Journal: Nucleic Acids Research

Article Title: H3K27 acetylation activated-long non-coding RNA CCAT1 affects cell proliferation and migration by regulating SPRY4 and HOXB13 expression in esophageal squamous cell carcinoma

doi: 10.1093/nar/gkw1247

Figure Lengend Snippet: CCAT1 promotes HOXB13 expression by competing for miR-7 in cytoplasm, thus facilitating ESCC cell growth and migration. ( A ) Western blot assays detected the expression of HOXB13 after knockdown of CCAT1 and overexpression of CCAT1 in Eca-109 cells. qRT-PCR assays showed that the level of HOXB13 was obviously upregulated in 90 pairs ESCC tissues. The level of CCAT1 in ESCC tissues showed a statistically positive correlation with the relative level of HOXB13 expression (N = 90). ( B ) CCAT1 displays no effect on the transactivity of HOXB13 promoter. The promoter of HOXB13 was cloned upstream of a luciferase reporter gene, and the resultant construct was co-transfected with si-NC/si-CCAT1 in Eca-109 and TE-1 cells. The RNAup algorithm predicted potential binding of miR-7 to CCAT1 and to HOXB13, with considerable sequence complementary in the indicated regions. qRT-PCR assays detected the expression of miR-7 after knockdown of CCAT1 and overexpression of CCAT1 in Eca-109 and TE-1 cells. qRT-PCR assays detected the expression of CCAT1/HOXB13 after overexpression of miR-7 in Eca-109 and TE-1 cells. Western blot assays detected the expression of HOXB13 after knockdown of overexpression of miR-7 in Eca-109 and TE-1 cells. ( C ) Both CCAT1 and HOXB13 are targeted by miR-7. The 3΄UTR of HOXB13 mRNA and full length of CCAT1 were respectively inserted downstream of a luciferase gene. The reporter vector was co-transfected with a renilla luciferase vector (for normalization) to Eca-109 and TE-1, which were treated by miR-7 mimics or control mimics. The luciferase signals of both reporter genes were significantly decreased when cells were treated with miR-7 mimics. Mutant 3΄UTR of HOXB13 and CCAT1 is not affected by miR-7. The 3΄UTR of HOXB13 and CCAT1 was mutated on the predicted binding site that is shown in (B), and was tested in the luciferase assay as described above. The results showed that miR-7 did not alter the luciferase signal. CCAT1 is required for the stability of HOXB13 3΄UTR. The reporter vector was co-transfected with a renilla luciferase vector (for normalization) to Eca-109 and TE-1 cells, which were treated by CCAT1 siRNA or control siRNA. The luciferase signal of reporter gene was significantly decreased when knockdown of CCAT1. ( D ) RIPs experiments showed that CCAT1 was obviously enriched in Ago2-immunoprecipitation relative to control IgG. Similarly, miR-7 was also detected in Ago2-immunoprecipitation relative to control IgG control. Successful immunoprecipitation of Ago2-associated RNA was verified by qRT-PCR. ( E ) The promotion of HOXB13 by CCAT1 was significantly reversed by miR-7, by using qPCR assays. MTT and Transwell assays showed that knockdown HOXB13 could reverse CCAT1-mediated growth and migration promotion. ( F ) Eca-109 cells transfected with Vector/pcDNA-CCAT1 and pcDNA-CCAT1-Mutation. After transfection, cells were analyzed by MTT, BrdU and Transwell assays. * P < 0.05, ** P < 0.01. n.s., not significant.

Article Snippet: The HOXB13 siRNA was from Santa Cruz (sc-43851).

Techniques: Expressing, Migration, Western Blot, Knockdown, Over Expression, Quantitative RT-PCR, Clone Assay, Luciferase, Construct, Transfection, Binding Assay, Sequencing, Plasmid Preparation, Control, Mutagenesis, Immunoprecipitation