miR-1246 releases RTKN2-dependent resistance to UVB-induced apoptosis in HaCaT cells

Abstract

MicroRNAs are a kind of small non-coding RNAs that play important roles in various biological processes such as cell proliferation, differentiation, and apoptosis. Cellular responses to UV-induced apoptosis have been suggested to be regulated by microRNAs at the posttranscriptional level, while the detailed mechanisms underlying this process remain unclear. Our aim in this study was to investigate the effects of miR-1246 in UVB-induced apoptosis and to identify the functional targets of miR-1246 in keratinocyte HaCaT cells. The expression of miR-1246 and apoptotic genes in HaCaT cells experiencing UVB stress was determined using quantitative real-time PCR. miR-1246 functions in UVB-induced apoptosis were quantified via fluorescence-activated cell sorter analysis of miR-1246 mimic or inhibitor-transfected cells. Additionally, the regulatory relationship between RTKN2 and miR-1246 was identified by Western blot and luciferase reporter assays. miR-1246 was upregulated accompanying with UVB-irradiated apoptosis in HaCaT cells. Overexpression of miR-1246 promoted UVB-induced apoptosis, while knockdown of miR-1246, using a specific inhibitor, resulted in a significant reduction in UVB-elicited apoptosis. We further demonstrate that miR-1246 negatively regulated the expression of RTKN2 through binding to the 3'-untranslated region of RTKN2 at the posttranscriptional level. Moreover, RTKN2 was observed to be resistant to UVB-induced apoptosis and RTKN2 antagonized the pro-apoptotic effects of miR-1246 during UVB-induced apoptosis in HaCaT cells. These findings suggested that miR-1246 promotes UVB-induced apoptosis by downregulating RTKN2 expression and that UVB-upregulated miR-1246 released RTKN2-dependent resistance to UVB-induced apoptosis by targeting RTKN2 post-transcriptionally in keratinocyte cells.

Knockdown of Rhotekin 2 expression suppresses proliferation and invasion and induces apoptosis in hepatocellular carcinoma cells

“…Conversely, suppressing expression of RTKN by short interfering RNA (siRNA) greatly sensitized cells to apoptosis (7). Similarly, several studies have been conducted concerning the anti-apoptotic effects of RTKN2 (9)(10)(11)(12). RTKN2 was originally identified in a promyelocytic cell line resistant to 25-OHC-induced apoptosis (12).…”

Hsa-miR-520d-5p promotes survival in human dermal fibroblasts exposed to a lethal dose of UV irradiation

“…After UV-induced damage, the expression of genes involved in DNA repair was silenced universally; 30 conversely, many miRNAs were upregulated by UVB (280–320 nm) irradiation. 31,32 In this study, OTUB1 (OTU deubiquitinase, ubiquitin aldehyde binding 1) was expressed following UVB irradiation, causing p53 stabilization, which was not downregulated even by lethal UVB irradiation 33 (Figure 3). It may be very important in the DNA repair process that ‘ubiquitination and p53 upregulation’ precedes ‘c-Abl-mediated ATR-BCRA1 upregulation’ in the DNA repair process.…”

MiR-26a Mediates Ultraviolet B-Induced Apoptosis by Targeting Histone Methyltransferase EZH2 Depending on Myc Expression

“…Protein samples were prepared and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation as previously reported [15]. Anti-EZH2 (1: 1000, BD Bioscience) and anti-GAPDH (1: 4000, Santa Cruz Biotechnology, USA) primary antibodies were used with horseradish peroxidase (HRP)-conjugated secondary antibody (1: 7000, Sigma).…”

“…The 3′-untranslated region (3′-UTR) of human EZH2 was amplified from genomic DNA and cloned into an miRNA luciferase reporter vector, as previously reported [15]. The luciferase reporter construct pRL-TK was co-transfected with RNA oligonucleotides or expression vectors into HaCaT cells for 48 h, after which luciferase reporter activity was determined using a dual luciferase reporter assay system (Promega, USA).…”

“…They are pivotal for sensing environmental stresses, such as UV radiation [9-11]. Previously, we identified dozens of miRNAs that are transcriptionally altered in response to UVA irradiation [12], and found that miR-23a, miR-1246, and miR-141 have roles in UV-induced apoptosis and aging [13-15]. miR-26a is a well-established tumor suppressor that targets multiple oncoproteins, such as CDC6, HMGA1, FGF9, and AKT [16-19].…”