Long noncoding RNA LINC00520 prevents the progression of cutaneous squamous cell carcinoma through the inactivation of the PI3K/Akt signaling pathway by downregulating EGFR

Abstract

Background: Long noncoding RNAs (lncRNAs) play pivotal roles in various malignant tumors. Epidermal growth factor receptor (EGFR) signaling is associated with the pathogenesis of cutaneous squamous cell carcinoma (cSCC). This study aimed to explore the role of LINC00520 in the development of cSCC via EGFR and phosphoinositide 3-kinase-protein kinase B (PI3K/Akt) signaling pathways.

Methods: A microarray analysis was applied to screen differentially expressed lncRNAs in cSCC samples. The A431 cSCC cell line was transfected and assigned different groups. The expression patterns of LINC00520, EGFR, and intermediates in the PI3K/Akt pathway were characterized using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting analysis. Cell proliferation, migration, and invasion were detected using the MTT assay, scratch test, and Transwell assay, respectively. Cell-based experiments and a tumorigenicity assay were conducted to assess the effect of LINC00520 on cSCC progression. This study was ended in September 2017. Comparisons between two groups were analyzed with t-test and comparisons among multiple groups were analyzed using one-way analysis of variance. The nonparametric Wilcoxon rank sum test was used to analyze skewed data. The enumerated data were analyzed using the chi-square test or Fisher exact test.

Results: Data from chip GSE66359 revealed depletion of LINC00520 in cSCC. Cells transfected with LINC00520 vector and LINC00520 vector + si-EGFR showed elevated LINC00520 level but decreased levels of the EGFR, PI3K, AKT, VEGF, MMP-2 and MMP-9 mRNAs and proteins, and inhibition of the growth, migration and adhesion of cSCC cells, while the si-LINC00520 group showed opposite trends (all P < 0.05). Compared with the LINC00520 vector group, the LINC00520 vector + si-EGFR group showed decreased levels of the EGFR, PI3K, AKT, VEGF, MMP-2 and MMP-9 mRNAs and proteins, and inhibition of the growth, migration and adhesion of cSCC cells, while the LINC00520 vector + EGFR vector group showed opposite results (all P < 0.05).

Conclusion: Based on our results, LINC00520-targeted EGFR inhibition might result in the inactivation of the PI3K/Akt pathway, thus inhibiting cSCC development.

Figure 1

LINC00520 targets the EGFR-3’UTR in cSCC. (A) Analysis of data from chip GSE66359. (B) Predicted relationship between EGRF and LINC00520 from the MEM website (has:04151 is the PI3K-Akt signaling pathway). (C) Subcellular localization of LINC00520 predicted by the bioinformatics website. (D) FISH images and immunofluorescence staining showing the subcellular localization of LINC00520 and EGFR (scale bar = 25 μm). (E) Binding sites for LINC00520 in the EGFR gene predicted by the bioinformatics website. (F) Verification of the relationship between LINC00520 and the EGFR gene using the dual luciferase reporter gene assay. ^∗P < 0.05 vs. the NC group. cSCC: Cutaneous squamous cell carcinoma; EGFR: Epidermal growth factor receptor; FISH: Fluorescence in situ hybridization; LINC00520: Long intergenic nonprotein coding RNA 520; NC: Negative control.

Figure 2

LINC00520 downregulates EGFR expression to inactivate the PI3K/AKT signaling pathway. (A) RT-qPCR detection of the expression of LINC00520 and the EGFR, PI3K, AKT, VEGF, MMP-2, and MMP-9 mRNAs. (B) Western blotting analysis showing the levels of the EGFR, PI3K, AKT, VEGF, MMP-2, MMP-9 and phosphorylated AKT proteins. (C) Bands for the EGFR, PI3K, AKT, VEGF, MMP-2, MMP-9 and phosphorylated AKT proteins; ^∗P < 0.05 vs. the blank and NC groups; ^†P < 0.05 vs. the LINC00520 vector or si-EGFR groups. EGFR: Epidermal growth factor receptor; LINC00520: Long intergenic nonprotein coding RNA 520; MMP: Matrix metalloproteinase; NC: Negative control; PI3K: Phosphatidylinositol-3-Kinase; RT-qPCR: Reverse transcription quantitative polymerase chain reaction; VEGF: Vascular endothelial growth factor.

Figure 3

The proliferation of transfected cells is inhibited by LINC00520. ^∗P < 0.05 vs. the blank and NC groups; ^†P < 0.05 vs. the LINC00520 vector group. EGFR: Epidermal growth factor receptor; LINC00520: Long intergenic nonprotein coding RNA 520; MTT: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide; NC: Negative control.

Figure 4

The invasion of transfected cSCC cells is attenuated by LINC00520 overexpression and EGFR silencing. (A) Representative images of invasion cells in the Transwell assay (scale bar = 25 μm). (B) The number of invasion cells among transfected cSCC cells. ^∗P < 0.05 vs. the blank and NC groups; ^†P < 0.05 vs. the LINC00520 vector group. cSCC: cutaneous squamous cell carcinoma; EGFR: Epidermal growth factor receptor; LINC00520: Long intergenic nonprotein coding RNA 520; NC: Negative control.

Figure 5

The migration of transfected cSCC cells is reduced by LINC00520 overexpression and EGFR silencing. (A) Representative images of migrating cells in the scratch test (×100). (B) Distance migrated by transfected cSCC cells. ^∗P < 0.05 vs. the blank and NC groups; ^†P < 0.05 vs. the LINC00520 vector group. cSCC: cutaneous squamous cell carcinoma; EGFR: Epidermal growth factor receptor; LINC00520: Long intergenic nonprotein coding RNA 520; NC: Negative control.

Figure 6

The inhibition rate of adhesion in transfected cells is increased by LINC00520 overexpression and EGFR silencing. ^∗P < 0.05 vs. the blank and NC groups; ^†P < 0.05 vs. the LINC00520 vector group. cSCC: cutaneous squamous cell carcinoma; EGFR: Epidermal growth factor receptor; LINC00520: Long intergenic nonprotein coding RNA 520; NC: Negative control.

Figure 7

The tumor volumes and weights, as well as lymph node metastasis, in nude mice are reduced by LINC00520 overexpression and EGFR silencing in vivo. (A) Volumes of transplanted tumors in nude mice over time. (B) Tumor weights in nude mice after the implantation of transfected cells. (C) Representative images of hematoxylin and eosin (H&E)-stained lymph node sections (scale bar = 25 μm). (D) Statistical analysis of the number of metastatic lymph nodes. ^∗P < 0.05 vs. the blank and NC groups; ^†P < 0.05 vs. the LINC00520 vector group. EGFR: Epidermal growth factor receptor; LINC00520: Long intergenic nonprotein coding RNA 520; NC: Negative control.

Figure 8

LINC00520 suppresses EGFR expression and inactivates the PI3K/Akt signaling pathway, thereby suppressing cSCC migration, invasion and metastasis. cSCC: Cutaneous squamous cell carcinoma; EGFR: Epidermal growth factor receptor; LINC00520: Long intergenic nonprotein coding RNA 520; PI3K/Akt: Phosphoinositide 3-kinase/protein kinase B.