The MiRNA Let-7 And Its Target Genes In Hela And Human Endometrium Stromal Cells

MicroRNAs are a class of endogenous non-coding small RNA, which can regulate gene expression through specific degradation of mRNAs or inhibiting its translation in post-transcriptional levels. MicroRNA let-7 family was first discovered in C. elegans where it was shown to regulate development, cellular proliferation and differentiation. There are 13 kinds of let-7 precursor sequence in the human genome, which can produce 10 kinds of mature let-7 miRNAs. Our previous study showed that let-7 was decreased in miscarriage decidua. We hypothesized that let-7 is likely involved in early pregnancy maintenance by regulating the expression of target genes.Objective:1. To predict the target genes of let-7f-5p, let-7g-5p and let-7i-5p respectively; 2. To confirm the relationship between let-7f-5p and IGF2BP-1, let-7g-5p and IGF-2R as well as let-7i-5p and LIF, IL-6 in vitro.Methods:target genes of let-7f-5p, let-7g-5p and let-7i-5p were predicted by informatics tools online, such as TargetScan, PicTar, MiRanda and so on. Then overexpression or inhibit the expression of let-7f-5p, let-7g-5p and let-7i-5p by transfecting mimics or inhibitors, and analysis the expression of IGF2BP-1, IGF-2R, LIF and IL-6 using qRT-PCR and iTRAQ (ELISA).Results:1. The seed sequences of let-7f-5p matched the 3’UTR of IGF2BP-1 mRNA. Overexpression of let-7f-5p in three different kinds of cells, the expression of IGF2BP-1 mRNA was significantly down-regulated (P<0.0.5). When inhibit the expression of let-7f-5p, the expression of IGF2BP-1 mRNA was significantly up-regulated (P<0.05). Furthermore, Overexpression of let-7f-5p in Hela cells, the expression of IGF2BP-1 protein was significantly down-regulated (P<0.05). When inhibit the expression of let-7f-5p in Hela cells, the expression of IGF2BP-1 protein was significantly up-regulated (P<0.05).2. The seed sequences of let-7g-5p matched the 3’UTR of IGF-2R mRNA. Overexpression of let-7g-5p in Hela cells, the expression of IGF-2R mRNA and protein were significantly up-regulated (P<0.05). When inhibit the expression of let-7g-5p, the expression of IGF-2R mRNA and protein were significantly down-regulated (P<0.05).3. The seed sequences of let-7i-5p matched the 3’UTR of LIF mRNA and IL-6 mRNA. Overexpression of let-7i-5p in HEK293T, Hela and H-ESC cells, the expression of LIF and IL-6 mRNA were significantly up-regulated (P<0.05). When inhibit the expression of let-7i-5p, the expression of LIF and IL-6 mRNA were significantly down-regulated (P<0.05).Conclusion:The expression of let-7f-5p and IGF2BP-1 on mRNA and protein level were significant negative correlation in vitro. Significant positive correlation was found between let-7i-5p and LIF and between let-7i-5p and IL-6 on mRNA levels in vitro. Significant positive correlation was found between let-7g-5p and IGF-2R in Hela cells.