Ma J, Chen S, Hao L, Sheng W, Chen W, Ma X, Zhang B, Ma D, Huang G.J Cell Mol Med. 2020 May 5. doi: 10.1111/jcmm.15298. Online ahead of print.PMID: 32368852 Free article.
Tetralogy of Fallot (TOF) is the most common complex congenital heart disease (CHD) with uncertain cause. Although long non-coding RNAs (lncRNAs) have been implicated in heart development and several CHDs, their role in TOF is not well understood. This study aimed to investigate how dysregulated lncRNAs contribute to TOF. Using Gene Expression Omnibus data mining, bioinformatics analysis and clinical heart tissue sample detecting, we identified a novel antisense lncRNA TBX5-AS1:2 with unknown function that was significantly down-regulated in injured cardiac tissues from TOF patients. LncRNA TBX5-AS1:2 was mainly located in the nucleus of the human embryonic kidney 293 (HEK293T) cells and formed an RNA-RNA double-stranded structure in the overlapping region with its sense mRNA T-box transcription factor 5 (TBX5), which is an important regulator in heart development. Knock-down of lncRNA TBX5-AS1:2 via promoter hypermethylation reduced TBX5 expression at both the mRNA and protein levels by affecting its mRNA stability through RNA-RNA interaction. Moreover, lncRNA TBX5-AS1:2 knock-down inhibited the proliferation of HEK293T cells. In conclusion, these results indicated that lncRNA TBX5-AS1:2 may be involved in TOF by affecting cell proliferation by targeting TBX5.
FIGURE 1 LncRNA TBX5‐AS1:2 was selected by data mining of the GEO database and bioinformatics analysis. A, CNC network of 19 lncRNAs and 26 mRNAs with a standard of PCC ≥0.9 or ≤−0.9 and P ≤ .05. Blue rhombus represents lncRNAs, red circular nodes represent mRNAs, and lines indicate gene co‐expression relationship between lncRNA and mRNA. B, Higher expression of lncRNA TBX5‐AS1:2 in foetal compared with adult hearts. Expression values represented by red and yellow shades, indicating expression above and below the median across all samples, respectively. C, Relative positions of lncRNA TBX5‐AS1:2 and its nearby gene TBX5 on the chromosome. LncRNA TBX5‐AS1:2 is localized at the antisense chain of the coding gene TBX5, with overlapping and complementary regions. Black highlighted region indicates the overlapping region (92 bp), and its sequence is presented in the above box. Blue boxes indicate exons. D, CNC network of lncRNA TBX5‐AS1:2 and 11 mRNAs. Rose V represents lncRNA TBX5‐AS1:2, green circular nodes represent mRNAs, and node size indicates the gene expression level (larger dot, higher expression level). Lines represent the gene co‐expression relationship between lncRNA TBX5‐AS1:2 and mRNA (full lines, positive correlation; dashed lines, negative correlation). E and F, LncRNA TBX5‐AS1:2 and TBX5 were significantly down‐regulated in TOF cardiac tissue samples compared with normal control (NC) by qPCR analysis. Values are mean ± SEM, n = 3, ***P < .0001. CNC, coding and non‐coding; GEO, Gene Expression Omnibus; lncRNAs, long non‐coding RNAs; PCC, Pearson’s correlation coefficient; qPCR, quantitative polymerase chain reaction; TOF, Tetralogy of Fallot
FIGURE 2 LncRNA TBX5‐AS1:2 affected proliferation of HEK293T cells. A, Three shRNAs targeting the non‐overlapping regions of lncRNA TBX5‐AS1:2 were designed to knock‐down lncRNA TBX5‐AS1:2 expression. The shRNA2 with the greatest interference efficiency was used to establish stable cell lines. B, LncRNA TBX5‐AS1:2 was successfully overexpressed. C and D Positive regulation of cell proliferation by lncRNA TBX5‐AS1:2 was revealed by CCK8 assays. E and F, LncRNA TBX5‐AS1:2 had no effect on cell apoptosis according to flow cytometry analysis. Values are mean ± SEM, n = 3, ***P < .0001. HEK293T, human embryonic kidney 293; lncRNAs, long non‐coding RNAs; shRNAs, short hairpin RNAs
FIGURE 3 LncRNA TBX5‐AS1:2 regulated expression of TBX5 in mRNA and protein levels by forming an RNA duplex with TBX5 to increase its stability. A, Nucleus cytoplasm separation indicated that lncRNA TBX5‐AS1:2 was mainly located in the nucleus of HEK293T cells, similar to U1. B, The result of a RNA‐FISH assay also showed that lncRNA TBX5‐AS1:2 was almost nuclear in HEK293T cells. Centre DAPI was used to stain nuclei (blue); left red fluorescence was from the biotin fusions; right the merged image. C and E, Dysregulation of lncRNA TBX5‐AS1:2 positively regulated TBX5 protein levels according to WB results. D and F, Dysregulation of lncRNA TBX5‐AS1:2 positively regulated TBX5 mRNA levels by qPCR analysis. Values are mean ± SEM, n = 3, ***P < .0001. G, Reduced cell proliferation caused by down‐regulation of lncRNA TBX5‐AS1:2 was rescued by TBX5 overexpression in HEK293T cells. H, RPA and RT‐PCR revealed that the overlapping region of lncRNA TBX5‐AS1:2 and TBX5 mRNA could not be digested by RNAse, suggesting the formation of an RNA‐RNA duplex. RNAse(+) indicates RNAse treatment; RNAse(‐) indicates no RNAse treatment. I, RNA‐RNA pull‐down assay showed the in vitro interaction between lncRNA TBX5‐AS1:2 and TBX5. J, LncRNA TBX5‐AS1:2 increased the stability of TBX5 mRNA in HEK293T cells during 10 h after blocking new RNA synthesis with actinomycin D. HEK293T, human embryonic kidney 293; lncRNAs, long non‐coding RNAs; qPCR, quantitative polymerase chain reaction; RPA, ribonuclease protection assay; WB, Western blotRNA‐FISH
FIGURE 4 Hypermethylation of CPG island 2 in the promoter caused down‐regulation of lncRNA TBX5‐AS1:2. A, Four CpG islands were predicted in the regulatory sequence of lncRNA TBX5‐AS1:2 by MethPrimer online. B, Every CpG site in lncRNA TBX5‐AS1:2 CpG island 2 was hypermethylated in TOF cardiac tissue samples compared with NC, as detected by BSP for clones. The colour of circles for each CpG site represents methylation percentage. C, The methylated reporter construct pGL3‐Basic‐lncRNA TBX5‐AS1:2‐i2 could not be digested by MSRE whereas the unmethylated construct could be digested. D, The methylation percentage of each CpG site was significantly increased in HEK293T cells transfected with methylated pGL3‐Basic‐lncRNA TBX5‐AS1:2‐i2 according to BSP. E, Overall hypermethylation of CpG island 2 was significant in HEK293T cells transfected with methylated pGL3‐Basic‐lncRNA TBX5‐AS1:2‐i2. (F) Dual‐luciferase reporter assay showed that hypermethylation of CpG island 2 decreased the transcriptional activity of lncRNA TBX5‐AS1:2 in HEK293T cells. Values are mean ± SEM, n = 3, ***P < .0001. BSP, bisulphite sequencing PCR; HEK293T, human embryonic kidney 293; lncRNAs, long non‐coding RNAs; NC, negative control; TOF, Tetralogy of Fallot