Effects of RNAi on mRNA expression of VEGF and VEGF receptor during granule cell in vitro culture of ovine

ObjectiveThe paper studied effects of RNA interference(RNAi) on mRNA expression of vascular en-dothelial growth factor(VEGF) and its receptors in the process of granule cells cultured in vitro, in order to lay a foundation for research in signaling pathway of VEGF during process of oocyte in vitro maturation and embryonic development.MethodThis research used electroporation to import two double chain small interfering RNA fragments which were de-signed for VEGF receptors Flt-1 and KDR/Flk-1 into ovine granule cells, and detected Flt-1 mRNA and KDR/Flk-1 mR-NA levels variation of ovine granule cells cultured in vitro at different times by real-time quantitative PCR. The researchers named the groups imported with Flt-1 interference fragment and KDR/Flk-1 interference fragment which were screened out in early experiment as treatment group Ⅰand treatment group Ⅱrespectively, and the group imported with both Flt-1 in-terference fragment and KDR/Flk-1 interference fragment as treatment group Ⅲ, and the group imported with interference fragment which had no RNAi effect as control group. ResultResults indicated that, during the process of ovine granule cells cultured in vitro, mRNA expression level of VEGF was 102 times higher than that of Flt-1 and 103 times higher than that of KDR/Flk-1. On the 1st day, the VEGF mRNA expression levels of treatment groupⅠ, treatment groupⅡand treat-ment group Ⅲ were 2.62×10-2, 3.54×10-2 and 2.94×10-2 respectively, which were significantly higher than that of control group(P<0.01, the same below). The expression levels of the three treatment groups reduced since the 2nd day and tended to be similar to that of control group on the 6th day. During the culture process, Flt-1 mRNA level of treatment group Ⅱdropped from 1.02×10-1(on the 1st day) to 8.99×10-4(on the 7th day), and it had been significantly higher than that of other groups. Flt-1 mRNA expressions of both treatment groupⅠand treatment groupⅢcould be barely detected, then, as time passing by, they rose and tended to be similar to those of other groups on the 8th day. KDR/Flk-1 mRNA level of treatment groupⅠwas significantly lower than that of control group since the 1st day. KDR/Flk-1 mRNA in both treatment group Ⅱand treatment group Ⅲ could be barely detected from the 1st day to the 3rd day, and they rose with time passing by and tended to be similar to those of other groups on the 9th day. ConclusionTwo pairs of effective interference fragments have good performance on interfering during the process of ovine granule cells in vitro culture, which results in change of mRNA expression levels of VEGF, Flt-1 and KDR mRNA, and affects transmission of growth signal concerning granule cells.