Glycolytic reprogramming is a typical feature of many cancers; however, key regulators of glucose metabolism reengineering are poorly understood, especially in cutaneous squamous cell carcinoma (cSCC). Here, Homeobox A9 (HOXA9), a direct target of onco-miR-365, is identified to be significantly downregulated in cSCC tumors and cell lines. HOXA9 acts as a tumor suppressor and inhibits glycolysis in cSCC in vitro and in vivo by negatively regulating HIF-1α and its downstream glycolytic regulators, HK2, GLUT1 and PDK1. Mechanistic studies show that HOXA9-CRIP2 interaction at glycolytic gene promoters impeds HIF-1α binding, repressing gene expression in trans. Our results reveal a miR-365-HOXA9-HIF-1α regulatory axis that contributes to the enhanced glycolysis in cSCC development and may represent an intervention target for cSCC therapy.

The expression levels of miR-365 vary in different malignancies. Herein, we found that miR-365 was overexpressed in both cells and clinical specimens of cutaneous squamous cell carcinoma (SCC). We demonstrated that the HaCaT ^{pre-miR-365-2} cell line, which overexpressed miR-365, could induce subcutaneous tumors in vivo . Antagomir-365, an anti-miR-365 oligonucleotide, inhibited cutaneous tumor formation in vivo , along with G ₁ phase arrest and apoptosis of cancer cells. These findings suggest that miR-365 may act as an onco-miR in cutaneous SCC both in vitro and in vivo . The present study provides valuable insight into the role of miR-365 in cutaneous SCC formation, which can help develop new drug and miR-365 target-based therapies for cutaneous SCC.

Aberrant expression of microRNAs plays vital roles in tumor development and progression. As transcription factors (TFs) are the critical components of signaling cascades, specific targeting effects of microRNAs to specific TFs may determine the role of microRNAs in different cancers. In this study, we identified Nuclear Factor I/B (NFIB) as one of the targets of miR-365 which was previously verified as an onco-miR in cutaneous squamous cell carcinoma (CSCC). Down-regulation of NFIB was a general feature in both CSCC cell lines and tumors from patients which show drastically up-regulated miR-365 expression levels. The siRNA-based knockdown of NFIB mimic the carcinogenic transformation of normal cells by ectopically expression of miR-365 which indicates depletion of NFIB is necessary for miR-365 to exert its pro-carcinogenic function. NFIB may represent a functional barrier targeted by miR-365 to the development of CSCC. Further studies also discovered a conserved feedback regulatory circuitry formed by NFIB and miR-365 in CSCC development which may be potentially utilized as therapeutic target to improve the clinical CSCC treatment.

Long noncoding RNAs (LncRNAs), including MALAT1, are critical regulators of tumor development. However, the roles and molecular mechanisms of LncRNAs in cutaneous squamous cell carcinoma (cSCC) remain underexplored. In this study, functional studies using in vitro cellular and in vivo xenograft models confirmed the pro-carcinogenic roles of MALAT1 in cSCC. Further, MALAT1 was identified to regulate epidermal growth factor receptor (EGFR) protein expression but did not affect EGFR mRNA expression. Transcriptomic sequencing identified kinectin 1 (KTN1) as the key mediator for MALAT1 regulation of EGFR. Mechanistic study revealed that MALAT1 interacts with c-MYC to form a complex and directly binds to the promoter region of KTN1 gene and enhances its transactivation to positively regulate EGFR protein expression. Our findings, therefore, establish a novel c-MYC-assisted MALAT1-KTN1-EGFR axis, which contributes to cSCC development and may serve as novel target for therapeutic intervention.

Pro-apoptotic BCL2 associated X (BAX) is traditionally thought to be regulated by anti-apoptotic BCL-2 family members, like BCL2-like 1 (BCL-XL), at the protein level. However, the posttranscriptional regulation of BAX is under explored. In this study, we identified BAX as the novel downstream target of miR-365, which is supported by gain- and loss-of-function studies of onco-miR-365. Loss of BAX by either RNA interference or highly-expressed miR-365 in cells of cutaneous squamous cell carcinoma (CSCC) enhanced the tumor resistance against apoptosis, while repressing cell proliferation, migration, and invasiveness. In vivo experiment confirmed that BAX knockdown promotes the growth of CSCC xenografts. Collectively, our results find a miR-365-BAX axis for alleviating the pro-apoptotic effects of BAX, which promotes CSCC development and may facilitate the generation of novel therapeutic regimens to the clinical treatment of CSCC.