The Expression Of Let-7 MicroRNA During The Process Of The Mouse Embryonic Lung Development And The Construction Of The Adenovirus Shuttle Plasmid

MicroRNAs (miRNAs) are a class of small non-coding RNAs, about 20～24 nucleotides in length. They can inhibit target genes translation or degrade them directly through combination with the complementary sequences in the 3'-UTRs of target mRNAs. The let-7 miRNAs were originally found in the nematode Caenorhabditis elegans, where they mainly regulated the timing of cell division. Increasing evidences indicate that let-7 miRNAs are associated with animal organs development and human diseases. In this study, real-time PCR was used to compare the transcription levels of the eight members of let-7 family during the process of the mouse embryonic lung development.We found that a increase in mRNA level of these eight genes was detected with increasing age, from the early of pseudoglandular stage to the early of saccular stage, but until the late of saccular stage, the expression of let-7a,let-7d,let-7e,let-7f,let-7i decreased slightly, while let-7b, let-7c did not have the obvious change.Then we examined the expression patterns of let-7a and let-7d by in situ hybridization. Section in situ hybridization indicated that let-7a and let-7d expression in the developing murine lung were strongly both in the mesenchyme and the respiratory epithelium, but the expression of let-7a and let-7d were then downregulated gradually at the three days after birth(P3), in a similar manner to the expressions of let-7a and let-7d which were detected by real-time PCR. Moreover, we constructed three adenovirus shuttle plasmids:pDC316-mCMV-mK-ras,pDC316-CMV-control and pDC316-CMV-let-7a.They were transfected into cells, then using RT-PCR and western blot to detect the expression of the constructed plasmids.We showed that pDC316-CMV-control as the control group, pDC316-CMV-let-7a and pDC316-mCMV-mK-ras can achieve over-expression in vitro in both mRNA level and protein level,which provided the basis for constructing adenovirus,studying the negative regulator relationship between let-7 miRNAs and mK-ras and the possible molecular mechanism of let-7 miRNAs regulating the developmental processes of mammal embryonic lung.