To Explore The Mechanism Of Action Of Bielongruangan Decoction In Inhibiting The Proliferation And Migration Of HBV Primary Liver Cancer Cells Based On Regulating METTL3-mediated M~6A Modificatio

Objective:By studying the regulation of Bielong Ruangan Decoction on METTL3 mediated m~6A modification,to explore its influence on the proliferation and migration of HBV-related primary liver cancer cells,and to clarify the potential mechanism of Bielong Ruangan Decoction in treating HBV-related primary liver cancer.Methods:Fifteen healthy adult SD rats were divided into blank serum group,low dose of Bielong Ruangan Decoction medicated serum group,and medium dose of Bielong Ruangan Decoction medicated serum group.According to human and animal body surface area-equivalent dose conversion method,the blank group was administrated with normal saline according to dose,and the Bielong Ruangan Decoction group was administrated with Bielong Ruangan Decoction according to dose,twice a day for 3 consecutive days.After anesthesia with 1%pentobarbital sodium,blood samples were collected from abdominal aorta to prepare drug-containing serum.The inhibitory effect of drug-containing serum on proliferation of HepG2.2.15 cells was detected by CCK-8 method,the effect of drug-containing serum on migration ability of HepG2.2.15 cells was detected by cell scar assay,and the expressions of METTL3 and SOCS2 proteins were detected by WesternBlot.The expression of METTL3 and SOCS2mRNA was detected by qPCR,and the m~6A modification level was detected by m~6A RNA methylation kit.Results:1.CCK-8 detection:Compared with the blank serum group,HepG2.2.15 cells were treated with the medicated serum(low dose and medium dose)of Bielong Ruangan Decoction for 24h and 4 8h,and the cell proliferation rate of Hepg2.2.15 cells was significantly different among different concentration groups(p<0.05).After 72h,the cell proliferation rate of the low-dose group was significantly different(p<0.001).There was no significant difference in cell proliferation rate between the medium dose group(p>0.05).In addition,the proliferation rate of Bielong Ruangan Decoction medicated serum treatment group(low dose,medium dose)was lower than that of the control group at each time point,while the proliferation rate of the low dose group was lower than that of the medium dose group at each time point,especially the lowest in the low dose group after 48h intervention.2.Cell scratch test:HepG2.2.15 cells were treated with Bielong Ruangan Decoction medicated serum for 48h,and the cell mobility decreased slightly,but there was no statistical significance among different concentration groups(p>0.05).Compared with blank serum group,after 72h treatment with Bielong Ruangan Decoction medicated serum,the mobility of low dose group and medium dose group decreased,the difference of low dose group was statistically significant(P<0.05),while the mobility of medium dose group decreased,but the difference was not statistically significant(p>0.05).Cell scratch test showed HepG2.2.15 cells could inhibit cell migration after 72h treatment with the medicated serum of low-dose group Bielong Ruangan Decoction.3.qPCR detection:Compared with blank serum group,METTL3 mRNA expression level was significantly decreased after treatment with Bielong Ruangan Decoction medicated serum,the difference was statistically significant(P<0.0001),but there was no significant difference among medicated serum groups(P>0.05).In contrast,SOCS2 increased significantly,with a statistically significant difference(P<0.0001)and was higher in the low-dose group than in the medium-dose group(P<0.0001).4.WB detection:Compared with blank serum group,METTL3 protein expression level was significantly decreased after treatment with medicated serum of Bielong Ruangan Decoction,the difference was statistically significant(P<0.0001).Meanwhile,histone expression level of low dose medicated serum was significantly higher than that of medium dose group(P<0.0001).On the contrary,SOCS2 protein expression increased,with statistical significance(P<0.05).5.Quantitative detection of m~6A methyl ati on:Compared with blank serum group,MettL3-mediated m~6A modification level decreased significantly in HBV-related primary liver cancer cells treated with low dose drug-containing serum(P<0.001),and decreased in HBV-related primary liver cancer cells treated with medium dose drug-containing serum(P<0.01).The modification level of m~6A in the low dose group was lower than that in the medium dose group(P<0.01).Conclusion:1.The medicated serum of Bielong Ruangan Decoction can inhibit the proliferation and migration of HBV-related primary liver cancer cells.2.The medicated serum of Bielong Ruangan Decoction can down-regulate the expression of METTL3 in HBV-related primary liver cancer cells,up-regulate the expression level of SOCS2,and reduce the modification level of m~6A.3.The inhibitory effect of medicated serum of Bielong Ruangan Decoction on the proliferation and migration of HBV-related primary liver cancer cells may be related to the decrease of MettL3-mediated m~6A modification level.