| Hepatocellular carcinoma(HCC), the most common primary liver cancer, is the fifth most frequent cancer and the third cause of cancer-related mortality worldwide. HCC normally develops because of an underlying liver disease and is often associated with cirrhosis. Most of the HCC cases arise from liver cirrhosis secondary to persistent infection with hepatitis B virus(HBV) or hepatitis C virus(HCV) in the area of east-south Asia.The mechanism of HCC development and progression is very complicated. A number of cellular phenomena, including inflammation and oxidative stress, and molecular events, such as inactivation of the tumor suppressor gene p53, beta-catenin mutations, overexpression of various ErbB receptor family members, overexpression of the MET receptor, and abnormal expression of micro RNA, have been reported to facilitate tumor initiation, progression, and metastasis.A lot of studies have shown that microRNA play an important role in HCC development.Victor Ambros’ group and Gary Ruvkum’ group discovered a class of micronucleartides, which blongs to RNA, in plant cell almost at the same time in 1993, and they found these micronucleartides can regulate gene’s expression, they named them as microRNA(miRNA). These original studyies clarified the important function of non-coding RNAs in cell biological process; therefore, new area of microRNA study is opened since then. The microRNAs are cleaved into 22-24 nucleotides in length, and they are double strand RNAs(dsRNA), and they are highly conserved in evolution, and studies showed that they control gene expression through the way of post-transcriptional regulation, and the genes targeted by microRNA are not remarkably specific, which means that one microRNA can bind with even hundred of genes to regulate their expression. Generally, microRNA can bind with 3’-untranslational region(3’ UTR) of target mRNAs to inhibit translation or induce mRNA degradation. Recent studies confirmed that some mciroRNAs also can bind to 5’-untranslational region(5’ UTR) or translational region of target mRNA to regulate their expression, and recent studies further showed that microRNAs can interation with long non-coding RNA(lncRNA) or cricle RNA(cRNA) to control genes expression indirectly, and further studies confirmed that microRNA not only inhibit but also promote genes expression at level of mRNA or protein by stabilization of mRNA or promote translation. More and more studies show that microRNAs participate in crucial biological processes, such as development, differentiation, apoptosis and proliferation, therefore abnormal expression of microRNA could lead to many kinds of diseases, including cancer.In recent years, miR-1246 has been identified as a transcriptional target of p53 in Down syndrome and may provide a new p53-miR-1246-DYRK1A-NFAT pathway in cancer. As so far, there is no miR-1246-related study about its function in HCC development or progression. The present study aimed to explore the expression of miR-1246 and the role of mi R-1246 in the tumorigenesis of human hepatocellular carcinoma(HCC), we anticipate finding new molecular mechanism in HCC development, which can provide novel theoretical basis for diagnosis and treatment in clinic.Based on the above mentioned background and the question we raised, we performed investigations to clarify the expression of miR-1246 and its function in HCC and its molecular mechanism, we used six HCC cell lines including Huh7, C3 A, PLC, HepG2, Hep3 B, SUN387 and normal hepatic cell line LO2, and 50 paired HCC tissues and matched adjacent tissue and 50 serum from same patients as our objects of our study, the relationship between expression of p53 and miR-1246 was identified by overexpression or RNAi method, and then qRT-PCR was performed to test expression of miR-1246 in seven HCC cell lines and 28 HCC tissues which has differantial expression of p53, and the relationship between miR-1246 and p53 in these HCC cell lines and HCC tissues was further analysed. Hep3 B and HepG2 was selected to serve as cell models for further investigation of miR-1246 function in HCC development, the effects of mi R-1246 on cell proliferation, the ability of colony formation and invasion was clarified by up-regulating or down-regulating of miR-1246 expression in Hep3 B and HepG2 respectively. Based on the study in cell lines, we established transplanted tumor model of nude mice to study the function of mi R-1246 in HCC development in vivo.The target of miR-1246 was indentified as NFIB by biosoftware analysis and luciferase reporter system, and the function of NFIB in HCC was identified by RNAi, MTT, colony formation, and cell apoptosis assay. The expression of p53, NFIB and miR-1246 were examined by western blot, ISH or QRT-PCR in 50 HCC tissues and macthced adjacent tissues and 50 serums from the same patients, and statistical analysis to clarify the relationships among p53, miR-1246 and NFIB, which further confirmed the clinical significance of p53-miR-1246-NFIB pathway.The main contents of this article are as follows: Part one The expression of miR-1246 induced by p53 and the functions of miR-1246 in HCC cellsObjective: 1 Whether p53 gene could induce miR-1246 expression in HCC cell lines HepG2 and C3 A. 2 The profile of miR-1246 was expressed in HCC cell lines, including HepG2,C3 A,Hep3B,PLC,Huh7 and SUN387, and HCC tissues, which express different type of p53 gene, and HCC tissues, which express different level of p53 mRNA. 3 Experiments of in vitro or in vivo was performed to clarify the biological functions of mi R-1246.Methods:1 we generated pcDNA 3.1-p53 eukaryotic expression plasmid, then HCC cell lines HepG2 and C3 A were transfected with pcDNA 3.1-p53 plasmid or pcDNA 3.1 empty vector as control by using Lipofectamine 2000 reagent, and at same time, we synthesized p53 specific small interfere RNA(siRNA-p53), and this siRNA-p53 and control siRNA were also tansfected into HCC cell lines HepG2 and C3 A, then qRT-PCR was performed to examine the expression of miR-1246 in the HCC cell lines after overexpession or silence of p53 gene. 2 We also tested the miR-1246 expression in six HCC cell lines HepG2, C3 A, Hep3 B, Huh7, PLC and SUN387 by qRT-PCR. We further performed qRT-PCR to identify miR-1246 expression in clinical HCC tissue samples which had differential p53 expression. 3 we over express or silence miR-1246 expression in HCC cell lines HepG2 and Hep-3B by transfection of miR-1246 mimics and miR-1246 antisense oligonucleotides(micro-RNA ASO), and then MTT assay and colony formation assay were performed to identify the biological function of miR-1246. 4 We generated HepG2 cells stably overexpressed shRNA-1246 or shRNA-control, and wound scratch assay to be performed to clarify the ability of migration of HepG2 stable overexpressed miR-1246 compared with control. Lastly, HepG2 stable overexpressed miR-1246 or control cells were injected subcutaneously into nude mice to examine whether overexpression of miR-1246 could inhibit the tumorigenicity.Results: 1 mi R-1246 expression was significantly inhibited in the HCC cell lines HepG2 and C3 A silenced p53 by siRNA(P<0.05), in contrast, mi R-1246 expression was significantly increased in the HCC cell lines HepG2 and C3 A overexpressed p53 gene(P<0.05). 2 miR-1246 expression was significantly decreased in the the HCC cell lines expressed mutated p53 or not expressed p53,compared with the cells expressed WT p53(P<0.05); at same time, miR-1246 expression was increased in clinical HCC tissue samples which had higher p53 expression compared with the tissues with lower p53 expression(P<0.05). 3 we confirmed the ability of proliferation and colony formation of HCC cells was significantly inhibited after overexpression of mi R-1246(P<0.05), in contrary the ability of proliferation and colony formation of HCC cells was significantly promoted after transfection of anti-sense miR-1246(P<0.05). 4 Wound scratch assay confirmed that the ability of migration of HepG2 stable overexpressed miR-1246 significantly decreased compared with control. 5 Nude mice experiments showed that overexpression of miR-1246 could significantly inhibit the tumorigenicity of HCC cells, the weight of tumor from HCC cells overexpressed miR-1246 was 0.32±0.07 g, while the weight of tumor from HCC cells was 0.56±0.10 g from the miR- control group(P<0.05).Conclusion: 1 p53 gene can induce miR-1246 expression in HCC cell lines, and there was consistance between miR-1246 and p53 expression in clinical HCC tissue samples. p53 gene has a role in regulation of miR-1246 expression. 2 miR-1246 has a role in regulation of cell proliferation, colony formation and migration. 3 Nude mice experiments show that mi R-1246 has a role in inhibiting the tumorigenicity of HCC cells. 4 Our results indicate that mi R-1246 induced by p53 inhibits the tumorigenesis and tumor progression of HCC cells in vitro and in vivo. Part two Investigation of the mechanism of NFIB targeted by miR-1246in HCC cell linesObjective: 1 Prediction and confirmation of the target gene of miR-1246 by using HCC cell lines HepG2, C3 A, Hep3 B, PLC, Huh7, SUN387 and normal hepatic cell line LO2. 2 The correlation between miR-1246 and NFIB, and the effect of miR-1246 on NFIB were investigated by experiments. 3 The effect of NFIB on cell proliferation, clonning formation assay and apoptosis was investigated in HCC cell lines.Methods:1 The putative targets of miR-1246 were predicted using the miRanda, PicTar and TargetScan target algorithms, and the oncogene NFIB is predicted the target gene of miR-1246, then we constructed a luciferase reporter vector including the putative binding sequence and mutated putative binding site of miR-1246, was inserted downstream of the luciferase coding region, and then the HepG2 cells were transfected with the reporter vector together with either miR-1246 ASO or miR-1246 to test the binding between mi R-1246 and 3’UTR fragment of NFIB. QRT-PCR and Western blot were performed to determine whether mi R-1246 regulates NFIB expression at the mRNA or protein level. 2 We assessed endogenous NFIB expression in HepG2 and Hep3 B cells with altered miR-1246 expression. Both cell lines were transfected with miR-1246 or miR-1246 ASO to overexpress or block mi R-1246, respectively, then western blot and qRT-PCR were performed to examine protein and mRNA expression of NFIB. 3 siRNA targeting NFIB(siR-NFIB) or control siRNA were used for the present study to clarify the function of NFIB gene in HCC cell line HepG2, then MTT assay, clonning formation assay and apoptosis assay by FACS were performed to analyze the biological function of NFIB in HCC cell line HepG2. We transfected HepG2 cells with the mi R-control, mi R-1246 or miR-1246/NFIB expression plasmid(pcDNA3/NFIB), respectively, and then MTT assay, clonning formation assay and apoptosis assay by FACS were performed to explore the miR-1246 induced inhibition of HCC cell proliferation targeted on NFIB. 4 Western blot and QRT-PCR were performed to explore the relevance of NFIB in relation to mi R-1246 in six HCC cell lines HepG2, C3 A, Hep3 B, Huh7, PLC and SUN387.Results: 1 Bioinformatics and luciferase reporter confirmed that mi R-1246 binds directly to the 3’UTR of NFIB. 2 QRT-PCR and western blot confirmed that mi R-1246 inhibited the protein expression of NFIB but not mRNA expression of NFIB after overexpression or blocking of miR-1246 expression in HepG2 and Hep3B;Western blot confirmed that miR-1246 inhibited expression of NFIB protein after alteration of miR-1246 expression in LO2, HepG2, C3 A, Hep3 B, Huh7 and PLC. 3 MTT assay, colony formation analysis and apoptosis assay by FACS showed that the ability of proliferation and colony formation was significantly inhibited, and the apoptosis increased after deletion of NFIB gene by si RNA(P<0.05). the mi R-1246 which targeted NFIB gene can inhibit formation of HCC. 4 Expression of NFIB in HepG2 and C3 A expressing wild type p53 was significantly lower than its expression in Hep3 B, PLC, Huh7 and SUN387 expressing mutated p53 or not expressing p53, and statistical analysis by Graph Pad software showed that the expression of NFIB was negatively correlated with expression of miR-1246 in HCC cell lines(R=0.8940, proliferation and inhibit apoptosis of HepG2 cells. 3 There is negative correlation between NFIB protein and mi R-1246 expression, which suggested the function of miR-1246 was carried out by targeting on NFIB. 4 p53-miR-1246-NFIB might be a novel signaling parthway in development and progression of HCC. Part three The significance of p53-miR-1246-NFIB parthway in clinicalHCCP<0.05).Conclusion: 1 Our results showed that mi R-1246 binds directly to the 3’UTR of NFIB to regulate the protein expression of NFIB but not m RNA expression of NFIB. 2 Our results showed that NFIB could promote cellproliferation and inhibit apoptosis of Hep G2 cells. 3 There is negative correlation between NFIB protein and mi R-1246 expression, which suggested the function of mi R-1246 was carried out by targeting on NFIB. 4 p53-mi R-1246-NFIB might be a novel signaling parthway in development and progression of HCC.Part three The significance of p53-mi R-1246-NFIB parthway in clinical HCCObjective: we used 50 clinical HCC and matched adjacent tissues and serum of HCC patients as objects of study, expression of p53, mi R-1246 and NFIB, and associations among them were analyzed to explore the significance of p53-mi R-1246-NFIB pathway on HCC development and progression. Methods:1 Expression of p53 and NFIB protein in clinical HCC tumor tissues and matched adjacent normal tissues was examined by western blot and IHC staining analysis. 2 QRT-PCR was performed to examine of mi R-1246 expression clinical HCC tumor tissues and matched adjacent normal tissues. Statistical analysis was performed to clarify the relationship between p53 protein and mi R-1246 expression, or between mi R-1246 expression and NFIB protein expression. 3 QRT-PCR was performed to examine of mi R-1246 expression in serum of HCC patients, relationship between mi R-1246 in serum and in its expression in HCC tumor tissues, which confirm the significance of p53-mi R-1246-NFIB parthway in clinical HCC.Results: 1 Western blot combined with phosphorimager quantification analysis showed that significant higher level expression of p53 protein in 50 HCC matched adjacent tissues compared with its expression in HCC tissues(t test, P<0.01). 2 QRT-PCR results showed that significant higher level expression of mi R-1246 in 50 HCC matched adjacent tissues compared with its expression in HCC tissues(P<0.01), and further analysis by SPSS software showed that there was positive correlation between expression of p53 protein and expression of mi R-1246(r=0.734, P<0.001). 3 IHC staining of 50 HCC and mached adjacent tissues showed that the percentage of higer expression ofNFIB is 54%(27/50), while expression of NFIB is significantly decreased in HCC mached adjacent tissues, 24%(12/50)(P<0.01), and futher analysis by SPSS software showed that there was negative correlation between expression of NIFB protein and expression of mi R-1246(r=-0.419, P=0.002). 4 Detection of mi R-1246 expression by q RT-PCR showed that the percentage of higer level expression of mi R-1246 in serums HCC patients is 30%(13/43), while 70%(30/43) was lower expression of mi R-1246, and statistical analysis showed that here was positive correlation between expression of mi R-1246 in HCC tissues and its expression in serums of HCC patients(r=0.323, P=0.035).Conclusion: 1 We confirmed that both p53 protein and mi R-1246 expression were higher in matched adjacent normal tissues compared with in HCC tumor tisses, and statistical analysis showed that there was closed positive correlationship between p53 protein and mi R-1246 expression, which further suggested mi R-1246 expression was induced by p53 protein in clinical HCC samples. 2 The expression of NFIB was higher in HCC tumor tissues compared with in matched adjacent tissues, and statistical analysis showed that there was closed negative correlationship between NFIB protein and mi R-1246 expression, which indicated mi R-1246 inhibited NFIB protein expression in clinical HCC samples. 3 Then we further confirmed mi R-1246 tested in serum of HCC patients was positively correlated with its expression in HCC tumor tisssues, which might have potential significance in diagnosis or treatment of HCC in clinic. |
PDF Full Text Request