EXPERIMENTAL AND THERAPEUTIC MEDICINE 21: 116, 2021

MicroRNA‑451a prevents cutaneous squamous cell carcinoma progression via the 3‑phosphoinositide‑dependent protein ‑1‑mediated PI3K/AKT signaling pathway

JIXING FU*, JIANHUA ZHAO*, HUAMIN ZHANG, XIAOLI FAN, WENJUN GENG and SHAOHUA QIAO

Department of Dermatology, Liaocheng Second People's Hospital, Shandong First Medical University Affiliated Liaocheng Second Hospital, Linqing, Shandong 252601, .R.P China

Received February 12, 2020; Accepted August 11, 2020

DOI: 10.3892/etm.2020.9548

Abstract. The role of microRNAs (miRNAs/miRs) in pathway, which may offer potential therapeutic targets for the governing the progression of cutaneous squamous cell carci‑ treatment of cSCC. noma (cSCC) has been the focus of recent studies. However, the functional role of miR‑451a in cSCC growth remains Introduction poorly understood. Therefore, the present study aimed to determine the expression levels of miR‑451a in cSCC cell Skin or cutaneous carcinoma is a major worldwide lines and the involvement of miR‑451a in cSCC progression. public health burden which is highly prevalent and demon‑ The results revealed that the expression levels of miR‑451a strates an ever‑increasing incidence with ~108,420 new cases were downregulated in cSCC tissues and cell lines, and that and 11,480 death in the United States in 2020 (1,2). The this subsequently upregulated 3‑phosphoinositide‑dependent majority of diagnosed skin cancer cases are non‑melanoma‑ protein kinase‑1 (PDPK1) expression levels. PDPK1 was tous, consisting of basal cell carcinoma and cutaneous cell validated as a direct target of miR‑451a in cSCC using carcinoma (cSCC), which originate from keratinized epithelial bioinformatics software Starbase, dual‑luciferase reporter cells (3). Common risk factors for most non‑melanomatous assays and western blotting. Additionally, CCK‑8, EdU include ultraviolet light exposure, radiation exposure, and Transwell assays, as well as flow cytometry and Hoechst immunosuppression and genetic factors (4). The most effective 3325 staining, were performed to assess the malignant aggres‑ treatment for the majority of lesions is surgery; however, other siveness of cSCC cells. Overexpression of miR‑451a was interventions, including radiation, topical immunomodulators demonstrated to impair the proliferation, migration, invasion and novel systemic drugs are also applied (5). The incidence and epithelial‑mesenchymal transition (EMT), and promoted rates of cSCC are much higher in populations with fairer apoptosis in cSCC cells by interacting with PDPK1, possibly by skin compared with individuals with darker skin and notably direct targeting. Furthermore, the western blotting results indi‑ higher, still, in regions with high ambient ultraviolet radiation cated that miR‑451a overexpression may block the PI3K/AKT levels (6). For instance, incidence rates have ranged from 60 per signaling pathway by interacting with PDPK1. In conclusion, 100,000 people annually in Canada (latitude, 54˚N; 2006) to the findings of the present study suggested that miR‑451a may 290 per 100,000 people annually in the United States (latitude, prevent the proliferation, migration, invasion and EMT of 31‑37˚N; 1991); these rates were higher compared with those cSCC cells through the PDPK1‑mediated PI3K/AKT signaling in Norway (annually, 20 per 100.000 men and 15 per 100,000 women; 2008‑11) (7‑9). cSCC is characterized by neoplastic squamous epithelial cells invading into the dermis, which may present as nodules, squamous islands or cystic structures (10). The past decade has seen significant advancements in the Correspondence to: Dr Jixing Fu, Department of Dermatology, development of diagnostic and prognostic biomarkers, which Liaocheng Second People's Hospital, Shandong First Medical may help elucidate the mechanisms that promote tumor initia‑ University Affiliated Liaocheng Second Hospital, 306 Health Street, tion, progression and metastasis (11). Linqing, Shandong 252601, P.R. China MicroRNAs (miRNAs/miRs) are a group of non‑coding E‑mail: [email protected] RNAs of ≤25 nucleotides in length (12). miRNAs modu‑ *Contributed equally late gene expression post‑transcriptionally and numerous miRNAs, including miR‑34a, miR‑181a and miR‑148a, have Key words: cutaneous squamous cell carcinoma, microRNA‑451a, been revealed to function as tumor suppressors in patients 3‑phosphoinositide‑dependent protein kinase‑1, PI3K/AKT with cSCC (13). A previous study revealed that miR‑451a signaling pathway, migration, invasion expression levels were markedly downregulated in human basal cell carcinoma tissues and mouse models (14), indicating its potential role in skin cancer. The present 2 FU et al: TUMOR SUPPRESSIVE ROLE OF miR-451a IN CSCC study predicted that 3‑phosphoinositide‑dependent protein RT‑qPCR. Total RNA was extracted from HaCaT, A431, kinase‑1 (PDPK1) may be a target gene of miR‑451a, which HSC‑5, SCC‑12 and SCL‑1 cells using TRIzol® reagent was discovered by performing a dual‑luciferase reporter gene (Invitrogen; Thermo Fisher Scientific, Inc.), according to the assay. PDPK1 was previously illustrated to serve a pivotal manufacturer's protocol. Total RNA was reverse transcribed role in the proliferation of angiosarcoma cells, indicating its into cDNA using a PrimeScript™ RT reagent kit according to potential use as a target against angiosarcoma, an aggressive the manufacturer's protocol with a gDNA Eraser (Takara Bio, malignancy of endothelial cells (15). In non‑small cell lung Inc.) at 70˚C for 5 min, ice‑bathed for 3 min, at 37˚C for 60 min cancer, miR‑503 upregulation was revealed to downregulate and at 95˚C for 10 min. qPCR was subsequently performed PDPK1 expression levels, thus blocking the PI3K/AKT using a SYBR Green PCR Master mix (Invitrogen; Thermo signaling pathway (16). The present study determined the Fisher Scientific, Inc.). The thermocycling conditions were expression profiles of miR‑451a and PDPK1 in cSCC tissues as follows: Pre‑denaturation at 95˚C for 5 min; denaturation using bioinformatics analysis and cSCC cell behavioral at 94˚C for 45 sec; annealing at 56˚C for 45 sec and extension examination. The association between miR‑451a and PDPK1 at 72˚C for 45 sec for a total of 35 cycles. The primer sequences was also investigated using bioinformatics analysis and a used for qPCR are provided in Table II. GAPDH and U6 were dual‑luciferase reporter gene assay. A431 and SCC‑12 cell used as the internal loading controls. Fold changes in the rela‑ lines overexpressing miR‑451a were generated to determine tive expression of the targets were calculated using the 2-ΔΔCq the regulatory role of miR‑451a in the cSCC malignant method (20). phenotype and the PI3K/AKT signaling pathway. Cell Counting Kit‑8 (CCK‑8) assay. The proliferative ability Materials and methods of cSCC cells was evaluated using a CCK‑8 assay (Roche Diagnostics). At 48 h post‑transfection, A431 and SCC‑12 were Bioinformatics analysis. The cSCC‑associated dataset seeded into 96‑well plates (5x103 cells/well) at 37˚C. At the GSE57768, which was comprised of 30 cSCC tissues and baseline, 24, 48 and 72 h post‑incubation, 50 µl CCK‑8 reagent 18 normal skin tissues (17), was downloaded from the Gene was added to determine the optical density values at 490 nm Expression Omnibus (GEO) database (ncbi.nlm.nih.gov/geo). with a Multiskan Sky microplate reader. The differentially expressed (DEG) between control and cancerous samples were identified using the limma package of 5‑ethynyl‑2'‑deoxyuridine (EdU) staining. Additionally, the R software (version no. 3.6.2; master.bioconductor.org/pack‑ proliferative ability of cSCC cells was analyzed using EdU ages/release/bioc/html/limma.html). The cut‑off values for staining, as previously described (21). Briefly, stably transfected DEG selection were P<0.05 and |Log FoldChange|>1, which A451 and SCC‑12 cells (5x103 cells/well) were seeded into were used to plot a heatmap of DEGs using the heatmap 96‑well petri dishes for a 24‑h culture and fixed with 100 µl package (version no. 1.0.12) of R software (cran.r‑project. 4% paraformaldehyde for 30 min at room temperature. Then, org/web/packages/pheatmap/index.html). RPMI‑1640 medium (100 µl) containing 20 µM EdU (Beijing The target mRNAs of miR‑451a were subsequently Solarbio Science & Technology Co., Ltd.) was added into predicted using the StarBase version 2.0 website (http://star‑ each well, after which cells were cultured at 37˚C for 2 h. The base.sysu.edu.cn). Signaling pathway enrichment analysis was EdU positive cells were stained red. Nuclei were subsequently conducted with the target mRNAs identified by Starbase using counterstained with 4',6‑diamidino‑2‑phenylindole at 37˚C the Kyoto Encyclopedia of Genes and Genomes (KEGG) for 30 min. The number of EdU positive cells was observed database (kegg.jp) through The Database for Annotation, in five different visual fields using a fluorescence microscope Visualization and Integrated Discovery (version no. 6.8; david. (magnification, x200; Olympus Corporation). The EdU positive ncifcrf.gov) (18,19). cell index (EdU positive cells/total cells) was measured using ImageJ (version no. 1.52; National Institutes of Health). Cell lines and culture. The human keratinocyte cell line HaCaT (cat. no. GDC106) and cSCC cells, A431, HSC‑5, Flow cytometric analysis of apoptosis. Apoptotic cSCC SCC‑12 and SCL‑1, were all purchased from The Cell Bank of cells were analyzed following miR‑451a transfection using Type Culture Collection of the Chinese Academy of Sciences. an Annexin V‑fluorescein isothiocyanate (FITC)/propidium Cells were cultured in RPMI‑1640 medium (Gibco; Thermo iodide (PI) apoptosis detection kit (Best‑Bio, Ltd.) and flow Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; cytometry as previously reported (22). Briefly, cells were resus‑

Thermo Fisher Scientific, Inc.) at 37˚C with 5% CO2. pended in 200 µl binding buffer and dual‑stained in the dark Subsequently, A431 and SCC‑12 cells in the logarithmic for 15 min at room temperature using PI and Annexin V‑FITC growth phase were seeded into 6‑well plates (2x105 cells/well) (both, 100 µl). Late apoptotic cells (positive for PI and and transfected with 100 nmol miR‑451a mimics or mimic Annexin V‑FITC) were analyzed using a BD Accuri™ C6 controls (synthesized by Shanghai GenePharma Co., Ltd.) Plus flow cytometer and CellQuest software (version no; 6.1; using Lipofectamine® 2000 reagent (Thermo Fisher Scientific, both from BD Biosciences). Inc.), according to the manufacturer's protocol. Following 48 h of transfection at 37˚C, miR‑451a expression levels were Hoechst 33258 staining. cSCC cells in the logarithmic growth subsequently analyzed using reverse transcription‑quantitative phase were subjected to Hoechst 33258 staining. Briefly, PCR (RT‑qPCR) to determine the transfection efficiency. The following transfection, 5x103 cSCC cells were seeded into sequences for the miR‑451a mimics and mimic controls are six‑well plates and incubated overnight at 37˚C. Following presented in Table I. permeabilization with 50 µl cold 4% formaldehyde for 30 min EXPERIMENTAL AND THERAPEUTIC MEDICINE 21: 116, 2021 3

Table I. Sequence information for miR‑451a mimic and mimic The migration assay was performed in a similar manner, control. except for the following modifications: The Transwell plates were not precoated in Matrigel and the cells were only incu‑ Name Sequence (5'‑3') bated for 24 h at 37˚C. miR‑451a mimic AAACCGUUACCAUUACUGAGUU Dual‑luciferase reporter gene assay. The PDPK1 wild‑type mimic control AUCUGCCGGUGGUAACUGACUA (WT) and mutant (MT) 3'untranslated region (UTR) binding sequences were synthesized by Shanghai GenePharma Co., Ltd. miR, microRNA. and inserted into pMIR‑REPORTTM plasmids (Thermo Fisher Scientific, Inc.). 293T cells (1x104 cells/well) were incubated overnight and transfected with 50 nmol PDPK1‑WT/MT and miR‑451a mimics or mimic controls using Lipofectamine® 2000 Table II. Primer sequences used for reverse transcription‑ (Invitrogen Thermo Fisher Scientific, Inc.) for 48 h. The relative quantitative PCR. luciferase activity was determined using a Dual‑Luciferase Reporter assay system (Promega Corporation) as previously Gene Primer sequence (5'‑3') described (24). Renilla luciferase activity was used for normal‑ miR‑451a F: GAGGGGAGCAGAGTTCAAGT ization of the firefly luciferase activity. R: TGGGAGGCAGCAATAGACAA Western blotting. Western blotting analysis was performed PDPK1 F: CTGGACGACTTTGTTCTGGGG to analyze the status of PI3K/AKT signaling R: GCTCAGGAGCGTATGAAGTGG pathway‑associated proteins in transfected A431 and SCC‑12 U6 F: CTCGCTTCGGCAGCACA cells. The experimental protocol was performed according R: AACGCTTCACGAATTTGCGT to a previously described study (21). Briefly, total protein GAPDH F: GCACCGTCAAGGCTGAGAAC was extracted from the cells using an SDS lysis buffer (cat. no. P0013G; Beyotime Institute of Biotechnology) on ice R: GGATCTCGCTCCTGGAAGATG for 30 min. The concentration of total protein was measured miR, microRNA; PDPK1, 3‑phosphoinositide‑dependent protein using a BCA protein assay kit (Pierce; Thermo Fisher kinase‑1; F, forward; R, reverse. Scientific, Inc.). Total protein (50 µg/lane) was subjected to 10% SDS‑PAGE and transferred onto PVDF membranes. Membranes were blocked with 10% non‑fat milk at 25˚C for ≥1 h. The membranes were incubated with the following at room temperature, the cells were incubated for ≥20 min primary antibodies: Anti‑PDPK1 (1:2,000; cat. no. ab52893; at room temperature with 20 µg/ml Hoechst. The cells were Abcam), anti‑PI3K (1:5,000; cat. no. ab151549; Abcam), visualized using a Leica confocal laser‑scanning microscope anti‑phosphorylated (p)‑PI3KY607 (1:5,000; cat. no. ab182651; (TCS SP8; Leica Microsystems GmbH) at 365 nm in five Abcam), anti‑AKT1 (1:5,000; cat. no. ab235958; Abcam), different visual fields. The apoptotic cell rate (%) was calcu‑ anti‑pAKT1S473 (1:5,000; cat. no. ab81283; Abcam) and lated using the following formula: (Number of apoptotic cells anti‑β‑actin (1:10,000; cat. no. ab8226; Abcam) for 1 h at room per field/total number of cells per field) x100. temperature. Following the primary antibody incubation, the membranes were incubated with a horseradish peroxi‑ ELISAs. The concentrations of Bax (cat. no. ab199080), dase‑conjugated goat anti‑rabbit secondary antibody (1:10,000; Bcl‑2 (cat. no. ab202411), E‑cadherin (cat. no. ab197751) and cat. no. ab7090; Abcam) for 2 h at room temperature. Signals N‑cadherin (cat. no. ab254512) were determined in A431 were detected using ECL reagents (cat. no. SW2030; Beijing and SCC‑12 cell lysates using ELISA kits (all from Abcam), Solarbio Science & Technology Co., Ltd.). Densitometry was according to the manufacturers' protocols. analyzed using Image‑Pro Plus software (version no. 6.0; Media Cybernetics, Inc.). Transwell assays. The invasion and migration of A431 and SCC‑12 cells following miR‑451a mimic transfection were Statistical analysis. Statistical analysis was performed using analyzed using Transwell assays, as described previously (23). SPSS 21.0 software (IBM Corp.). All data and results were Briefly, for the invasion analysis, 3x104 cells/well were calculated from ≥3 replicate measurements and are presented as added to 200 µl FBS‑free RPMI‑1640 medium and plated the mean ± SD. The Kolmogorov‑Smirnov method was used to into the upper chamber of Transwell plates precoated with analyze whether the data were normally distributed. Multigroup Matrigel at 4˚C until the Matrigel was solidified (80 µl; 1:4; comparisons were performed using a one‑way or two‑way Sigma‑Aldrich; Merck KGaA). A total volume of 500 µl (factorial) ANOVA with a Tukey's post hoc test. P<0.05 was RPMI‑1640 medium supplemented with 10% FBS was plated considered to indicate a statistically significant difference. into the lower chamber. Following incubation at 37˚C for 48 h, the cells were fixed with 2.5% glutaraldehyde at room temper‑ Results ature for 30 min and stained with 0.1% crystal violet at room temperature for 30 min. The number of cells was counted in miR‑451a is poorly expressed in cSCC. Following the five different randomly selected fields of view using a light analysis of the GSE57768 dataset, 20 DEGs were selected microscope (magnification, x200). and plotted in a heat map (Fig. 1A). The results revealed that 4 FU et al: TUMOR SUPPRESSIVE ROLE OF miR-451a IN CSCC

Figure 1. miR‑451a expression levels are significantly downregulated in cSCC tissues and cell lines. (A) Heatmap representing the differentially expressed miRs in the GSE57768 dataset obtained from the GEO database. (B) miR‑451a expression levels in normal skin and cSCC tissues in the GSE57768 dataset obtained from the GEO database. **P<0.01. (C) miR‑451a expression levels in HaCaT, A431, HSC‑5, SCC‑12 and SCL‑1 cells were determined using RT‑qPCR. *P<0.05 vs. HaCaT cells. (D) miR‑451a expression levels in A431 and SCC‑12 cells transfected with the miR‑451a mimic or mimic control were determined by RT‑qPCR. *P<0.05. The data are presented as the mean ± SD. One‑way ANOVA and Tukey's multiple comparison test were used to determine statistical significances. miR, microRNA; cSCC, cutaneous squamous cell carcinoma; GEO, gene expression omnibus; RT‑qPCR, reverse transcription‑quantitative PCR. compared with the normal skin tissues, the expression levels miR‑451a mimics or mimic controls were subsequently of miR‑451a were downregulated in cSCC tissues (Fig. 1B). transfected into A431 and SCC‑12 cells to verify the effect A previous study indicated that miR‑451a may serve as a of miR‑451a on cSCC cells. These cell lines were chosen tumor suppressor in cutaneous basal cell carcinoma (14). for further experiments due to their relatively low miR‑451a Therefore, the current study determined the expression expression. RT‑qPCR analysis revealed that miR‑451a expres‑ levels of miR‑451a in human keratinocyte and cSCC cells; sion levels were significantly upregulated in cSCC cells the expression levels of miR‑451a were discovered to be following transfection with miR‑451a mimics compared significantly downregulated in cSCC cells compared with the with mimic controls, indicating that the transfection was HaCaT cells (Fig. 1C). successful (Fig. 1D). EXPERIMENTAL AND THERAPEUTIC MEDICINE 21: 116, 2021 5

Figure 2. miR‑451a overexpression inhibits cutaneous squamous cell carcinoma cell proliferation. (A) Cell proliferation rate in A431 and SCC‑12 cells transfected with the miR‑451a mimic or mimic control was determined using CCK‑8 assays. (B) Cell proliferation in A431 and SCC‑12 cells transfected with miR‑451 mimics or mimic controls was analyzed using EdU staining. Scale bar, 50 µm. The data are presented as the mean ± SD. One‑way ANOVA and Tukey's multiple comparison tests were used to determine statistical significances. *P<0.05. miR, microRNA; EdU, 5‑ethynyl‑2'‑deoxyuridine. miR‑451a overexpression suppresses cSCC cell viability. inhibited the EMT process in A431 and SCC‑12 cells by The CCK‑8 assay revealed that the proliferation of miR‑451a enhancing E‑cadherin expression and lowering N‑cadherin mimic‑transfected A431 and SCC‑12 cells was significantly expression (Fig. 4A). A431 and SCC‑12 cell invasion and inhibited compared with their respective mimic control‑treated migration were subsequently analyzed using Transwell assays. cells (Fig. 2A). Additionally, the results of the EdU staining The results demonstrated that the overexpression of miR‑451a assay illustrated that the transfection with miR‑451a mimics significantly inhibited the invasion and migration of both significantly decreased the proliferation of A431 and SCC‑12 cell lines compared with the mimic control‑transfected cells cells compared with mimic control‑transfected cells (Fig. 2B). (Fig. 4B and C). miR‑451a overexpression promotes cSCC cell apoptosis. miR‑451a blocks the PI3K/AKT signaling pathway by targeting The levels of apoptosis in A431 and SCC‑12 cells labeled PDPK1 in cSCC cells. The possible target genes of miR‑451a with PI/Annexin V were detected via flow cytometry. The were predicted using StarBase to determine the downstream results revealed that the transfection with miR‑451a mimics signaling pathways of miR‑451a in cSCC, which were further significantly increased the levels of apoptosis in both cell lines analyzed using KEGG signaling pathway enrichment analysis. compared with transfection with mimic controls (Fig. 3A), The results revealed that the ‘PI3K‑Akt signaling pathway’ was which was further validated by the results of Hoechst 33258 enriched (data not shown; Fig. 5A), which has been reported to staining (Fig. 3B). The concentrations of various apop‑ be associated with the malignant behavior of cSCC cells (26,27). tosis‑associated proteins, including Bax and Bcl‑2, in A431 Moreover, the PDPK1 gene was located upstream of the and SCC‑12 cell lysates were subsequently analyzed using PI3K/AKT signaling pathway (Fig. S1). The Starbase prediction ELISAs. The results demonstrated that the transfection with illustrated that miR‑451a shared a complementary binding site the miR‑451a mimic significantly increased the concentration with the 3'UTR sequence of PDPK1 (Fig. 5B). Subsequently, the of Bax, while decreasing that of Bcl‑2, compared with the data were verified by performing a dual‑luciferase reporter gene mimic control‑transfected cells in both cell lines (Fig. 3C). assay, which revealed that luciferase activity was significantly decreased in 293T cells transfected with miR‑451a mimics miR‑451a overexpression inhibits cSCC cell epithelial‑mesen‑ and PDPK1‑WT; however, there was no significant difference chymal transition (EMT), migration and invasion. EMT is a in cells transfected with miR‑451a mimics and PDPK1‑MT or common malignant biological process of tumor cells (25). mimic control and PDPK1‑WT (Fig. 5C). RT‑qPCR and western Therefore, the current study determined whether miR‑451a blotting were subsequently performed to analyze the mRNA and the EMT process were associated in cSCC cells. The and protein expression levels of PDPK1 in A431 and SCC‑12 concentrations of the specific epithelial marker, E‑cadherin cells. The results revealed that miR‑451a mimics significantly and mesenchymal marker, N‑cadherin, in the cell lysates of downregulated the mRNA and protein expression levels of A431 and SCC‑12 cells were analyzed via ELISAs. The results PDPK1 in both cell lines compared with the mimic control revealed that the overexpression of miR‑451a significantly group (Fig. 5D and E). The extent of PI3K/AKT signaling 6 FU et al: TUMOR SUPPRESSIVE ROLE OF miR-451a IN CSCC

Figure 3. miR‑451a promotes cSCC cell apoptosis. (A) Apoptotic A431 and SCC‑12 cells transfected with miR‑451a mimics or mimic controls were deter‑ mined by flow cytometric analysis. (B) Hoechst 33258 staining of A431 and SCC‑12 cells transfected with the miR‑451a mimic or mimic control. Scale bar, 50 µm. (C) Bax and Bcl‑2 concentrations in A431 and SCC‑12 cells transfected with the miR‑451a mimic or mimic control were analyzed using ELISAs. The data are presented as the mean ± SD. One‑way ANOVA and Tukey's multiple comparison tests were used to determine statistical significances. *P<0.05. miR, microRNA; cSCC, cutaneous squamous cell carcinoma; PI, propidium iodide. pathway phosphorylation was subsequently determined using analysis of miRNA expression profiles using the NanoString western blotting. The miR‑451a mimic was discovered to platform by Latchana et al (31) revealed a downregulation of significantly reduce PI3K/AKT signaling pathway phosphoryla‑ miR‑451a expression levels in patients with metastatic mela‑ tion compared with the mimic control group in both cell lines noma following resection. Additionally, the upregulation of (Fig. 5F), which may suggest the inhibition of the malignant miR‑451a expression levels in melanoma cells were reported to biological behavior of cSCC cells. markedly decrease cell migration and invasion (32). The EMT process may explain the invasiveness and aggressiveness of Discussion cSCC, which metastasizes by depleting the epithelial marker E‑cadherin and acquiring the mesenchymal marker N‑cadherin, Perineural invasion is a risk factor for cSCC, with an incidence amongst others (33). In the present study, the overexpression rate ranging from 2.5‑14% (28). miRNAs are considered to of miR‑451a significantly repressed cSCC cell proliferation, be vital genetic modulators of different biological processes, invasion, migration and EMT, while promoting apoptosis, including cell proliferation, invasion and apoptosis (22). Since indicating that miR‑451a may be a crucial negative modulator miR‑451a was identified to be involved in cell proliferation and of cSCC cell growth by serving as a tumor suppressor. Similar migration (29,30), it was predicted that the downregulation of to these findings, a previous study determined that miR‑451a miR‑451a may contribute to cSCC pathogenesis. expression levels were downregulated in osteosarcoma, while The current study determined that compared with HaCaT the restoration of miR‑451a expression suppressed the viability cells, the expression levels of miR‑451a in cSCC cells were and invasion of osteosarcoma cell lines by binding to tripartite significantly downregulated. Consistent with these results, the motif‑containing (34). EMT is a crucial biological event for EXPERIMENTAL AND THERAPEUTIC MEDICINE 21: 116, 2021 7

Figure 4. miR‑451a suppresses cSCC cell epithelial‑mesenchymal transition, migration and invasion. (A) E‑cadherin and N‑cadherin protein concentrations in A431 and SCC‑12 cells transfected with miR‑451a mimics or mimic controls were analyzed using ELISAs. (B) Migratory ability of A431 and SCC‑12 cells transfected with miR‑451a mimics or mimic controls was determined using Transwell assays. Scale bar, 50 µm. (C) Invasive ability of A431 and SCC‑12 cells transfected with miR‑451a mimics or mimic controls was analyzed using Transwell Matrigel assays. Scale bar, 50 µm. One‑way ANOVA and Tukey's multiple comparison tests were used to determine statistical significances. *P<0.05. miR, microRNA; cSCC, cutaneous squamous cell carcinoma. the invasion and migration of epithelial‑derived tumor cells PI3K signaling pathway and has been identified to induce a and represents a crucial step in the malignant progression plethora of downstream effectors, representing a crucial hub of tumors (35). Furthermore, miR‑451a overexpression was for synchronizing signals from extracellular cues towards demonstrated to decrease cell proliferation and EMT processes, the cytoskeletal machinery (39). Furthermore, PDPK1 was and induce papillary thyroid cancer cell apoptosis (36). During previously demonstrated to enhance cell proliferation, migra‑ early diabetic kidney injury, microvesicle‑delivered miR‑451a tion, invasion, tumor growth and metastasis, and to interact promoted the proliferation and viability of HK2 cells, while with the Notch1 intracellular domain, thus repressing its the injection of miR‑451a upregulated the expression levels of ubiquitin‑modulated degradation (40). Although the present E‑cadherin (37). Similar findings were observed in the current study did not provide experimental results regarding the study, whereby the transfection with the miR‑451a mimic involvement of PDPK1 in the malignant phenotype of cSCC increased the levels of E‑cadherin and decreased those of cells due to the funding limitations, the association between N‑cadherin in the two cSCC cell lines. Consequently, further miR‑451a and the PI3K/AKT signaling pathway was vali‑ investigations of the mechanisms underlying the miR‑451a‑in‑ dated. The inhibition of the heat shock protein 90/PI3K duced downregulation of cSCC cell growth are required to signaling pathway has been previously associated with further determine cSCC development. reduced melanoma growth (41). Furthermore, the treatment The relative luciferase activity of 293T cells containing the with himachalol significantly promoted skin carcinogenesis PDPK1‑WT 3'UTR with complementary miR‑451a binding cell apoptosis and cell cycle arrest in skin carcinogenesis cells sites was significantly decreased upon co‑transfection with through inhibiting the PI3K/AKT signaling pathway (42). miR‑451a mimics, which was determined using dual‑luciferase Additionally, Riquelme et al (43) identified a decrease in reporter gene assays. These results indicated that miR‑451a gastric cancer cell migration and invasion following miR‑451a may interact with the PDPK1 3'UTR and inhibit its expression. mimic transfection, which was achieved by the regulation of PDPK1 was first discovered in 1997 as the kinase responsible the PI3K/AKT/mTOR axis. Furthermore, it was reported that for AKT phosphorylation (38). PDPK1 is a transducer of the the upregulation of miR‑451a expression levels impaired the 8 FU et al: TUMOR SUPPRESSIVE ROLE OF miR-451a IN CSCC

Figure 5. miR‑451a downregulates the PI3K/AKT signaling pathway via targeting PDPK1. (A) Target mRNAs of miR‑451a were predicted using StarBase and Kyoto Encyclopedia of Genes and Genomes signaling pathway enrichment analysis was conducted using The Database for Annotation, Visualization and Integrated Discovery software. The bubble plot represents the top 10 enriched pathways. (B) Identified complementary binding site between miR‑451a and PDPK1 mRNA was predicted using StarBase. (C) Relative luciferase activity of PDPK1 MT or WT following the transfection of miR‑451a mimics or mimic controls was analyzed using dual luciferase reporter assays. PDPK1 (D) mRNA and (E) protein expression levels in A431 and SCC‑12 cells following the transfection of the miR‑451a mimic or mimic control were determined using reverse transcription‑quantitative PCR or western blotting, respectively. (F) Extent of PI3K/AKT signaling pathway phosphorylation in A431 and SCC‑12 cells following the transfection of miR‑451a mimic or mimic control was analyzed using western blotting. The data are presented as the mean ± SD. One‑way ANOVA and Tukey's multiple comparison tests were used to determine statistical significances.* P<0.05. miR, microRNA; PDPK1, 3‑phosphoinositide‑dependent protein kinase‑1; MT, mutant; WT, wild‑type; p‑, phosphorylated. proliferation and migration of papillary thyroid carcinoma Availability of data and materials cells, downregulated the expression levels of its target gene, AKT1 and attenuated AKT/mTOR signaling pathway activa‑ The datasets used and/or analyzed during the current study tion (44). The results of the present study determined that are available from the corresponding author on reasonable miR‑451a blocked the induction of the PI3K/AKT signaling request. The GEO datasets generated and/or analyzed during pathway by reducing the extent of PI3K and AKT phosphory‑ the current study are available from the GSE57768 dataset lation. (ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE57768). In conclusion, the results of the present study indicated that miR‑451a may bind to PDPK1 and subsequently down‑ Authors' contributions regulate the activity of the PI3K/AKT signaling pathway. The overexpression of miR‑451a significantly reduced cSCC cell JF and JZ conceived and designed the current study. HZ and XF proliferation, migration, invasion and EMT, while promoting performed all the experiments. WG and SQ analyzed and inter‑ apoptosis. However, further studies analyzing the association preted data. All authors read and approved the final manuscript. between PDPK1 and the PI3K/AKT signaling pathway in cSCC are required. The results of the current study provided Ethics approval and consent to participate insight into the molecular mechanism by which miR‑451a may impair the cSCC malignant phenotype. Not applicable.

Acknowledgements Patient consent for publication

Not applicable. Not applicable.

Funding Competing interests

No funding was received. The authors declare that they have no competing interests. EXPERIMENTAL AND THERAPEUTIC MEDICINE 21: 116, 2021 9

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