Inhibition Immune Evasion Of Colorectal Cancer Cells In Vitro With Small Interfering RNA Of FLIP

Objective:Colorectal cancer is one of the most general intestinal tumors and none of therapies can effectually prevent it from metastasis and recurrence by now. The most possible reason about this phenomenon is that tumor cells reduce their apoptotic sensitivity, evade immune surveillance and survive upon many kinds of signals. Thus, how to exclude the mechanism of tumor evasion and find out new targets of tumor therapy has been the hotspot. Here, we focus on FLIP, one . of the inhibitory proteins of Fas-mediated apoptotic pathway. After RNA interfering, the mRNA expression level of FLIP is decreased and the apoptotic sensitivity is increased in the transfected colorectal cancer cells. We hope our study could exclude whether there is FLIP-associated apoptotic evasion in colorectal cancer cells and put a new way on tumor gene therapy. Methods:Four colorectal cancer cell lines were cultured in vitro. After direct immunofluoscent FACScan and semi-quantitative RT-PCR analysis, the optimal experimental model was selected. HT-29 was transfected with siRNA by electroporation. The inhibitory of FLIP mRNA was detected by relative semi-quantitative PCR. After that, the transfected cells were treated with agonistic anti-Fas antibody in order to induce apoptosis. We used Annexin V and cell cycle analysis to determine the apoptotic sensitivity of transfected HT-29. Results:1. Combining the levels of Fas and FLIP, HT-29 was selected as the best experimental model.2. After transfecting siRNA into HT-29, the expression level of FLIP mRNA wasdecreased from 78.55 % to 27.48 % and the interfering effectiveness is 65.02 %. 3. After treated with agonistic anti-Fas antibody, the apoptotic rate of transfected HT-29 was increased from 1.76 % to 29.5 % and we can find an "apoptotic peak" on the cell cycle analysis. Conclusions:1 . There is FLIP-associated immune evasion mechanism in colorectal cancer cell lines exactly.2. As an important factor in Fas-mediated apoptotic pathway, FLIP could be one of the effective targets in tumor gene therapy.3. Since the mRNA level of FLIP is effectually reduced after transfecting siRNA into HT-29 cell line, RNAi will possess expansive applicable prospect in gene silencing area.