| Background and Objective BTG2 is a p53 induced anti-proliferation tumor suppressor gene that has the characteristic of hepatocyte(liver)-specific. BTG2 is involved in so many important biological functions during carcinogenesis, including cell differentiation, anti-proliferation, cell apoptosis, cell cycle and DNA damage repair and so on. BTG2 is an important regulator in the signal transduction pathway of mechanism of carcinogenesis. mi RNAs are small non-coding RNA that exist widely in human and play an important role in biological function. mi RNAs have such a great clinical significance in early diagnosis and prognosis of tumors and mi RNAs regulate biological behavior on the upstream of target genes by binding to the 3’-untranslated region(3’-UTR) of target genes. In our previous study, mi R-21 and mi R-29 b were expressed aberrantly in hepatocellular carcinoma according to micro RNA array and bioinformatics analysis, and the expression of mi R-21 and mi R-29 b were associated with aberrant expression of BTG2. Therefoce, the purpose of this study is to provide further insight into the functions and significances of mi R-21 and mi R-29 b on regulation BTG2 protein during hepatocarcinogenesis, and to discuss the mechanism of biological behavior of liver cancer cells in which mi RNAs were involoved, and to look for novel biomarkers that could be used to assess the diagnosis, treatment and prognosis of hepatocellular carcinoma.Materials and Methods1. Immunohistochemistry and tissue microarray were used to discuss the relationship between BTG2 protein expression and the prognosis of hepatocellular carcinoma followed by statistical methods.2. q RT-PCR, MTT assay, apoptosis analysis, flow cytometry cell cycle, cell migration, wound scratch assay and transwell invasion assay were used to validate the expression of mi R-21 and its biological functions in liver cancer cells. Bioinformatic analysis, dual luciferase report gene system, vector construction and western blot were used to perform that BTG2 gene was a direct target gene of mi R-21.3. q RT-PCR, MTT, apoptosis analysis, flow cytometry cell cycle and transwell invasion assay were used to validate the expression of mi R-29 b and its biological functions in liver cancer cells. Bioinformatic analysis, dual luciferase report gene system, vector construction and western blot were used to perform that BTG2 gene was a direct target gene of mi R-29 b.4. The clinical data of patients with hepatocellualr carcinoma, and 50 plasma samples from patients and 12 normal human plasma samples were collected to identify the relationship between mi R-21/mi R-29 b and stage, distant metastasis and AFP of hepatocellular carcinoma patients by q RT-PCR(SYBR Green)on the basis of statistical analysis.Results1. Compared with gastric cancer tissue, cervical cancer tissue and their paracarcinoma, BTG2 expression has the characteristic of hepatocyte(liver)-specific by immunohistochemistry. BTG2 expression staining in cytoplasm was scored as strong(+++) in paracarcinoma of liver cancer. According to the analysis of tissue microarray, the stronger BTG2 expression stained in cytoplasm, the worse survival patients had, and when patients with BTG2 expression stained both in cytoplasm and nucleus, and mainly staining in nucleus, then those patients had better survival.2. The expression level of mi R-21 in liver cancer cells was significantly higher than that in normal liver cells. Inhibiting mi R-21 increased apoptosis and reduced cell proliferation, migration, invasion and arrested G2/M cell cycle in liver cancer cells. BTG2 protein expression was inhibited by high mi R-21 expression and increased by inhibition of mi R-21 expression in liver cancer cells. BTG2 was a direct target gene of mi R-21, and mi R-21 mainly could regulate cell cycle by BTG2 in liver cancer cells.3. The expression level of mi R-29 b in liver normal cells was significantly higher than that in liver cancer cells. Increasing mi R-29 b inhibited invasion and arrested cell cycle in S phase in liver cancer cells but not to affected proliferation and apoptosis in liver cancer cells. BTG2 protein expression was inhibited by high mi R-29 b expression and increased by inhibition of mi R-29 b expression in liver cancer cells. BTG2 was a direct target gene of mi R-29 b, and mi R-29 b could mainly regulate invasion by BTG2 in liver cancer cells.4. The expression level of mi R-29 b was aberreantly expressed in the plasma of HCC patients, and it was decreased sharply in the plasma of liver cancer patients comparing with control ones. With the analysis of clinical data, the expression level of mi R-29 b was associated with stage(p=0.001) and distant metastasis(p<0.001), however, there was no relationship between mi R-29 b and AFP. Compared with the control group, the expression level of mi R-21 in the plasma of liver cancer patients was a little higher, but there was no statistical difference(p=0.087). With the analysis of clinical data, the expression level of mi R-21 was not associated with stage and distant metastasis but was associated with AFP(p=0.007).Conclusion1. We first discovered that BTG2 expression has the characteristic of hepatocyte(liver)-specific. The stronger BTG2 expression stained in cytoplasm, the worse survival patients had, and when patients with BTG2 expression stained both in cytoplasm and nucleus, and mainly staining in nucleus, then those patients had better survival, which indicated that BTG2 could be used as an important biomarker in the prognosis of hepatocellular carcinoma.2. The expression level of mi R-21 was significantly increased in liver cancer cells, and it was involved in the regulation of cell proliferation, migration, invasion, apoptosis and cell cycle. BTG2 was the direct target gene of mi R-21 and it has a negative correlation with expression of mi R-21, and BTG2 may be involved in the biological effects of mi R-21 on liver cancer Hep G2 cells.3. The expression level of mi R-29 b was sharply decreased in liver cancer cells, and it was involved in the regulation of invasion and cell cycle. BTG2 was the direct target gene of mi R-29 b and it has a negative correlation with expression of mi R-29 b, and BTG2 was regulated by mi R-29 b and may be involved in the biological effects of mi R-29 b on liver cancer Hep G2 cells.4. BTG2 could be regulated simultaneously by mi R-21 and mi R-29 b, and the mechanism of the cross-over regulation needs further investigation.5. The expression level of mi R-29 b and mi R-21 were aberreantly expressed in the plasma of liver cancer patients. The expression level of mi R-21 was associated with AFP, and the expression level of mi R-29 b was inversely related to stage and distant metastasis, which indicated that both mi R-21 and mi R-29 b may be used as biomarkers for early diagnosis, stage and distant metastasis of hepatocellualr carcinoma, and they provide a basis for screening biological markers on diagnosis, treatment evaluation and prognosis of hepatocellular carcinoma. |
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