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. 2023 Jun;29(6):e13321.
doi: 10.1111/srt.13321.

Hsa_circ_0005085 may suppress cutaneous squamous cell carcinoma growth and metastasis through targeting the miR-186-5p/LAMC1 axis

Lipeng Wang  1 , Yuxin Liu  1 , Qiong Gao  1 , Rongying Hu  1
Affiliations

Hsa_circ_0005085 may suppress cutaneous squamous cell carcinoma growth and metastasis through targeting the miR-186-5p/LAMC1 axis

Lipeng Wang et al. Skin Res Technol. 2023 Jun.

Retraction in

Abstract

Background: Cutaneous squamous cell carcinoma (CSCC) is a severe malignancy derived from the skin. Mounting evidence suggests that circular RNAs (circRNAs) participate in diverse biological functions in human cancers, containing CSCC. However, the biological functions and underlying mechanism of hsa_circ_0005085 in CSCC have not been clearly studied.

Methods: Expression levels of hsa_circ_0005085, microRNA-186-5p (miR-186-5p), and Laminin subunit gamma 1 (LAMC1) were detected by reverse transcription-quantitative polymerase chain reaction. Cell counting kit-8 assay, colony formation assay, and 5-Ethynyl-2'-deoxyuridine assay were used to assess cell proliferation. Transwell assay was conducted to detect cell migration and invasion. Cell apoptosis was analyzed by flow cytometry. Protein expression of LAMC1, E-cadherin, Snail, and slug were assessed using western blot assay. Using bioinformatics software, the binding between miR-186-5p and hsa_circ_0005085 or LAMC1 was predicted, followed by verification using a dual-luciferase reporter and RNA-Immunoprecipitation. The mouse xenograft model was established to investigate the role of hsa_circ_0005085 in vivo.

Results: Hsa_circ_0005085 level was downregulated in CSCC tissues and cells. Overexpression of hsa_circ_0005085 inhibited cell proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), and promoted cell apoptosis in CSCC. MiR-186-5p could restore the effect of hsa_circ_0005085 overexpression on CSCC cells, and the knockdown of LAMC1 reversed the regulation of the miR-186-5p inhibitor. In mechanism, hsa_circ_0005085 served as a sponge for miR-186-5p to regulate LAMC1 expression. Overexpression of hsa_circ_0005085 reduced growth of tumor via miR-186-5p/LAMC1 axis in vivo.

Conclusion: In our study, hsa_circ_0005085 might inhibit CSCC development by targeting the miR-186-5p/LAMC1 axis, which might provide a promising therapeutic target for CSCC.

Keywords: Hsa_circ_0005085; LAMC1; cutaneous squamous cell carcinoma; miR-186-5p.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

FIGURE 1
FIGURE 1
Hsa_circ_0005085 expression was boosted in cutaneous squamous cell carcinoma (CSCC) tissues and cells. (A) GSE74758 microarray analyzed the circRNA expression profiling between CSCC tissues and normal tissues. (B) The ring structure of has_hsa_circ_0005085. (C and D) Reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) analyzed has_hsa_circ_0005085 and linear ASAP2 expression after RNase R digestion in HSC‐1 and A431 cells. (D) Analyzed hsa_circ_0005085 expression in CSCC tissues and normal tissues. (E) Analyzed the expression of hsa_circ_0005085 in HaCaT, HSC‐1, and A431 cells. *p < 0.05.
FIGURE 2
FIGURE 2
Overexpression of hsa_circ_0005085 could influence the progression of cutaneous squamous cell carcinoma (CSCC). (A) Reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) analyzed the expression of hsa_circ_0005085 in HSC‐1 and A431 cells after transfected with vector or oe‐has_hsa_circ_0005085. (B) CCK8 assay analyzed cell viability of HSC‐1 and A431 cells after transfected with vector or oe‐has_hsa_circ_0005085. (C) Colony formation assay analyzed the colonies number of HSC‐1 and A431 cells after transfected with vector or oe‐has_hsa_circ_0005085. (D) EdU assay analyzed cell proliferation of HSC‐1 and A431 cells after transfected with vector or oe‐has_hsa_circ_0005085. (E and F) Transwell analyzed cell migration and invasion of HSC‐1 and A431 cells after transfected with vector or oe‐has_hsa_circ_0005085. (G) Flow cytometry assay analyzed cell apoptosis of HSC‐1 and A431 cells after transfected with vector or oe‐has_hsa_circ_0005085. (H) Western blot analyzed E‐cadherin, snail1 and slug protein expression in HSC‐1 and A431 cells after transfected with vector or oe‐has_hsa_circ_0005085. *p < 0.05.
FIGURE 3
FIGURE 3
Hsa_circ_0005085 could target miR‐186‐5p. (A) Circinteractome predicted the binding site between hsa_circ_0005085 and miR‐186‐5p. (B) Dual‐luciferase reporter assay was used to analyze luciferase activities of hsa_hsa_circ_0005085 wt and has_hsa_circ_0005085 mut in 293T cells after transfected with mimic NC or miR‐186‐5p mimic. (C and D) RNA immunoprecipitation (RIP) assay analyzed the interaction between hsa_circ_0005085 and miR‐186‐5p in HSC‐1 and A431 cells. (E) Reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) analyzed the expression of miR‐186‐5p in cutaneous squamous cell carcinoma (CSCC) and normal tissues, HaCaT, HSC‐1, A431 cells. (F) Pearson's correlation analysis of the correlation between miR‐186‐5p and hsa_circ_0005085. *p < 0.05.
FIGURE 4
FIGURE 4
MiR‐186‐5p mimic could restore the effect of oe‐has_hsa_circ_0005085 on the process of cutaneous squamous cell carcinoma (CSCC). (A) Reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) analyzed miR‐186‐5p in HSC‐1 and A431 cells after transfected with mimic NC or miR‐186‐5p mimic. (B) RT‐qPCR analyzed miR‐186‐5p in HSC‐1 and A431 cells after transfected with vector, oe‐has_hsa_circ_0005085, oe‐has_hsa_circ_0005085 + mimic NC, oe‐has_hsa_circ_0005085+miR‐186‐5p mimic. (C, D, and E) CCK8 assay, colony formation assay, and EdU assay analyzed cell proliferation of HSC‐1 and A431 cells after transfected with vector, oe‐has_hsa_circ_0005085, oe‐has_hsa_circ_0005085 + mimic NC, oe‐has_hsa_circ_0005085+miR‐186‐5p mimic. (F and G) Transwell analyzed cell migration and invasion in HSC‐1 and A431 cells after transfected with vector, oe‐has_hsa_circ_0005085, oe‐has_hsa_circ_0005085 + mimic NC, oe‐has_hsa_circ_0005085+miR‐186‐5p mimic. (H) Flow cytometry analyzed cell apoptosis in HSC‐1 and A431 cells after transfected with vector, oe‐has_hsa_circ_0005085, oe‐has_hsa_circ_0005085 + mimic NC, oe‐has_hsa_circ_0005085+miR‐186‐5p mimic. (I) Western blot analyzed E‐cadherin, snail1, and slug protein expression in HSC‐1 and A431 cells after transfected with vector, oe‐has_hsa_circ_0005085, oe‐has_hsa_circ_0005085 + mimic NC, oe‐has_hsa_circ_0005085+miR‐186‐5p mimic. *p < 0.05.
FIGURE 5
FIGURE 5
MiR‐186‐5p could target LAMC1. (A) STARBASE3.0 predicted the binding sits of miR‐186‐5p on LAMC1. (B) Dual‐luciferase reporter assay analyzed luciferase activities of LAMC1 3′UTR wt and LAMC1 3′UTR mut in 293T cells after transfected mimic NC or miR‐186‐5p. (C) RNA immunoprecipitation (RIP) assay analyzed the interaction of miR‐186‐5p and LAMC1 in HSC‐1 and A431 cells using beads of anti‐IgG or anti‐Ago2. (D) Analyzed LAMC1 expression by reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) in normal and CSCC tissues. (E and F) Western blot analyzed protein LAMC1 in HaCaT, HSC‐1, A431 cells and normal tissues, CSCC tissues. (G) Pearson's correlation analysis of the correlation of miR‐186‐5p and LAMC1. (H) Western blot analyzed the expression level of LAMC1 in HSC‐1 and A431 cell after transfection with vector, oe‐has_hsa_circ_0005085, oe‐has_hsa_circ_0005085+mimic NC, oe‐has‐hsa_circ_0005085+miR‐186‐5p mimic. *p < 0.05.
FIGURE 6
FIGURE 6
Knockdown of LAMC1 could overturn the effect of the miR‐186‐5p inhibitor on the process of cutaneous squamous cell carcinoma (CSCC). (A) Analyzed miR‐186‐5p expression in HSC‐1 and A431 cells after transfected with inhibitor NC or miR‐186‐5p inhibitor. (B) Analyzed protein LAMC1 in HSC‐1 and A431 cells after transfected with inhibitor NC, miR‐186‐5p inhibitor, miR‐186‐5p inhibitor +si‐NC, miR‐186‐5p inhibitor+ si‐LAMC1. (C, D, and E) CCK8 assay, colony formation assay, and EdU assay analyzed cell proliferation in HSC‐1 and A431 cells after transfected with inhibitor NC, miR‐186‐5p inhibitor, miR‐186‐5p inhibitor +si‐NC, miR‐186‐5p inhibitor+ si‐LAMC1. (F and G) Transwell assay analyzed cell migration and invasion in HSC‐1 and A431 cells after transfected with inhibitor NC, miR‐186‐5p inhibitor, miR‐186‐5p inhibitor +si‐NC, miR‐186‐5p inhibitor+ si‐LAMC1. (H) Flow cytometry analyzed cell apoptosis in HSC‐1 and A431 cells after transfected with inhibitor NC, miR‐186‐5p inhibitor, miR‐186‐5p inhibitor +si‐NC, miR‐186‐5p inhibitor+ si‐LAMC1. (I) Western blot analyzed proteins of E‐cadherin, snail1 and slug in HSC‐1 and A431 cells after transfected with inhibitor NC, miR‐186‐5p inhibitor, miR‐186‐5p inhibitor +si‐NC, miR‐186‐5p inhibitor+ si‐LAMC1. *p < 0.05.
FIGURE 7
FIGURE 7
Overexpression of hsa_circ_0005085 could inhibit tumor growth in vivo. (A) Analyzed the volume of tumors in 3, 6, 9, 12, 15, 18, and 21 days. (B) Detected tumor weight. (C) Analyzed the expression of has_0005085, miR‐186‐5p, and LAMC1 in tumor tissues. (D) Western blot analyzed the proteins of LAMC1, E‐cadherin, snail1 and slug in tumor tissues. (E) Immunohistochemical (IHC) analyzed LAMC1, E‐cadherin, snail1, and slug in tumor tissues. *p < 0.05.
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References

    1. Cheng J, Yan S. Prognostic variables in high‐risk cutaneous squamous cell carcinoma: a review. J Cutan Pathol. 2016;43(11):994‐1004. - PubMed
    1. Lomas A, Leonardi‐Be J, Bath‐Hextall FA. A systemic review of worldwide incidence of nonmelanoma skin cancer. Br J Dermatol. 2012;166:1069‐1080. - PubMed
    1. Watson M, Holman DM, Maguire‐Eisen M. Ultraviolet radiation exposure and its impact on skin cancer risk. Semin Oncol Nurs. 2016;32(3):241‐254. - PMC - PubMed
    1. Waldman A, Schmults C. Cutaneous squamous cell carcinoma. Hematol Oncol Clin North Am. 2019;33(1):1‐12. - PubMed
    1. Que SKT, Zwald FO, Schmults CD. Cutaneous squamous cell carcinoma: incidence, risk factors, diagnosis, and staging. J Am Acad Dermatol. 2018;78(2):237‐247. - PubMed

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