| Part OneCleft lip with or without palate (CL/P) is among the most frequent of all major birth defects that affect1/1000live births. There are two types of orofacial clefts:cleft lip (CL) and cleft palate (CP). According to whether or not associated with other birth defects, CL/P can be also divided nonsyndromic CL/P (NSCL/P) and syndromic CL/P (SCL/P). About80to85%of CP cases are nonsyndromic (NSCP) according to most studies. The etiology of CL/P is complex, including multiple genetic and environmental factors and the pathogenesis is unclear now. Most research has been performed with animal model, to our best of knowledge, there have been no reports about human soft palate miRNAs associated with CL/P.NSCP with a prevalence of1.3-25.3per10000births is among the most common craniofacial anomalies in humans. NSCP is a multifactorial disorder influenced by both genetic and environmental factors. Several genes have been found to contribute to NSCP. For example, WANG et al. found that significant evidence of linkage and association was shown for3SNPs (rs7858435, rs10820914and rs3905385) among57Asian non-syndromic cleft palate trios in Family Based Association Tests. Sairee et al. found the mutations of PDGFa in humans with isolated cleft palate. FitzPztrick et al. identified that SATB2as the cleft palate gene on2q32-q33. However, the share of positive family history was relatively small, foreign reports, the proportion of positive family history of more than20%, and polygenic inheritance, domestic less than foreign, usually5%to10%. Most cases do not use genetics to explain, so many scholars to study other factors such as environmental factors, quality of life and so on. Epigenetic has become a hot research during the palate development. Abbott et al. revealed hormone drugs such as prednisone, dexamethasone and so on, can cause the distribution changes of epidermal growth factor (EGF) and transforming growth factor (TGF) and its receptor during the development of palate shelf, and cause the occurrence of cleft palate. Domestic Shi et al. also showed the results that dexamethasone can induce the formation of cleft palate in mice, while vitamin B6, vitamin B12can prevent cleft palate. On the other hand, some research reported the effect of miRNA during the development of palate. For example, miR-140regulates pdgfra signal during palate development in zebrafish. miR-200b and Tgf-β signaling are important for proper palatogenesis and especially for palate fusion. Whereas all results base on animal experiments, no miRNA profile has, to our knowledge, yet been shown in human soft palate tissues.The epigenetics refers to the alteration of gene expression and function without the change of DNA sequence, including DNA methylation, covalent modification of histones, noncoding RNAs, X chromosome inactivation and so on. The main argument is that, the most traits of organism are controlled by DNA sequence encoding proteins, but the epigenetic code encoded by chemical markers outside the DNA sequences also has a profound effect for lift organism health and phenotypic characteristics. Epigenetics has becom hot spot in the research of diseases which etiology include genetic and environmental factors. MicroRNAs (miRNAs) are endogenous22～25nucleotides, non-protein-encoding RNAs. Mature miRNA combines with the complementary base of mRNA to regulate gene expression through inhibiting translation or degrading specific mRNA. MiRNAs are believed to participate in variedly regulation approaches, including cell development, proliferation and apoptosis. Some study found that miRNAs play pivotal roles during embryonic development, including palate development.Therefore, we study the altered miRNA and the target genes associated with NSCP which shows high incidence and consistent with a single gene regulation. We hope to reveal NSCP possible pathogenesis from epigenetic perspective, to provide direct basis for human cleft lip with or without palate prevention.Objection Find out the NSCP-associated miRNA and determine the target genes.Methods and results(1)50NSCP patients’soft palate tissues were obtained from the Handan Central Hospital of Hebei Province of Northern China and cleft lip and palate surgery funded by smile train in Yunnan Province.20control samples were collected from the deceased in traffic accidents by Forensic Department of Fudan University. Samples were collected during orthopedic surgery and were immediately transferred to RNAlater (Ambion, USA). Then samples were stored at-80℃until extraction of total RNA.(2) MiRNAs microarray was used to detect miRNA expression of6normal and10NSCP soft palate tissues. Among the screened microRNAs,12miRNAs have different experssion between normal control and NSCP samples. (3) Expression of all the selected microRNAs by real-time RT-PCR from more NSCP patients (n=50) and normal persons (n=20). The analysis confirmed that4microRNAs have differently expression patterns and contain3up-regulated and1down-regulated.(4) The risk analysis identified that odds ratio (95%CI) of four miRNAs can be used as the risk factors for NSCP.(5) Because most miRNAs are expressed in a highly temperal-space manner, and there may have differences in miRNAs expression with age changes. The correlation between miRNA expression and age was analysed. We found no association of four miRNAs expression levels with age.(6) The correlation between miRNA expression and gender was analysed and displayed that significant association of the selected miRNAs expression levels with NSCP was discovered in the male group but not in the female group support the epidemic investigation that NSCP more occurred in male.(7) To analysis the biological significance of miRNA deregulation, we used bioinformatics tools for target genes prediction of altered miRNAs and verified by real-time RT-PCRConclusions and outlook:The miR-1246, miR-1323and miR-93were up-regulated, whereas miR-143*was down-regulated in NSCP samples. The four altered miRNAs may be the high risk factor of NSCP. Two target genes (WNT5A and FGFR2) were validated that WNT5A is upregulated which is regulated by hsa-miR-143*, while FGFR2is downregulated which is regulated by hsa-miR-1246, hsa-miR-1323and hsa-miR-93in NSCP samples compare with control samples.Further research will be needed to elucidate the mechanism. Part TwoThe determination of the postmortem interval (PMI) is a major focus of investigation in forensic science. The precise estimation of PMI is a critical step in many death investigations. Traditional methods to estimate PMI generally based on the physiological changes such as algor mortis, livor mortis, rigor mortis and supravital activity, but these methods only can provides a rough estimation of PMI, and more frustrated, people can find one indication often contradicts the results of another ones. The rapid development of molecular biology provides a new idea for this research. The nucleic acid analysis technology application in body sample, particularly by real-time RT-PCR method for the study of the relationship between PMI and nucleic acid degradation, has become the focus of research. Because the results obtained by real-time RT-PCR method must depend on the accurate reference gene standardization to eliminate the differences between individuals. Therefore, finding a stable reference gene for PMI research by real-time RT-PCR is very necessary.In this study, we chosed and compared six commonly used reference biological markers-β-actin, GAPDH, B2M, U6,18S rRNA and hsa-mir-1using real-time RT-PCR and genormPLUS*program to analysis the stability of reference biological markers in the early PMIs. The aim was to provide an accurate and stable reference biological marker for the application of real-time RT-PCR in the determination of the early PMIs.Objection:To study the stability of commonly used reference biological markers and find the most stable marker.Methods and results(1) Samples were collected from the heart from ten individuals with different postmortem intervals (PMI) ranging from4.3to22.3h referring to the time of autopsy. Individuals with similar environmental conditions (room temperature in hospital autopsy room storage before necropsy, the average temperature:25℃and with various causes of death were chosen for this study. Samples were collected during routine autopsy and were immediately transferred to RNAlater (Ambion, USA). Then samples were stored at-80℃until extraction of total RNA.(2) To chose six commonly used reference biological markers which are housekeeping genes or expressed specifically in heart and design the primers.β-actin, GAPDH, B2M, U6,18S rRNA and hsa-mir-1, respectively.(3) Total RNA was isolation by a modified TRIzol reagent (Invitrogen, USA); cDNA synthesis; and mRNA expression was analyzed by the Sybr Green qRT-PCR according to the manufacturer’s instructions (Applied Biosystems).(4) The stability analysis of six chosed reference biological markers was performed using genormPLUS*and indicated that U6was the most stable in early PMIs.(5) The correlation between U6expression and age, gender and cause of death was performed in order to further verify the stability of U6. The result showed that U6was stable and an ideal reference marker.Conclusions and outlookDuring the early PMIs, expression of U6in human heart is the most stable, not influenced by age, gender and cause of death, and is an ideal reference biological marker for real-time RT-PCR. |
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