The Effects Of Silencing Galectin-3Expression By Lentiviral-mediated RNA Interference On The Prolactinoma Cells

Objective：With Galectin-3gene knockdown in the prolactinoma cells in vitro, it provideexperimental basis for the treatment of prolactinomas for the next body prolactinomas and animportant basis for the preliminaryexplore the Galectin-3in the occurrence of the mechanismin the development of prolactinomas.Methods:1. Prolactinoma cell culture and hormone levels of measurement: select the surgicalremoval of prolactinomas, using the suspension cell culture, regular observation of the growthof the prolactinoma cells, and hormone secretion determination.2. The effects of prolactinoma cells by recombinant lentiviral vector-mediated genesilencing of Galectin-3in vitro.Galectin-3-shRNA lentiviral vector (LV-shRNA-Galectin-3) Packaging by Shanghai JiKai Technology Co., Ltd.Cultured prolactinoma primary cells are grouped, divided into experimental divided intotransferred group (LV-shRNA-Expression of Galectin-3), the negative control group(independent of the sequence of the hairpin structure of the virus carrier, provided byShanghai Ji Kai) and The blank control group (without adding any plasmid). Lentiviral vectortransfected target cells and detect the reduction efficiency of Galectin-3knock by Real-timePCR and Western blot methods, the MTT assay prolactinoma cell viability. Then we candetermine the effect of prolactinoma cell proliferation and apoptosis by theLV-shRNA-Galectin-3mediated by the RNAi.3. Statistical methodsThe experimental data were analyzed using SPSS16.0statistical software, the groupswere compared using analysis of variance, the two groups were compared using the SNKmethod.Results:1. Prolactinoma cells in culture and hormone levels were measured.Observed under an inverted microscope, primary cultures of prolactinoma cells are ovalor spindle-shaped, part of prolactinomas start semi-adherent growth after24hours, most weresuspended growth. Cultured for about10days, fibroblasts began to increase in the numberand gradually replace the prolactinoma cells. The replaced culture medium was used by a double antibody radioimmunoassay method for detection. The assay showed that culturedprolactinoma cells secrete the hormone levels in about the seventh day.2. shRNA knock on reduction efficiency of Galectin-3in the cells of prolactinomas.Real-time PCR results showed that after the LV-shRNA-Galectin-3transfected withprolactinoma cells their expression of Galectin-3mRNA was lower than knockdown groupnot more than70%, p <0.05, the negative control group and blankgroup expression levelswere not decreased significantly.Western blot test results are that shRNA significantly reduced the prolactinoma cellsGalectin-3protein expression, the inhibition efficiency of more than70%, p <0.05, while thenegative control group and blank group compared with no significant difference, p>0.05.3. The vitality of the cells of prolactinomas was detected by MTT.Compared with negative control group and blank control group, transfection group has astatistically significant difference between groups, p <0.05. Knockdown of Galectin-3caninhibit the cell viability.Conclusion: Initially lentivirus-mediated Galectin-3gene silencing in vitro knockdown theprolactinoma cells mRNA and protein, the cell viability decreased. Galectin-3plays animportant role in the occurrence and development of the pituitary adenoma.