Biological Function Of LAMA5 During Mice Secondary Palate Development

Objective:In this study,the best interference sequence of Laminin-α5(LAMA5)screened out in previous experiments was used to interference with the LAMA5 gene,to observe the effect of LAMA5 expression downregulation on palatal development and fusion,and to study whether LAMA5 interferes with palatal development through proliferation,apoptosis,epithelial-mesenchymal transition(EMT)related biological behaviors and SHH signaling pathway.Methods:1.The C57BL/6J pregnant mice were sacrificed at embryonic day 13.5(E13.5)by neck broken dislocation,and the embryonic palatal tissue was dissected.The palatal organ culture model was established in vitro.The LAMA5-sh RNA adenovirus vectors with green fluorescent marker was constructed and transfected into E13.5 palatal tissue.After 48 h in vitro culture,the virus transfection and palatal fusion were observed under fluorescence microscope.Real-time fluorescence quantitative PCR(RT-q PCR)and Western blotting were used to test the expression of LAMA5 at m RNA and protein level,respectively.Immunohistochemical(IHC)staining was used for LAMA5 localization and semiquantitative detection.Hematoxylin-eosin(HE)staining was used to observe the histological morphology of mice palatal process after virus transfection.2.The expression of proliferation,apoptosis and EMT markers in the blank control group,the negative control group and the LAMA5-sh RNA group were detected respectively.The expression levels of ki67,cyclin D1,caspase 3,cleaved caspase 3,E-cadherin and vimentin were detected by RT-q PCR and Western blotting at m RNA and protein level.IHC staining was used for localization and semi-quantitative detection.3.The expression level of SHH signaling pathway-related factors was detected in blank control group,negative control group and LAMA5-sh RNA group,respectively.RT-q PCR and Western blotting tested the expression levels of Shh,ptch1,and gli1 after virus transfection,respectively.IHC staining was used for localization and semi-quantitative detection.Results:1.Fluorescence microscope observation after virus transfection showed green fluorescence in palatal process of negative control group and LAMA5-sh RNA group.In the negative control group,the two palatal processes moved toward the center and fused,while in the LAMA5-sh RNA group,the two palatal processes failed to fuse and there was still a large gap.The results indicate that,compared with the blank control group,the m RNA expression of LAMA5 in the negative control group showed no significant change(P>0.05),and the LAMA5 m RNA expression in the LAMA5-sh RNA group decreased by 74.48%(P <0.05).The results of Western blotting were compatible with PCR.IHC staining showed that LAMA5 was mainly located in the epithelial basement membrane,and no difference was found in the positive rate of LAMA5 between the blank control group and the negative control group(P>0.05).LAMA5 m RNA expression was decreased in LAMA5-sh RNA group(P <0.05).HE staining showed normal development of palatal processes in the blank control group and the negative control group.The palatal processes on both sides contacted and fused with each other to form a complete secondary palatal.However,in LAMA5-sh RNA group,the palatal processes were underdeveloped and the two sides of the palatal processes failed to contact and fuse with each other,which was manifested as cleft palate.2.PCR results showed that the mRNA expression levels of ki67 and cyclin D1,caspase 3,E-cadherin and vimentin in negative control group were not significantly changed compared with blank control group(P >0.05).In the LAMA5-sh RNA group,the m RNA expression levels of both ki67 and cyclin D1 were decreased by 56.88% and 83.63%(P <0.05),respectively,while the m RNA expression levels of caspase 3 increased by 157.47%(P <0.05).There was no significant change in the m RNA expression levels of E-cadherin and vimentin(P >0.05).The results of Western blotting were consistent with PCR.The protein expression levels of cleaved caspase 3 increased(P <0.05).The results of IHC staining showed that there was no significant difference in the positive rates of ki67,cyclin D1 and caspase 3 between the blank control group and the negative control group(P >0.05).Compared with the blank control group,expression levels of ki67 and cyclin D1 in LAMA5-sh RNA group were significantly decreased,and caspase 3 was significantly increased(P <0.05).3.PCR results showed that,compared with blank control group,m RNA expression levels of Shh,ptch1 and gli1 in negative control group were not significantly changed(P >0.05).The expressions of Shh and ptch1 in LAMA5-sh RNA group did not change significantly(P >0.05),but the expression of gli1 m RNA was decreased by 67.73%(P<0.05).The results of Western blotting and PCR were consistent.IHC staining showed that gli1 positive rate in blank control group was not significantly different from that in negative control group(P >0.05).The gli1 positive rate in LAMA5-sh RNA group was significantly lower than that in blank control group(P <0.05).Conclusion:1.LAMA5 plays an important role in palate development,and LAMA5 gene silencing leads to cleft palate in mice.2.LAMA5 gene silencing leads to cleft palate by inhibiting palatal cell proliferation and promoting cell apoptosis in mice,and EMT may not be involved in this process.3.LAMA5 gene silencing leads to cleft palate by interfering with normal SHH signaling pathway,and there may be related signaling pathway crossover.