HOTAIR/Sp1/miR-199a critically regulates cancer stemness and malignant progression of cutaneous squamous cell carcinoma.

The long non-coding RNA (lncRNA), HOX antisense intergenic RNA (HOTAIR) is a well-characterized oncogene in multiple human cancers, but not in cutaneous squamous cell carcinoma (CSCC). In this study, we focused on investigating the potential role of HOTAIR in stemness of CSCC. By measuring its expression using RT-qPCR in CSCC vs. normal tissues, as well as in CSCC cell lines A431 or SCC13, A431- or SCC13-derived CSCC stem cells (CSCSCs), and normal skin fibroblasts (HSFs), we detected higher expression of HOTAIR in CSCC than in normal tissues, in recurrent than in non-recurrent CSCC tissues, in CSCCs and CSCSCs than in HSFs, and particularly, in CSCSCs than in CSCCs. Kaplan-Meier analysis suggested that higher expression of HOTAIR was positively correlated with worse overall survival of CSCC patients. Functional assays on colony formation, EdU incorporation, sphere formation, western blot on stem-cell biomarkers, and in vivo models showed that HOTAIR was essential in maintaining multiple stem cell phenotypes of CSCSCs in vitro and in vivo xenograft growth as well as metastasis. Mechanistically, HOTAIR directly interacted with and up-regulated Sp1. Sp1 then induced DNMT1-mediated promoter methylation and direct transcriptional repression of miR-199a-5p. Targeting Sp1 or DNMT1 further boosted the in vivo anti-tumor and anti-metastasis activities of targeting HOTAIR. In conclusion, HOTAIR, by up-regulating Sp1 and targeting miR-199a, promotes stemness and progression of CSCC. Targeting HOTAIR, Sp1 or the underlying mechanisms may thus benefit CSCC treatment.

Prognostic signature and immune efficacy of m1 A-, m5 C- and m6 A-related regulators in cutaneous melanoma.

Cutaneous melanoma (CM) is an aggressive cancer; given that initial and specific signs are lacking, diagnosis is often late and the prognosis is poor. RNA modification has been widely studied in tumour progression. Nevertheless, little progress has been made in the signature of N1 -methyladenosine (m1 A), 5-methylcytosine (m5 C), N6 -methyladenosine (m6 A)-related regulators and the tumour microenvironment (TME) cell infiltration in CM. Our study identified the characteristics of m1 A-, m5 C- and m6 A-related regulators based on 468 CM samples from the public database. Using univariate, multivariate and LASSO Cox regression analysis, a risk model of regulators was established and validated by a nomogram on independent prognostic factors. The gene set variation analysis (GSVA) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) clarified the involved functional pathways. A combined single-sample gene set enrichment analysis (ssGSEA) and CIBERSORT approach revealed TME of regulator-related prognostic signature. The nine-gene signature stratified the patients into distinct risk subgroups for personalized prognostic assessment. Additionally, functional enrichment, immune infiltration and immunotherapy response analysis indicated that the high-risk group was correlated with T-cell suppression, while the low-risk group was more sensitive to immunotherapy. The findings presented here contribute to our understanding of the TME molecular heterogeneity in CM. Nine m1 A-, m5 C- and m6 A-related regulators may also be promising biomarkers for future research.

MiR-199a-5p represses the stemness of cutaneous squamous cell carcinoma stem cells by targeting Sirt1 and CD44ICD cleavage signaling.

Tumorigenic cancer stem cells (CSCs) exist in various tumors including the cutaneous squamous cell carcinoma (cSCC) as a minor subpopulation and are tightly associated with metastasis and therapeutic resistance. Better understanding of CSCs properties is essential for the novel therapeutic strategy targeted toward these cancers. The cSCC stem cells (cSCCSCs) were enriched from a cSCC cell line A431 by repeated sphere culture, and identified via the expression analysis of stemness marker genes and CD44 proteolysis. MiR-199a-5p was previously reported to be related with the proteolysis modulation of CD44, so the specific regulation mechanisms were verified by overexpression in vitro and in vivo. MiR-199a-5p is under-expressed in cSCCSCs and functions as a tumor suppressive molecule. Overexpression of miR-199a-5p reduced the stemness of cSCCSCs and inhibited cell proliferation. By targeting the deacetylase Sirt1, miR-199a-5p inhibited cellular proteolysis of CD44 and reduced the CD44 intracellular domain (CD44ICD) release and nuclear translocation. Overexpression of CD44ICD reversed the effects of miR-199a-5p overexpression or Sirt1 silencing, and increased the transcriptional expression of stemness genes. Our results revealed that the miR-199a-5p/Sirt1/CD44ICD signaling pathway regulates cSCCSCs progression by affecting its migration ability and tumorigenicity, therefore can be utilized to develop a curative approach for cSCC.Abbreviations: CSCs: cancer stem cells; cSCC cutaneous squamous cell carcinoma; cSCCSCs: cSCC stem cells; CD44ICD: CD44 intracellular domain; HA: hyaluronic acid; HNSCC: hand and neck squamous cell carcinoma; ESCC: esophageal squamous cell carcinoma;MMPs: matrix metalloproteinases; SFM: sphere formation medium; EGF: epidermal growth factor; bFGF: basic fibroblast growth factor; BSA: bovine serum albumin; CCK-8: cell counting kit-8.

LncRNA TUG1 regulates FGF1 to enhance endothelial differentiation of adipose-derived stem cells by sponging miR-143.

Adipose-derived stem cells (ADSCs) have emerged as a cell source for regeneration medicine. ADSCs possess the capacity to differentiate into endothelial cells and serve an essential role in vascular development and function. LncRNA taurine upregulated gene 1 (TUG1) has recently been linked with angiogenesis in hepatoblastoma. However, the roles of TUG1 in endothelial differentiation of ADSCs remain unidentified. Human adipose-derived stem cells (hADSCs) were obtained and characterized by flow cytometry, Oil red O and Alizarin Red staining. HADSCs were maintained in the endothelial differentiation medium and the expressions of TUG1, miR-143, and FGF1 were examined by qRT-PCR. To assess endothelial differentiation, the expressions of CD31, von Willebrand factor (vWF), VE-cadherin were examined by Western blot analysis, qRT-PCR, and immunofluorescence. Tube formation in Matrigel was examined. The interactions between TUG1 and miR-143, miR-143 and FGF1 were validated by luciferase assays. During the endothelial differentiation process, TUG1 and FGF1 were upregulated, whereas miR-143 was downregulated. TUG1 overexpression downregulated miR-143, upregulated FGF1, CD31, vWF, and VE-cadherin, and enhanced capillary tube formation. Luciferase assays showed that TUG1 interacted with miR-143, and FGF1 was a direct target of miR-143. Furthermore, the enhancement of endothelial differentiation induced by TUG1 overexpression was abolished by miR-143 overexpression. Our findings implicated that lncRNA TUG1 promoted endothelial differentiation of ADSCs by regulating the miR-143/FGF1 axis.