| 【Backgrounds】 The occurrence of hepatocellular carcinoma(HCC) is occult, and it is difficult for early diagnosis. Moreover, it progresses rapidly, and the mortality is high. Thus, it is very important for early diagnosis in order to improve the survival of patients with HCC. Pathologically, HCC is also obvious multistage and multistep process of tumor progression. During the process, an important change is that precancerous lesions develop gradually into HCC. To date, several different lesions have been suggested to represent precancerous conditions in human liver. They include dysplastic foci(DF), nodules of altered hepatocyte(NAH), and dysplastic nodules(DN). The majority of them appear in the cirrhotic liver. Moreover, some studies have demonstrated that molecular aberration with high frequency in cirrhotic liver, precancerous lesions, and small HCC might represent early molecular event of HCC. It will be helpful for early diagnosis and therapy of HCC to study on these early molecular aberration.Micro RNAs(mi RNAs) are a class of endogenous non-coding small RNAs with 17-25 nucleotides in length. Its expression level shows significant differences in different stage during carcinogenesis, and relates to the occurrence and development of various tumors. mi RNAs regulate negatively genes expression by sequence-specific binding to the 3’ untranslated region(UTR) of protein-coding m RNA. Thus, they can behave as oncogenes or tumor suppressor genes depending on the cellular function of their targets. In addition, mi RNAs are stable in circulation because they can repress the degradation of endogenous RNA enzyme, and makes them perfect biomarkers of tumors, especially for detection of early stage. Many studies have been reported about the relationship between mi RNAs and HCC, and some mi RNAs associated with the occurrence, development, invasion, and migration of HCC have been demonstrated. However, few of researchs have been described about mi RNAs related to small HCC or precancerous lesions, and mi RNAs expression alteration from precancerous lesions to HCC. Moreover, it is only studied in animal HCC models.Based on previous mi RNAs array expression profiles and English literatures, we noticed that the expression levels of mi R-202, mi R-1271-5p, mi R-1271-3p, and mi R-204 were down-regulated in HCC. However, the expression alteration and function in hepatocarcinogenesis have not been further studied. mi R-211, which is a homogeneous mi RNA of mi R-204, has been showed to be down-regulated in some malignant tumors, but it has not been documented in HCC. In addition, mi RNAs behave as the corresponding function by regulating their downstream targets, and its mechanism may be elucidated by differentiating their targets. According to the biological message software, we found a possible target gene glypican-3(GPC3), which may be regulated by the above five mi RNAs. GPC3 is an oncofetal antigen and a member of the glypican family of the membrane-bound heparan sulfate proteoglycans. It plays an important role in the development and regulation of cellular proliferation and malignant transformation. Recently, several studies have demonstrated that GPC3 is positive in most HCC, but not in healthy livers. This pattern of expression suggests great promise for GPC3 as a diagnostic marker of HCC. In addition, some authors have demonstrated positive staining in precancerous lesions, and considered that it has an important value of early warning. Some other authors have demonstrated that the transition from premalignant lesions to small HCC is associated with a sharp increase in GPC3 expression in a majority of cases. Simultaneously, the occurrence of small focal positivity for GPC3 in liver cirrhosis indicated strongly HCC regardless of the percentage of positive cells for GPC3. Our previous Affymetrix SNP6.0 array has showed that GPC3 is amplified in HCC. The above views make us recgonize that there may be a certain relatinship between the downregulation of mi R-202, mi R-1271-5p, mi R-1271-3p, mi R-204, and mi R-211 and upregulation of GPC3 in HCC. Namely, these five mi RNAs may be related to GPC3.【Aims】 1. To observe the expression levels of five mi RNAs, including mi R-202, mi R-1271-5p, mi R-1271-3p, mi R-204, and mi R-211 in HCC cell lines and progressed HCC, and the impact of mi R-202 and mi R-211 on biological characters of HCC cells in vivo and vitro; To confirm further their function of tumor suppressor genes;2. To filter out GPC3-positive DN with molecular aberration by immunohistochemical methods, clonal analysis, and chromosomal loss of heterozygosity(LOH), and observe the expression levels of GPC3 in GPC3-positive DN, small HCC, and progressed HCC, and confirm that GPC3 plays an important role in hepatocarcinogenesis.3. To investigate the expression levels of mi R-202 and mi R-211 in GPC3-positive DN, small HCC, and progressed HCC, and observe their roles in hepatocarcinogenesis.4. To confirm the negative regulated relationship between mi R-202 and GPC3, and elucidate its mechanism in human hepatocarcinogenesis in order to provide new theory and evidence for monitoring the occurrence of HCC, early diagnosis, and therapy.【Methods】 1. We first observed the expression levels of five mi RNAs in human HCC cell lines and progressed HCC by RT-q PCR. Then, we examined the function of mi R-202 and mi R-211 in HCC cells by MTT assay, colony formation assay, transwell, and flow cytometry analysis. Moreover, we established animal HCC models, and observed the impact of micr ONTMagomir-202 on HCC growth.2. We examined the expression levels of GPC3 in HCC cell lines, progressed HCC, small HCC, and DN: 1) To examine the expression of GPC3 using immunohistochemical methods, and filter out further DN with GPC3-positive immunoreactivity and genetic changes using clonal analysis and chromosomal LOH: To investigate the expression of GPC3 in 136 cases of HCC and 103 DN by Max Vision immunohistochemical methods, and observe the relationship between GPC3 expression and the clinical pathological parameters of patients with HCC; To analyze the clonality of 81 DN from female patients by clonal assay based on X-chromosome inactivation mosaicism and polymorphism at androgen receptor(AR) loci in female somatic cells; To observe the chromosomal LOH of all DN at 11 microsatellite loci, and filter out DN with GPC3-positive immunoreactivity and molecular genetic changes; 2) To observe the expression levels of GPC3 m RNA in HCC cell lines, 26 cases of progressed HCC, 10 cases of small HCC and 8 cases of GPC3-positive DN by RT-q PCR.3. We observed the expression levels of mi R-202 and mi R-211 in 10 cases of small HCC and 8 cases of DN with GPC3-positive immunoreactivity by RT-q PCR, and compared with the expression level in progrossed HCC, and filter out mi R-202, which might involve in hepatocarcinogenesis.4. Finally, we investigated the relationship between mi R-202 and GPC3. We first observed the influence of overexpression of mi R-202 on GPC3 m RNA and GPC3 protein by RT-q PCR and Western blotting, respectively. And then, we confirmed further the negative regulated relationship between mi R-202 and GPC3 by Dual-Fluorescence Report assay.【Results】 1. The expression levels of five mi RNAs were down-regulated in HCC cell lines and progressed HCC. Among them, mi R-202(P=0.002) and mi R-211(P=0.000) were significantly down-regulated, and the mean difference folds were more than 2. However, the expression downregulation did not relate to the clinical pothological parameters of patients with HCC.2. The cellular function experiment demonstrated that overexpression of mi R-202 and mi R-211 repressed cell proliferation, colony formation, invasion, and migration, but did not influence cell cycle. Overexpression of mi R-202 promoted cell apoptosis, but mi R-211 did not influence cell apoptosis. The animal experiment results demonstrated that HCC growth was obviously slow after micr ONTMagomir-202 was injected in HCC tissues.3. The expression levels of GPC3 were significantly up-regulated in HCC cells lines, progressed HCC, small HCC, and DN. 1) Immunohistochemically, 103 out of 136 cases of HCC were positive for GPC3, and the GPC3 expression correlated with HBs Ag positivity(P=0.000). Among 103 DN, 19 DN, including 15 high grade dysplastic nodules(HGDN) and 4 low grade dysplastic nodules(LGDN), were positive for GPC3. The ratio of HGDN(15/30, 50%) was obviously higher than that of LGDN(4/73, 5.48%), and this difference is statistically significant; The clonality assay demonstrated that 15 GPC3-positive DN and 28 out of 66 GPC3-negative DN were monoclonal; Chromosomal LOH showed that the frequency of LOH was differentially higher in GPC3-positive DN than GPC3-negative DN at D8S262(P=0.014), D11S1301(P<0.001), and D6S1008(P<0.001). 2) Compared with human normal hepatocyte line HL-7702, the expression levels of GPC3 m RNA were significantly up-regulated in Huh7 and Hep G2. 3) The expression levels of GPC3 m RNA increased in DN with GPC3-positive immunoreactivity, small HCC, and progressed HCC in turn. Among them, the expression between DN with GPC3-positive immunoreactivity and small HCC was significantly different(P=0.003), but not between small HCC and progressed HCC(P=0.145).4. The expression levels of mi R-202 and mi R-211 were significantly down-regulated in small HCC and DN with GPC3-positive immunoreactivity. Among them, the expression level of mi R-202 was decreased in DN with GPC3-positive immunoreactivity, small HCC, and progressed HCC in turn. Moreover, the expression difference between DN with GPC3-positive immunoreactivity and small HCC was significantly statistic importance(P=0.038), but no difference between small HCC and progressed HCC(P=0.432). mi R-211 did not show the significant difference among the above three lesions.5. The expression level of GPC3 m RNA and GPC3 protein were down-regulated in HCC cell lines after mi R-202 was overexpressed. In addition, Dual-Fluorescence Report assay demonstrated mi R-202 regulated negative directly GPC3.【Conclusions】 1. The expression level of five m RNAs related to GPC3, including mi R-202, mi R-1271-5p, mi R-1271-3p, mi R-204, and mi R-211, were down-regulated in HCC cell lines and progressed HCC. Among them, the expression level of mi R-202 was decreased in DN with GPC3-positive immunoreactivity, small HCC, and progressed HCC in turn. Moreover, the expression difference between DN with GPC3-positive immunoreactivity and small HCC was significantly statistic importance. The results indicated that mi R-202 might involve in hepatocarcinogenesis, and be an early molecular event. The downregulation of mi R-211 expression in HCC was firstly reported by us.2. The ectopic expression of mi R-202 and mi R-211 in HCC cells repressed cell proliferation, colony formation, invasion, and migration, but did not influence cell cycle. Overexpression of mi R-202 promoted cell apoptosis, but mi R-211 did not influence cell apoptosis in vitro. Moreover, micr ONTMagomir-202 repressed significantly HCC growth in vivo.3. The expression levels of GPC3 m RNA increased in DN with GPC3-positive immunoreactivity, small HCC, and progressed HCC in turn. Moreover, the expression difference between DN with GPC3-positive immunoreactivity and small HCC was significantly statistic importance, indicated that GPC3 play a key role in hepatocarcinogenesis.4. mi R-202 might target GPC3 directly by post-transcriptional regulation, and repress cell proliferation, colony formation, invasion, and migration by canonical Wnt pathway. Thus, it might repress the occurrence of HCC. |
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