Long Noncoding RNA LINC00520 Prevents the Progression of Cutaneous Squamous Cell Carcinoma Through the Inactivation of the PI3K/Akt Signaling Pathway by Downregulating EGFR
Cutaneous squamous cell carcinoma (cSCC) is the second most frequently diagnosed type of nonmelanoma skin cancer, with a high annual mortality rate. It primarily occurs in keratinocytes of the epidermis due to sun exposure and is associated with risk factors such as ultraviolet (UV) radiation, older age, male sex, chronic skin ulcers, burn scars, and immunosuppression. cSCC is highly metastatic, often spreading to lymph nodes, and patients with regional lymphatic or distant metastases have a less than 20% 10-year survival rate. Despite advances in treatment, the genetic mutations driving aggressive cSCC remain poorly understood, highlighting the need for targeted therapeutic approaches.
Long noncoding RNAs (lncRNAs) have emerged as critical regulators in various malignant tumors, including cSCC. These RNAs play pivotal roles in tumor cell proliferation, differentiation, invasion, and metastasis. Among them, long intergenic nonprotein coding RNA 520 (LINC00520) has been implicated in cell migration, invasion, and metastasis in cancers such as breast cancer. The epidermal growth factor receptor (EGFR) and the phosphoinositide 3-kinase-protein kinase B (PI3K/Akt) signaling pathway are also known to be involved in the pathogenesis of cSCC. EGFR, in particular, is a potential therapeutic target for cSCC, as its overexpression is associated with tumor progression and poor prognosis. This study aimed to explore the role of LINC00520 in cSCC development through its interaction with EGFR and the PI3K/Akt signaling pathway.
The study began with a microarray analysis to screen differentially expressed lncRNAs in cSCC samples. Data from chip GSE66359 revealed that LINC00520 was significantly downregulated in cSCC. The Multi Experiment Matrix (MEM) website was used to predict the target genes of LINC00520, and EGFR was identified as a key target. The KEGG pathway analysis further confirmed that EGFR is involved in the PI3K/Akt signaling pathway, which is critical for cSCC progression.
To investigate the functional role of LINC00520, the A431 cSCC cell line was transfected with various plasmids, including LINC00520 overexpression, LINC00520 siRNA (si-LINC00520), EGFR siRNA (si-EGFR), and EGFR overexpression plasmids. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting were used to characterize the expression patterns of LINC00520, EGFR, and intermediates in the PI3K/Akt pathway. The results showed that LINC00520 overexpression led to a significant increase in LINC00520 levels and a corresponding decrease in EGFR, PI3K, AKT, VEGF, MMP-2, and MMP-9 mRNA and protein levels. Conversely, silencing LINC00520 resulted in increased levels of these molecules. These findings suggest that LINC00520 negatively regulates EGFR expression, thereby inactivating the PI3K/Akt signaling pathway.
Cell proliferation, migration, and invasion assays were conducted to assess the functional impact of LINC00520 on cSCC cells. The MTT assay revealed that LINC00520 overexpression significantly inhibited cell proliferation, while silencing LINC00520 promoted proliferation. The scratch test and Transwell assay demonstrated that LINC00520 overexpression reduced cell migration and invasion, whereas LINC00520 knockdown enhanced these processes. Additionally, a cell adhesion assay showed that LINC00520 overexpression weakened cell adhesion ability, while its knockdown increased adhesion. These results collectively indicate that LINC00520 plays a suppressive role in cSCC cell proliferation, migration, invasion, and adhesion.
To further validate these findings in vivo, a tumorigenicity assay was performed using nude mice. Cells transfected with LINC00520 overexpression plasmids or si-EGFR were subcutaneously inoculated into the axilla of nude mice. After six weeks, the tumor volumes and weights were significantly reduced in the LINC00520 overexpression and si-EGFR groups compared to the control groups. Hematoxylin and eosin (H&E) staining of lymph nodes revealed fewer metastatic tumor cells in the LINC00520 overexpression and si-EGFR groups, indicating that LINC00520 and EGFR silencing inhibited tumor growth and lymph node metastasis.
The study also explored the molecular mechanisms underlying the effects of LINC00520. Bioinformatics analysis predicted that LINC00520 binds to the 3′ untranslated region (3’UTR) of EGFR, which was confirmed by a dual luciferase reporter gene assay. The luciferase activity in the EGFR-Wt group was significantly decreased compared to the negative control group, indicating that LINC00520 specifically binds to the EGFR gene. RNA fluorescence in situ hybridization (RNA-FISH) and immunofluorescence staining further confirmed the subcellular localization of LINC00520 in the nucleus and EGFR in the cytoplasm and nucleus.
The PI3K/Akt signaling pathway, which is activated in cSCC, was found to be inactivated by LINC00520 overexpression and EGFR silencing. The expression of key molecules in this pathway, including PI3K, AKT, VEGF, MMP-2, and MMP-9, was significantly reduced in the LINC00520 overexpression and si-EGFR groups. These findings suggest that LINC00520 inhibits the PI3K/Akt signaling pathway by downregulating EGFR, thereby suppressing cSCC progression.
In conclusion, this study demonstrates that LINC00520 is downregulated in cSCC and plays a critical role in inhibiting tumor progression. LINC00520 suppresses EGFR expression, leading to the inactivation of the PI3K/Akt signaling pathway. This, in turn, inhibits cSCC cell proliferation, migration, invasion, and adhesion, as well as tumor growth and lymph node metastasis. These findings provide novel insights into the molecular mechanisms of cSCC and highlight LINC00520 as a potential therapeutic target for the treatment of this aggressive skin cancer.
doi.org/10.1097/CM9.0000000000000070
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