| Background and objective:Hepatocellular carcinoma is one of the most common cause of cancer death in the world.Risk factors include hepatitis B virus,hepatitis C virus,fatty liver disease,alcohol-related cirrhosis,smoking,obesity,diabetes.The prognosis of hepatocellular carcinoma is very poor.Only a small part of hepatocellular carcinoma has the chance to receive surgical resection.Although the systemic treatment of hepatocellular carcinoma has been improved in the past 10 years,the overall effect is still not ideal.Therefore,it is necessary to conduct further research to find a better way to treat hepatocellular carcinoma.Long non-coding RNA(LncRNA)is a type of RNA transcript that is greater than 200 nucleotides in length.Generally,LncRNA does not encode proteins or peptides.In addition to the size of other types of non-coding RNAs(microRNAs,small interfering RNAs,small nucleolar/nuclear RNAs),LncRNAs also have secondary and three-dimensional structures,making them both RNA-like and protein-like functions.Recent studies have shown that dysregulated LncRNA profiles are widely involved in the pathogenesis of tumors,including cell proliferation,migration,invasion,epithelial mesenchymal transition(EMT),apoptosis and anti-tumor drug resistance.These findings indicate that some LncRNAs are potential targets and biomarkers for the diagnosis and prognosis of malignant tumors.Part Ⅰ miR503HG plays a tumor suppressive role in HCC cellsMethods:(1)Real-time fluorescent quantitative PCR(RT-PCR)was used to detect the expression of miR503HG in hepatocellular carcinoma tissues,and survival analysis was performed.(2)Expression of miR503HG in five hepatoma cell lines(HepG2,SMMC-7721,Hugh-7,Bel-7404 and HCC)and human normal liver cells(LO2)was detected by RT-PCR.(3)Overexpression and knockdown of miR503HG in Bel-7404 and Huh-7 HCC cells,respectively.The effects of miR503HG on migration,invasion and EMT of HCC cells and angiogenesis of HUVECs cells were detected by scratch,Transwell,RT-PCR,Western blot and matrix gel tube formation assay.Results:(1)The expression of miR503HG was significantly lower in HCC tissues and cells,and the overexpression of miR503HG could significantly improve the overall survival rate of patients;(2)Overexpression of miR503HG inhibited angiogenesis in Huvecs cells,and also inhibited migration,invasion and EMT in Bel-7404 and Huh-7 cells.Brief sum-up:The down-regulated expression of miR503HG in HCC tissues and cells is closely related to the poor prognosis of patients and the malignant characteristics of HCC cells.Part Ⅱ The tumor inhibition of miR503HG is mediated by miR-15b/PDCD4Methods:(1)The expression of miR-15b and PDCD4 in hepatocellular carcinoma tissues was detected by RT-PCR,and the correlation between the expression of miR503HG,miR-15b and PDCD4 was analyzed.(2)miR503HG was silenced or overexpressed in Bel-7404 and Huh-7 cells,and the expression of miR-15b and PDCD4 was detected by RT-PCR and/or Western blot,(3)RIP,RNA pulldown and dual luciferase reporter assay were used to detect the interaction of miR-15b,miR503HG and PDCD4 in Bel-7404 and Huh-7 cells.(4)In Bel-7404 and Huh-7 HCC cells,miR503HG was silenced or both miR503HG and miR-15b were silenced.The regulation of miR503HG on migration,invasion,EMT and angiogenesis of HCC cells by miR-15b was detected by scratch,Transwell,RT-PCR,Western blot and matrix gel tubule formation assay.(5)PDCD4 was overexpressed or both PDCD4 and miR-15b were overexpressed in Bel-7404 and Huh-7 hepatocellular carcinoma cells.The regulation of miR-15b on migration,invasion,EMT and angiogenesis of hepatocellular carcinoma cells by PDCD4 was detected by scratch,Transwell,RT-PCR,Western blot and matrix gel tubule formation assay.Results:(1)RT-PCR results showed that miR-15b was significantly high expressed in hepatocellular carcinoma tissues,and PDCD4 was significantly low expressed in hepatocellular carcinoma tissues.In addition,miR503HG was positively correlated with PDCD4 expression,while miR503HG was negatively correlated with miR-15b,and miR-15b was negatively correlated with PDCD4 expression.(2)Silencing of miR503HG promoted the expression of miR-15b and inhibited the expression of PDCD4 in Bel-7404 and Huh-7 cells by RT-PCR and/or Western blot.(3)RIP results showed that miR-15b,miR503HG and PDCD4 were significantly enriched in AGO2 group compared with the IgG group;(4)RNA pulldown results showed that compared with the scram probe group,miR503HG was significantly enriched in the miR-15b probe group;(5)The results of dual luciferase reporter gene assay showed that the luciferase activity was significantly reduced in Bel-7404 and Huh-7 cells without the mutation of miR503HG or PDCD4.However,the luciferase activity was not significantly affected by mimic-miR-15b after the mutation of miR503HG or PDCD4,indicating that miR503HG and PDCD4 were competent to bind miR-15b;(6)Compared with the silenced miR503HG group,in the simultaneous silenced miR503HG and miR-15b group,the tumor-promoting effect of low-expressing miR503HG on BEL-7404 and Huh-7 cells was reversed;(7)Compared with the PDCD4 overexpression group,in the PDCD4 and miR-15b over-expression group,the anti-tumor effect of PDCD4 on hepatocellular carcinoma cells were both reversed.Brief sum-up:(1)In Bel-7404 and Huh-7 cells,miR503HG inhibits the expression of miR-15b by directly binding to miR-15b,and miR-15b inhibits the expression of PDCD4 by directly binding to PDCD4;(2)miR503HG inhibits the malignant characteristics of Bel-7404 and Huh-7 cells by miR-15b/PDCD4.Part Ⅲ The tumor inhibition effect of miR503HGin vivoMethods:(1)The effect of miR503HG on tumor growth was studied in vivo using a xenograft model of hepatocellular carcinoma in nude mice;(2)The expressions of miR-15b,PDCD4,E-CAD,N-CAD,Vim and Snail-1 in tumor tissues were detected by RT-PCR and/or WB assay,and the expressions of Ki-67 and CD31 were detected by immunohistochemistry.Results:(1)Compared with the control group,the overexpression of miR503HG significantly inhibited the tumor volume in the xenograft model of HCC in nude mice;(2)RT-PCR results showed that the expression of miR-15b was significantly decreased,while the expression of PDCD4 significantly increased in tumor tissues with overexpression of miR503HG.(3)Western blot results showed that the expressions of PDCD4 and E-CAD were significantly increased in miR503HG overexpressing tumor tissues,while the expressions of N-CAD,Vim and Snail-1 were significantly decreased.(4)Immunohistochemical results showed that the positive rates of Ki-67 and CD31 were significantly decreased in the tumor tissues of the overexpressed miR503HG group.Brief sum-up:miR503HG significantly inhibits the growth of liver cancer in nude mice.Conclusions:(1)The expression of miR503HG was lower in HCC tissues and cells,and the low expression of miR503HG was closely associated with poor overall survival of HCC patients;(2)In vitro experiments showed that miR-15b directly bound to miR503HG and PDCD4,and miR503HG promoted PDCD4 expression by inhibiting the expression of miR-15b;(3)The miR503HG inhibits the growth,invasion,migration and angiogenesis of HCC cells in vitro through the miR-15b/PDCD4 axis;(4)The vivo experiments showed that overexpression of miR503HG inhibit the growth of HCC cells. |
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