Effects Of Phyllanthus Urinaria Compound Ⅱ On Liver Cancer-specific MiRNAs And Downstream Target Proteins

ObjectiveHepatocellular carcinoma(HCC),one of the most common malignant tumors in the clinical,is extremely harmful to human and called as“king of cancer”.The causes of HCC are numerous and complex,involving in multiple gene mutations or inactivation.The formation of HCC goes through the three stages of hepatitis-liver cirrhosis-liver cancer,commonly known as the“trilogy of liver cancer”.At present,the molecular mechanism of the development and progression of non-viral HCC remains unknown.We postulated here that small RNTA(miRNAs)exert tumorigenesis effects by inhibiting oncogene,and established a chronic inflammatory mouse model with lipopolysaccharide(LPS)to observe the effect of LPS on the expression of pro-inflammatory cytokines,antioxidant enzymes and HCC markers.The Phyllanthus Urinaria Compound Ⅱ(CPUⅡ)was used to regulate miRN As and target protein in inflammatory liver injury model;A nude mouse xenograft model of HepG2 hepatoma cells was est.ablished to explore the pro-inflammatory cytokines with nitric oxide synthase(NOS)inhibitors NG-methyl-L-arginine(L-N MMA)on regulation of miRNAs and target protein,and to reveal the mechanism of inflammation leading to the initiation of tumors and enrich the anti-hepatoma mechanism of CPUII.MethodsIn present study,Balb/c mice were intraperitoneally injected with LPS at 0.25 mg/kg every other day for eight weeks and subcutaneously injected with D-galactose at 120 mg/kg every day to establish a chronic inflammatory mouse model.We quantitatively measured pro-inflammatory cytokines:IL-1β,IL-6,TN F-α and antioxidant enzymes:GSH-Px,SOD,oxidation products:MDA,liver cancer-specific markers:AFP,CEA expression levels in liver tissues,to determine the best inflammatory tumorigenic model.Simultaneously,we measured the hepatic miR-21 miR-24,miR-122,miR-195 levels and determined the hepatic levels of PTEN,MEN1,FOXO1,AEG-1 in LPS-induced mice and analyzed the correlation between liver cancer-specific miRNAs(miR-21,miR-24,miR-122,miR-195)and downstream target proteins(PTEN,MEN1,FOXO1,AEG-1).After intragastric administration of CPU Ⅱ,we measured the expression of inflammatory pathway pr oteins:TLR4,COX-2,assessed liver pathological changes,and analyzed the correlation between miRNA and target proteins.A nude mouse xenograft model of HepG2 liver cancer cells was established to detect the causal relationship between inflammatory pathway proteins,miRNA and target protein expressions,and colon and liver tissue lesions.Results1、LPS significantly up-regulated the pro-inflammation cytokines,IL-1β,IL-6,TNF-α,as well as liver cancer-specific tumor marker,AFP and CEA,significantly decreased GSH-px and SOD,elevated MDA without significance.The same results did in LPS and D-galactose combined application group.However,there was no significant.difference between LPS and LPS and D-galactose combined application.2、LPS dramatically up-regulated hepatic miR-21,miR-122 and miR-24 levels and significantly down-regulated tumor suppressor genes,PTEN,FOXO1 and MEN1.However,LPS significantly reduced miR-195 and increased oncogene,AEG-1.3、CPU Ⅱ significantly reduced pro-inflammatory proteins,COX-2 and TLR4,and ameliorated the inflammatory infiltration and liver necrosis.4、CPU Ⅱ significantly down-regulated the levels of miR-21 and miR-24,and increased the levels of MENl,PTEN and FOX01 in liver tissues.However,it up-regulated the levels of miR-195 but failed to down-regulate the oncogene AEG-1.5、L-NMMA significantly down-regulated the levels of pro-inflammatory proteins COX-2,iNOS and TLR4,significantly improved the pathological changes of inflammatory infiltration and necrosis in liver and colon,but failed to affect the expression of E-cadherin.6、L-NMMA could impact on the levels of miRNAs,but their induction was modulated in xenografts differentially from the liver tissue.L-NMMA had no effect on tumor suppressor genes and oncogenes in liver tissue of nude mouse,but up-regulated the expression of tumor suppressor miRNAs and inhabited the levels of oncogenes in transplanted tumors,and had no significant effect.on oncogenic miRNAs.Conclus ion1、Long-term administration of LPS induced inflammatory response and oxidative stress,eventually led to the occurrence of liver cancer.2、LPS plays a role in promoting cancer by targeting miRNAs to inhibit the expression of tumor suppressor genes and up-regulate the expression of tumor suppressor gene.3、Administration with CPU Ⅱ up-regulated the tumor suppressor gene by targeting oncogenic miR-21 and miR-24,but didn’t down-regulated the oncogene,AEG-1 to exert anti-inflammatory effects.Simultaneously,CPU Ⅱ downregulated the pro-inflammatory proteins and ameliorate LPS-induced hepatic hyperplasia and inflammatory infiltration.4、L-NMMA blocked the activation of pro-inflammatory cytokines and nitric oxide(NO)-induced signal transduction by targeting tumor suppressor miRNA to down-regulate oncogenes,to play a role in inhibiting tumor growth,but failed to affect tumor metastasis.