TCP1 Regulates Snail1 Expression Through P65 And Erk/GSK-3β/Snail1 Pathways To Promote The Proliferation And Metastasis Of Hepatocellular Carcinoma

Objective: Molecular chaperone CCT plays an important role in tumorigenesis,but the specific mechanism of it is unknown.Our research group performed protein mass spectrometry technology to screen out the α subunit of CCT,namely TCP1,which is significantly up-regulated in leukemia cell lines.Early experiments showed that TCP1 leads to a poor prognosis in a variety of malignant tumors and affects tumor proliferation and metastasis.In view of the high fatality rate of liver cancer and the role of TCP1 in liver cancer is rarely explored,we plan to further study the effect of TCP1 on the proliferation and migration of hepatocellular carcinoma and its related molecular mechanism.Methods:(1)Use the TCGA database to find the relationship between TCP1 expression and survival rate in liver cancer.(2)Lentivirus is packaged and infected to construct a liver cancer cell line that stably knocks down TCP1.The effects of knockdown of TCP1 on the morphogenesis,proliferation,clone formation and migration of liver cancer cells were studied by observing cell morphology changes,clone formation experiments,and Transwell chamber experiments.(3)TCP1 was knocked down or overexpressed in liver cancer cells Huh7 and SMMC-7721,respectively.Western blot was used to detect the effect of knock down TCP1 on the Erk/GSK-3β/Snail1 pathway.The Erk inhibitor Trametinib was used to treat Huh7 and SMMC-7721 cells to detect the effect of inhibiting Erk activity on the Erk/GSK-3β/Snail1 pathway.(4)Use RT-q PCR to detect the effect on the level of Snail1 m RNA expression with knockdown TCP1,and further detect the expression of the upstream transcription factor P65 of Snail1.(5)Using wild-type and TCP1 overexpressing transgenic mice(TCP1-OE),the mice primary liver cancer model was induced by DEN.The liver tissue pathological changes and tumor proliferation were detected by HE staining and immunohistochemistry respectively.Western blot and RT-q PCR were used to further verified the in vitro results.(6)In wild-type and TCP1 overexpression transgenic mice,mouse-derived hepatocarcinoma cells Hepa1-6/luciferase were used to construct subcutaneous transplantation tumor models,and small animal live imaging was used to detect the growth of subcutaneous transplantation tumors.(7)Using SMMC-7721-sh V/luciferase and SMMC-7721-sh T/luciferase cells to construct a nude mouse metastasis model through tail vein injection,live imaging of small animals dynamically detect the formation of liver cancer metastases to further explore the effect on in vivo metastasis of liver cancer cells of knockdown TCP1 in nude mice.Results:(1)TCGA database analysis showed that the expression of TCP1 in liver cancer tissue was significantly higher than that in the normal group,and the decreased survival rate.(2)The cells morphology of Huh7 and SMMC-7721 transform from mesenchymal to epithelial by knock-down TCP1,and the cell proliferation,clone formation and migration was inhibited.(3)Knockdown of TCP1 in liver cancer cells Huh7 and SMMC-7721 can downregulate protein expression of Erk/GSK-3β/Snail1 pathway;overexpression of TCP1 can up-regulate protein expression of Erk/GSK-3β/Snail1 pathway.(4)Knockdown of TCP1 can down-regulate the expression of P65 protein and Snail1 m RNA level.(5)In the DEN-induced primary liver cancer model,compared with the wild type,the HE staining results of TCP1 overexpression mice showed obvious liver histopathological characteristics,and the immunohistochemical results showed that the tumor proliferation related molecular Ki67 and c-Myc expression were significantly up-regulated.Western blot and RT-q PCR results show that TCP1 overexpression mice can up-regulate the expression of Erk/GSK-3β/Snail1 pathway protein,and the expression of P65 protein and Snail1 m RNA level are also significantly up-regulated.(6)In the subcutaneous xenograft model constructed by Hepa1-6/luciferase cells,small animal live imaging results showed that the bioluminescence signal of TCP1 overexpression mice was stronger than that of wild-type mice,and the bioluminescence signal difference between the two groups was significant.(7)The results of the tail vein metastasis model showed that the bioluminescence signal of nude mice in the control group increased within 2-8 weeks of tail vein injection of liver cancer cells,and the metastases increased significantly and were mostly distributed in the lungs,while the group of knockdown TCP1 could hardly see the bioluminescence signal,and the difference between the two groups was extremely significant,indicating that knocking down TCP1 can inhibit the formation of liver cancer metastases in nude mice.Further analysis of the survival rate of the two groups,we found that the nude mice survival rate of the TCP1 knockdown group was significantly higher than that in the control group,P<0.01,and the difference was extremely significant,further confirming that knockdown of TCP1 can improve the survival rate of in vivo liver cancer metastasis in nude mice.Conclusions:(1)TCGA database analysis shows that TCP1 is highly expressed and the prognosis is poor in liver cancer tissues.(2)TCP1 can regulate the proliferation and migration of Huh7 and SMMC-7721 cells and the occurrence of EMT.(3)It was verified that TCP1 regulates the expression of Snail1 protein through P65 and Erk/GSK-3β/Snail1 pathway to promote the proliferation of hepatocellular carcinoma at the cell and animal model levels,respectively.(4)TCP1 can promote the growth of subcutaneous tumors constructed by Hepa1-6/luciferase cells.(5)Knockdown of TCP1 can improve the survival rate of nude mice by significantly inhibiting the metastasis of liver cancer cells in nude mice.