Effects Of Scutellaria Barbata And Hedyotis Diffusa On Doxorubicin-resistant Hepatocellular Carcinoma And Their Effects On Differential Expression Of CircRNA

Effects of Herb Pair of Scutellaria Barbata and Oldenlandia Diffusa on Adriamycin-resistant Hepatocellular Carcinoma and Differential Expression of Circ RNAsObjective: Hepatocellular carcinoma has a high morbidity and mortality rate in China.As a commonly used drug in the treatment of hepatocellular carcinoma,adriamycin is prone to drug resistance after repeated chemotherapy.This experiment is to investigate the inhibitory effects of ethanol extracts of Scutellaria barbata and Oldenlandia diffusa on adriamycin resistant cells of human liver cancer;to investigate the effects of ethanol extracts of Scutellaria barbata and Oldenlandia diffusa on the expression of circRNA in HepG2/ADM cells.Methods:1.The ethanol extracts of Scutellariae Scutellariae and Hedyotis diffusa were prepared by ethanol immersion.The chemical constituents of Scutellaria barbata and Oldenlandia diffusa were analyzed by UPLC-Q-TOF-MS/MS.2.CCK-8 method was used to detect the effects of different concentrations of ethanol extracts of Scutellaria barbata and Oldenlandia diffusa on HepG2/ADM;clonal formation assay was conducted to determine the effects of ethanol extracts from Scutellaria barbata and Oldenlandia diffusa on the clonal formation ability of HepG2/ADM;flow cytometry was used to detect the effects of ethanol extracts of Scutellaria barbata and Oldnlandia diffusa on apoptosis and cell cycle of HepG2/ADM;cell migration and invasion assay was conducted to detect the effects of ethanol extracts of Scutellaria barbata and Oldenlandia diffusa on migration and invasion of HepG2/ADM;western blot analysis was performed to detect the effects of ethanol extracts of Scutellaria barbata and Oldenlandia diffusa on the protein expressions of Bcl-2,Cyclin D1 and Ki-67 in HepG2/ADM cells.The HepG2/ADM subcutaneous xenograft was used as a model in nude mice,to observe the effects of ethanol extracts of Scutellaria barbata and Oldenlandia diffusa on tumor,the expression of Bcl-2,Cyclin D1 and Ki-67 protein was detected by immunohistochemistry.3.The differential expression of circRNAs was explored by high-throughput sequencing technology.To observe the changes of circRNA differential expression profiles in HepG2/ADM cells treated with ethanol extracts of Scutellaria barbata and Oldenlandia diffusa,bioinformatics was used to predict target genes,GO functions and KEGG signaling pathways.Bioinformatics software was used to predict circRNA-targeted miRNAs.The differentially expressed mRNAs were screened and the miRNAs were predicted by bioinformatics software.The intersection of the two was selected to screen the target mRNA.PPARG was selected and bioinformatics analysis and luciferase reporting analysis were performed to predict and verify circRNA-miRNA-mRNA signaling pathway.The effect of circRNA-miRNA-mRNA signaling pathway on adriamycin resistant hepatocellular carcinoma was further verified by cell proliferation,apoptosis and cell migration experiments.Results:1.A total of 70 chemical constituents were identified from the ethanol extracts of Scutellaria barbata and Oldenlandia diffusa,including 35 flavonoids,7 iridoid glycosides,6 phenolic acids,3 phenylethanol glycosides,2 anthraquinones,2triterpenes,2 alkaloids,11 fatty acids and other constituents.2.In vitro tests have shown that,the ethanol extracts of Scutellaria barbata and Oldenlandia diffusa inhibited the proliferation of HepG2/ADM,blocked the cell cycle of HepG2/ADM,promoted apoptosis of HepG2/ADM,inhibited migration and invasion of HepG2/ADM(P<0.05).Western blot results showed that,the ethanol extracts of Scutellaria barbata and Oldenlandia diffusa could down-regulate the protein expressions of Bcl-2,Cyclin D1 and Ki-67(P<0.05).In vivo studies show that,the ethanol extracts of Scutellaria barbata and Oldenlandia diffusa could inhibit the growth of HepG2/ADM subcutaneous graft tumor bodies in nude mice(P<0.05).The expression of Bcl-2 and Ki-67 protein was down-regulated(P<0.05).There was no significant effect on cyclin D1 protein(P>0.05).3.The differential expression profiles of circRNA in HepG2/ADM cells were analyzed and identified before and after the ethanol extracts of Scutellaria barbata and Oldenlandia diffusa.GO function and KEGG signaling pathway analysis also revealed the function of differentially expressed circRNA.Bioinformatics software was used to predict circRNA-targeted miRNAs.The differentially expressed mRNAs were screened and the miRNAs were predicted by bioinformatics software.The intersection of the two was selected,and the target mRNA was screened to determine the co-expression regulation of circRNA-miRNA-mRNA.4.The coexpression of circ-0005420-miR-137-PPARG was confirmed.The up-regulation of circ-0005420 and PPARG and down-regulation of miR-137 can inhibit the proliferation,promote the apoptosis and inhibit the migration of adriamycin resistant hepatocellular carcinoma.The up-regulation of miR-137 and down-regulation of PPARG can promote the proliferation,inhibit the apoptosis and promote the migration of adriamycin resistant hepatocellular carcinoma.Conclusion:1.The ethanol extracts of Scutellaria barbata and Oldenlandia diffusa showed inhibitory effects on HepG2/ADM cells.2.Ethanol extracts of Scutellariae Scutellariae and Hedyotis diffusa could affect the differential expression of circRNAs in adriamycin-resistant hepatocellular carcinoma.