* < 0.05, ** < 0.01. 3.6. 5-Hydroxydopamine hydrochloride the corresponding author on request. 3. Results 3.1. HOXA9 Is Predicted to Regulate Apoptotic- and Autophagic-Genes in cSCC HOXA9 has been identified as a tumor suppressor in cSCC by inhibiting glycolysis while promoting apoptosis . Yet, the exact roles of HOXA9 in regulating apoptosis process is still unclear. To understand how HOXA9 Rabbit Polyclonal to HTR2B regulates the molecular events related with apoptosis and other cellular processes in cSCC cells, we repeated the bioinformatic analysis of the previous transcriptome sequencing after HOXA9 knockdown . Gene Ontology (GO) analysis with the list of significantly up-regulated genes revealed that the top-ranked lists of enriched Gene Ontology categories includes Positive regulation of apoptotic process, Apoptotic process, Regulation of apoptotic process, Regulation of extrinsic apoptotic signaling pathway via death domain receptors, Positive regulation of programmed cell death, Positive regulation of NF-kappaB transcription factor activity, Positive regulation of I-kappaB kinase/NF-kappaB signaling, Macroautophagy and Positive regulation of transcription, DNA-templated, etc. (< 0.05, Table 1, Supplementary Data 1). Markedly, Kyoto Encyclopedia 5-Hydroxydopamine hydrochloride of Genes and Genomes (KEGG) pathway analysis indicated that molecular signaling pathways including NF-kappaB signaling pathway, Apoptosis and Autophagy are significantly influenced (< 0.05, Table 1, Supplementary Data 1). Among the genes affected in the above three pathways, significantly-upregulated genes including NF-B, BCL2L1 (BCL-XL), ULK1 (ATG1), ATG3, and ATG12 were functionally relevant to apoptosis or autophagy. Table 1 Transcriptomic analysis of HOXA9-regulated genes by RNA-Seq in cutaneous squamous cell carcinoma (cSCC) cells. Over-represented 5-Hydroxydopamine hydrochloride categories by GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis of differently-expressed genes. BP: biological process. The NF-kappaB signaling pathway, Apoptosis and Regulation of autophagy were highlighted. = 3) (b) and apoptosis assay by Annexin V/PI double staining (= 3) (c) were performed in cSCC cells treated with siRNAs targeting HOXA9. (d) HOXA9 protein expression was detected by western blot after overexpression of HOXA9 in cSCC cells. Measurements of cell proliferation by CCK-8 assay (= 3) (e) and apoptosis assay by Annexin V/PI double staining (= 3) (f) were performed in cSCC cells overexpressing HOXA9. (g) The autophagy in cSCC cells following HOXA9 knockdown was evaluated by LC3 staining. * < 0.05, ** < 0.01, *** < 0.001. The status of autophagy in response to HOXA9 variation was also checked. Knockdown of HOXA9 enhances autophagy as shown by the increased LC3B II modification, P62 expression (Figure 1a) and LC3B immunofluorescence (Figure 1g), while overexpression of HOXA9 significantly inhibited autophagy (Figure 1d). Thus, we concluded that loss of HOXA9 inhibits apoptosis but promotes autophagy in cSCC cells. 3.3. HOXA9 Negatively Regulated the Expression of RELA, BCL-XL, ULK1, ATG3, and ATG12 To further validate the pro-apoptotic and anti-autophagic functions of HOXA9, (encoding P65 subunit of NF-B), were selected from the significantly varied genes owing to their critical roles in apoptosis and autophagy. qRT-PCR and western blot detection confirmed that the upregulation of RELA, BCL-XL, ULK1, ATG3, and ATG12 in response to HOXA9 knockdown (Figure 2a,b). Conversely, overexpression of HOXA9 inhibited the expression of RELA, BCL-XL, ULK1, ATG3, and ATG12 (Figure 2c,d). Collectively, HOXA9 regulates apoptosis and autophagy by negatively regulating anti-apoptotic and pro-autophagic genes. Open in a separate window Figure 2 HOXA9 represses the expression of NF-B and its downstream apoptotic and autophagic genes by directly binding to the promoter region of NF-B. (aCd) The mRNA or.