| Objective:This study is based on miRNA omics to explore the effects of applying and preparing chemical formulas on the intervention of liver cancer stem cells in the progression of liver cancer,aiming to provide new methods and choices for the study of traditional Chinese medicine prevention and treatment of liver cancer.Methods:Experiment 1:Isolation and purification of liver cancer stem cells(CD133~+Hep G2):use CD133 magnetic bead antibody to isolate and purify to ensure the high positive rate of CD133.After separation and purification,use flow cytometry to identify the purity of CD133 positive cells;Experiment 2:40 SPF grade male nude mice were selected for 4 to 6 weeks.The nude mice in each group were divided into four groups according to the method of composite etiology:liver depression and spleen deficiency.The mice were divided into liver cancer cell blank group,stem cell liver cancer blank group,positive drug control group(dimethyl sulfoxide),and application and preparation of traditional Chinese medicine group(referred to as traditional Chinese medicine group),a total of 4 groups,with 10 mice in each group.Hep G2 or CD133~+Hep G2 cells were injected into groups to establish tumor models.After tumorigenesis,they were administered in groups for 4weeks.The general survival status of nude mice in each group was observed and recorded.The tumor was killed on the 30th day,blood and tumor tissue were taken,tumor weight was weighed,and tumor inhibition rate was calculated;Experiment 3:The tumor tissue miRNAs were sequenced,and the sequencing results were analyzed by bioinformatics.Results:Experiment 1:Isolation and purification of CD133~+Hep G2hepatoma stem cells:CD133~+Hep G2 hepatoma stem cells were successfully separated and cultured by immunomagnetic bead method.The cells grew well and the cell structure was intact under microscope.The positive rate of cells identified by flow cytometry reached 99.4%,which can be used for subsequent experiments.Experiment 2:(1)Comparison of the severity of the decline in the general survival status of mice in each group:stem cell liver cancer blank group>liver cancer cell blank group>positive drug control group(all-trans retinoic acid)>traditional Chinese medicine group(application and preparation of chemical formula)(P<0.01).(2)Comparison of tumor growth rate of mice in each group:stem cell liver cancer blank group>liver cancer cell blank group>Chinese medicine group>positive drug group(P<0.01).(3)Comparison of tumor diameter of mice in each group:stem cell liver cancer blank group>liver cancer cell blank group>positive drug control group>application and preparation of Chinese herbal medicine group(P<0.01).(4)Comparison of average tumor body weight of mice in each group:stem cell liver cancer blank group>liver cancer cell blank group>traditional Chinese medicine group>positive drug group(P<0.01),the inhibition rate of traditional Chinese medicine group was 65.7%,and the inhibition rate of positive drug group was 68.7%.(5)Comparison of AFP values in serum of mice in each group:stem cell liver cancer blank group>liver cancer cell blank group>positive drug group>traditional Chinese medicine group(P<0.01);Comparison of ALP values in serum of mice in each group:stem cell liver cancer blank group>liver cancer cell blank group>Chinese medicine group>positive drug group(P<0.01).Experiment 3:(1)Compared with the F sample(liver cancer cell blank group),there were a total of 193 miRNAs differentially expressed between the ZFG sample(traditional Chinese medicine group)and the F sample(liver cancer cell blank group),with 77 upregulated and 116 downregulated;Compared with the SG sample(blank group for stem cell liver cancer),a total of 170 miRNAs were differentially expressed between the two samples,with69 upregulated and 101 downregulated;Compared with the ZG sample(positive drug group),a total of 163 miRNAs were differentially expressed between the two samples,with 100 upregulated and 63 downregulated.(2)Based on the screening criteria of P<0.05,there are 344 newly predicted miRNAs and 306148 target genes in total.In the ZFG/F sample group,the GO function of the differentially expressed target gene was significantly enriched in 192 items,including 138 biological processes,36cell components and 18 molecular functions;In the ZFG/SG sample group,there are 215 items with significantly enriched GO functions,including 147biological processes,42 cell components and 26 molecular functions;In the ZG/ZFG sample group,there are 178 items with significantly enriched GO functions,including 122 biological processes,29 cell components and 27molecular functions.(3)With P<0.05 as the screening criteria,the new predicted miRNA of ZFG/F sample enriched to 154 pathways,the new predicted miRNA of ZFG/SG sample enriched to 163 pathways,and the new predicted miRNA of ZG/ZFG sample enriched to 155 pathways.The differentially expressed miRNA target genes are mainly concentrated in MAPK signal pathway,AMPK signal pathway,Hippo signal pathway,c AMP signal pathway,Wnt signal pathway,etc.Conclusion(s):(1)For liver cancer of liver depression and spleen deficiency type,the Chinese medicine group and the positive drug group can significantly reduce the tumor size and inhibit the tumor development,and the Chinese medicine group has more obvious effects;(2)The development of stem cell carcinoma is more rapid than that of hepatocellular carcinoma;(3)Fuhe Beihua Prescription can cure liver cancer by regulating the expression of miRNA in liver cancer stem cells.(4)Fu He Bei Hua Fang may act on nude mice bearing hepatocellular carcinoma of liver depression and spleen deficiency type by regulating the expression of mmu-let-7a-5p,mmu-let-7b-5p,mmu-let-7f-5p,mmu-let-7g-5p,mmu-miR-150-5p,mmu-miR-466i-5p,mmu-miR-6946-5p,mmu-miR-126a-3p,mmu-miR-574-3p,mmu-miR-6238 etc,which may involve MAPK signal pathway,AMPK signal pathway,Hippo signal pathway,c AMP signal pathway,Wnt signal pathway,etc.,and participate in the biological process,cell composition and molecular function of hepatocellular carcinoma stem cells. |
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