Part Ⅰ:Study On The Therapeutic Effect And Mechanism Of Lenvatinib In The Treatment Of Regorafenib-Resistant Hepatocellular Carcinoma Part Ⅱ:Study On The Mechanism Of LKB1 Regulating TNIK Expression

Objective:To investigate the therapeutic effect and mechanism of lenvatinib on regorafenib-resistant hepatocellular carcinoma cells.Methods:Cell Counting Kit-8(CCK-8)and clone formation assay were used to observe the inhibitory effect of lenvatinib on the growth of hepatocellular carcinoma cells.Flow cytometry was used to detect the apoptosis of regorafenib-resistant hepatocellular carcinoma cells treated with lenvatinib.The expression levels of related proteins were detected by western blot and immunohistochemical staining.The inhibitory effect of lenvatinib on the tumor formation ability of regorafenib-resistant hepatocellular carcinoma cells in vivo was observed by subcutaneous tumor formation experiment in mice.Results:CCK-8 and clone formation assay showed that lenvatinib could inhibit the proliferation of regorafenib-resistant hepatocellular carcinoma cells.Flow cytometry showed that lenvatinib could promote apoptosis of regorafenib-resistant hepatocellular carcinoma cells.After lenvatinib treatment,the ratio of apoptosis-related protein Bcl2/Bax decreased,suggesting that the cell apoptosis increased.Lenvatinib can inhibit the growth of regorafenib-resistant cells in vivo.Immunohistochemistry and Western blot results showed that lenvatinib could down-regulate the abnormally activated IGFIR/Mek/Erk signaling pathway in regorafenib-resistant cells.CCK-8 assay showed that IGF1R knockdown by RNAi markedly reversed regorafenib resistance in regorafenib-resistant hepatocellular carcinoma cell lines.Conclusions:Lenvatinib can reverse regorafenib resistance in hepatocellular carcinoma,possibly by down-regulating IGF1R/Mek/Erk signaling pathway.Objective:The aim of this study is to explore the key transcriptional regulatory mechanism of LKB1 affecting TNIK expression,so as to provide new ideas for the treatment of colorectal cancer patients with LKB1 low expression.Methods:Real-time PCR and Western blot were used to detect the expression levels of related proteins in colorectal cancer cells.PROMO and GeneCards databases predicted transcription factors and transcription factor binding sites regulating TNIK expression.Chromatin immunoprecipitation(ChIP)assay was used to explore the predicted binding sites of transcription factors in the promoter region of TNIK.Luciferase reporter assay was used to explore the regulation of TNIK expression by transcription factors.Results:Real-time PCR and Western blot analysis of colorectal cancer HCT116 cells and HCT116/LKBI-KO cells showed that LKB1 negatively regulated the expression of TNIK.The PROMO and GeneCards databases were used to predict the transcription factors regulating TNIK expression.The PROMO website predicted 69 transcription factors upstream of TNIK,and the GeneCards website predicted 10 transcription factors in the promoter region of TNIK,of which 3 transcription factors were supported by the two databases.The correlation between the expression of 3 transcription factors and TNIK was analyzed by GEPIA database,and it was found that c-Jun had the best correlation with TNIK expression(P<0.05,R=0.34),and the difference was statistically significant.Western blot analysis showed that the c-Jun protein level in HCT116/LKB1-KO cells was significantly higher than that in HCT116 cells.Western blot showed that the level of TNIK protein in c-Jun overexpressed HCT116 cells was significantly higher than that in control HCT116 cells.ChIP assay and luciferase reporter assay showed that LKB1 knockout upregulated TNIK transcription through c-Jun binding to the c-Jun binding site(GTGGTCA)in the promoter region of TNIK.Conclusions:LKB1 knockout up-regulates the transcription of TNIK through the transcription factor c-Jun via binding to its promoter.