Study On The Role And Mechanism Of TRPM8 In Liver Cancer

Background: TRPM8 is a non-selective cation channel protein located on the cell membrane and endoplasmic reticulum membrane,widely distributed in multiple tissues and organs of mammals,especially in the prostate and liver.In recent years,studies have shown that TRPM8 plays an important role in solid tumors such as prostate cancer and breast cancer,but its role in liver cancer and its potential molecular mechanism are still completely unknown.This study aims to reveal the expression,biological effects,and specific mechanisms of TRPM8 in liver cancer through a series of clinical samples,cell and animal experiments.Methods:(1)Collect human liver cancer tissue and peri-tumor tissue samples,detect the differential expression of TRPM8 between them by Western blot,qPCR,immunohistochemistry,and analyze the relationship between the expression level of TRPM8 and the clinicopathological characteristics of hepatocellular carcinoma patients.(2)Wild-type mice and TRPM8 gene knockout mice were raised,and DEN was injected intraperitoneally to establish a mouse model of primary liver cancer.After successful modeling,mouse liver and serum samples were collected,and the number of liver tumors,tumor proliferation indicators,blood biochemical indicators,and ultrastructure of mouse liver in different treatment groups were tested to explore the effect and mechanism of TRPM8 on the occurrence of liver cancer.(3)Hep G2 and Huh7 cancer cell lines were cultured,and intervention on the activity or expression level of TRPM8 channel are conducted by the agonist WS12 and inhibitor M8 B of the TRPM8 channel or corresponding lentivirus.Subsequently,the role of TRPM8 in the development of HCC was further explored through experiments such as plate cloning,CCK8,flow cytometry,EdU staining,and transplanted tumor models.(4)A series of high-throughput sequencing methods such as m RNA sequencing,Hi-C sequencing,and histone Ch IP-seq were performed on TRPM8 knockdown group and control group liver cancer cells.The corresponding data were analyzed jointly to find intermediate molecules that may mediate the role of TRPM8 in promoting cancer progression in HCC.(5)Through a series of recovery experiments to confirm that intermediate molecules mediated the carcinogenic effect of TRPM8 in HCC.Results:(1)Compared with peri-tumor tissues,TRPM8 is significantly overexpressed in liver cancer tissues,and overexpression of TRPM8 is a poor prognostic factor in HCC patients.(2)In the DEN induced primary liver cancer model,TRPM8 knockout can significantly inhibit the occurrence of liver cancer,and can cause abnormalities in the morphology of mitochondria,nucleoli,and chromatin.In vivo and in vitro experiments further confirmed that TRPM8 knockout can cause mitochondrial dysfunction.(3)In liver cancer,TRPM8 can cause changes in the expression level of nucleolar related molecule SNORA55 by regulating changes in chromatin conformation and histone modification types.SNORA55 can migrate to mitochondria and serve as a communication bridge for nuclear mitochondrial communication.(4)A series of in vitro experiments have confirmed that SNORA55 plays a carcinogenic role in liver cancer.Its effect on mitochondrial function is achieved by binding to the key components of ATP synthase ATP5A1 and ATP5B and influencing their expression levels.SNORA55 mediates the effect of TRPM8 on mitochondrial function.Conclusions:(1)TRPM8 is highly expressed in HCC and its expression level is associated with poor prognosis in patients with HCC.(2)TRPM8 promotes the occurrence and development of HCC by regulating mitochondrial function.(3)TRPM8 affects the expression and localization of nucleolar related molecule SNORA55 by regulating the epigenetic modification process.(4)TRPM8 can induce SNORA55 mediated nuclear-mitochondrial communication,which in turn leads to mitochondrial dysfunction and promotes the progression of HCC.