| ObjectiveTo explore the effect of Di Wu Yang Gan Capsule on DNA methylation of liver cancer tissue in solt Farber two-step liver cancer rats,and to analyze the mechanism of multi-target,multidimensional and multi-channel effects of Di Wu Yang Gan Capsule on the prevention and treatment of liver cancer from the perspective of DNA methylation,and to study the mechanism of the effect of Di Wu Yang Gan Capsule on single gene DNA methylation.Combining the whole with the local,we can provide the experimental basis for Di Wu Yang Gan capsule to affect DNA methylation and improve the microenvironment of liver regeneration of liver cancer.Methods48 male SPF SD rats,4-5 weeks old,weighing(150±50)g.The rats were randomly divided into model group,dwyg group and normal group with 16 rats in each group.The liver cancer model of rats was made by solt Farber two-step method.The rats in the treatment group were treated with di Wu Yang Gan capsule 750 mg /kg/D by gavage,and the rats in the normal group and the model group were treated with the same amount of normal saline by gavage.The liver cancer tissue samples were collected after 16 weeks of continuous intervention.DNA methylation chip was used to analyze the changes of DNA methylation in the model group,the treatment group and the normal group.Go and pathyway comprehensive analysis were used to explore the characteristics of liver cancer tissue biological process(BP),cell component(CC),molecular function(MF)and signal pathway in the process of liver cancer occurrence and development,and further study the multi-dimensional effect of Di Wu Yang Gan Capsule on DNA methylation of liver cancer tissue.The expression of adck2 in the experimental rat liver cancer tissues was detected by MeDIP-PCR,Western blot and fluorescence quantitative PCR.Hep3 B and Huh7 hepatoma cell lines were used to carry out cytological experiments.The cell lines were divided into blank control group(control),5-fluorouracil group(5-FU)and diwuyangan capsule group(DWYG).The treatment concentration of diwuyangan capsule was 50?g/ ml,the treatment concentration of 5-fluorouracil was 5?g/ml,and the intervention treatment time was 24 hours.Hep3 B and Huh7 hepatoma cell lines were detected by CCK8 kit Proliferation,Transwell and scratch test were used to detect the migration and invasion of Hep3 B and Huh7 hepatoma cell lines.The expression of adck2 in Hep3 B and Huh7 hepatoma cell lines was detected by blotting and fluorescence quantitative PCR.pcDNA3.1-adck2 interference plasmids were constructed.Hep3 B and Huh7 hepatoma cell lines were divided into three groups: control group(control),Di Wu Yang Gan Capsule group(DWYG)and Di Wu Yang Gan Capsule+pcDNA3.1-adck2 group(DWYG+ pcDNA3.1-adck2).The intervention time was 24 hours.Transwell and scratch test were used to detect the migration and invasion of Hep3 B and Huh7 hepatoma cell lines.Immunofluorescence and fluorescence quantitative PCR were used to detect the expression of HIF-1? in Hep3 B and Huh7 hepatoma cell lines.ResultsThe quality control of DNA extracted from liver cancer tissue samples of experimental rats in each group was qualified,the coprecipitation DNA met the requirements of methylation chip scanning,and the quality control of DNA methylation chip scanning data was qualified.In the analysis of gene promoter methylation difference,a total of 2422 gene promoters in the model group were different from those in the normal group,of which 948 were hypermethylation(197HCP,284 ICP,467 LCP),1474 hypomethylation(995 HCP,295 ICP,184 LCP),and the different gene promoter methylation participated in the occurrence and development of liver cancer.Compared with the model group,there were 1650 gene promoter methylation differences in the treatment group,935 hypermethylation(588 HCP,219 ICP,128 LCP),and 715 hypomethylation(216 HCPs,186 ICP,313 LCP)were synthesized.The methylation regions of these different gene promoters are likely to be the targets of Di Wu Yang Gan capsule to block the occurrence and development of liver cancer by affecting DNA methylation.According to go analysis of the promoter methylation gene of the difference between the model group and the normal group,the changes of DNA methylation in the model group were mainly reflected in the biological processes such as immune response process,bacterial source molecular response and biological stimulation stress,the molecular functions such as oxidoreductase activity,molecular function regulation and protein dimer activity were significantly different from those in the normal group.At the same time,there are significant differences in cell components such as plasma membrane receptor complex,endoplasmic reticulum and T cell receptor complex.These differences in biological process,molecular function and cell components involved in the occurrence and development of liver cancer.According to go analysis of the promoter methylation gene in the treatment group and the model group,the effect of Di Wu Yang Gan Capsule on DNA methylation of liver cancer in solt Farber liver cancer rats is mainly in the biological process of cell metabolism,cell localization and organelle tissue activity,in the molecular function of protein binding,ribonucleotide binding and ion channel binding.There were significant differences in cell components such as organelle binding membrane,cytoplasm and organelle.These differences in biological processes,molecular functions and changes in cell components may be one of the therapeutic mechanisms of Di Wu Yang Gan capsule in the prevention and treatment of liver cancer.The results of pathway analysis showed that there were significant differences in the signal pathways of hematopoietic cells,steroid biosynthesis pathway,Th1 and Th2 cell differentiation pathway,toll like receptor signaling pathway,selenium complex and metabolism pathway and retinoic acid metabolism pathway between the model group and the normal group The different signal pathways caused by the changes are involved in the occurrence and development of liver cancer.The results of pathway analysis of promoter methylation gene showed that there were significant differences between the model group and the model group in calcium signaling pathway,cAMP signaling pathway,cgmp-pkg signaling pathway,cancer signaling pathway,cell fat regulatory signaling pathway and HIF-1 signaling pathway It is possible that di Wu Yang Gan capsule can prevent and treat liver cancer by affecting DNA methylation.Detection of ADCK2 expression in liver cancer experimental tissue of rat by Western Blot and real-time fluorescence quantitative PCR.The expression of ADCK2 in the model group(0.3867 ± 0.0893)was significantly higher than that in the control group(0.1724±0.6863),The expression of ADCK2 protein in the Di Wu Yang Gan capsule(DWYG)treatment group was decreased(0.1031±0.0513),which was significantly different from that in the model group(P <0.05);Similarly,the expression of ADCK2 mRNA in the model group(4.0800±0.2126)was higher than that in the control group(0.8500±0.0987),The expression of ADCK2 mRNA in the treatment group of DWYG decreased(2.3860±0.2601).DWYG can reduce the expression of ADCK2 protein and mRNA in experimental rat LC.Detection of the methylation level of ADCK2 in liver tissue of rats with hepatocarcinoma by MeDIP-PCR.The results showed that the methylation level of ADCK2 in model group(25.7200± 5.8500)was lower than that in DWYG group(77.61±6.156),and the difference was statistically significant(P<0.05).DWYG could improve the methylation level of ADCK2 in LC tissue of rats.The proliferation of Hep3 B and HuH7 cell lines was detected by CCK8.The value-added rate of Hep3 B cell line in the treatment group of DWYG(0.5584±0.0168)was lower than that in the control group(0.6828 ± 0.0137).The difference was statistically significant(P<0.05),there was no significant difference between 5-FU group(0.5734±0.0150)(P>0.05);The value-added rate of HuH7 cell line in the treatment group of DWYG(0.6034± 0.0162)was lower than that in the control group(0.7696± 0.0181),the difference was statistically significant(P<0.05),and there was no significant difference between the treatment group and the 5-FU group(0.6354±0.0055)(P>0.05).The results of Transwell experiment showed that the number of Hep3 B cells in the treatment group of DWYG(65.4000±7.9870)was lower than that in the control group(106.4000±8.5030),the difference was statistically significant(P<0.05),and there was no significant difference compared with that in the 5-FU group(63.6000±4.0990)(P>0.05);The number of HuH7 cells in the treatment group of DWYG(11.8000±2.4900)was lower than that in the control group(25.2000 ± 8.4970),the difference was statistically significant(P<0.05),and there was no significant difference between the treatment group and the 5-FU group(14.2000±2.5880)(P>0.05).The results of cell scratch test showed that the relative migration rateof Hep3 B cell line in the DWYG group(0.4265± 0.0056)was lower than that in the control group(0.6919± 0.0389),and the difference was statistically significant(P < 0.05),but not statistically significant(P> 0.05)compared with that in the 5-FU(0.4378 ± 0.0207).Similarly,the relative migration rate of HuH7 cell line(0.1710±0.0673)in the wuyangan capsule group was lower than that in the blank control group(0.2597±0.0555).The difference was statistically significant(P<0.05),but not statistically significant compared with that in the 5-FU(0.1905±0.0816)(P>0.05).Detection of ADCK2 mRNA expression in Hep3 B cell line by real-time fluorescence quantitative PCR.The results showed that the ADCK2 mRNA expression of Hep3 B cell line in the treatment group of DWYG(0.5167±0.0305)was lower than that in the control group(1.0100 ± 0.0854),the difference was statistically significant(P<0.05),and there was no significant difference compared with that in the 5-FU(0.4767±0.0305)(P>0.05).In HuH7 cell line,ADCK2 mRNA expression in the treatment group of DWYG(0.5033±0.0057)was also lower than that in the control group(1.1100 ± 0.1249),the difference was statistically significant(P<0.05),and there was no significant difference between the treatment group and the 5-FU group(0.5233±0.0907)(P>0.05).Detection of ADCK2 protein expression in Hep3 B cell line by Western Blot.The ADCK2 protein expression of Hep3 B cell line in the treatment group of DWYG(0.0995±0.0224)was lower than that in the control group(0.5288 ± 0.0187).The difference was statistically significant(P<0.05),but not statistically significant(P>0.05)compared with that in the 5-FU group(0.1008 ±0.0158).The ADCK2 protein expression of HuH7 cell line in the treatment group of DWYG(0.0977±0.0186)was lower than that in the control group(0.5209 ± 0.0350).The difference was statistically significant(P<0.05),but not statistically significantcompared with that in the 5-FU group(0.0927±0.0239)(P>0.05).The results of Transwell experiment showed that the number of Hep3 B cell lines in the treatment group of DWYG(54.1100 ± 7.3560)was lower than that in the control group(107.6000 ± 6.7660),the difference was statistically significant(P<0.05).After the introduction of pcDNA3.1-ADCK2 plasmid,the number of cells in the treatment group of DWYG increased(70.4400 ± 6.0440).The difference was statistically significant(P<0.05);The number of HuH7 cells in the treatment group of DWYG(16.3300 ± 2.5980)was lower than that in the control group(38.0000 ± 4.6900),the difference was statistically significant(P<0.05).The number of cells in the treatment group of DWYG + pcDNA3.1-ADCK2 increased(25.6700±3.6060),compared with that in the treatment group of DWYG(25.6700±3.6060)(P < 0.05).The results of cell scratch test showed that the relative migration rate of Hep3 B cell line in the treatment group of DWYG(0.2547±0.0030)was lower than that in the control group(0.6833 ± 0.0211),and the difference was statistically significant(P <0.05).After the introduction of pcDNA3.1-ADCK2 plasmid,the relative migration rate in the treatment group of DWYG increased(0.4250 ± 0.0045),compared with that in the treatment group of DWYG.The difference was statistically significant(P<0.05);The relative migration rate of HuH7 cell line in the treatment group of DWYG(0.1746±0.0479)was lower than that in the control group(0.2642 ± 0.0532),and the difference was statistically significant(P<0.05).After pcDNA3.1-ADCK2 plasmid was introduced,the relative migration rate of the treatment group was increased(0.2258 ± 0.0198),which was statistically significant(P<0.05).Detection of HIF-1 ? expression in Hep3 B and HuH7 cell lines by immunofluorescence.The results showed that the intensity of HIF-1 ? expression of Hep3 B cell line in the treatment group of DWYG(8634.58±1457.47)was significantly lower than that in the control group(23758.50 ± 502.25),and the difference was statistically significant(P<0.05).After the introduction of pcDNA3.1-ADCK2 plasmid to over express ADCK2,the effect of DWYG on HIF-1 ? expression was reversed(18549.81 ± 2775.33).The difference was statistically significant(P<0.05).In HuH7 cell line,the expression intensity of HIF-1 ? decreased in treatment group of DWYG(6616.14±1087.15)and that in control group(15624.25±1554.99)were statistically significant(P<0.05).After pcDNA3.1-ADCK2 plasmid was overexpressed into ADCK2,the inhibition effect of DWYG on HIF-1 ? expression was reversed(9532.74±752.45),which was statistically significant compared with that in DWYG accounting significance(P<0.05).Detection of HIF-1 ? mRNA expression in Hep3 B and HuH7 cell lines by real-time fluorescence quantitative PCR.The results showed that the expression of HIF-1 ? mRNA in Hep3 B cell line decreased significantly in the treatment group of DWYG(0.2600 ±0.1153)compared with that in the control group(1.047± 0.2631),and the difference was statistically significant(P<0.05).After the pcDNA3.1-ADCK2 plasmid was overexpressed with ADCK2,the inhibition of HIF-1 ? mRNA was reversed(0.5700±0.0435),The difference was statistically significant(P<0.05);In HuH7 cell line,the intensity of HIF-1 ? mRNA expression decreased in DWYG(0.1967±0.0321)compared with that in control group(1.077 ± 0.0929)(P < 0.05).After pcDNA3.1-ADCK2 plasmid overexpressing ADCK2,the inhibition effect of DWYG on HIF-1 ? mRNA expression was reversed(0.6400 ± 0.1179),which was significantly different from that in DWYG group Statistical significance(P<0.05).Conclusion: DWYG can affect the DNA methylation of liver tissue in Solt-Farber hepatoma rats,and play a multi-target,multi-dimensional and multi-channel role in the prevention and treatment of hepatocarcinogenesis and development.DWYG inhibits the proliferation,migration and invasion of Hep3 B and HuH7 hepatoma cell lines.One of the mechanisms of DWYG in the prevention and treatment of LC may be to increase the methylation degree of ADCK2 gene in solt Farber LC tissues,reduce the expression intensity of ADCK2 in solt Farber LC tissues and Hep3 B,HuH7,and inhibit the expression of hypoxia inducible factor HIF-1 ? by reducing the expression of ADCK2 Under the pressure,the proliferation,migration and invasion ability of tumor cells can be improved,the malignant regeneration state induced by hypoxia and pressure can be improved,and the continuous deterioration of liver regeneration microenvironment can be blocked to prevent the occurrence and development of LC. |
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