LAMA5 Expression Changes During The Critical Period Of Palate Development And Gene Silencing Interferes With The Development Of Palate In Vitro

Objective: The blood samples were taken from 30 children with non-syndromic cleft lip with or without palate to search for possible candidate pathogenic genes by whole exon sequencing and bioinformatics technology in preliminary experiment.The highest mutation rate of LAMA5 gene rs145192286 sites were screened.This study relied on the experimental basis of the previous stage to explore the relationship between the LAMA5 gene and the non-syndromic cleft lip with or without palate.This experiment observed the expression changes in the development fusion of the mouse embryo’s palate,and then used RNAi technology to target interference with the LAMA5 gene to observe the fusion of palate and to see whether the palate fusion was under the specific inhibition target gene.Methods: 1.The C57BL/6J pregnant mice were sacrificed on day 12.5(E12.5),E13.5,E14.5,E15.0 and E15.5 by neck broken dislocation method.The embryonic heads of mice only were preserved.The expression of the LAMA5 protein in embryonic palate was located and semi-quantitatively detected using Immunohistochemical staining and Western Blotting.2.The shRNA adenovirus vectors with green fluorescent markers are designed to inhibit the expression of the LAMA5 gene and then it was introduced into E13.5 palatal process tissue mass which were cultured in vitro in an animal model of palatal organ.The fusion of palatal process was cultured for 48 hours and observed under inverted fluorescence microscope.Finally,the changes of gene LAMA5 expression after interference were definitely detected by real-time fluorescence quantitative PCR and Western blotting,respectively.Results: 1.Immunohistochemistry showed that LAMA5 was mainly located on the basement membrane,which belongs to a special extracellular matrix.It expressed on the basement membrane of the middle palatal ridge epithelium at E12.5 and E13.5,and the expression of E14.5 which was only in the extracellular matrix of palatal process and adjacent oral cavity and nasal cavity was significantly lower than that of E13.5.At E15.0,it was only expressed in the contact middle palatal suture,however no expression of LAMA5 was found with the fusion of palatal process at E15.5.Combined with semi quantitative absorbance value to analyze the results of immunohistochemical staining,the absorbance value gradually decreased with the development of palatal process to maturity,and the expression amount also decreased.So,there was significant difference between each group and the former group(P < 0.05).Western Blotting showed that the expression of LAMA5 was the most at E12.5 and the least at E15.5.2.Both sides of the palatal frames on the LAMA5-shRNA3 experimental group and the EGFP-NC control group converged to the middle and gradually fused after transfection for 48 hours.There was still a gap between bilateral palatal processes and were not fused on LAMA5-shRNA1 group and LAMA5-shRNA2 group after interference.The PCR results showed that compared with the HBAD-EGFP-NC control group,the LAMA5-shRNA1 group and the LAMA5-shRNA2 group decreased by 63.7% and 48.1% respectively,which was statistically significant.However,there was no significant decrease expression on the LAMA5-shRNA3 experimental group,and there was no significant difference in the expression level between the two groups(P>0.05).The trend of Western blotting was consistent with that of PCR test.Conclusion: LAMA5 has expression changes at a critical stage in the development of palate.The experiment successfully silently inhibited LAMA5 by building adenovirus vectors and eventually leading to cleft palate.Therefore,we speculated that LAMA5 may be a new susceptible candidate for NSCL/P.It also lies the foundation for the study of late-stage mechanisms and signaling pathways.