| Objectives:To investigate the regulatory role of Prohibitin (PHB) in proliferation, apoptosis, and cycle of colorectal carcinoma (CRC) cell lines based on RNA interference (RNAi) technology, and provide theoretical and experimental basis for the treatment of CRC by RNAi.Methods:Western blot was used to assay the expression of PHB in human CRC cell lines to screen cell lines which highly expressed PHB protein. The tumor cells were transiently transfected by three specific small interfering RNA (siRNA) named PHB 138, PHB 173, and PHB539 via vehicle Lipofectamine2000. Then, two fragments which reduced the expression of PHB were screened after the transfection for 72 h. Meanwhile, the negative control was selected in the present study. Cell Counting Kit-8 (CCK-8), clone formation, and soft agar clone formation assay were used to determine the cell proliferation. Further, the PHB was silenced on the basis of oxaliplatin induction. Subsequently, fluorescence-activated cell sorting (FACS) was employed to assay the cell cycle and apoptosis. The activities of apoptosis related molecules such as Caspase-3 and Caspase-9 were also measured. And, the typical cell apoptotic morphous and nuclear alternatios were also recorded under a fluorescence microscope.Results:Western blot result revealed that the PHB protein was expressed in seven CRC cell lines and the expression was highest in HCT 116. The specific three RNAi fragments targeting PHB displayed significant suppression on the expression of PHB protein after the HCT 116 was transiently transfected. And, the suppression efficiency of fragments PHB138 and PHB539 were better than PHB173. The cell proliferation ability assay suggested siRNA targeting PHB could suppress the growth of HCT 116 cells. Clone formation, and soft agar clone formation assay showed that the number of HCT 116 cell clones after the trasfection was less than that in the negative group. The plate cloning efficiency was NC (43.4±2.11)%,PHB138 (31.1±3.05)%,PHB539 and (12.4±1.26)%, PHB138, and PHB539, respectively. Correspondingly, soft agar cloning efficiency was (8.03±0.912)%,(4.22±0.569)%, and (2.82±0.621) %o in NC, PHB138, and PHB539, respectively. Cell cycle assay demonstrated that the proportion of HCT 116 cells in phase S was significantly decreased after the transfection and the apoptotic peak was raised. The proportion in phase S was (47.7±8.75)%, (33.4±0.510)%, and (31.7±1.91)% in NC, PHB138, and PHB539, respectively. The activity of Caspase-3 was (1.50±0.392) and (1.81±0.416) in PHB138, PHB539 respectively. The activity of Caspase-9 was (2.02±0.435) and (2.81±0.517) in PHB138 and PHB539, respectively. Obvious apoptosis was observed under a fluorescence microscope and the proportion and density was stronger than that in the negative control, respectively.Conclusions:Human CRC HCT 116 cells have higher expression of PHB; The three siRNAs targeting PHB could efficiently silence the PHB expression in HCT 116; Blocking the PHB expression of HCT 116 in vitro could inhibit the proliferation and induce apoptosis; PHB gene in CRC gene therapy may be a potential target. In our study, we screened two fragements PHB138 and PHB539 which displayed a better suppression effect on CRC cells. RNAi provided a theoretical foundation for gene therapy of CRC.
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