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