| Liver tumors are mainly divided into the following categories: hepatocellular carcinoma(HCC),malignant angioendothelioma,intrahepatic cholangiocarcinoma(ICC),the most common of which is HCC,often Referred to as liver cancer,accounting for 90% of all liver tumors.HCC is one of the most common malignant tumors with high metastatic rate and recurrence rate,resulting in a high mortality rate.HCC is a major tumor disease that endangers human health.With the development of biological science and technology,great progress has been made in the treatment of HCC.However,radical resection of liver cancer and liver transplantation are still the main means of treating HCC at present.However,due to postoperative recurrence,the proportion of metastasis is still high,more than 50% of patients with HCC recurrence and metastasis within 5 years.So far,the most effective HCC targeted therapy in the world is sorafenib,and the effect of improving prognosis is not satisfactory either.Therefore,it is important for early diagnosis and treatment of HCC to search for markers for diagnosis,recurrence and metastasis of HCC at the level of molecular gene for the early diagnosis and development of new,truly effective and low side-effect precise targeted therapies.Micro RNAs(miRNAs)are a group of small,non-coding RNAs about 22 nucleotides in length that bind primarily to seed sequences located within the 3 ’untranslated region(3’UTR)of a target gene m RNA and can promote the target Degradation or inhibition of translation of the gene m RNA ultimately inhibits the biological function of the target gene.The human genome contains thousands of micro RNAs.Nearly33% of the protein-coding genes in the human body are regulated by micro RNAs.Each micro RNA can directly regulate about 200 target genes and play an important role in various physiological and pathological processes,Such as embryonic development,cell proliferation,differentiation,cell cycle progression,apoptosis,autophagy,angiogenesis and metabolism.Thus,micro RNA has a very important position in human gene expression.Over the past decade,the multiple roles of micro RNAs in the development and progression of human cancers have been established,and about 50% of known micro RNAs are associated with a variety of human cancers.Micro RNAs are involved in the regulation of almost all biological behaviors such as proliferation,migration,invasion,metastasis,angiogenesis,cell cycle progression,apoptosis and autophagy by regulating the expression of target genes at the transcriptional or translational level.If a micro RNA inhibits the expression of a tumor suppressor gene,it functions as an oncogene,and if it inhibits oncogene expression,it becomes a tumor suppressor micro RNA.With the deepening of related micro RNA research in recent years,its role in the development of HCC has become increasingly prominent.In addition.Several recent studies have shown that the expression of some micro RNAs is closely related to the clinical phenotype and clinical prognosis of HCC,confirming the validity of micro RNA as a predictor of HCC prognosis.Micro RNA-126(micro RNA-126,miR-126)is a closely related micro RNA.It has been reported that miR-126 levels are significantly reduced in many tumor tissues compared to the corresponding normal tissues,including colon cancer,Gastric cancer,oral cancer,pancreatic cancer,lung cancer,etc.,and in most cases low expression of miR-126 is associated with tumor invasion and metastatic phenotype.Studies have shown that in vivo or in vitro experiments to correct the low expression of miR-126(or overexpression of miR-126)on the biological behavior of HCC cells were significantly inhibited,on the mechanism,some studies screening and identification of miR-126 Possible partial target genes: for example,Sox2,but the specific effects and molecular mechanisms are not yet fully understood.In order to further study the molecular mechanism of miR-126 playing a role in tumor suppressor genes in hepatocellular carcinoma,we used gene chip technology and bioinformatics methods to screen and identify another possible target gene of miR-126: PLK-4.The Polo-like kinase(PLK)family,a highly conserved family of serine / threonine kinases,plays an important role in cell cycle progression and DNA damage response and has recently been identified as a potential anticancer target.PLK-4 is located on chromosome 4q28 and is a conserved upstream regulator of centrosome replication.Studies have shown that in mammalian cell lines,the PLK-4 protein is located in the nucleolus during G2 phase,localized to the centrosome in the early stages of M phase,and located in the cleavage ditch during cell division.PLK-4 gradually increased from the G1 phase,and peaked in mitosis,indicating that its expression is regulated by the cell cycle.Has recently been reported to play an important role in centrosome replication,cytokinesis and maintenance of chromosomal stability in the cell cycle.Thus,the imbalance of PLK-4 expression leads to the loss of the integrity of the number of centrosomes,thus contributing to genomic instability and eventually tumorigenesis.It has been reported that PLK-4 is abnormally expressed in many human tumors.For example,overexpression in colon and breast cancer.The level of PLK-4 was consistent with centrosome replication,with a lower level in G1,a gradual increase in S,and a maximum in G2 / M.Further destruction of centrosome replication by inhibiting PLK-4 activity can exacerbate genomic instability in cancer cells and cause their death.Therefore,PLK-4 inhibition may also affect G1 or S-phase tumor cells that are refractory to mitotic specific drugs.For example,it was found that RNA interference(RNAi)-mediated depletion of PLK-4in human breast cancer and cell lines can prevent cancer cell centriole duplication and inhibit tumor cell growth.All these interesting findings undoubtedly make PLK-4 a potential target for cancer treatment.As a potent antitumor oral PLK-4 inhibitor,CFI-400945 stands out among recently discovered PLK-4 inhibitors and is well tolerated in breast cancer xenograft models,particularly in the tumor suppressor PTEN Lack of model is more prominent.This inhibition relies on precise control of the number of centrosomes through a balance between phosphorylation of downstream substrates and autophosphorylation that regulates PLK-4 levels.CFU-400945 fumarate has been approved as a single agent by the Health Canada and the FDA for phase I clinical trials(NCT01954316)for the treatment of solid tumors,especially breast cancer.The expression and role of PLK-4 in hepatocellular carcinoma rarely reported,and its upstream regulatory mechanism is not yet clear.The aim of this study is to investigate the role and molecular mechanism of miR-126 /PLK-4 regulatory axis in the development and progression of hepatocellular carcinoma(HCC).In the era of accurate tumor therapy,miR-126 / PLK-4 regulatory axis may be potential and novel,Effective therapeutic target for liver cancer.Part 1: the expression and clinical relevance of miR-126 in tumor tissues from HCC patientsObjective:To determine the expression of miR-126 in tumor tissue from HCC patients,and evaluate the clinical relationship between miR-126 expression and clinical characteristics including prognosis of patients,and then further study the biological function of miR-126 in the progress of HCC.Methods:1.By using gene chip analysis software BRB-mining Arraytools for liver cancer gene expression profile,download and analyze TCGA public database of liver cancer mirna gene chip data,analysis of miR-126 expression in liver cancer tissue and normal liver cancer.Combined with the clinical follow-up data,analysis of miR-126 expression in liver cancer tissue and the relationship between the liver cancer clinical phenotype.Based on bioinformatics,microarray analysis software(BRB-Arraytools),TCGA public databases were used to analyze the miR-126 expression in HCC tissues and normal liver tissues.We also analyzed the relationship between the expression of miR-126 in tissues and clinical phenotype of HCC by combined with the clinical follow-up data,.2.The expression of miR-126 was detected in human normal liver cell lines(chang liver and LO2 cell lines),HCC cell lines(Hep G2,Hep-3b,SMMC-7721,the MHCC97-L,HCCLM3,Bel7402).3.Cancer tissue and corresponding adjacent tissues from 75 HCC cases were collected from the first affiliated hospital of Zhengzhou university.The expression levels of miR-126 were determined.Moreover,the relationship between the degree of miR-126 expression and the clinical features and prognosis of patients was analyzed.4.The effect of miR-126 overexpression on cell proliferation of HCC was determined by MTT.5.The effect of miR-126 overexpression on cell migration and invasion of HCC was determined by scratch repair and matrix hit transwell experiments.6.The effect of miR-126 overexpression on cell proliferation of HCC was determined by clone forming experiment.7.All data was present by mean+/-standard deviation.The comparisons between two groups were analyzed by Student’s t-test or the Mann-Whitney’s inspection.Disease-free survival and overall survival were analyzed by Kaplan Meier-survival curves,alpha <0.05 as the inspection level.Results:1.Compared with the normal liver tissue(50 cases),the expression of miR-126(TCGA and GEO gene database analysis)in HCC tissue(375 cases)have a significant increase(P <0.05).Kaplan-Meier analysis showed that the survival time of patients with high miR-126 was significantly better than patients with low miR-126,suggesting that the expression ofwas closely related to the prognosis of HCC patients.2.Compared with human normal liver cell lines(chang liver and LO2 cell lines),HCC cell lines(Hep G2,Hep-3b,SMMC-7721,the MHCC97-L,HCCLM3,Bel7402)had a low expression of miR-126.3.The expression levels of miR-126 were low in cancer tissue from 75 HCC cases compared with corresponding adjacent tissues.Moreover,the relationship between the degree of miR-126 expression and the clinical features and prognosis of patients was analyzed.There were a close correlation between the expression of the prognosis and miR126(P = 0.001),as well as metastasis(P = 0.000),TNM stage(p =0.006),recurrence(p=0.041)of HCC patients,while no correlation were found between the expression of the prognosis and age,sex and AFP level.Low miR-126 suggested a poor prognosis of HCC patients(p<0.01).Kaplan-Meier analysis showed that the survival time of patients with high miR-126 was significantly better than patients with low miR-126,suggesting that the expression of miR-126 was closely related to the prognosis of HCC patients.4.The growth of HCC cells was significantly suppressed by overexpression of miR-126 compared with the blank control group and nonspecific scramble transfection blank control group(P < 0.05)5.Transfection of miR-126 into HCC cells suppressed the repair ability significantly 48 h after migration compared with the blank control group and nonspecific scramble transfection blank control group(P < 0.05)6.Transfection of miR-126 into HCC cells inhibited HCC cells penetrate matrix compared with the blank control group and nonspecific scramble transfection blank control group(P < 0.05)7.Transfection of miR-126 into HCC cells inhibited HCC cells clone formation compared with the blank control group and nonspecific scramble transfection blank control group(P < 0.05).Conclusion:Lower expression of miR-126 was closely related to clinical phenotypes and clinical prognosis of HCC patients and miR-126 overexpression significantly inhibit HCC proliferation,migration and invasion.These data indicated that miR-126 could be considered as a tumor suppressor genes in the development of HCC.Part 2: screening the target gene of miR-126 and investigating the molecular mechanism of miR-126 on HCC developmentObjective:Bioinformatics techniques were used to Screening and identified the miRNA gene targeting miR-126,and the molecular mechanisms involved in HCC progression were investigated.Methods:1.Using a lentivirus was constructed overexpression of miR-126 in HCC cell line and RT-PCR was used to identify successful overexpression of miR-126.2.Changes of cell transcriptome m RNA gene expression profile were analyzed by gene chip technology and bioinformatics for differences in gene analysis(GO analysis,KEGG pathway analysis).3.Online database(targetscan miRbase miRwalk)was used to predict the target genes of miR-126.4.Using RT-PCR,Westernblot and immunofluorescence technique to detect the pecific target of miR-126 for predicted target genes PLK-4.5.The effect of miR-126 overexpression and target genes overexpression were detected by MTT via building overexpression plasmid of PLK-4.6.By using gene chip analysis software BRB-mining Arraytools for liver cancer gene expression profile,download and analyze TCGA public database of liver cancer miRNA gene chip data,analysis of PLK-4 expression in liver cancer tissue and normal liver cancer.Combined with the clinical follow-up data,PLK-4 expression in liver cancer tissue and the relationship between the liver cancer clinical phenotype and miR-126PLK-4 were performed.Regulating shaft role of miR-126-PLK-4 in liver disease progression were analysis.7.All data was present by mean+/-standard deviation.The comparisons between two groups were analyzed by Student’s t-test or the Mann-Whitney’s inspection.Disease-free survival and overall survival were analyzed by Kaplan Meier-survival curves,alpha <0.05 as the inspection level.Results:1.RT-PCR and Western blot were used to identify successful overexpression of miR-126 in the HCC cell line.2.Gene analysis found that 254 genes were changed(2 folds change or higher,P< 0.05)after miR126 overexpression,125 increased expression and 129 decrease expression.3.Mi R-126 and PLK-4 m RNA 3 ’-nucleotides can be combined by analysis and prediction of miRbase and Target Scan databases shows PLK-4 may be target genes of miR-126.4.miR-126 overexpression in HCC cell line could induce the decrease of PLK-4expression by RT-PCR,Westernblot and immunofluorescence technique.5.miR-126 overexpression or PLK-4 gene silencing could reduce the proliferation of HCC cell lines;the combination of P PLK-4 gene and miR-126 overexpression present a poor inhibition of HCC proliferation compared with miR-126 overexpression alone.These data indicated that miR-126 could inhibit HCC proliferation by regulation the expression of PLK-4.6.Compared with normal liver group(50 cases),PLK-4 expression in HCC group(375 cases)were significantly increased(P<0.05)in TCGA public database.Kaplan and Meier analysis showed survival time of HCC patients with low PLK-4expression were long compared with HCC patients with high PLK-4 expression,which indicated that PLK-4 expression was closely related to the clinical prognosis of HCC patients.Conclusion:Mi R-126 could regulated the expression of PLK-4,resulting in the inhibition of HCC proliferation.PLK-4 expression was closely related to the clinical prognosis of HCC patients.These data suggested that miR-126 involved in the progression of HCC by regulating the expression of PLK-4.Part 3: the effect of miR-126 on biology behavior of HCC in nude miceObjective:To observe the effect of miR-126 on biology behavior of HCC in vivo by building miR-126 stable expression of HCC,and subcutaneously injecting into nude mice,Methods:1.miR-126 stable expression of HCC were build and subcutaneously injected into nude mice.Survival time and tumor growth were observe and nude mice survival curve were analyzed by Kaplan Meier.2.The expression of miR-126,PLK-4 and Ki67 in nude mice tumor tissue was detected.Results:1.RT-PCR and Western blot were used to identify successful overexpression of miR-126 in HCC cell line.In vivo experiments confirmed that miR-126 overexpression in HCC could inhibit the proliferation,tumor volume growth,and increase the survival time compared with transfection empty virus in nude mice,2.The immunohistochemical confirmed overexpression of miR-126 reduced the expression of PLK-4.PLK-4 expression was negative correlated with miR-126 expression.Meanwhile,the expression of tumor proliferation marker Ki67 decreased.Conclusion:Overexpression of miR-126 can inhibit the expression of PLK-4,resulting in the inhibition of HCC proliferation in nude mice.The full text conclusion:1.In the cell line,local samples and TCGA database large samples,mir-126 was detected significantly decreased in liver cancer.Mi R-126 abnormally low expression and metastasis and recurrence in patients with clinical malignant phenotype closely related to the survival time of patients with miR-126 high expressed significantly longer than miR-126 patients with lower expression,prompt miR-126 in liver disease progression may have the effect of tumor suppressor genes,can be a clinical phenotype to predict and judge the prognosis of malignant potential targets.2.In vitro study results showed that the proliferation ability,migration ability,invasion ability and cloning ability of the liver cancer were significantly decreased after mir-126 expression.In the study of body,miR-126 expression inhibition of liver cancer tumor capacity has been further validation,prompt miR-126 in the process of liver disease tumor-suppressor gene effect,may be potential targets for targeted therapy of liver cancer.3.Experimental results in vivo and in vitro showed that mir-126 overexpression can inhibit plk-4 expression,suggesting that mir-126 can target the expression of plk-4 in liver cancer.4.PLK-4 expression level increased significantly in the liver,TNM staging late and PLK-4 patients with recurrent higher expression level,and high expression of PLK-4 patients survival time significantly shorter than the lower expression of PLK-4 patients,prompt PLK-4 May play a role in cancer gene in liver cancer,can be a clinical phenotype to predict and judge the prognosis of malignant potential targets.5.In liver cancer tissues,the expression level of PLK-4 markers and tumor proliferation Ki67 expression levels were positively correlated,silent PLK-4 genes can make the proliferation of liver cancer dropped significantly,and the results are consistent,after expressing miR-126 tips miR-126 / PLK-4 adjusting shaft can through regulating liver cancer cell proliferation,affect the progress of the liver disease,may become the new target for the treatment of liver cancer. |
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