Expression Of PLSCR1in Colorectal Cancers And Its Biological Function

Phospholipid scramblase1(PLSCR1) is a calcium-binding, multiple-palmitoylated type Ⅱ endofacial plasma membrane protein. Initial studies revealed that PLSCR1participates in the transbilayer movement of the phospholipids, but new research has shown that PLSCR1contributes to cell signaling pathways. More and more studies propose that PLSCR1not only plays an important role in cell proliferation, differentiation, and apoptosis, but also contributes to the pathogenesis and progression of cancers. The expression level of PLSCR1is significantly elevated in malignant adenocarcinoma compared with normal colorectal mucosa, and the plasma level of PLSCR1was increased in colorectal cancer (CRC) patients, especially in patients with early stage CRC. A univariate analysis with the Cox regression model also indicated that increased PLSCR1expression was associated with a poor prognosis, suggesting that over expression of PLSCR1may be an early but important marker in CRC. This also suggests that PLSCR1over expression may have a significant role in tumorigenesis and tumor progression. However, the function of the PLSCR1protein in the pathogenesis and progression of CRC is still unclear.Objective:To detect the expression of PLSCR1in colorectal cancers and its biological function. To study the possibility of PLSCR1as a potential gene therapy target for CRC.Methods:The expressions of PLSCR1in CRC cells were assayed by immunofluorescent cytochemistry. The paraffin-embedded specimens from cases of CRC and normal mucosa and liver metastatic carcinoma were also examinaed using immunohistochemistry. Western-blot was used to detect the expression of PLSCR1in both CRC tissues and corresponded normal colorectal mucosa. Reverse transcriptional PCR was used to detect the mRNA of PLSCR1in CRC tissues and normal colorectal mucosa. Primary culture of CRC cells was carried out in vitro, and Transwell assay was used to detect the invasion ability of CRC cells with different expression levels of PLSCR1. We designed three siRNA oligonucleotide segments targeted at PLSCR1. Successful transfection was confirmed. The biological behavior of the cells in proliferation, adhesion, migration and invasion was determined. In addition, we generated a Lovo-subcutaneous mice model to observe anti-tumor effects and its mechanism from RNA interference (RNAi).Results:First, PLSCR1protein was mainly located in the cell membrane of the CRC cells. The expression level of PLSCR1was significantly elevated in CRC, liver metastatic carcinoma compared with normal colorectal mucosa. The elevated PLSCR1expression was correlated with metastasis and indicated a poor prognosis for colorectal carcinoma.Second, primary culture of frozen-thawed CRC tissue which was reserved by the cryopreserved agent can be carried out. The expression level of PLSCR1protein and the mRNA level of PLSCR1were significant increased in CRC tissues compared with normal colorectal mucosa. In invasion test, the number of invaded cells in group of high level of PLSCR1was significantly higher than the number in group of relative low level.Third, the siRNA-390oligonucleotide segment had the best silencing effect. After transfection, Lovo cell proliferation was significantly inhibited compared with the controls in MTT assay. The Ln and Fn adhesion assay showed Lovo cell adhesion was also significantly inhibited. In the migration assay, the number of migrating cells in the PLSCR1siRNA group was significantly lower than the number in the siRNA-N group and in the control group. In an invasion test, the number of invading cells in PLSCR1siRNA group was significantly lower than the number in the siRNA-N group and in the control group. Silencing of PLSCR1by siRNA inhibits the proliferation, adhesion, migration and invasion of Lovo CRC cells.Finally, the tumor masses in PLSCR1siRNA-treated mice were significantly smaller than those in control mice. The expression of epidermal growth factor receptor were down-regulated in PLSCR1siRNA treated tumors. Our results demonstrated that PLSCR1targeted RNAi was able to successfully suppress PLSCR1gene expression and inhibit cell growth and invasion of Lovo CRC in vitro and in vivo.Conclusion:PLSCR1may be a potential gene therapy target for for the treatment of CRC. The anti-tumor effects of PLSCR1targeted RNAi may inhibit the signaling pathways via down-regulate the expression of EGFR.