Original Article

Integrated analysis identifying long non-coding RNAs (lncRNAs) for competing endogenous RNAs (ceRNAs) network-regulated palatal shelf fusion in the development of mouse cleft palate

Xuan Shu, Zejun Dong, Mingjun Zhang, Shenyou Shu


Background: Cleft palate results from the defective palatal fusion of the medial-edge epithelium after cells undergo epithelial-mesenchymal transition, a process that involves regulation by microRNAs (miRNAs). However, in palatal shelf fusion, miRNA regulation by long non-coding RNAs (lncRNAs) when acting as competing endogenous RNAs (ceRNAs) or miRNA sponges, remains unclear.
Methods: We systematically analyzed the correlation between lncRNAs, miRNAs, and mRNAs from RNA sequencing profiling in embryonic gestation day 14.5 (E14.5) mouse embryos from control (n=3) and all- trans retinoic acid (ATRA)-treated (n=3) mice. We then constructed a lncRNA-associated ceRNA network. The expression profiles of mRNA, lncRNA, and miRNA were verified by quantitative polymerase chain reaction (qPCR).
Results: In total, 18 aberrantly expressed miRNAs, 861 mRNAs, and 583 lncRNAs were identified from palate samples of control and ATRA-treated samples. Bioinformatics data and integrative analysis identified 69 lncRNAs, 18 miRNAs, and 78 mRNAs that were aberrantly expressed, and a ceRNA network was then constructed. Finally, we identified a NONMMUT004850.2/NONMMUT024276.2-miR-741-3p/miR-465b- 5p-Prkar1α ceRNA network associated with palatal shelf fusion at E14.5. The qPCR results showed that NONMMUT004850.2 (P=5E−05), NONMMUT024276.2 (P=0.0012), and Prkar1α (P=3E−05) were up-regulated, whereas miR-741-3p (P=0.006) and miR-465b-5p (P=1E−04) were down-regulated in ATRA-treated mice compared to the control samples. The qPCR results were in concordance with the RNA sequencing profiling.
Conclusions: Our study demonstrated that NONMMUT004850.2/NONMMUT024276.2-miR-741-3p/ miR-465b-5p-Prkar1α could potentially serve as an important regulatory mechanism of palatal fusion in the development of the cleft palate.

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