Analysing the relationship between lncRNA and protein-coding gene and the role of lncRNA as ceRNA in pulmonary fibrosis - PubMed (original) (raw)

Analysing the relationship between lncRNA and protein-coding gene and the role of lncRNA as ceRNA in pulmonary fibrosis

Xiaodong Song et al. J Cell Mol Med. 2014 Jun.

Abstract

Long non-coding RNAs (lncRNAs) are involved in various pathophysiologic processes and human diseases. However, their dynamics and corresponding functions in pulmonary fibrosis remain poorly understood. In this study, portions of lncRNAs adjacent or homologous to protein-coding genes were determined by searching the UCSC genome bioinformatics database. This was found to be potentially useful for exploring lncRNA functions in disease progression. Previous studies showed that competing endogenous RNA (ceRNA) hypothesis is another method to predict lncRNA function. However, little is known about the function of ceRNA in pulmonary fibrosis. In this study, we selected two differentially expressed lncRNAs MRAK088388 and MRAK081523 to explore their regulatory mechanisms. MRAK088388 and MRAK081523 were analysed as long-intergenic non-coding RNAs (lincRNAs), and identified as orthologues of mouse lncRNAs AK088388 and AK081523, respectively. qRT-PCR and in situ hybridization (ISH) showed that they were significantly up-regulated, and located in the cytoplasm of interstitial lung cells. We also showed that MRAK088388 and N4bp2 had the same miRNA response elements (MREs) for miR-200, miR-429, miR-29, and miR-30, whereas MRAK081523 and Plxna4 had the same MREs for miR-218, miR-141, miR-98, and let-7. Moreover, the expression levels of N4bp2 and Plxna4 significantly increased in fibrotic rats, and were highly correlated with those of MRAK088388 and MRAK081523, respectively. Among their shared miRNAs, miR-29b-3p and let-7i-5p decreased in the model group, and were negatively correlated with the expression of MRAK088388 and MRAK081523, respectively. MRAK088388 and MRAK081523 could regulate N4bp2 and Plxna4 expression by sponging miR-29b-3p and let-7i-5p, respectively, and possessed regulatory functions as ceRNAs. Thus, our study may provide insights into the functional interactions of lncRNA, miRNA and mRNA, and lead to new theories for the pathogenesis and treatment of pulmonary fibrosis.

Keywords: MRAK081523; MRAK088388; ceRNA; lncRNA; pulmonary fibrosis.

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Figures

Figure 1

The 2D structure of the miRNA-binding sites on lncRNAs. 2D structure: the specific location of the binding sites on the full-length sequence of lncRNA and the types of binding sites (8mer, 7mer-m8, 7mer-A1, 6mer, offset 6mer, imperfect), as well as the base pairing. ‘|’ Indicates an exact match; ‘:’ indicates G: U pairing. The base pairings at 2–7 and 13–16 are particularly important for site recognition, highlighted in brown. Local AU: weighting of the AU. This feature affects accessibility of binding sites. The red bar indicates that the location is A: U. The redder the bar is, the higher the weighting of the AU. Position: relative position of binding sites on lncRNA. The location closer to the sides is better. Conservation: relatively conservative estimates of seed complementary region and UTR between species. A higher weighting occurs when the conservative seed region is located on the non-conservative UTR. Currently, data for constructing the phylogenetic tree of different species for lncRNAs are not sufficient. The ‘Conservation’ section is meaningless for lncRNA. All sites on lncRNA are treated as non-conserved ones. Predicted By: whether this locus is in accordance with the threshold criterion of prediction algorithms (miRanda, TargetScan). (A) The 2D structure of miR-194-5p on MRAK088388. (B) The 2D structure of miR-6321 on MRAK088388. (C) The 2D structure of miR-326-5p on MRAK081523. (D) The 2D structure of let-7i-5p on MRAK081523.

Figure 2

Expression of MRAK088388 and MRAK081523 as well as related protein-coding genes and miRNAs by qRT-PCR. (A) The expression of MRAK088388 was up-regulated in the model group compared with that in the normal group. (B) The expression of N4bp2 was increased in the model group compared with that in the normal group. (C) The expression of miR-29b-3p was reduced in the model group compared with that in the normal group. (D) The expression of MRAK081523 was up-regulated in the model group compared with that in the normal group. (E) The expression of Plxna4 was increased in the model group compared with that in the normal group. (F) The expression of let-7i-5p was decreased in the model group compared with that in the normal group. (G) MRAK088388 and MRAK081523 levels were positive correlation with their respective related protein-coding genes N4bp2 and Plxna4, however, inversely correlated with their respective shared miRNAs miR-29b-3p and let-7i-5p. And statistical analysis was performed by Pearson correlation coefficient respectively; n = 6 (rats) with three replicates.

Figure 3

Location and expression of MRAK088388 (A, C and E) and MRAK081523 by in situ hybridization (B, D and F). MRAK088388 and MRAK081523 was stained blue in the plasma of interstitial lung cells. (A) Location and expression of MRAK088388 in the normal groups. (B) Location and expression of MRAK081523 in the normal groups. (C) Location and expression of MRAK088388 in the model group. MRAK088388 was up-regulated. (D) Location and expression of MRAK081523 in the model group. MRAK081523 was up-regulated. (E) Density analysis of MRAK088388. (F) Density analysis of MRAK081523. Each bar represents the mean ± SD, n = 6. Original magnification, 400×; *P < 0.05.

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Analysing the relationship between lncRNA and protein-coding gene and the role of lncRNA as ceRNA in pulmonary fibrosis - PubMed (original) (raw)