Open Access

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

Retraction in: /10.3892/etm.2024.12568

  • Authors:
    • Jixing Fu
    • Jianhua Zhao
    • Huamin Zhang
    • Xiaoli Fan
    • Wenjun Geng
    • Shaohua Qiao
  • View Affiliations

  • Published online on: December 3, 2020     https://doi.org/10.3892/etm.2020.9548
  • Article Number: 116
  • Copyright: © Fu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

The role of microRNAs (miRNAs/miRs) in governing the progression of cutaneous squamous cell carcinoma (cSCC) has been the focus of recent studies. However, the functional role of miR‑451a in cSCC growth remains poorly understood. Therefore, the present study aimed to determine the expression levels of miR‑451a in cSCC cell lines and the involvement of miR‑451a in cSCC progression. The results revealed that the expression levels of miR‑451a were downregulated in cSCC tissues and cell lines, and that this subsequently upregulated 3‑phosphoinositide‑dependent protein kinase‑1 (PDPK1) expression levels. PDPK1 was validated as a direct target of miR‑451a in cSCC using bioinformatics software Starbase, dual‑luciferase reporter gene assays and western blotting. Additionally, CCK‑8, EdU and Transwell assays, as well as flow cytometry and Hoechst 3325 staining, were performed to assess the malignant aggressiveness of cSCC cells. Overexpression of miR‑451a was demonstrated to impair the proliferation, migration, invasion and epithelial‑mesenchymal transition (EMT), and promoted apoptosis in cSCC cells by interacting with PDPK1, possibly by direct targeting. Furthermore, the western blotting results indicated that miR‑451a overexpression may block the PI3K/AKT signaling pathway by interacting with PDPK1. In conclusion, the findings of the present study suggested that miR‑451a may prevent the proliferation, migration, invasion and EMT of cSCC cells through the PDPK1‑mediated PI3K/AKT signaling pathway, which may offer potential therapeutic targets for the treatment of cSCC.

Introduction

Skin cancer or cutaneous carcinoma is a major worldwide public health burden which is highly prevalent and demonstrates an ever-increasing incidence with ~108,420 new cases and 11,480 death in the United States in 2020 (1,2). The majority of diagnosed skin cancer cases are non-melanomatous, consisting of basal cell carcinoma and cutaneous cell carcinoma (cSCC), which originate from keratinized epithelial cells (3). Common risk factors for most non-melanomatous cancers include ultraviolet light exposure, radiation exposure, immunosuppression and genetic factors (4). The most effective treatment for the majority of lesions is surgery; however, other interventions, including radiation, topical immunomodulators and novel systemic drugs are also applied (5). The incidence rates of cSCC are much higher in populations with fairer skin compared with individuals with darker skin and notably higher, still, in regions with high ambient ultraviolet radiation levels (6). For instance, incidence rates have ranged from 60 per 100,000 people annually in Canada (latitude, 54˚N; 2006) to 290 per 100,000 people annually in the United States (latitude, 31-37˚N; 1991); these rates were higher compared with those 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 development of diagnostic and prognostic biomarkers, which may help elucidate the mechanisms that promote tumor initiation, progression and metastasis (11).

MicroRNAs (miRNAs/miRs) are a group of non-coding RNAs of ≤25 nucleotides in length (12). miRNAs modulate gene expression post-transcriptionally and numerous miRNAs, including miR-34a, miR-181a and miR-148a, have been revealed to function as tumor suppressors in patients with cSCC (13). A previous study revealed that miR-451a expression levels were markedly downregulated in human basal cell carcinoma tissues and mouse models (14), indicating its potential role in skin cancer. The present study predicted that 3-phosphoinositide-dependent protein kinase-1 (PDPK1) may be a target gene of miR-451a, which was discovered by performing a dual-luciferase reporter gene assay. PDPK1 was previously illustrated to serve a pivotal role in the proliferation of angiosarcoma cells, indicating its potential use as a target against angiosarcoma, an aggressive malignancy of endothelial cells (15). In non-small cell lung cancer, miR-503 upregulation was revealed to downregulate PDPK1 expression levels, thus blocking the PI3K/AKT signaling pathway (16). The present study determined the expression profiles of miR-451a and PDPK1 in cSCC tissues using bioinformatics analysis and cSCC cell behavioral examination. The association between miR-451a and PDPK1 was also investigated using bioinformatics analysis and a dual-luciferase reporter gene assay. A431 and SCC-12 cell lines overexpressing miR-451a were generated to determine the regulatory role of miR-451a in the cSCC malignant phenotype and the PI3K/AKT signaling pathway.

Materials and methods

Bioinformatics analysis

The cSCC-associated dataset GSE57768, which was comprised of 30 cSCC tissues and 18 normal skin tissues (17), was downloaded from the Gene Expression Omnibus (GEO) database (ncbi.nlm.nih.gov/geo). The differentially expressed genes (DEG) between control and cancerous samples were identified using the limma package of R software (version no. 3.6.2; master.bioconductor.org/packages/release/bioc/html/limma.html). The cut-off values for DEG selection were P<0.05 and |Log FoldChange|>1, which were used to plot a heatmap of DEGs using the heatmap package (version no. 1.0.12) of R software (cran.r-project.org/web/packages/pheatmap/index.html).

The target mRNAs of miR-451a were subsequently predicted using the StarBase version 2.0 website (http://starbase.sysu.edu.cn). Signaling pathway enrichment analysis was conducted with the target mRNAs identified by Starbase using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (kegg.jp) through The Database for Annotation, Visualization and Integrated Discovery (version no. 6.8; david.ncifcrf.gov) (18,19).

Cell lines and culture

The human keratinocyte cell line HaCaT (cat. no. GDC106) and cSCC cells, A431, HSC-5, SCC-12 and SCL-1, were all purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. Cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37˚C with 5% CO2.

Subsequently, A431 and SCC-12 cells in the logarithmic growth phase were seeded into 6-well plates (2x105 cells/well) and transfected with 100 nmol miR-451a mimics or mimic controls (synthesized by Shanghai GenePharma Co., Ltd.) using Lipofectamine® 2000 reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Following 48 h of transfection at 37˚C, miR-451a expression levels were subsequently analyzed using reverse transcription-quantitative PCR (RT-qPCR) to determine the transfection efficiency. The sequences for the miR-451a mimics and mimic controls are presented in Table I.

Table I

Sequence information for miR-451a mimic and mimic control.

Table I

Sequence information for miR-451a mimic and mimic control.

NameSequence (5'-3')
miR-451a mimic AAACCGUUACCAUUACUGAGUU
mimic control AUCUGCCGGUGGUAACUGACUA

[i] miR, microRNA.

RT-qPCR

Total RNA was extracted from HaCaT, A431, HSC-5, SCC-12 and SCL-1 cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Total RNA was reverse transcribed into cDNA using a PrimeScript™ RT reagent kit according to the manufacturer's protocol with a gDNA Eraser (Takara Bio, Inc.) at 70˚C for 5 min, ice-bathed for 3 min, at 37˚C for 60 min and at 95˚C for 10 min. qPCR was subsequently performed using a SYBR Green PCR Master mix (Invitrogen; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: Pre-denaturation at 95˚C for 5 min; denaturation at 94˚C for 45 sec; annealing at 56˚C for 45 sec and extension at 72˚C for 45 sec for a total of 35 cycles. The primer sequences used for qPCR are provided in Table II. GAPDH and U6 were used as the internal loading controls. Fold changes in the relative expression of the targets were calculated using the 2-ΔΔCq method (20).

Table II

Primer sequences used for reverse transcription-quantitative PCR.

Table II

Primer sequences used for reverse transcription-quantitative PCR.

GenePrimer sequence (5'-3')
miR-451aF: GAGGGGAGCAGAGTTCAAGT
 R: TGGGAGGCAGCAATAGACAA
PDPK1F: CTGGACGACTTTGTTCTGGGG
 R: GCTCAGGAGCGTATGAAGTGG
U6F: CTCGCTTCGGCAGCACA
 R: AACGCTTCACGAATTTGCGT
GAPDHF: GCACCGTCAAGGCTGAGAAC
 R: GGATCTCGCTCCTGGAAGATG

[i] miR, microRNA; PDPK1, 3-phosphoinositide-dependent protein kinase-1; F, forward; R, reverse.

Cell Counting Kit-8 (CCK-8) assay

The proliferative ability of cSCC cells was evaluated using a CCK-8 assay (Roche Diagnostics). At 48 h post-transfection, A431 and SCC-12 were seeded into 96-well plates (5x103 cells/well) at 37˚C. At the baseline, 24, 48 and 72 h post-incubation, 50 µl CCK-8 reagent was added to determine the optical density values at 490 nm with a Multiskan Sky microplate reader.

5-ethynyl-2'-deoxyuridine (EdU) staining

Additionally, the proliferative ability of cSCC cells was analyzed using EdU staining, as previously described (21). Briefly, stably transfected A451 and SCC-12 cells (5x103 cells/well) were seeded into 96-well petri dishes for a 24-h culture and fixed with 100 µl 4% paraformaldehyde for 30 min at room temperature. Then, RPMI-1640 medium (100 µl) containing 20 µM EdU (Beijing Solarbio Science & Technology Co., Ltd.) was added into each well, after which cells were cultured at 37˚C for 2 h. The EdU positive cells were stained red. Nuclei were subsequently counterstained with 4',6-diamidino-2-phenylindole at 37˚C for 30 min. The number of EdU positive cells was observed in five different visual fields using a fluorescence microscope (magnification, x200; Olympus Corporation). The EdU positive cell index (EdU positive cells/total cells) was measured using ImageJ (version no. 1.52; National Institutes of Health).

Flow cytometric analysis of apoptosis

Apoptotic cSCC cells were analyzed following miR-451a transfection using an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (Best-Bio, Ltd.) and flow cytometry as previously reported (22). Briefly, cells were resuspended in 200 µl binding buffer and dual-stained in the dark for 15 min at room temperature using PI and Annexin V-FITC (both, 100 µl). Late apoptotic cells (positive for PI and Annexin V-FITC) were analyzed using a BD Accuri™ C6 Plus flow cytometer and CellQuest software (version no; 6.1; both from BD Biosciences).

Hoechst 33258 staining

cSCC cells in the logarithmic growth phase were subjected to Hoechst 33258 staining. Briefly, following transfection, 5x103 cSCC cells were seeded into six-well plates and incubated overnight at 37˚C. Following permeabilization with 50 µl cold 4% formaldehyde for 30 min at room temperature, the cells were incubated for ≥20 min at room temperature with 20 µg/ml Hoechst. The cells were visualized using a Leica confocal laser-scanning microscope (TCS SP8; Leica Microsystems GmbH) at 365 nm in five different visual fields. The apoptotic cell rate (%) was calculated using the following formula: (Number of apoptotic cells per field/total number of cells per field) x100.

ELISAs

The concentrations of Bax (cat. no. ab199080), Bcl-2 (cat. no. ab202411), E-cadherin (cat. no. ab197751) and N-cadherin (cat. no. ab254512) were determined in A431 and SCC-12 cell lysates using ELISA kits (all from Abcam), according to the manufacturers' protocols.

Transwell assays

The invasion and migration of A431 and SCC-12 cells following miR-451a mimic transfection were analyzed using Transwell assays, as described previously (23). Briefly, for the invasion analysis, 3x104 cells/well were added to 200 µl FBS-free RPMI-1640 medium and plated into the upper chamber of Transwell plates precoated with Matrigel at 4˚C until the Matrigel was solidified (80 µl; 1:4; Sigma-Aldrich; Merck KGaA). A total volume of 500 µl RPMI-1640 medium supplemented with 10% FBS was plated into the lower chamber. Following incubation at 37˚C for 48 h, the cells were fixed with 2.5% glutaraldehyde at room temperature for 30 min and stained with 0.1% crystal violet at room temperature for 30 min. The number of cells was counted in five different randomly selected fields of view using a light microscope (magnification, x200).

The migration assay was performed in a similar manner, except for the following modifications: The Transwell plates were not precoated in Matrigel and the cells were only incubated for 24 h at 37˚C.

Dual-luciferase reporter gene assay

The PDPK1 wild-type (WT) and mutant (MT) 3'untranslated region (UTR) binding sequences were synthesized by Shanghai GenePharma Co., Ltd. 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 (Invitrogen Thermo Fisher Scientific, Inc.) for 48 h. The relative luciferase activity was determined using a Dual-Luciferase Reporter assay system (Promega Corporation) as previously described (24). Renilla luciferase activity was used for normalization of the firefly luciferase activity.

Western blotting

Western blotting analysis was performed to analyze the phosphorylation status of PI3K/AKT signaling pathway-associated proteins in transfected A431 and SCC-12 cells. The experimental protocol was performed according to a previously described study (21). Briefly, total protein was extracted from the cells using an SDS lysis buffer (cat. no. P0013G; Beyotime Institute of Biotechnology) on ice for 30 min. The concentration of total protein was measured using a BCA protein assay kit (Pierce; Thermo Fisher 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 primary antibodies: Anti-PDPK1 (1:2,000; cat. no. ab52893; Abcam), anti-PI3K (1:5,000; cat. no. ab151549; Abcam), anti-phosphorylated (p)-PI3KY607 (1:5,000; cat. no. ab182651; Abcam), anti-AKT1 (1:5,000; cat. no. ab235958; Abcam), anti-pAKT1S473 (1:5,000; cat. no. ab81283; Abcam) and anti-β-actin (1:10,000; cat. no. ab8226; Abcam) for 1 h at room temperature. Following the primary antibody incubation, the membranes were incubated with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:10,000; cat. no. ab7090; Abcam) for 2 h at room temperature. Signals were detected using ECL reagents (cat. no. SW2030; Beijing Solarbio Science & Technology Co., Ltd.). Densitometry was analyzed using Image-Pro Plus software (version no. 6.0; Media Cybernetics, Inc.).

Statistical analysis

Statistical analysis was performed using SPSS 21.0 software (IBM Corp.). All data and results were calculated from ≥3 replicate measurements and are presented as the mean ± SD. The Kolmogorov-Smirnov method was used to analyze whether the data were normally distributed. Multigroup comparisons were performed using a one-way or two-way (factorial) ANOVA with a Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Results

miR-451a is poorly expressed in cSCC

Following the analysis of the GSE57768 dataset, 20 DEGs were selected and plotted in a heat map (Fig. 1A). The results revealed that compared with the normal skin tissues, the expression levels of miR-451a were downregulated in cSCC tissues (Fig. 1B). A previous study indicated that miR-451a may serve as a tumor suppressor in cutaneous basal cell carcinoma (14). Therefore, the current study determined the expression levels of miR-451a in human keratinocyte and cSCC cells; the expression levels of miR-451a were discovered to be significantly downregulated in cSCC cells compared with the HaCaT cells (Fig. 1C).

miR-451a mimics or mimic controls were subsequently transfected into A431 and SCC-12 cells to verify the effect of miR-451a on cSCC cells. These cell lines were chosen for further experiments due to their relatively low miR-451a expression. RT-qPCR analysis revealed that miR-451a expression levels were significantly upregulated in cSCC cells following transfection with miR-451a mimics compared with mimic controls, indicating that the transfection was successful (Fig. 1D).

miR-451a overexpression suppresses cSCC cell viability

The CCK-8 assay revealed that the proliferation of miR-451a mimic-transfected A431 and SCC-12 cells was significantly inhibited compared with their respective mimic control-treated cells (Fig. 2A). Additionally, the results of the EdU staining assay illustrated that the transfection with miR-451a mimics significantly decreased the proliferation of A431 and SCC-12 cells compared with mimic control-transfected cells (Fig. 2B).

miR-451a overexpression promotes cSCC cell apoptosis

The levels of apoptosis in A431 and SCC-12 cells labeled with PI/Annexin V were detected via flow cytometry. The results revealed that the transfection with miR-451a mimics significantly increased the levels of apoptosis in both cell lines compared with transfection with mimic controls (Fig. 3A), which was further validated by the results of Hoechst 33258 staining (Fig. 3B). The concentrations of various apoptosis-associated proteins, including Bax and Bcl-2, in A431 and SCC-12 cell lysates were subsequently analyzed using ELISAs. The results demonstrated that the transfection with the miR-451a mimic significantly increased the concentration of Bax, while decreasing that of Bcl-2, compared with the mimic control-transfected cells in both cell lines (Fig. 3C).

miR-451a overexpression inhibits cSCC cell epithelial-mesenchymal transition (EMT), migration and invasion

EMT is a common malignant biological process of tumor cells (25). Therefore, the current study determined whether miR-451a and the EMT process were associated in cSCC cells. The concentrations of the specific epithelial marker, E-cadherin and mesenchymal marker, N-cadherin, in the cell lysates of A431 and SCC-12 cells were analyzed via ELISAs. The results revealed that the overexpression of miR-451a significantly inhibited the EMT process in A431 and SCC-12 cells by enhancing E-cadherin expression and lowering N-cadherin expression (Fig. 4A). A431 and SCC-12 cell invasion and migration were subsequently analyzed using Transwell assays. The results demonstrated that the overexpression of miR-451a significantly inhibited the invasion and migration of both cell lines compared with the mimic control-transfected cells (Fig. 4B and C).

miR-451a blocks the PI3K/AKT signaling pathway by targeting PDPK1 in cSCC cells

The possible target genes of miR-451a were predicted using StarBase to determine the downstream signaling pathways of miR-451a in cSCC, which were further analyzed using KEGG signaling pathway enrichment analysis. The results revealed that the ‘PI3K-Akt signaling pathway’ was enriched (data not shown; Fig. 5A), which has been reported to be associated with the malignant behavior of cSCC cells (26,27). Moreover, the PDPK1 gene was located upstream of the PI3K/AKT signaling pathway (Fig. S1). The Starbase prediction illustrated that miR-451a shared a complementary binding site with the 3'UTR sequence of PDPK1 (Fig. 5B). Subsequently, the data were verified by performing a dual-luciferase reporter gene assay, which revealed that luciferase activity was significantly decreased in 293T cells transfected with miR-451a mimics and PDPK1-WT; however, there was no significant difference in cells transfected with miR-451a mimics and PDPK1-MT or mimic control and PDPK1-WT (Fig. 5C). RT-qPCR and western blotting were subsequently performed to analyze the mRNA and protein expression levels of PDPK1 in A431 and SCC-12 cells. The results revealed that miR-451a mimics significantly downregulated the mRNA and protein expression levels of PDPK1 in both cell lines compared with the mimic control group (Fig. 5D and E). The extent of PI3K/AKT signaling pathway phosphorylation was subsequently determined using western blotting. The miR-451a mimic was discovered to significantly reduce PI3K/AKT signaling pathway phosphorylation compared with the mimic control group in both cell lines (Fig. 5F), which may suggest the inhibition of the malignant biological behavior of cSCC cells.

Discussion

Perineural invasion is a risk factor for cSCC, with an incidence rate ranging from 2.5-14% (28). miRNAs are considered to be vital genetic modulators of different biological processes, including cell proliferation, invasion and apoptosis (22). Since miR-451a was identified to be involved in cell proliferation and migration (29,30), it was predicted that the downregulation of miR-451a may contribute to cSCC pathogenesis.

The current study determined that compared with HaCaT cells, the expression levels of miR-451a in cSCC cells were significantly downregulated. Consistent with these results, the analysis of miRNA expression profiles using the NanoString platform by Latchana et al (31) revealed a downregulation of miR-451a expression levels in patients with metastatic melanoma following resection. Additionally, the upregulation of miR-451a expression levels in melanoma cells were reported to markedly decrease cell migration and invasion (32). The EMT process may explain the invasiveness and aggressiveness of cSCC, which metastasizes by depleting the epithelial marker E-cadherin and acquiring the mesenchymal marker N-cadherin, amongst others (33). In the present study, the overexpression of miR-451a significantly repressed cSCC cell proliferation, invasion, migration and EMT, while promoting apoptosis, indicating that miR-451a may be a crucial negative modulator of cSCC cell growth by serving as a tumor suppressor. Similar to these findings, a previous study determined that miR-451a expression levels were downregulated in osteosarcoma, while the restoration of miR-451a expression suppressed the viability and invasion of osteosarcoma cell lines by binding to tripartite motif-containing (34). EMT is a crucial biological event for the invasion and migration of epithelial-derived tumor cells and represents a crucial step in the malignant progression of tumors (35). Furthermore, miR-451a overexpression was demonstrated to decrease cell proliferation and EMT processes, and induce papillary thyroid cancer cell apoptosis (36). During early diabetic kidney injury, microvesicle-delivered miR-451a promoted the proliferation and viability of HK2 cells, while the injection of miR-451a upregulated the expression levels of E-cadherin (37). Similar findings were observed in the current study, whereby the transfection with the miR-451a mimic increased the levels of E-cadherin and decreased those of N-cadherin in the two cSCC cell lines. Consequently, further investigations of the mechanisms underlying the miR-451a-induced downregulation of cSCC cell growth are required to further determine cSCC development.

The relative luciferase activity of 293T cells containing the PDPK1-WT 3'UTR with complementary miR-451a binding sites was significantly decreased upon co-transfection with miR-451a mimics, which was determined using dual-luciferase reporter gene assays. These results indicated that miR-451a may interact with the PDPK1 3'UTR and inhibit its expression. PDPK1 was first discovered in 1997 as the kinase responsible for AKT phosphorylation (38). PDPK1 is a transducer of the PI3K signaling pathway and has been identified to induce a plethora of downstream effectors, representing a crucial hub for synchronizing signals from extracellular cues towards the cytoskeletal machinery (39). Furthermore, PDPK1 was previously demonstrated to enhance cell proliferation, migration, invasion, tumor growth and metastasis, and to interact with the Notch1 intracellular domain, thus repressing its ubiquitin-modulated degradation (40). Although the present study did not provide experimental results regarding the involvement of PDPK1 in the malignant phenotype of cSCC cells due to the funding limitations, the association between miR-451a and the PI3K/AKT signaling pathway was validated. The inhibition of the heat shock protein 90/PI3K signaling pathway has been previously associated with reduced melanoma growth (41). Furthermore, the treatment with himachalol significantly promoted skin carcinogenesis cell apoptosis and cell cycle arrest in skin carcinogenesis cells through inhibiting the PI3K/AKT signaling pathway (42). Additionally, Riquelme et al (43) identified a decrease in gastric cancer cell migration and invasion following miR-451a mimic transfection, which was achieved by the regulation of the PI3K/AKT/mTOR axis. Furthermore, it was reported that the upregulation of miR-451a expression levels impaired the proliferation and migration of papillary thyroid carcinoma cells, downregulated the expression levels of its target gene, AKT1 and attenuated AKT/mTOR signaling pathway activation (44). The results of the present study determined that miR-451a blocked the induction of the PI3K/AKT signaling pathway by reducing the extent of PI3K and AKT phosphorylation.

In conclusion, the results of the present study indicated that miR-451a may bind to PDPK1 and subsequently downregulate the activity of the PI3K/AKT signaling pathway. The overexpression of miR-451a significantly reduced cSCC cell proliferation, migration, invasion and EMT, while promoting apoptosis. However, further studies analyzing the association between PDPK1 and the PI3K/AKT signaling pathway in cSCC are required. The results of the current study provided insight into the molecular mechanism by which miR-451a may impair the cSCC malignant phenotype.

Supplementary Material

KEGG signaling pathway enrichment analysis revealed that PDPK1 is upstream of the PI3K/AKT signaling pathway. Differentially expressed genes are indicated by red stars. KEGG, Kyoto Encyclopedia of Genes and Genomes; PDPK1, 3-phosphoinositide-dependent protein kinase 1.

Acknowledgements

Not applicable.

Funding

No funding was received.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. The GEO datasets generated and/or analyzed during the current study are available from the GSE57768 dataset (ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE57768).

Authors' contributions

JF and JZ conceived and designed the current study. HZ and XF performed all the experiments. WG and SQ analyzed and interpreted data. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References

1 

Gordon R: Skin cancer: An overview of epidemiology and risk factors. Semin Oncol Nurs. 29:160–169. 2013.PubMed/NCBI View Article : Google Scholar

2 

Siegel RL, Miller KD and Jemal A: Cancer statistics, 2020. CA Cancer J Clin. 70:7–30. 2020.PubMed/NCBI View Article : Google Scholar

3 

Linares MA, Zakaria A and Nizran P: Skin Cancer. Prim Care. 42:645–659. 2015.PubMed/NCBI View Article : Google Scholar

4 

Lomas A, Leonardi-Bee J and Bath-Hextall F: A systematic review of worldwide incidence of nonmelanoma skin cancer. Br J Dermatol. 166:1069–1080. 2012.PubMed/NCBI View Article : Google Scholar

5 

Dubas LE and Ingraffea A: Nonmelanoma skin cancer. Facial Plast Surg Clin North Am. 21:43–53. 2013.PubMed/NCBI View Article : Google Scholar

6 

Green AC and Olsen CM: Cutaneous squamous cell carcinoma: An epidemiological review. Br J Dermatol. 177:373–381. 2017.PubMed/NCBI View Article : Google Scholar

7 

Harris RB, Griffith K and Moon TE: Trends in the incidence of nonmelanoma skin cancers in southeastern Arizona, 1985-1996. J Am Acad Dermatol. 45:528–536. 2001.PubMed/NCBI View Article : Google Scholar

8 

Jung GW, Metelitsa AI, Dover DC and Salopek TG: Trends in incidence of nonmelanoma skin cancers in Alberta, Canada, 1988-2007. Br J Dermatol. 163:146–154. 2010.PubMed/NCBI View Article : Google Scholar

9 

Robsahm TE, Helsing P and Veierod MB: Cutaneous squamous cell carcinoma in Norway 1963-2011: Increasing incidence and stable mortality. Cancer Med. 4:472–480. 2015.PubMed/NCBI View Article : Google Scholar

10 

Parekh V and Seykora JT: Cutaneous squamous cell carcinoma. Clin Lab Med. 37:503–525. 2017.PubMed/NCBI View Article : Google Scholar

11 

Konicke K, Lopez-Luna A, Munoz-Carrillo JL, Servín-González LS, Flores-de la Torre A, Olasz E and Lazarova Z: The microRNA landscape of cutaneous squamous cell carcinoma. Drug Discov Today. 23:864–870. 2018.PubMed/NCBI View Article : Google Scholar

12 

Horsburgh S, Fullard N, Roger M, Degnan A, Todryk S, Przyborski S and O'Reilly S: MicroRNAs in the skin: Role in development, homoeostasis and regeneration. Clin Sci (Lond). 131:1923–1940. 2017.PubMed/NCBI View Article : Google Scholar

13 

García-Sancha N, Corchado-Cobos R, Pérez-Losada J and Cañueto JJ: MicroRNA dysregulation in cutaneous squamous cell carcinoma. Int J Mol Sci. 20(2181)2019.PubMed/NCBI View Article : Google Scholar

14 

Sun H and Jiang P: MicroRNA-451a acts as tumor suppressor in cutaneous basal cell carcinoma. Mol Genet Genomic Med. 6:1001–1009. 2018.PubMed/NCBI View Article : Google Scholar

15 

Wada M, Horinaka M, Yasuda S, Masuzawa M, Sakai T and Katoh N: PDK1 is a potential therapeutic target against angiosarcoma cells. J Dermatol Sci. 78:44–50. 2015.PubMed/NCBI View Article : Google Scholar

16 

Wei Y, Liao Y, Deng Y, Zu Y, Zhao B and Li F: MicroRNA-503 Inhibits non-small cell lung cancer progression by targeting PDK1/PI3K/AKT Pathway. Onco Targets Ther. 12:9005–9016. 2019.PubMed/NCBI View Article : Google Scholar

17 

Gillespie J, Skeeles LE, Allain DC, Kent MN, Peters SB, Nagarajan P, Yu L, Teknos TN, Olencki T and Toland AE: MicroRNA expression profiling in metastatic cutaneous squamous cell carcinoma. J Eur Acad Dermatol Venereol. 30:1043–1045. 2016.PubMed/NCBI View Article : Google Scholar

18 

Kanehisa M and Goto S: KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 28:27–30. 2000.PubMed/NCBI View Article : Google Scholar

19 

Kanehisa M: Toward understanding the origin and evolution of cellular organisms. Protein Sci. 28:1947–1951. 2019.PubMed/NCBI View Article : Google Scholar

20 

Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001.PubMed/NCBI View Article : Google Scholar

21 

Yu GJ, Sun Y, Zhang DW and Zhang P: Long non-coding RNA HOTAIR functions as a competitive endogenous RNA to regulate PRAF2 expression by sponging miR-326 in cutaneous squamous cell carcinoma. Cancer Cell Int. 19(270)2019.PubMed/NCBI View Article : Google Scholar

22 

Tian J, Shen R, Yan Y and Deng L: miR-186 promotes tumor growth in cutaneous squamous cell carcinoma by inhibiting apoptotic protease activating factor-1. Exp Ther Med. 16:4010–4018. 2018.PubMed/NCBI View Article : Google Scholar

23 

Song H, Tao Y, Ni N, Zhou X, Xiong J, Zeng X, Xu X, Qi J and Sun J: miR-128 targets the CC chemokine ligand 18 gene (CCL18) in cutaneous malignant melanoma progression. J Dermatol Sci. 91:317–324. 2018.PubMed/NCBI View Article : Google Scholar

24 

Zhang Y, Guo L, Li Y, Feng GH, Teng F, Li W and Zhou Q: MicroRNA-494 promotes cancer progression and targets adenomatous polyposis coli in colorectal cancer. Mol Cancer. 17(1)2018.PubMed/NCBI View Article : Google Scholar

25 

Fernandez-Figueras MT and Puig L: The role of epithelial-to-mesenchymal transition in cutaneous squamous cell carcinoma: Epithelial-to-mesenchymal transition in cutaneous SCC. Curr Treat Options Oncol. 21(47)2020.PubMed/NCBI View Article : Google Scholar

26 

Ci C, Wu C, Lyu D, Chang X, He C, Liu W, Chen L and Ding W: Downregulation of kynureninase restrains cutaneous squamous cell carcinoma proliferation and represses the PI3K/AKT pathway. Clin Exp Dermatol. 45:194–201. 2020.PubMed/NCBI View Article : Google Scholar

27 

Mei XL and Zhong S: Long noncoding RNA LINC00520 prevents the progression of cutaneous squamous cell carcinoma through the inactivation of the PI3K/Akt signaling pathway by downregulating EGFR. Chin Med J (Engl). 132:454–465. 2019.PubMed/NCBI View Article : Google Scholar

28 

Karia PS, Morgan FC, Ruiz ES and Schmults CD: Clinical and incidental perineural invasion of cutaneous squamous cell carcinoma: A systematic review and pooled analysis of outcomes data. JAMA Dermatol. 153:781–788. 2017.PubMed/NCBI View Article : Google Scholar

29 

Gao Z, Zhang P, Xie M, Gao H, Yin L and Liu R: miR-144/451 cluster plays an oncogenic role in esophageal cancer by inhibiting cell invasion. Cancer Cell Int. 18(184)2018.PubMed/NCBI View Article : Google Scholar

30 

Wei GY, Hu M, Zhao L and Guo WS: MiR-451a suppresses cell proliferation, metastasis and EMT via targeting YWHAZ in hepatocellular carcinoma. Eur Rev Med Pharmacol Sci. 23:5158–5167. 2019.PubMed/NCBI View Article : Google Scholar

31 

Latchana N, Abrams ZB, Howard JH, Regan K, Jacob N, Fadda P, Terando A, Markowitz J, Agnese D, Payne P and Carson WE III: Plasma MicroRNA levels following resection of metastatic melanoma. Bioinform Biol Insights Feb 23, 2017 (Epub ahead of print). doi: 10.1177/1177932217694837.

32 

Babapoor S, Fleming E, Wu R and Dadras SS: A novel miR-451a isomiR, associated with amelanotypic phenotype, acts as a tumor suppressor in melanoma by retarding cell migration and invasion. PLoS One. 9(e107502)2014.PubMed/NCBI View Article : Google Scholar

33 

Hodorogea A, Calinescu A, Antohe M, Balaban M, Nedelcu RI, Turcu G, Ion DA, Badarau IA, Popescu CM, Popescu R, et al: Epithelial-mesenchymal transition in skin cancers: A review. Anal Cell Pathol (Amst). 2019(3851576)2019.PubMed/NCBI View Article : Google Scholar

34 

Ma X, Li D, Gao Y and Liu C: miR-451a Inhibits the growth and invasion of osteosarcoma via targeting TRIM66. Technol Cancer Res Treat. 18(1533033819870209)2019.PubMed/NCBI View Article : Google Scholar

35 

Lin JX, Xie XS, Weng XF, Qiu SL, Yoon C, Lian NZ, Xie JW, Wang JB, Lu J, Chen QY, et al: UFM1 suppresses invasive activities of gastric cancer cells by attenuating the expres7sion of PDK1 through PI3K/AKT signaling. J Exp Clin Cancer Res. 38(410)2019.PubMed/NCBI View Article : Google Scholar

36 

Fan X and Zhao Y: miR-451a inhibits cancer growth, epithelial-mesenchymal transition and induces apoptosis in papillary thyroid cancer by targeting PSMB8. J Cell Mol Med. 23:8067–8075. 2019.PubMed/NCBI View Article : Google Scholar

37 

Zhong L, Liao G, Wang X, Li L, Zhang J, Chen Y, Liu J, Liu S, Wei L, Zhang W and Lu Y: Mesenchymal stem cells-microvesicle-miR-451a Ameliorate early diabetic kidney injury by negative regulation of P15 and P19. Exp Biol Med (Maywood). 243:1233–1242. 2018.PubMed/NCBI View Article : Google Scholar

38 

Di Blasio L, Gagliardi PA, Puliafito A and Primo L: Serine/threonine kinase 3-phosphoinositide-dependent protein kinase-1 (PDK1) as a key regulator of cell migration and cancer dissemination. Cancers (Basel). 9(25)2017.PubMed/NCBI View Article : Google Scholar

39 

Gagliardi PA, di Blasio L and Primo L: PDK1: A signaling hub for cell migration and tumor invasion. Biochim Biophys Acta. 1856:178–188. 2015.PubMed/NCBI View Article : Google Scholar

40 

Jing P, Zhou S, Xu P, Cui P, Liu X, Liu X, Liu X, Wang H and Xu W: PDK1 promotes metastasis by inducing epithelial-mesenchymal transition in hypopharyngeal carcinoma via the Notch1 signaling pathway. Exp Cell Res. 386(111746)2020.PubMed/NCBI View Article : Google Scholar

41 

Zhao Q, Zhu HP, Xie X, Mao Q, Liu YQ, He XH, Peng C, Jiang QL and Huang W: Novel HSP90-PI3K dual inhibitor suppresses melanoma cell proliferation by interfering with hsp90-egfr interaction and downstream signaling pathways. Int J Mol Sci. 21(1845)2020.PubMed/NCBI View Article : Google Scholar

42 

Shebaby W, Elias A, Mroueh M, Nehme B, El Jalbout ND, Iskandar R, Daher JC, Zgheib M, Ibrahim P, Dwairi V, et al: Himachalol induces apoptosis in B16-F10 murine melanoma cells and protects against skin carcinogenesis. J Ethnopharmacol. 253(112545)2020.PubMed/NCBI View Article : Google Scholar

43 

Riquelme I, Tapia O, Leal P, Sandoval A, Varga MG, Letelier P, Buchegger K, Bizama C, Espinoza JA, Peek RM, et al: miR-101-2, miR-125b-2 and miR-451a act as potential tumor suppressors in gastric cancer through regulation of the PI3K/AKT/mTOR pathway. Cell Oncol (Dordr). 39:23–33. 2016.PubMed/NCBI View Article : Google Scholar

44 

Minna E, Romeo P, Dugo M, De Cecco L, Todoerti K, Pilotti S, Perrone F, Seregni E, Agnelli L, Neri A, et al: miR-451a is underexpressed and targets AKT/mTOR pathway in papillary thyroid carcinoma. Oncotarget. 7:12731–12747. 2016.PubMed/NCBI View Article : Google Scholar

Related Articles

Journal Cover

February-2021
Volume 21 Issue 2

Print ISSN: 1792-0981
Online ISSN:1792-1015

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Fu J, Zhao J, Zhang H, Fan X, Geng W and Qiao S: MicroRNA‑451a prevents cutaneous squamous cell carcinoma progression via the 3‑phosphoinositide‑dependent protein kinase‑1‑mediated PI3K/AKT signaling pathway Retraction in /10.3892/etm.2024.12568. Exp Ther Med 21: 116, 2021.
APA
Fu, J., Zhao, J., Zhang, H., Fan, X., Geng, W., & Qiao, S. (2021). MicroRNA‑451a prevents cutaneous squamous cell carcinoma progression via the 3‑phosphoinositide‑dependent protein kinase‑1‑mediated PI3K/AKT signaling pathway Retraction in /10.3892/etm.2024.12568. Experimental and Therapeutic Medicine, 21, 116. https://doi.org/10.3892/etm.2020.9548
MLA
Fu, J., Zhao, J., Zhang, H., Fan, X., Geng, W., Qiao, S."MicroRNA‑451a prevents cutaneous squamous cell carcinoma progression via the 3‑phosphoinositide‑dependent protein kinase‑1‑mediated PI3K/AKT signaling pathway Retraction in /10.3892/etm.2024.12568". Experimental and Therapeutic Medicine 21.2 (2021): 116.
Chicago
Fu, J., Zhao, J., Zhang, H., Fan, X., Geng, W., Qiao, S."MicroRNA‑451a prevents cutaneous squamous cell carcinoma progression via the 3‑phosphoinositide‑dependent protein kinase‑1‑mediated PI3K/AKT signaling pathway Retraction in /10.3892/etm.2024.12568". Experimental and Therapeutic Medicine 21, no. 2 (2021): 116. https://doi.org/10.3892/etm.2020.9548