Effects Of MiR-340 Targeted DcR3 Gene Pathway On The Machenisms Of Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is one of the most common malignant tumors which severely threaten the human health due to the high morbidity and mortality.At present,the effective treatment methods are explored including surgery resection,liver transplantation,transcatheter arterial chemoembolization(TACE),radiotherapy,chemotherapy,targeted therapy,immunotherapy and gene therapy,etc.Although there are varied therapeutic methods have been developed,the overall 5 years survival rate is still no significant increased due to its features of easier recurrence and metastasis.It is a disease that apparently cannot be cured.Recently,alone with the rapid development of HCC investigation,studies which focusing on the molecular diagnosis and therapy have drawn more and more people's attention.It was proposed to be a hope of curative therapy for HCC.And molecular diagnosis is also indicated that are good approaches to earlier diagnosis and prevention of HCC.Accumulating studies have shown that miRNAs are a class of small molecule fragments,which involved in a variety of biological processes.MiR-340,one of the importment mi RNAs,involves in the regulation of tumorigenesis and progression,plays an important regulatory role in the metastasis of breast,gastric and ovarian cancer cells.Meanwhile,the Dc R3 gene was discovered that could recognize with miR-340 from bioinformatics prediction website.And it is involved in the proliferation and apoptosis of various cancer cells.Dc R3 is up-regulated in many tumor cells and inflammatory tissues,which is considered as a potential biomarker to predict the development of inflammatory diseases and tumor metastasis.Therefore,the aims of our study are to further investigate the relationship between miR-340 and DcR3 genes,and to explore their important regulatory role in HCC.We hypothesize that the mi R-340 and DcR3 genes expressed differently in the tumor and the adjacent tissues,and are both involved on the development of HCC.The mechanism of miR-340 on the development of HCC is through the targeted gene Dc R3 pathway.The results from our study will provide the molecular basis for the earlier diagnosis and therapy of HCC and yield large potential of clinical values.Part 1: Analysis of miR-340 expression on HCC tissues and the detection of the target gene for miR-340Aims:To analyze of the differential expression of miR-340 and DcR3 genes on HCC and adjacent tissues.To further determine the relationship between miR-340/DcR3 and TGF-?/Smad signaling pathways.Methods:1.To predict the binding sites of miR-340 and DcR3 genes by the websites of target scan,micrriorna.org,NCBI and mi Rbase.2.To determine the difference and relevant of miR-340 with DcR3 gene using real-time fluorescent quantitative method in random collection of patients.3.To analyze the TGF-?/Smad signaling pathways by qPCR and western blot,to analyze the correlation by SPSS 19.0.Results:1.Specific recognition site was founded in DcR3 gene 3'UTR(100-106bp),which could be combination with mi R-340 seed sequence.2.mi R-340 has a low expression in liver cancer tissue.In contrast,the DcR3 gene showed the opposite expression.In addition,the miR-340 and DcR3 was significant negative correlation.3.TGF-? and Smad were both showed higher expression in part of liver cancer and significantly correlated with the DcR3 gene.Conclusions:1.According to the biological analysis,DcR3 gene was target gene of miR-340.2.There is a negative regulation between miR-340 and DcR3 genes in normal and adjunct carcinomas tissues,indicating that DcR3 may be controlled by miR-340.3.DcR3 may activate the TGF-?/Smad regulatory pathway in the liver.Part 2:Determination of the relationship of miR-340 and DcR3 genesAims:To verify the target relationship between miR-340 and DcR3 genes.Methods:1.Transfer the miR-340 maturity sequence and DcR3 gene 3'UTR,respectively,using miRBase and NCB I databases.2.Mimics co-transfect of miR-340 with DcR3-WT,DcR3-mut,and empty vector into HepG2 cells.To detect the luciferase activity in various cells after 24 h transfection for combination ability of miR-340 and DcR3 3'UTR(100-106bp).Result:The vectors were successfully constructed,including miR-340 mimics,miR-340 inhibitor,miR-shNC,DcR3-WT and DcR3-mut.And luciferase activity was significantly lower in group(miR-340 mimics+DcR3-WT)than group(miR-340mimics+DcR3-mut)and group(miR-340 mimics+ empty vector)(P<0.01).Group(miR-340 mimics+DcR3-mut)and group(miR-340 mimics+ empty vector)had no significant difference(P>0.05).Conclusion:The DcR3 gene was target gene of miR-340,and could be combined with miR-340 seed sequences.Part 3: The mechanism of miR-340 effects on the proliferation and apoptosis of HCC cells Aims:1.Determine the effect of miR-340 on proliferation and apoptosis of HCC cells.2.Verify the effects of the DcR3 gene on TGF-?/Smad 2pathways.Methods:1.Transfect the mi R-340 mimics,miR-340 inhibitor and miR-shNC into the cultured HepG2 cells,the relative expression of miR-340 were detected by qPCR.And then the mRNA and protein of DcR3 gene were analyzed in cells that transfected plasmid by qPCR and western blot.2.To examine the effects of miR-340 on proliferate and migration of HCC cells using MTT and flow cytometry.3.TGF-?/Smad2 pathways were analyzed by qPCR and western blot.Results:1.The relative expression of miR-340 was significantly higher in group of miR-340 mimics than miR-340 inhibitor and miR-shNC(P<0.05);But the mRNA and protein of DcR3 gene was significantly down-regulated in cells that transfected mi R-340 mimics(P<0.05).2.The proliferation capacity of the hepatocellular carcinoma cells was significantly lower in cells overexpression mi R-340(P<0.05)than interference group.3.The apoptosis rate significant increased in cells overexpression miR-340(P<0.05).4.DcR3 gene could be reduced by mi R-340,and then DcR3 gene could activate the TGF-?/Smad2 pathway.DcR3 high expression group could lead TGF-? and Smad2 genes significant increases in m RNA and protein.Conclusions:1.miR-340 could down-regulate the mRNA and protein levels of DcR3.2.miR-340 could promote apoptosis of hepatoma cells,inhibit cell proliferation.3.Higher exression of DcR3 gene could activate the TGF-?/Smad pathway.Part 4:Effects of DcR3 on tumor formation and deterioration of liver cancer in rats Aim:To evaluate the effects of DcR3 gene on tumor formation of hepatocarcinoma in rats.Methods:1.The rats were chosen with similar ages and weight,then treat HepG2 cells into the abdominal cavity,finally,DcR3 were delivered through tail vein inject.2.DcR3 and TGF-?/Smad pathways were detected by real-time PCR and Western blot.3.The effects of DcR3 on the tumor growth was assessed.Results:1.The DcR3,TGF-? and Smad were significantly higher in DcR3 overexpression group than the negative control group(P<0.05).2.The tumor volumes were significantly larger in the group of DcR3 overexpression.Conclusions:1.DcR3 can promote the tumor formation effectively.2.The mechanism of DcR3 regulation is dependent on the TGF-?/Smad signaling pathway in development of hepatocellular carcinoma.