| Background and Objective:Primary liver cancer is one of the most common malignant tumors clinically.The malignancy degree of this cancer is very high,during the onset hidden,faster progress,easy to relapse and metastasis.The global incidence of this cancer increases year by year,and this cancer has very high incidence rate of morbidity and mortality,has great influences on people's lives and health,has certain harmfulness and severely threatens the human health.There are many factors that can induce liver cancer,such as aflatoxin intake,virus infection,genetic inheritance,smoking,drinking water and drinking alcohol,however,the induction mechanism of liver cancer is not yet entirely clear so far.Lipids are a class of compounds that are difficult to be dissolved in water but easy to be dissolved in non-polar organic solvents,such as lipid solvents.Their chemical composition and structure are non-uniform,and lipids are mainly composed of carbon,hydrogen and oxygen via non-polar covalent bonds.Lipids are abundant and widely distributed in human body.More and more studies have shown that the synthesis or metabolism disorders of lipids are closely associated with cancer incidence.Recently,lipids have been identified as important mediators of tumor-stromal communication in tumor microenvironment.The purpose of this study was to analyze the changes of glycerol phospholipids and sphingolipids in the process of liver cancer metastasis by using liquid chromatography-electrospray ionization mass spectrometry and multi-reaction monitoring combined technology(LC-ESI-MRM/MS).In addition,the lipids that have large changes were screened out in this study,and added these lipids to liver cancer cells with different metastatic potential cultured in vitro to observe the effects of lipids on the biological function of liver cancer cells,aiming to seek a new scheme for the diagnosis and treatment of liver cancer and provide a basis for improving the patients' prognosis.Methods:In the first part of this study,the liver cancer cells were cultured under the condition developed by the M2 tumor associated macrophage,then the quantitative analysis of two kinds of fat lipids,glycerol phospholipids and the sphingolipids,in the cancer cells was performed by liquid chromatography-electrospray ionization mass spectrometry and multi-reaction monitoring combined technology,hoping to explicit the lipid changes in liver cancer cells in the process of liver cancer metastasis promoted by M2CM.In the second part of this study,the proliferation ability of cells was verified by detecting cell vitality via CCK-8 method,and the appropriate concentration of lipid molecules were selected,then the MHCC-97L and MHCC-97H liver cancer cells with different metastatic potential were treated by lipid molecules with the selected concentration.The metastatic and invasion ability of liver cancer cells were evaluated using the method of cell scratch experiment and Transwell cell invasive experiments,finally the cells cycle of liver cancer cells with different metastatic potential was detected through the cell cycle experiment.SPSS 16.0 statistical software was used for statistical analysis,and P<0.05 was considered as statistically significant.Results:1.By liquid chromatography-electrospray ionization mass spectrometry and multi-reaction monitoring combined technology,the changes of glycerophospholipids and sphingolipids in liver cancer cells stimulated by M2CM for 48 hours were quantitatively analyzed Under the positive ion scanning mode,the lysoPC and PC contents of MHCC-97H treated by M2CM were significantly higher than those of the control group(Fold Change was 2.45 and 1.71,respectively),while the contents of Cer and sphingolipid with D-type structure decreased significantly(Fold Change was 0.26 and 0.45,respectively),while under the negative ion scanning mode,CIP increased significantly(Fold Change=0.35)in MHCC-97H treated by M2CM.Further quantitative analysis of all lipids detected in liver cancer cells was carried out after different treatments.According to the criterion of signal intensity change multiple>1.5 or<0.67,and P<0.05 in comparing different groups of lipid molecules,77 differential lipid molecules(49 significantly increased and 28 significantly decreased)were found in MHCC-97H stimulated by M2CM.The up-regulated lipids were mainly lysoPC,PC and some long-chain PE,while the down-regulated lipids were mainly short-chain PE and PS.2.In MCHCC-97L and MCHCC-97H liver cancer cells,CCK-8 assay showed that LysoPC 14:0 could enhance the cell viability of MHCC-97L and MCHCC-97H cells at 50 ?mol/L.With the prolongation of the intervention time,the promotion effect of L ysoPC14:0 was more stable than that of other concentrations.Cell scratch assay was used to observe cell migration at 12 h and 24 h after scratch and calculate the migration rate.The results showed that the average migration rates of experimental group were 0.34±0.01 and 0.54±0.02 at 12 h and 24 h for MHCC-97L liver cancer cells,respectively.For MHCC-97H liver cancer cells,the average migration rates of experimental group were 0.34±0.04 and 0.56±0.04 at 12 h and 24 h,respectively.In two kinds of cells,the cell migration rate of LysoPC14:0 lipid group was higher than that of control group(P<0.05).Transwell experiment showed that LysoPC14:0 lipid group had stronger invasion ability than control group(P<0.05).Flow cytometry showed that there was no significant difference between the control group and the experimental group in MICC-97L cells(P>0.05).Compared with the control group,LysoPC14:0 group had higher G1 phase ratio(P<0.05),lower S phase number(P<O.OS),and lower G2/M phase number(P<0.05)in MHCC-97H cells.In liver cancer cells with different metastatic potential,the specific mechanism of LysoPC 14:0 on cell cycle is different.Conclusions:1.M2CM stimulation can significantly affect the lipid composition of liver cancer cells,and its role in promoting the migration and invasion of liver cancer may be through changing the lipid composition of h liver cancer cells.2.A certain concentration of LysoPC 14:0 can promote the proliferation,migration and invasion of liver cancer cells,indicating that lipid molecules participate in the growth,development,metastasis,invasion and other links in liver cancer cells,which provide a theoretical basis for the occurrence and development of liver cancer. |
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