Long Non-Coding RNAs MALAT1 and NEAT1 in Non-syndromic Cleft Lip and Palate. Long Non-Coding RNAs MALAT1 and NEAT1 in Non-syndromic Cleft Lip and Palate

The etiology and pathogenesis of non-syndromic cleft lip and palate (NSCL/P) are largely unknown. Long non-coding RNAs (lncRNA) are thought to play important roles in NSCL/P, but reports on the underlying processes are currently unavailable. Our study focused on children diagnosed with NSCL/P alone. Based on the morphology, patients were categorized as either cleft lip with or without cleft palate (CL/P) or cleft palate-only (CPO). When patients received surgery for NSCL/P, tissue excised from the trimmed wound edge was reserved to serve as experimental samples; adjacent normal tissue was used as a positive control. Target lncRNAs in the collected tissues were identified using microarray and quantitative reverse transcription PCR (RT-qPCR). Immunohistochemical (IHC) staining and RT-qPCR were used to verify the target mRNAs. Pathway, gene ontology (GO) enrichment, and TargetScan prediction were employed to construct endogenous RNA networks (ceRNA networks) and explore their potential functions. RNA-Seq analysis revealed 24 upregulated and 43 downregulated lncRNAs in the CL/P and CPO groups compared with those in the control group; of these, MALAT1and NEAT1 were screened and validated using RT-qPCR. Common NSCL/P risk factors positively correlated with MALAT1 and NEAT1 expression (ORMALAT1 = 28.111, 95% CI: 4.054-194.923; ORNEAT1 = 30.556, 95% CI: 4.422-211.142; P < 0.05). Bioinformatics predicted four ceRNA networks: MALAT1-hsa-miR-1224-3p-SP1, MALAT1-hsa-miR-6734-5p/hsa-miR-1224-3p-WNT10A, NEAT1-hsa-miR-140-3p.1-CXCR4, and NEAT1-hsa-miR-3129-5p/hsa-miR-199a-3p/hsa-miR-199b-3p-ZEB1. GO enrichment focused on the potential functions of ceRNA networks, including biosynthesis of organic cyclic compounds, formation of membrane-enclosed and organelle lumens, and Wnt-protein binding. The results of RT-qPCR were consistent with those of IHC staining with regard to expression of related mRNAs. MALAT1 and NEAT1, which are upregulated in NSCL/P, are associated with the severity of NSCL/P. This study provides a new insight into NSCL/P pathogenesis and suggests that MALAT1 and NEAT1 act as potential therapeutic targets and prognostic biomarkers for NSCL/P. Overall design: When patients received surgery for NSCL/P, tissue excised from the trimmed wound edge was reserved to serve as experimental samples; adjacent normal tissue was used as a positive control. Target lncRNAs in the collected tissues were identified using microarray and quantitative reverse transcription PCR (RT-qPCR).

非综合征性唇腭裂（non-syndromic cleft lip and palate, NSCL/P）的病因与发病机制目前尚未完全明确。长链非编码RNA（long non-coding RNAs, lncRNA）被认为在NSCL/P的发生发展中发挥重要作用，但目前针对其潜在调控过程的相关研究仍较为匮乏。本研究聚焦于仅确诊为NSCL/P的儿童患者。根据患者的畸形形态，将其分为伴或不伴腭裂的唇裂（cleft lip with or without cleft palate, CL/P）组与单纯腭裂（cleft palate-only, CPO）组。当患者接受NSCL/P修复手术时，采集修整创缘时切除的组织作为实验样本，以邻近的正常组织作为阳性对照。通过基因芯片与定量反转录聚合酶链反应（quantitative reverse transcription PCR, RT-qPCR）鉴定收集组织中的目标lncRNA；采用免疫组织化学（immunohistochemical, IHC）染色与RT-qPCR验证目标mRNA的表达情况。通过通路分析、基因本体论（gene ontology, GO）富集分析以及TargetScan预测，构建内源RNA网络（competing endogenous RNA networks, ceRNA网络）并探讨其潜在生物学功能。RNA测序分析显示，与对照组相比，CL/P组与CPO组中共存在24个上调表达的lncRNA以及43个下调表达的lncRNA；其中，通过RT-qPCR筛选并验证了MALAT1与NEAT1。NSCL/P常见危险因素与MALAT1、NEAT1的表达水平呈正相关（MALAT1的比值比OR=28.111，95%置信区间CI：4.054~194.923；NEAT1的OR=30.556，95%CI：4.422~211.142；P<0.05）。生物信息学分析预测得到4条ceRNA调控网络：MALAT1-hsa-miR-1224-3p-SP1、MALAT1-hsa-miR-6734-5p/hsa-miR-1224-3p-WNT10A、NEAT1-hsa-miR-140-3p.1-CXCR4以及NEAT1-hsa-miR-3129-5p/hsa-miR-199a-3p/hsa-miR-199b-3p-ZEB1。GO富集分析聚焦于ceRNA网络的潜在生物学功能，涵盖有机环类化合物的生物合成、膜包被细胞器管腔的形成以及Wnt蛋白结合。RT-qPCR的检测结果与IHC染色中相关mRNA的表达结果一致。在NSCL/P患者中上调表达的MALAT1与NEAT1，与NSCL/P的病情严重程度呈显著相关。本研究为NSCL/P的发病机制提供了全新的研究视角，并提示MALAT1与NEAT1可作为NSCL/P潜在的治疗靶点与预后生物标志物。实验整体设计：当患者接受NSCL/P修复手术时，采集修整创缘时切除的组织作为实验样本，以邻近的正常组织作为阳性对照；通过基因芯片与定量反转录聚合酶链反应（RT-qPCR）鉴定收集组织中的目标lncRNA。