Research Paper Volume 14, Issue 21 pp 8839—8855

Figure 4. lncRNA HHIP-AS1 increases HHIP mRNA stability by ELAVL1. (A) BM-MSCs transfected with overlapping sequence or non-overlapping sequence of lncRNA HHIP-AS1 and HHIP mRNA were analyzed by PCR. (B) qPCR validation of HHIP enrichment versus input after RNA pull down by two different specific probes (Bio-HHIP-AS1 OL and Bio-HHIP-AS1 non-OL) as compared to a non-specific one (Bio-NC) in BM-MSCs. (C) Measurement of the stability of HHIP mRNA by RT-qPCR in the presence of transcriptional inhibitor (actinomycin D) at the indicated time as compared to an internal control 18S rRNA. (D, E) Using the web (http://service.tartaglialab.com/page/catrapid_group), we found that ELAVL1 has high affinity to the lncRNA HHIP-AS1, especially with the region that is complementary with HHIP mRNA. (F) RIP assay was performed in lncRNA HHIP-AS1-overexpressed and silenced BM-MSCs and the control cells. The qPCR assay was further performed to determine the enrichments of HHIP-AS1 and HHIP mRNA in ELAVL1 protein. IgG is as a negative control. (G) BM-MSCs were transfected with lncRNA HHIP-AS1 overexpression vector alone or in combination with ELAVL1 siRNA. Measurement of the stability of HHIP mRNA by RT-qPCR in the presence of transcriptional inhibitor (actinomycin D) at the indicated time as compared to an internal control 18S rRNA. The histogram data for each group are the average of three independent replicates; bars represent standard deviation; *P < 0.05, **P < 0.01 and ***P < 0.001 vs. NC or BM-MSCs without transfection; ^#P < 0.01 vs. BM-MSCs transfected with lncRNA HHIP-AS1 overexpression vector.