Knockdown Of Li-cadherin Increases Metastatic Behaviors Of LoVo Cells

Background:Colorectal cancers are the most common in malignant tumors, remains the second leading cause of cancer deaths worldwide with particular high incidences in developed country. Metastasis and invasion are the main factor to do with the prognosis and death rate of the patients with colorectal cancers. Tumor invasion involves two independent processes relevant to cell adhesion-detachment of cells from the primary tumor and reattachment to the targets. Cadherin belongs to one of the adhesion molecule families, which plays a crucial role in establishing cell-cell interaction. Li-cadherin is only expressed in liver and intestine of the rat, it was therefore given the name Li-cadherin. Some studies illustrate that Li-cadherin is involved in biological behaviors of tumor cells. Nevertheless, the exact roles and mechanisms of Li-cadherin in the invasion and metastasis of human cancer have not been fully established, and remains to be elucidated.Aims:Constructing a Li-cadherin siRNA expression vector and establishing a Li-cadheirn knock-down model of colorectal cancer LoVo cells. And then, to evaluate whether knock-down of Li-cadherin by RNAi can result in the process of invasion and metastasis in LoVo cells. To enlighten us that interference with Li-cadherin may serve as a new method in future therapeutic strategies of colorectal cancer.Methods:1. Li-cadherin siRNA expression vector was constructed in vitro.2. After transfection with pU6-Li-cadherin-shRNA and pU6-control-shRNA, hygromycin (G418) was added to select stable transfection cell lines.3. RT-PCR, western blot and immunofluorescence were performed to evaluate the expression and location of Li-cadherin.4. In vitro assays:Wound healing assay and in vitro cell migration assay using Millicell chamber were performed to investigate the effect of the Li-cadheirn on cellular motility. To evaluate the effect of downregulation of Li-cadherin expression on the invasion of cancer cells in vitro invasion assay, Millicell chamber was used. To assess the effect of down-regulation of Li-cadheirn expression on the adhesion to ECM of LoVo cells adhesion assay,96-well plates coated with BD MatriegelTM Matrix was employed.5. To evaluate the effect of downregulation of Li-cadherin expression on the expression levels and activities of MMP-2 and MMP-9, RT-PCR and gelatin zymography were employed.Results:1.Li-siRNA expression vector was confirmed to be successfully constructed with the assay of enzyme digestion and sequencing.2. Li-cadherin mRNA expression reduced to 43.61% in pU6-Li-cadherin-shRNA expressing Lovo cells (P<0.05), and Li-cadherin mRNA expression in pU6-control-shRNA expressing Lovo cells was not reduced (P>0.05).3.Li-cadherin protein expression reduced to 36.13% in pU6-Li-cadherin-shRNA expressing Lovo cells(P<0.05), and Li-cadherin protein expression in pU6-control-shRNA expressing Lovo cells was not reduced (P>0.05).4. Immunofluorescence staining showed that Li-cadherin was mainly present on the cell membrane, and Li-cadherin expression in LoVo cells was diminished by pU6-Li-cadherin-shRNA.5. Wound healing assay showed that the migrating distance of untreated LoVo cells was the same as that of stably expressing pU6-control-shRNA LoVo cells at 24 hours. However, a higher degree of wound healing in expressing pU6-Li-cadherin-shRNA LoVo cells was seen after 24 hours (P<0.05). Millicell chamber migration assay discovered that the expressing pU6-Li-cadherin-shRNA LoVo cells showed remarkably increased cellular migration compared with untreated LoVo cells and expressing pU6-control-shRNA LoVo cells(P<0.05).6. RT-PCR showed that the expression levels of MMP-2 and MMP-9 in untreated LoVo cells were the same as that in stably expressing pU6-control-shRNA LoVo cells. The higher mRNA expression level of MMP-2 and MMP-9 were detected in expressing pU6-Li-cadherin-shRNA LoVo cells (P<0.05).9. Gelatin zymography showed that expression levels of the proMMP-2, proMMP-9 and their active forms were all significantly enhanced in pU6-Li-cadherin-shRNA expressing LoVo cells compared with untreated LoVo cells and LoVo cells stably expressing pU6-control-shRNA (P< 0.05).Conclusion:1. Li-cadherin siRNA expression vector was successfully constructed in vitro.2. Li-cadherin siRNA expression vector was effectively inhibited the Li-cadherin expression, and the mRNA and protein inhibition ratios were 43.61% and 36.1% respectively.3. The present studies show that silencing Li-cadherin has positive actions in the processes of LoVo cells invasion and metastasis.4. Downregulation of Li-cadherin expression induced increasing expression levels and activities of MMP-2 and MMP-9 in LoVo cells. Li-cadherin siRNA could promote the invasive and metastasis potential of colorectal cancer LoVo cells, and gene interference targeted on Li-cadherin may be serve as a new method in future therapeutic strategies of colorectal cancer.