A Pilot Study Of Differential Genes In Nonsyndromic Cleft Lip With Cleft Lip With Or Without Cleft Palate(NSCL/P) By Expression Microarray

Objective:To analyze the differentially expressed genes between Non syndromic cleft lip with or without cleft palate(NSCL / P)patients and healthy control using Gene expression microarray technology and to screen NSCL / P related genes,as well as to lay a solid foundation for future study of the function of the syndrome-associated cleft lip and palate genes and exploring the role and mechanism of related genes in the development of the disease.Methods:1.13 cases of nonsyndromic cleft lip and palate(case group)and 6 cases of non-malformation children(control group)in the Department of Burn Plastic Surgery of Children’s Hospital of Chongqing Medical University were collected.2.Total RNA was extracted from 3NSCL / P patients and 3 controls by centrifugal column method.The RNA was puri fied and qualit atively quali fied,then reverse transcri bed into cDNA.The cDNA was transcribed into cRNA,fluorescence dye Cy3 was used to label the cRNA.The labeled cRNA was used to hybrid with theAgilent 4 × 44 k human genome-wide expression of the gene chip,the fluorescence signal was scanned by the computer and the original data was normalized,The differential expression genes was screened by t-test and fold Fold-change.And the differentially expressed genes were analyzed through GO and KEGG.And they were further classified into several categories according to their functions.The biological function of differential genes were preliminarily analyzed 3.Five differential expression genes were selected and the reliability of the chip results was verified by real-time PCR.Results:1.The significance analysis of microarray(SAM)software were applied to select differentially expressed genes((FC)≥ 2 or ≤ 0.5and T-test p-value < 0.05 as the significant difference criteria).254 differentially expressed genes were screened out,among which 151 had up-regulated expression and 103 had down-regulated expression.2.The selected differentially expressed genes were classified into several group according to their Go functions.The results showed that differentially expressed genes were mainly related to protein binding,cell material metabolism,the composition of the medial cavity,positive regulation of adaptive immune response based on somatic cell recombinant immune receptors,etc.It was showed by the The KEGG pathway analysis that the differential genes mainly involved cell phagocytosis,cell adhesion,and alcoholism.3.Five differential expression genes were selected for real-timePCR.The results showed that three genes had down-regulated expression and two genes had up-regulated expression,which was consistent with the result of chip expression.Conclusion:The gene chip is a highly efficient method for selecting differentially expressed genes in children with NSCL/P and normal children.In this study,we used gene chips to find that there is a significant difference in gene expression between NSCL/P children and normal children.The differential genes may be closely related to the development of non-syndromic cleft lip and palate.The large amount of information that was screened out can set a limit for the biological process of the disease,select the genes of interest within a limited range,and conduct in-depth molecular-level research to help elucidate the pathogenesis of NSCL/P.It provides a theoretical basis for disease prevention.