SphK1-targeted miR-6784 inhibits functions of skin squamous cell carcinoma cells

Sphingosine kinase 1 (SphK1) is overexpressed in skin squamous cell carcinoma (SCC). It has emerged as a novel therapeutic oncotarget. The current study identified a novel SphK1-targeting microRNA, microRNA-6784 (miR-6784). Here, we show that miR-6784 is located at the cytoplasm of A431 skin SCC cells. It directly binds to SphK1 mRNA. Ectopic overexpression of miR-6784 inhibited SphK1 3’-untranslated region (UTR) luciferase activity and downregulated its expression. Moreover, miR-6784 overexpression caused ceramide accumulation in skin SCC cells. Functional studies in established (A431 and SCC9) and primary skin SCC cells revealed that miR-6784 overexpression inhibited cell viability, proliferation, migration, and invasion. It also simultaneously provoked apoptosis activation. Conversely, miR-6784 silencing by antagomiR-6784 induced SphK1 elevation and augmented A431 cell proliferation, migration, and invasion. miR-6784 overexpression-induced anti-A431 cell activity was inhibited by the expression of an UTR-null SphK1 construct. CRISPR/Cas9-induced SphK1 knockout inhibited A431 cell growth. Importantly, miR-6784 was completely ineffective when treating SphK1-knockout A431 cells. Collectively, miR-6784 silences SphK1 and inhibits skin SCC cell progression.

Studies have shown that SphK1 expression is elevated in skin SCC tissues and cells [12,13]. It has emerged as an important prognostic marker and valuable therapeutic target for skin SCC [12,13]. SphK1 overexpression is vital for skin SCC cell proliferation, migration, and metastasis [12,13]. SphK1 inhibition, however, would lead to ceramide production that promotes cancer cell apoptosis [12,13]. The anti-skin SCC activity by I-BET726, a novel bromodomaincontaining protein 4 (BRD4) inhibitor, has been associated with SphK1 inhibition and ceramide accumulation [14]. These results indicated that SphK1 inhibition or silencing should produce significant antiskin SCC activity.

miR-6784 binds to and silences SphK1 in skin SCC cells
First, the miRNA database TargetScan (V7.2) [26] was utilized to search possible miRNAs that can target the 3'-UTR of SphK1. The candidate miRNAs with high binding scores to SphK1 mRNA were further verified in other miRNA databases. The bioinformatic studies discovered one particular miRNA, miR-6784, which putatively binds to SphK13'-UTR (at position 113-120) ( Figure 1A). The binding context score percentage is 99%, and the context ++ score is -0.60 ( Figure 1A). These parameters indicated a high percentage of possible direct binding between miR-6784 and SphK1 3'-UTR [26]. When analyzing subcellular localization of miR-6784, we found that over 92% of endogenous miR-6784 was located at the cytosol fraction of A431 cells ( Figure 1B). Only less than 8% was located at the nuclear fraction ( Figure 1B). By applying a RNA Pull Down assay, we found that the biotinylated-miR-6784 can directly associate with SphK1 mRNA in A431 cells ( Figure 1C). As a negative control, biotinylated-miR-155 failed to bind to SphK1 mRNA in A431 cells ( Figure 1C).
AGING cell activity. These results suggested that SphK1 silencing might be the primary mechanism of miR-6784-induced actions in skin SCC cells.
Studies have shown that skin SCC and various other types of human cancers acquire survival advantage, hyper-proliferative properties, aggressiveness, and chemotherapy resistance via non-oncogenic addiction to S1P signaling [30][31][32][33]. In oral SCC cells, downregulation of leucine-rich repeats and immunoglobulinlike domains 1 (LRIG1) induced the activation of EGFR-mediated SphK1 signaling to promote extracellular matrix (ECM) remodeling and cancer cell progression [34]. Tamashiro et al., showed that SphK1 was required for the invasion of head and neck SCC (HNSCC) cells [35]. Furthermore, SphK1 inhibition sensitized radiation-induced anti-HSNCC cell activity [36]. These results clearly indicated that SphK1 is a promising therapeutic target for skin SCC.
To our knowledge miR-6784 is a relatively novel miRNA with its functions largely unknown. Here, we found that miR-6784 is a novel SphK-targeting miRNA in skin SCC cells. miR-6784 is located at the cytoplasm of skin SCC cells and it binds directly to SphK1 mRNA. In established and primary skin SCC cells, SphK1's 3-UTR activity, as well as its mRNA and protein expression, decreased following the overexpression of miR-6784. Conversely, antagomiR-6784-mediated miR-6784 silencing elevated SphK1 mRNA and protein. Importantly, the mutant miR-6784 mimics, with mutations at the SphK1's 3-UTR-binding sites, failed to inhibit SphK1 expression. Therefore, miR-6784 directly binds to and silences SphK1 in skin SCC cells.
The results of this study indicated that SphK1 silencing should be the primary mechanism of miR-6784 in skin SCC cells. To restore SphK1 expression by an UTR-null SphK1 construct, completely reversed miR-6784-induced actions. CRISPR/Cas9-induced SphK1 KO also resulted in proliferation inhibition and apoptosis in A431 cells. Importantly, miR-6784 overexpression or silencing was ineffective in SphK1-KO A431 cells.

CONCLUSIONS
Nowadays, over one million cases of skin SCC are diagnosed each year as SCC incidence has increased up to two folds in the past three decades [1][2][3]. The advanced, recurrent, and metastatic skin SCC are still fatal to many affected patients [1][2][3]. Targeted molecular therapies are vital for better skin SCC treatments [1][2][3]. The results of this study showed that miR-6784 silenced SphK1 and inhibited skin SCC cell progression. Therefore, miR-6784 could be a novel therapeutic candidate for skin SCC.

Cell culture
Established skin SCC cell lines, A431 and SCC9, were provided by Dr. Liu at Wenzhou Medical University [14,27]. The primary skin SCC cells derived from two written-informed consent SCC patient ("C1/C2", with PTEN depletion and p53-null) were provided by Dr. Liu [14] as well. Cells were cultured as reported before [39]. Protocols were approved by the Ethics Committee of Nantong University, in accordance with Declaration of Helsinki.

Quantitative real time-PCR (qPCR)
As reported [22,40], TRIzol reagents (Thermo Fisher Scientific, Shanghai, China) were utilized to extract total cellular RNA that was reversely transcripted [41]. An ABI Prism 7500 system was applied to perform qPCR using a SYBR GREEN PCR Master Mix (Thermo Fisher Scientific). The product melting temperature was always calculated. Glyceraldehyde-3phosphatedehydrogenase(GAPDH) was tested as the internal control and reference gene for data quantification using an established 2 −∆∆Ct method. Expression of miR-6784 was normalized to U6. The primers of this study were listed in Table 1.

Western blotting
As reported before [42,43], total cellular protein lysates (40 μg proteins per treatment into each lane) were separated by 10-12% SDS-PAGE gels and then transferred to polyvinylidene fluoride (PVDF) membranes (Sigma-Aldrich, St. Louis, MO, USA). The membranes were blocked and immuno-blotted with indicated primary and secondary antibodies. An enhanced chemiluminescence (ECL) reagent kit (Bio-Rad, Shanghai, China) was utilized to visualize interested protein bands. The ImageJ software (NIH) was utilized for data quantification.

RNA-Pull down assay
The detailed protocol for RNA-Pull Down assay via a Pierce Magnetic RNA Pull-Down Kit has been described previously [46,47]. Briefly, A431 cells were transfected with biotinylated miR-6784 mimic or control mimic (Genechem, 200 nmol/L) for 24h, and the cells were harvested [47]. The quantified total cellular lysates were incubated with streptavidin-coated magnetic beads to pull-down biotin-captured RNA complex that was purified [46]. SphK1 mRNA was examined through qPCR and normalized to the input controls.

CCK-8 viability assay
Cells with applied genetic modifications were seeded into 96-well plates (exact 4, 000 cells per well) and cultured for 96h. Cell viability was examined by the CCK-8 assay kit, and in each well CCK-8's, optical density (OD) was tested at 450 nm.

Colony formation
A431 cells with applied genetic modifications were initially seeded into 10-cm dishes (exact 2×10 5 cells per dish). Complete medium was renewed every two days for a total of 10 days. Afterwards, the number of viable A431 colonies was manually counted.

In vitro cell migration and migration assays
Cells with applied genetic modifications were seeded into the upper surfaces of the "Transwell" chambers (BD Biosciences, San Jose, CA, USA) at a density of 40, 000 cells per chamber (maintained in 300 μL serum-free medium [48]). FBS (12%) complete medium was filled into the lower chambers. Cells were allowed to migrate for 24h. Migrated cells on the lower surface of the chamber were stained and counted manually. For each condition, five repeated views were included to calculate the average number of migrated cells. For cell invasion assays, the chamber surface was always coated with "Matrigel" (Sigma).

Caspase-3 activity assay
As described [27], total cellular protein lysates (30 μg for each treatment) were incubated with the 7-amino-4trifluoromethylcoumarin (AFC)-conjugated caspase-3 substrate into the caspase assay buffer. After 3h of incubation, the AFC activity was examined by an Infinite 200 PRO reader at 400 nm excitation and 505 nm emission.

TUNEL staining
Cells with applied genetic modifications were seeded into 96-well plates (4, 000 cells per well) and cultured for 72h. Cells were then stained with TUNEL and nuclear dye DAPI. TUNEL-positive nuclei ratio was calculated from five random views (1000 nuclei per treatment).

DNA breaks
Skin SCC cells with applied genetic modifications were seeded into 96-well plates (4 × 10 3 cells per well) and cultured for 48h. To test DNA breaks, a single strand DNA (ssDNA) ELISA kit (EMD Millipore, Burlington, MA, USA) was utilized. ELISA absorbance in each well was tested at 405 nm.

Mitochondrial depolarization assay
Cells with applied genetic modifications were initially seeded into 12-well plates (4 ×10 4 cells per well) for 48h and stained with JC-1 dye. The intensity of JC-1 green monomers was measured.

Ceramide assay
Cells with applied genetic modifications were initially seeded into six-well plates (1×10 5 cells per well) for 24h. The cellular ceramide content was examined through a protocol as previously described [49]. Ceramide content was expressed as fmol by nmol of phospholipid. Its level was normalized to the control.

Ectopic overexpression of SphK1
The GV369 lentiviral construct encoding the UTR-null SphK1 was received from Dr. Yao at Nanjing Medical University [22]. The construct was transduced to A439 AGING cells with miR-6784 overexpression. Stable cells were selected by puromycin. SphK1 expression was verified by qPCR and Western blotting assays.

CRISPR/Cas9-induced SphK1 knockout (KO)
A lentiCRISPR-Cas9-GFP SphK1 KO construct was from Dr. Yao at Nanjing Medical University [22]. A431 cells were cultured into six well-tissue plates at 50-60% confluence for 24h and then transfected with theSphK1-KO construct for another 48h. Afterwards, FACSmediated GFP sorting was applied and the transfected cells were distributed into 96-well plates for another 72h. Single stable SphK1 KO A431 cells were further screened by qPCR and Western blotting assays. The empty vector, "Cas9-C" [22], was transfected to control A431 cells.

Statistical analysis
Experiments were repeated for at least three times. Data were presented as mean ± standard deviation (SD). Statistics were analyzed by one-way ANOVA with SPSS software (21.0, Chicago, CA). To compare difference between two specific groups, a two tailed T Test was applied (Excel 2007). P<0.05 was considered for significant statistical difference.

CONFLICTS OF INTEREST
None of the authors have any conflicts of interest.