| ?Background and Objective?Colorectal cancer(CRC) is one of the most common malignant cancers. Metastasis, recurrence are the main cause of death of the disease, and its metastasis mechanism has become the current research focus. In the past decade, epithelial–mesenchymal transition(EMT) has been increasingly recognized to play a pivotal role in promoting colorectal carcinoma cells invasion and metastasis. Martin et al.found that histidine triad nucleotide binding 2(HINT2) displays suppression property on the growth of tumor tissue, and the protein can induce the apoptosis of tumor cells. Our previous study found that HINT2 expression level in the colorectal cancer tissue was significantly lower than that in normal colon tissue, and HINT2 has an inhibitory effect on migration and invasion ability of colorectal cancer cells. Therefore, the purpose of this study is exploring the role and related molecular mechanism of HINT2 in the epithelial-mesenchymal transition of colon carcinoma cells.?Method?1. Construction of lentivirus that downexpression of HINT2Colon carcinoma cell line SW480 was infected with Hint2 shRNA(h) Lentiviral Particles, and HINT2 was knocked out, and stable cell lines were established by the method of limited dilution cloning.2. The effect of HINT2 on EMT in SW480 colon cancer cell.2.1. After HINT2 was knockdown, western blot were used to evaluate the expression of HINT2 and EMT markers including E-cadherin, N-cadherin and Vimentin in SW480 cells. At the same time, morphologic change was observed with microscope.2.2. Lentivirus was used to down regulate HINT2 in SW480 cells.The effect of HINT2 on E-cadherin transcription was examined with reporter assay using p GL3-promoter plasmids containing E-cadherin promoter.3. The role of HINT2 in the migration and invasion abilities of SW480 cellsWith the HINT2 down-regulation methods,the changes of migration and invasion ability were tested by wound scratch assay and transwell assay.4. Molecular mechanism of HINT2 in the epithelial-mesenchymal transition of colon carcinoma cells.4.1. Real-time PCR assay was used to detect the effect of HINT2 downregulation on the expression of EMT related transcription factors SNAI1, SNAI2, TWIST2, ZEB1, ZEB2, TWIST1 in SW480 cells.4.2. Double silencing lentivirus HINT2+ZEB1 sh RNA Lentiviral Particles was constructed.4.3. SW480 cells were infected with lentivirus Hint2 sh RNA(h) Lentiviral Particles?HINT2+ZEB1 sh RNA Lentiviral Particles or Control sh RNA Lentiviral Particles-A. Western blot were used to evaluate the expression of HINT2 and EMT markers including E-cadherin, N-cadherin and Vimentin in SW480 cells.4.4. SW480 cells were infected with lentivirus above. The role of ZEB in HINT2 dependent regulation of E-cadherin transcription was examined withluciferase reporter assay using p GL3-promoter plasmids containing E-cadherin promoter.5. Statistical analysisStatistical analysis was performed using SPSS 17.0(SPSS Inc.), Data are shown as mean ± standard error of the mean(SD). Student's t-test was used for two-group comparisons. P < 0.05 was considered statistically significant.?Results?1. SW480 cell lines with low expression of HINT2 were successfully constructed.2. The knockdown of HINT2 significantly increased N-cadherin and vimentin levels,inhibited E-cadherin expression and resulted in striking morphological change in SW480 cells.3. Luciferase assay showed that HINT2 promoted the expression of E-cadherin through the transcriptional level.4. After downregulation of HINT2 expression, wound scratch assay and transwell assay showed that the invasion and migration ability of SW480 cells were significantly enhanced.5. Double knockdown of HINT2 and ZEB1 nearly canceled out the effect of HINT2 silencing on EMT. Inhibition of ZEB-1 expression did not resulted in decreased E-cadherin expression despite repression of HINT2 expression.?Conclusions?1. HINT2 can inhibit EMT in SW480 colorectal cancer cells.2. HINT2 can positivelyregulate the transcription of E-cadherin in SW480 colorectal cancer cells.3. HINT2 can effectively inhibit the migration and invasion of colorectal cancer cells.4. ZEB1 mediates HINT2-dependent regulation of the EMT program.5. The negative regulation of low expression of HINT2 on E-cadherin transcriptional activity was dependent on ZEB1... |
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