ASIC1a Promotes Acidic Microenvironment-induced HCC Cells Migration And Invasion By Inducing Autophagy

In recent years,hepatocellular carcinoma（HCC）has already emerged as one of the most serious cancers at both home and abroad,and the occurrence rate of hepatocellular carcinoma ranks fourth among all kinds of cancers,which is among the highest in the world.Currently,liver cancer treatments such as hepatectomy and targeted radiotherapy are developing rapidly,but the 5-year survival rate of liver cancer patients is still low and their prognosis is poor.Therefore,it is important to find safer and more effective methods to treat liver cancer and improve the quality of patients’survival.The acidic microenvironment of hepatocellular carcinoma cells is closely related to the high metastatic nature of hepatocellular carcinoma.Some solid tumors of tumor cells by their rapid proliferation of tumor cells and increased oxygen consumption,so in the absence of oxygen,the cells produce L-lactic acid and free H⁺by the metabolic way of glycolysis,and thus a weak acidic environment with p H 6.5～pH 6.9 localization is formed,while the pH of normal tissues is within the range of 7.3～7.4.Therefore,the acidity and alkalinity of the microenvironment of hepatocellular carcinoma cells may have an impact on the developmental process of hepatocellular carcinoma and provide conditions for the invasion and metastasis of cancer cells.Acid-sensitive ion channels（ASICs）are a class of cation-gated channels that can be transiently activated by extracellular H⁺.As a member of the epithelial sodium channel/regenerator family,they are expressed in large numbers in mammalian lung tissue,nervous system,and bone cells,etc.Meanwhile,several studies have found that ASICs are widely involved in the development of various diseases Several studies have found that ASICs are widely involved in the development of a variety of diseases,such as thromboembolism-induced cerebral ischemia,epilepsy,inflammation and tumors,and local tissue acidification is a common feature of these diseases.One of the subunits of ASIC1,acid-sensing ion channel 1a（ASIC1a）,can mediate the inward flow of extracellular Na⁺and Ca²⁺plasma,thus causing a series of intracellular physiological and pathological changes.The acid-sensing ion channel 1a（ASIC1a）mediates the inward flow of extracellular Na⁺.Autophagy is a process of degradation of its own organelles and cytoplasmic proteins by lysosomes and vesicles together.The process of autophagy is mainly that vesicles wrap their own cytoplasmic proteins or organelles,transport them to lysosomes and fuse with lysosomes to form autophagic lysosomes,and finally degrade the autophagic lysosomal contents.Autophagy can be induced to activate under some stress conditions,such as endoplasmic reticulum stress,starvation,hypoxia and acidification,but excessive activation of autophagy can lead to cell death.The relationship between tumor cells and autophagy is complex,and autophagy may play a dual role of promoting or inhibiting tumor cell development at different stages of tumorigenesis.Under different circumstances,cellular autophagy can not only be a target for tumor treatment and a direction to attack tumors,but also help cancer cells avoid apoptosis and become one of the mechanisms of cancer deterioration and development.Based on the group’s previous years of research on liver,ASIC1a and autophagy,this study investigated the effect of ASIC1a on Hep G2 cell invasion and metastasis under acidic microenvironment by culturing hepatocellular carcinoma cells Hep G2 in acidic environment,and also explored whether autophagy is involved and its role in hepatocarcinogenesis.This will help to clarify the pathogenesis of hepatocellular carcinoma in acidic microenvironment,which will help to further improve the study of related pathogenesis and provide new ideas for the prevention and treatment of hepatocellular carcinoma.In this study,the expression of ASIC1a protein in Hep G2 cells under acidic microenvironment at p H 7.4 and p H 6.5 was firstly observed by protein immunoblotting（Western Blot,WB）.The effect of ASIC1a on hepatocellular carcinoma cell invasion and migration was observed in vitro using the ASIC1a-specific blockers Psalmotoxin-1（Pc Tx-1）,which blocked ASIC1a protein expression and m RNA transcription,and the effect of ASIC1a on hepatocellular carcinoma cell invasion and migration was observed using Transwell invasion assay and scratch healing assay;laser confocal observation of intracellular calcium ions in hepatocellular carcinoma cells under transient acid stimulation conditions.The effect of ASIC1a on autophagic flux was observed by transfection with e GFP-m Cherry-LC3 dual fluorescent plasmid,and the effect of autophagy on hepatocellular carcinoma cell invasion and metastasis was observed by inhibition of autophagy expression with the autophagy inhibitor 3-MA,and finally the effect of blocking ASIC1a on the submicrostructure of autophagic vesicles in hepatocellular carcinoma cells was verified by transmission electron microscopy.The results revealed that ASIC1a and autophagy were increased in Hep G2 cells in an acidic microenvironment,and both promoted the migration and invasion of hepatocellular carcinoma cells.In addition,inhibition of ASIC1a with Pc Tx-1 or ASIC1a sh RNA decreased autophagic flux.In conclusion,ASIC1a promotes acidic microenvironment-induced migration and invasion of Hep G2 cells by inducing autophagy,which may be related to Ca²⁺inward flow.