used as a target to enhance the efficacy of immunotherapy with sorafenib and PD1. |
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