ABSTRACT
@#AIM: To investigate the effect of silencing SIAH1 gene on H2O2-induced apoptosis of human lens epithelial cells.<p>METHODS: The human lens epithelial cell line HLE-B3 was cultured and divided into normal group, H2O2 group(cultured with medium containing 400μmol/L H2O2)and H2O2+siR-SIAH1 group(transfected with SIAH1 interference sequence, followed by cultured with medium containing 400μmol/L H2O2)and siR-NC group(transfected with negative control sequence, followed by cultured with medium containing 400μmol/L H2O2). The expression of SIAH1 gene in cells was detected by real-time fluorescent quantitative PCR. The apoptosis rate was detected by flow cytometry. The expressions of p38 MAPK, p-p38 MAPK, Bcl-2 and Bax proteins were detected by Western blot.<p>RESULTS: The relative expression levels of SIAH1 mRNA in the H2O2 group, siR-NC group and H2O2+siR-SIAH1 group were higher than that in the normal group(<i>P</i><0.05). The relative expression level of SIAH1 mRNA in H2O2+siR-SIAH1 group was lower than those in the H2O2 group and siR-NC group(<i>P</i><0.05). The apoptosis rates in the H2O2 group, siR-NC group and H2O2+siR-SIAH1 group were higher than that in the normal group(<i>P</i><0.05). The apoptosis rate in the H2O2+siR-SIAH1 group was lower than those in the H2O2 group and siR-NC group(<i>P</i><0.05). The expression levels of p38 MAPK and Bcl-2 proteins in the H2O2 group, siR-NC group and H2O2+siR-SIAH1 group were lower than those in the normal group, while the expression levels of p-p38 MAPK and Bax proteins were higher than those in the normal group(<i>P</i><0.05). The expression levels of p38 MAPK and Bcl-2 proteins in the H2O2+siR-SIAH1 group were higher than those in the H2O2 group and siR-NC group, while the expression levels of p-p38 MAPK and Bax proteins were lower than those in the H2O2 group and siR-NC group(<i>P</i><0.05).<p>CONCLUSION: Down-regulation the expression of SIAH1 gene could inhibit H2O2-induced apoptosis of human lens epithelial cell line HLE-B3, which might be related to inhibition of p38 MAPK signaling pathway activation.
ABSTRACT
MiRNA-429 (MicroRNA-429) are small, endogenous, and noncoding RNA molecules in the cells, which contribute to the post-transcriptional regulation of target gene expression. Research shows that miRNA-429 affects the occurrence and development of colorectal cancer. E3 Ubiquitin-protein ligase-1 (seven in absentia homolog-1) is a kind of enzyme encoded by the SIAH1 gene. SIAH1 contributes to protein abundance through proteolytic degradation of proteins and plays an important role in colorectal cancer. At present, there is a lack of specific biomarkers to diagnosis and effective methods to cure late-stage colorectal cancer. This article aims to summarize the progression of the role of miRNA-429 and SIAH1 in colorectal cancer tumorigenesis and progression, and explore the possibility of its clinical application.
ABSTRACT
BACKGROUND: A human orthologue of mouse S100A6-binding protein (CacyBP), Siah- 1-interacting protein (SIP) had been shown to be a component of novel ubiquitinylation pathway regulating beta-catenin degradation. The role of the protein seems to be important in cell proliferation and cancer evolution but the expression pattern of SIP in actively dividing cancer tissues has not been known. For the elucidation of the role of SIP protein in carcinogenesis, it is essential to produce monoclonal antibodies specific to the protein. METHODS: cDNA sequence coding for ORF region of human SIP gene was amplified and cloned into an expression vector to produce His-tag fusion protein. Recombinant SIP protein and monoclonal antibody to the protein were produced. The N-terminal specificity of anti-SIP monoclonal antibody was conformed by immunoblot analysis and enzyme linked immunosorbent assay (ELISA). To study the relation between SIP and colon carcinogenesis, the presence of SIP protein in colon carcinoma tissues was visualized by immunostaining using the monoclonal antibody produced in this study. RESULTS: His-tag-SIP (NSIP) recombinant protein was produced and purified. A monoclonal antibody (Korea patent pending; #2003-45296) to the protein was produced and employed to analyze the expression pattern of SIP in colon carcinoma tissues. CONCLUSION: The data suggested that anti-SIP monoclonal antibody produced here was valuable for the diagnosis of colon carcinoma and elucidation of the mechanism of colon carcinogenesis.