The Phospholipid Flippase ATP9A Promotes Tumor Growth Under Nutrient Deletion Via Inducing Macropinocytosis In Hepatocellular Carcinoma

Backgroud and purposeHepatocellular carcinoma(HCC)is a rapidly proliferating tumor.In order to sustain the growth of HCC cells,HCC cells need to acquire a lot of nutrients to supply their synthesis metabolism.However,abnormal tumor vascular system,coupled with high interstitial pressure caused by liver cirrhosis,lead to insufficient nutritional supply of liver cancer.Therefore,tumor cells need to obtain nutrients from the microenvironment to maintain their proliferation.Macropinocytosis is an important way for cells to obtain nutrients from the surrounding microenvironment.A number of studies have revealed the growth-promoting effect of macropinocytosis in malignant tumors.Tumor cells take nutrients such as extracellular proteins and necrotic cell fragments through macropinocytosis,and then transport these substances to lysosomes for degradation to provide nutritional support for cell growth.However,it is not clear whether macropinocytosis plays a promoting role in hepatocellular carcinoma.Therefore,exploring the function and detailed molecular mechanism of macropinocytosis in the proliferation and growth of hepatocellular carcinoma will help to provide new ideas for the role of targeting macropinocytosis pathway in the treatment of hepatocellular carcinoma.Methods1.70kDa dextran uptake assay was used to detect the micropinocytosis ability of different hepatocellular carcinoma cell lines.2.70kDa dextran uptake in hepatoma tissue was used to detect the macropinocytosis ability of hepatoma cells.3.In vivo experiment to detect the effect of ATP9A and macropinocytosis inhibitor amiloride(EIPA)on the growth of hepatocellular carcinoma.4.The ability of cell proliferation was detected by CCK8,EDU assay and flow cytometry.5.Isolation of cell membrane proteins to detect the expression of Rac1 and ATP6V1 A.6.PBD-GST pull down assay to detect Rac1 activity.7.The distribution of free cholesterol was detected by filipin complex staining.8.The interaction between ATP9A and ATP6V1A was detected by protein co-immunoprecipitation assay.9.The membrane localization of ATP6V1A was detected by immunofluorescence assay.10.The membrane localization ofATP6V1A was detected by immunofluorescence assay.11.Real-time quantitative PCR assay and Western Blot assay were used to detect the expression of ATP9A in hepatocellular carcinoma cells,normal liver cells,fresh liver tissues and corresponding normal liver tissues.12.The expression of ATP9A in clinical paraffin specimens was detected by immunohistochemistry,and the relationship between ATP9A and the prognosis of hepatocellular carcinoma was analyzed.Resutls1.Macropinocytosis occur in HCC cells under the condition of nutrient deprivation,and can promote the proliferation and growth of HCC.2.The expression of ATP9A in HCC cells and HCC tissues was significantly higher than that in normal cells or tissues.Moreover,the overexpression of ATP9A is related to the poor prognosis of HCC and can be used as an independent predictor of the prognosis of HCC.3.In vivo and in vitro,it was found that the high expression of ATP9A could promote the macropinocytosis of HCC cells,and promote the proliferation and growth of HCC cells through macropinocytosis.Knockdown of ATP9A can inhibit the macropinocytosis and growth of HCC.4.Overexpression of ATP9A can enhance the membrane localization of cholesterol and promote the membrane localization of Racl,and then play the role of promoting macropinocytosis.Inhibition of Racl can inhibit the effect ofATP9A-mediated macropinocytosis.5.By combining with ATP6V1 A,ATP9A promotes the transport of ATP6V1A-positive endosomes to the plasma membrane,thus promoting cholesterol membrane localization;ATP6V1A silencing inhibits ATP9A-mediated cholesterol membrane localization.ConclusionsATP9A promotes the transport ofATP6V1A-positive endosomes to the plasma membrane,regulates the plasma membrane localization of cholesterol,and then promotes the membrane localization of Racl,induces macropinocytosis,and promotes the proliferation of HCC cells.ATP9A can be used as a new molecular marker to predict the prognosis of HCC.