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Function And Mechanism Study Of Non-coding RNA In Hepatocellular Carcinoma

Posted on:2014-02-06Degree:DoctorType:Dissertation
Country:ChinaCandidate:L ZhangFull Text:PDF
GTID:1264330398466376Subject:Genetics
Abstract/Summary:PDF Full Text Request
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors, and isone of the main factors of leading causes of tumor related death worldwide. There are80%of the new HCC cases occur in developing countries. Hepatocellular carcinoma notonly is one of the leading causes of cancer death in China, but also increased in othercountries slowly, especially in western countries. Although for hepatocellular carcinoma,local treatment such as operation and transcatheter arterial chemoembolization can bepartially successful, but the patients suffered from liver cancer still have a high rate ofrecurrence and metastasis. This is mainly due to the high frequency of tumor recurrenceafter resection of hepatocellular carcinoma operation, which lead to the poor prognosis.However, the molecular mechanism of HCC recurrence or metastasis remains largelyunclear.Some reports suggest that unfolded protein response activation is associated withclinical prognosis. The high expression levels of unfolded protein chaperone inhepatocellular carcinoma are correlated with higher tumor grade. And the activation ofunfolded protein pathway in vitro changes the sensitivity of tumor cells to chemotherapyagents. The endoplasmic reticulum (ER) is a place for cell to synthesize membrane andsecretion protein. Proteins synthesized in cells, must be correctly folded, and assisted bymolecular chaperones. The environmental stress such as hypoxia, nutrient deprivation,and chemotherapeutic drugs can make tumor cells produce extra unfolded protein, andthe unfolded protein will be gathered in the endoplasmic reticulum of tumor cells. So, theunfolded protein response (UPR) activated in the tumor cells to fight these stress reaction.The unfolded protein response is to restore a cascade effect of cell signal transductionand to make a more suitable environment for protein folding. This mechanism suggeststhat the unfolded protein response can be an important way to protect cancer cells fromadverse environmental conditions, so as to promote tumor growth and survival in the"poor environment ".In addition, epithelial-mesenchymal transition (EMT) and conversion of stromal andepithelial cells also play an important role in cancer metastasis process. Also, it has beenfound that EMT may be an important cause of the spread of of tumor metastasis and theresistance to treatment.Occurrence of EMT in tumor cells, experiencing the genetic andepigenetic changes in gene cloning, growth and the formation, is conducive to the localdevelopment of tumor. These changes, particularly through the influence of oncogenes and tumor suppressor genes, thereby promoted the EMT regulation pathway. Manystudies show that, the miR-200family has been found to inhibit cell EMT and tumor cellmigration. Cancer cells in the process of EMT, obtain the stronger invasion ability andmetastasis, which eventually develop into malignant degree of cancer so as to shorten life.However, the cancer cells may exist in inhomogeneous distribution. Some cells retainmany epithelial characteristics and part of interstitial phenotypic, some cells of epithelialorigin has the complete stromal cell characteristics.Recent data indicate that, epigenetics, such as non-coding RNA, histone modificationand DNA methylation, involved in the process of cancer metastasis. Therefore, our studystarts from the non-coding RNA, expounds the relationship between small RNA(microRNA) and long non-coding RNA (lncRNA) and endoplasmic reticulum stress andepithelial-mesenchymal transition, thus further explains various epigenetic mechanismsin the development of liver cancer.The human genome is only about20000protein coding genes, accounting for <2%oftotal genomic sequence. However, the whole genome chip, whole genome sequencingand transcriptome sequencing found that at least90%of the genome transcriptionallyactivated. The human transcriptome contains more complex sets of non-coding genes,many overlapping, splicing, variation and antisense non-coding RNA (ncRNA) has awider meaning expression. NcRNA was firstly thought of no significance transcription as"noise ", however, recent evidence show that, these genomic "noise " in cell growth andmetabolism, may play an important biological role. For example, the newly discoveredlong non-coding RNA (lncRNA) gene, with tissue-specific expression patterns, plays acritical role in the development of various diseases including cancer.Non-coding RNA according to the transcript size roughly divided into two categories,small non-coding RNA and long non-coding (lncRNAs).Many small non-coding RNAs, including microRNAs (miRNAs) are found in cells.The miRNA contains many known and newly discovered genes, and the non-coding geneis usually composed of22nucleotides (NT), which can bind to the5’ or3’ region in thecoding genes, leading to posttranscriptional regulation of the silencing, inhibition andactivation of protein coding genes. miRNA plays an important role in controlling manybiological process, the regulation of gene expression and in the regulation of complexnetwork. There is growing evidence that miRNA has a significant role in the occurrenceand development of tumor, and may be powerful biomarkers for cancer diagnosis and prognosis. Unlike miRNA, lncRNAs, range from200~1000000nucleotides in length,and lack of significant open reading frame. Many lncRNAs can be transcribed by RNApolymerase II (RNA pol II) and polyadenylated. And like protein coding genes, thelncRNA gene is involved in a series of biological process, for example, epigenetics,splicing, nucleocytoplasmic transport, transcriptional activation, regulating mRNAdegradation and as the molecular precursors of miRNAs, in addition, it is reported thatthe abnormal expression of the lncRNA plays a regulatory role in many types of cancer,and may be the main factor of tumor.The first part of our study is the in-depth discussion of the relationship of themicroRNA-122and of the the unfolded protein pathway endoplasmic reticulumstress and its effect on chemosensitivity in hepatoma cells. microRNA (miR-122) isthe most abundant in the liver, accounting for about70%of the total number of themiRNAs. Some studies show that miR-122of is very important to liver homeostasis.miR-122expresses abundantly in mouse and human liver cells, but have a very lowexpression in most HCC cell lines and transformed cells. Loss of miR-122expression isrelated to hepatic differentiation, invasion and intrahepatic metastasis. Recently, nudemice model confirmed that miR-122acted as a tumor suppressor and can have drugsensitization effect. Previous studies have found that miR-122affects sensitivity ofhepatocellular carcinoma cells to doxorubicin (DOX) through p53-independent apoptosispathway. However, the detailed mechanism of this phenomenon is still unclear. Theresults of previous studies did not fully explain the potential for miR-122. Therefore newtechnology and new methods are required to study the complex function of miR-122.Now, we apply proteomics to study effects of miRNA on protein. In our study,because the expression of miR-122has a relatively high level in Huh7cells, we silencethe miR-122expression in Huh7cells, and then study the alteration of the protein patternusing the method of differential proteomics. Results showed that, after the inhibition ofmiR-122expression in hepatocellular carcinoma cell line, a plurality of unfoldedmolecules upregulated and protein reaction pathways activated. After the transfection ofmiR-122inhibitors in Huh7cells, the expression of miR-122was inhibited, and at thesame time, the PSMD10protein was upregulated. PSMD10has been shown to increaseglucose regulated protein78(GRP78), which can improve the folding ability ofnon-folding protein in Huh7cell, to promote the recovery of the endoplasmic reticulumstress. The unfolded protein response pathway plays an important role in tumor biology, so the mechanism and relationships of miR-122and the endoplasmic reticulum stressresponse regulatory were fully investigated. Our results show that, miR-122changestumorigenic ability of the tumor cells at least or in part by the negative regulation ofnon-folding protein pathway. These results indicate that miRNA is a regulation factorand plays a crucial role in tumor development and chemotherapy resistance.However, with in-depth study of the regulatory mechanism of miRNA, we found that,in addition to miRNA, another type of non-coding RNA is also involved in the regulationof RNA network, the discovery of these lncRNAs enables us to further understand themolecular mechanism. The second part of our study is the discussion about therelationship between lncRNA and epithelial-mesenchymal transition and themutual regulatory mechanism of lncRNA and microRNA in the process of tumormetastasis.A recent report found that the lncRNA can act as a scaffold protein and guide theprotein to locate in the genomic DNA, which related to changes in gene expression. Butin our recent study, also found disregulation of lncRNAs in human hepatitis B virus(HBV) related hepatocellular carcinoma compared to corresponding adjacent tissue bymicroarray screening technology. Our results also show that specific expression spectrumof lncRNA can accurately distinguish tissues from HCC. Among these lncRNAs, wescreened the representative lncRNAs such as lncRNA-HEIH and H19, which play apotential role in the formation and development of hepatocellular carcinoma. Our studyshows that long non-coding RNA up-regulated in HCC ‘lncRNA-HEIH’ can interactwith EZH2, and guide PRC2complexes to locate in the genome so as to regulate theexpression of cell cycle related genes. And it is also showed that the up-regulatedexpression of lncRNA-HEIH in hepatocellular carcinoma is closely related with livercancer recurrence, and is significantly associated with patients’ survival time. The higherexpression level of lncRNA-HEIH in HCC tissues indicates a shorter survival time ofpatients. Early esearch has shown a strong association between H19and various types ofcancer including liver cancer. In HCC, the expression of H19is usually due to aberrantDNA methylation and losing imprinting. However, the relationship between thehepatoma prognosis and H19remains unknown. In our chip, H19with one of the mostobvious expression differences of all lncRNA molecules is screened out, and we find thatH19can up-regulate the expression of miR-200family, which inhibit the metastasis rateof advanced hepatocellular carcinoma. miR-200is the star molecule in the EMT process, and the high expression of miR-200can reverse the progress of EMT, and inhibit cancercell metastasis. Furthermore, our study finds that long non-coding RNA H19regulate theexpression of miRNA-200through histone acetylation, and play the same role asmiR-200in the progression of advanced HCC. It not only shows that the mutualregulation and synergy of lncRNA and miRNA in the development process of the disease,but also provides new targets for the treatment of liver cancer.In conclusion, the application of gene chip, proteomics and other means are involvedin liver carcinogenesis, development of functional non-coding regulatory mechanism ofRNA and histone modification epigenetic regulation in-depth and extensive research,provide new perspective for further understanding the signaling pathway in this morecomplicated process and new study target for early diagnosis and treatment inhepatocellular carcinoma.
Keywords/Search Tags:primary liver cancer, proteomics, endoplasmic reticulum stress, miRNA, long non-coding RNA, epigenetics, epithelial-mesenchymal transition
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