HOXA11-AS regulates NIPAL3 via hsa-miRNA-19a-3p, hsa-miR-141-3p and hsa-miR-140-5p in keloid fibroblast: stepwise bioinformatical screen for an integrated molecular network [RNA-Seq]

Purpose: To explore the potential integrated molecular network by long non-coding RNA HOXA11-AS in keloid fibroblast Methods: Using primary cultured keloid-derived fibroblasts (sample 0), small interfering RNAs were designed and transfected into two keloid fibroblast samples (samples 1 and 2) to knockdown HOXA11-AS. One nonspecific transfection control (sample 3) and one blank control (sample 4) were used to remove nonspecific overlap from the studied group. The lncRNAs, messenger RNAs (mRNAs), and microRNAs (miRNAs) of five samples were sequenced to identify differentially expressed (DE) profiles in HOXA11-AS-knockdown keloid fibroblasts in samples 1 and 2, which facilitated removal of overlap with the nonspecific controls (samples 3 and 4). Using stepwise bioinformatic analysis, DE lncRNA and mRNAs characterized by close and natural antisense relationship were constructed into an interactive network by bioinformatic analysis. Based on this network, a lncRNA–mRNA–protein interaction network was extended by integration of the human protein–protein interaction (PPI) network. The Page Rank algorithm was used to score each mRNA in the extended LMN network to reveal significant functioning genes. Next, screened genes were validated by real-time PCR and western blot ting. Validated genes were used to construct a competing endogenous network based on DE miRNA profiles. Results: SNED1, NIPAL3, and VTN were detected significantly changed in HOXA11-AS-knockdown keloid fibroblasts. Only NIPAL3 was predicted to be involved in an intergated network for HOXA11-AS via three competing endogenous miRNAs (hsa-miRNA-19a-3p, hsa-miR-141-3p, and hsa-miR-140- 5p). Conclusion: An interactive molecular network of HOXA11-AS–three miRNAs–NIPAL3 was predicted in keloid fibroblasts Using primary cultured keloid-derived fibroblasts (sample 0), small interfering RNAs were designed and transfected into two keloid fibroblast samples (samples 1 and 2) to knockdown HOXA11-AS. One nonspecific transfection control (sample 3) and one blank control (sample 4) were used to remove nonspecific overlap from the studied group. Comparison between sample 1 and sample 0, sample 2 and sample 0,sample 3 and sample 0 as well as between sample 4 and sample 0 was respectively performed for differentially experessed (DE) profiles of lncRNA, mRNA or miRNA.