Effects Of Ethanol On The Morphology, VASN Expression And Secretion Of HepG2 Cells

Primary liver cancer is one of malignant tumors and the second common cause of death from cancer following lung cancer in the world. In China, liver cancer patients accounted for more than half of the world’s burden, accompanied by high mortality. Several major established factors associated with hepatocarcinoma(HCC) include Hepatitis C virus(HCV),Hepatitis B virus(HBV), aflatoxin and alcohol. And new emerging risk factors contain diabetes, obesity, nonalcoholic fatty liver disease(NAFLD). Liver is the main organ of ethanol metabolism, and chronic excessive drinking seriously damages the liver, leading to fatty liver, liver cirrhosis, significantly increase the incidence of liver cancer.Part one: Ethanol promotes VASN protein secretion in HepG2 cells and its effect on HUVEC cell migrationVasorin(VASN)is a typical type I transmembrane protein, containing tandem arrays of a characteristic leucine-rich repeat motif, an epidermal growth factor-like motif, and a fibronectin type III-like motif, which can be shedded to the extracellular microenvironment as soluable VASN by metalloproteinase ADAM17. Human VASN protein is mainly expressed in vascular smooth muscle cells, kidney, placenta tissue, and the expression level is much low in other tissues. It was reported that breast cancer cells highly expressed VASN protein, and the secreted sVASN protein could inhibit the activation of TGFβ signaling pathway, playing a role in promoting tumor. The expression level of VASN is high in human brain tumor tissues and cells. When VASN knocked down with siRNA in glioblastoma cells,hypoxia or TNF-α-induced apoptosis was significantly increased, suggesting that VASN could protect them from hypoxia-induced apoptosis, thus contributing to tumor progression.Previous study in our laboratory showed that VASN was a potential serum biomarker which was selected from the serum of patients with hepatocellular carcinoma with SELEX technology. VASN was verified to be highly expressed in liver cancer cells and liver tissues.Further experiments showed that exosomal VASN protein in HepG2 cells promoted human umbilical vein endothelial cell migration.It was reported that long term ethanol exposure altered the expression of various genes,affecting several pathways, including acute phase response, amino acid metabolism,carbohydrate metabolism and lipid metabolism. In this part, ethanol was used to stimulate HepG2 cells to detect the effects on VASN expression and possible effects on HUVEC cells.For the simulation of moderate, heavy and binge drinking in the daily life, we selected three corresponding concentration(25, 50 and 100mM) of ethanol to stimulate HepG2 cells.After ethanol treatment at different times(24 h, 48 h, 72 h), total RNA of the cells was extracted and the expression of VASN mRNA was analyzed by qPCR. Results showed that the three concentrations of ethanol could up-regulate VASN mRNA level. Western Blot results showed that ethanol could promote the expression of VASN protein. The collected supernatant was concentrated after ethanol stimulation to detect VASN protein level. TheWestern Blot results showed that the VASN protein level in the supernatant was also increased. These results indicated that ethanol stimulation could promote the expression and secretion of VASN protein in HepG2 cells.In our previous work, we found that VASN protein could not only be shedded by metalloproteinases ADAM17 to be sVASN, but also be found in the exosomes derived from HepG2 cells. Ethanol stimulation can increase the level of VASN protein in the supernatant of HepG2 cell culture medium. We then explored what form of VASN secretion is increased in the supernatant of HepG2 cell culture media. We used the exosome extraction reagent to purify exosomes from ethanol-stimulated cell culture media, and collected the exosome-deprived medium. Based on the protein quantity analysis of exosomes, results showed that ethanol stimulation significantly increased the quantity of exosomes. At the same time, Western Blot was carried out to detect exosome biomarker Hsp70 protein, we found Hsp70 protein level increased significantly after ethanol stimulation, consisted with protein determination, suggesting that ethanol could promote HepG2 cells secrete exosomes. To detect VASN protein level in the exosomes, Western Blot results showed that the VASN protein level was increased in the ethanol treated group. We also examined sVASN protein levels in the media, and the results showed that ethanol stimulation also promoted sVASN secretion in HepG2 cells.To investigate the effect of increased sVASN in supernatant shedded from ethanol-stimulated HepG2 cells on HUVEC cells, we used ethanol to stimulate HUVEC cells.The results showed that the migration of HUVEC cells was inhibited significantly by ethanol.Subsequently, HUVEC cells were co-cultured with HepG2 cells in presence of 50 mM ethanol and the migration rate of HUVEC cells was increased in wound healing assay. So was the number of transwell migration cells. When VASN was knocked down by siRNA, co-culture had almost no effect on the migration rate and transwell migration number of HUVEC cells.In general, ethanol stimulation promoted VASN secretion, while VASN play an important role in HUVEC cells migration and chemotaxis.The above results suggested that ethanol could promote the expression and secretion of VASN protein in HepG2 cells, regulating VASN protein mediated intercellular communication between HepG2 and HUVEC cells. This study will help to address the role of ethanol in liver cancer progression. Meanwhile, as an important component of tumor microenvironment, VASN may become a candidate target in the prevention and treatment of liver cancer.Part Two: Preliminary observation of effects on the formation of blebs of HepG2 cells treated with lethal dose ethanol in short timeEthanol affect cell activities through a variety of mechanisms, and different concentrations of ethanol may have different effect. Ethanol of 100 mM could promote liver cancer cell migration, tumor angiogenesis and exosome secretion, while 1.5M ethanol can significantly enhance pancreatic ductal adenocarcinoma cell membrane permeability. Ethanol direct injection to center of the liver tumor could make tumor cells and nearby vascularendothelial cells dehydrate quickly, triggering the cellular protein coagulation and degeneration, which leads to tumor cell necrosis.The cell cortex is a layer of actin-rich structures, close to the cytoplasmic region of the plasma membrane, drawing near of actin microfilament, causing the formation of the outer membrane tension. When the junction between cortex and plasma membrane was destroyed or cortex dissolve, cytoplasm will flow to these sites, then fluid pressure leads to plasma membrane protusion and forms bubbles(blebs). Cell membrane blebs usually developed to 2microns, and then shrink to the origin of membrane bleb. Membrane blebs can be found in many cells life activities, important to the three-dimensional movement, such as cell movement, tumor cell invasion, apoptosis, stress response process.We found that 2M ethanol induced blebs formation in HepG2 cells in very short time.We observed the morphological characteristics of membrane blebs, some blebs fused and shrinked on the cell surface with elongated ethanol treatment time, and others shedded from the cell surface and was released in the culture medium. We explored what concentration of ethanol could induce membrane bleb formation. The results showed that treatment of 100 mM,400mM ethanol for hours could not induce membrane bleb formation, while, a large number of cell membrane blebs formded after 2M ethanol stimulated for 10 min.After 2M ethanol treated, membrane bleb cells were stained by trypan blue, and the cell membrane permeability was enhanced. Through the observation by scanning electron microscope, we found that cell surface became rough, microvilli disappeared, membrane bleb protruded, cell connection with the substrate weakened, and some cells exhited holes. The membrane blebs and actin were separately labeled with dye Dil and phalloidin. The results of fluorescent assay showed that cell membrane bleb become protrude and turn round after ethanol stimulation. Blebs contained cytoplasm components, but not contain cytoskeleton actin, which suggested the membrane bleb detached from the cytoskeleton and result in cell morphological changes.Finally, we detected the cell bleb rate and mortality. The experimental results showed that both the bleb rate and mortality increased with ethanol treatment time extended.Combined with analysis of cell morphology, we found that the ultimate fate of most membrane bleb cells were dead, but there were still a handful of cells survived, going to proliferate. In addition, we collected extracellular vesicles to detect, and the results showed that 2M ethanol could significantly promote the secretion of extracellular vesicles, but the biological functions of these vesicles were not clear.In this part, we explored the effects of lethal dose of ethanol on HepG2 cells. We found that 2M ethanol induced formation of membrane blebs, more secretion of extracellular vesicles, death of large number of cells, but there was still a few of cells survived. In the future, we will continue to study the biological characteristics and functions of the ethanol-tolerant membrane bleb cells, and biochemical composition and biological functions of the membrane blebs and extracellular vesicles.