Circ_0068631 sponges miR-139-5p to promote the growth and metastasis of cutaneous squamous cell carcinoma by upregulating HOXB7

Abstract

Background: Circular RNAs (circRNAs) are often dysregulated in cancers and closely related to cancer progression, including cutaneous squamous cell carcinoma (CSCC). However, the role and mechanism of circ_0068631 in CSCC progression have not been reported.

Methods: The expression of circ_0068631, microRNA-139-5p (miR-139-5p), and homeobox B7 (HOXB7) was measured by real-time quantitative polymerase chain reaction (RT-qPCR). Cell counting kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay, and colony formation assay were used to measure cell proliferation. Cell apoptosis was assessed by flow cytometry. Cell migration was detected by transwell assay. The interaction between miR-139-5p and circ_0068631 or HOXB7 was confirmed by dual-luciferase reporter assay. A xenograft tumor model was established to confirm the function of circ_0068631 in vivo.

Results: Circ_0068631 was upregulated in CSCC tissues and cells, and its silencing could inhibit CSCC cell proliferation and metastasis while promoting apoptosis in vitro, as well as restrain CSCC tumor growth in vivo. Circ_0068631 acted as a sponge of miR-139-5p, and miR-139-5p inhibition reversed the repressive effect of circ_0068631 knockdown on CSCC cell progression. Furthermore, HOXB7 was a target of miR-139-5p, and miR-139-5p inhibited the malignant behaviors by downregulating HOXB7 expression in CSCC cells. Further, circ_0068631 sponged miR-139-5p to regulate HOXB7 expression.

Conclusion: Circ_0068631 functioned as a novel oncogene in CSCC progression by regulating miR-139-5p/HOXB7 axis, suggesting that circ_0068631 may be a potential target for CSCC treatment.

Highlights: Circ_0068631 was overexpressed in CSCC tissues and cells. Circ_0068631 downregulation suppressed CSCC progression via miR-139-5p. Circ_0068631 regulated HOXB7 via sponging miR-139-5p.

Keywords: HOXB7; circ_0068631; cutaneous squamous cell carcinoma; miR-139-5p.

FIGURE 1

Circ_0068631 was increased in cutaneous squamous cell carcinoma (CSCC) tissues and cells. (A) The heat map of GSE74758 showed the expression of 12 circRNAs in CSCC tissues and normal tissues. (B) The expression of circ_0068631 in CSCC tissues and normal tissues was presented. (C and D) Circ_0068631 was detected via RT‐qPCR in tissue samples and cell lines. (E and F) The effects of RNase R treatment on circ_0068631 were assessed by RT‐qPCR. (G and H) Random or oligo (dT)18 primers were used for reverse transcription experiments, and the expression levels of circ_0068631 and GAPDH were examined using RT‐qPCR. *p < 0.05, ***p < 0.001, ****p < 0.0001

FIGURE 2

Circ_0068631 deletion inhibited cutaneous squamous cell carcinoma (CSCC) progression in vitro. SCL‐1 and A431 cells were transfected with sh‐NC or sh‐circ_0068631. (A) The transfection efficiency of sh‐circ_0068631 was confirmed by detecting circ_0068631 expression using RT‐qPCR. (B) Cell viability was measured by CCK‐8 assay. (C) DNA synthesis was determined using EdU assay. (D–F) Colony number, apoptosis rate, and invasion were assessed by colony formation, flow cytometry, and transwell assays. (G and H) Western blot assay was used to analyze E‐cadherin and N‐cadherin protein levels. **p < 0.01, ***p < 0.001, ****p < 0.0001

FIGURE 3

Circ_0068631 interacted with miR‐139‐5p in cutaneous squamous cell carcinoma (CSCC) cells. (A) The binding sequence between miR‐139‐5p and circ_0068631 was presented. (B) Overexpression efficiency of miR‐139‐5p was assessed by detecting miR‐139‐5p expression using RT‐qPCR. (C and D) Dual‐luciferase reporter assay was used to determine the interaction between miR‐139‐5p and circ_0068631. (E) The expression of miR‐139‐5p was examined by RT‐qPCR in normal and CSCC tissues. (F) Pearson correlation analysis was used to analyze the correlation between miR‐139‐5p and circ_0068631 in CSCC tissues. (G) The expression of miR‐139‐5p was determined by RT‐qPCR in HaCaT, SCL‐1 and A431 cells. ***p < 0.001, ****p < 0.0001

FIGURE 4

Circ_0068631 played an oncogenic role through miR‐139‐5p in cutaneous squamous cell carcinoma (CSCC) cells. SCL‐1 and A431 cells were transfected with sh‐NC, sh‐circ_0068631, sh‐circ_0068631+anti‐miR‐NC, or sh‐circ_0068631+ anti‐miR‐139‐5p. (A) The expression of miR‐139‐5p was examined by RT‐qPCR. (B–D) CCK‐8 assay, EdU assay and colony formation assay were used to detect cell proliferation. (E) Cell apoptosis was analyzed by flow cytometry analysis. (F) Transwell assay was applied to detect cell invasion ability. (G and H) The protein levels of E‐cadherin and N‐cadherin were detected by western blot assay. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

FIGURE 5

MiR‐139‐5p directly targeted HOXB7 in cutaneous squamous cell carcinoma (CSCC) cells. (A) The binding sites between miR‐139‐5p and HOXB7 were shown. (B and C) The interaction between miR‐139‐5p and HOXB7 was confirmed using dual‐luciferase reporter assay in SCL‐1 and A431 cells. (D) HOXB7 was measured by RT‐qPCR in normal tissues and CSCC tissues. (E) The correlation between miR‐139‐5p and HOXB7 expression in CSCC tissues was analyzed by Pearson correlation analysis. (F) Immunohistochemical staining revealed the expression of HOXB7 in CSCC tissues. (G and H) HOXB7 protein expression was determined using western blot assay in tissue samples and cell lines. **p < 0.01, ****p < 0.0001

FIGURE 6

MiR‐139‐5p hindered cutaneous squamous cell carcinoma (CSCC) progression via HOXB7. SCL‐1 and A431 cells were transfected with miR‐NC, miR‐139‐5p, miR‐139‐5p+pcDNA, or miR‐139‐5p+HOXB7. (A) HOXB7 protein expression was examined. (B–D) Cell proliferation was assessed by CCK‐8 assay, EdU assay, and colony formation assay. (E) Flow cytometry analysis was utilized to determine cell apoptosis. (F) Cell invasion was measured by transwell assay. (G and H) Western blot assay was employed to examine the protein levels of E‐cadherin and N‐cadherin. (I) The impacts of circ_0068631 and miR‐139‐5p on HOXB7 protein expression were analyzed by western blot assay. **p < 0.01, ***p < 0.001, ****p < 0.0001

FIGURE 7

Knockdown of circ_0068631 reduced tumor growth in vivo. A431 cells transfected with sh‐NC and sh‐circ_0068631 were injected into the nude mice. (A) Tumor volume was calculated. (B) Tumor weight was measured after 23 days of inoculation. (C and D) Circ_0068631 and miR‐139‐5p were detected by RT‐qPCR. (E) HOXB7 protein was analyzed using western blot assay. (F) Immunohistochemistry (IHC) analysis was applied to examine Ki67, E‐cadherin, N‐cadherin, HOXB7, MMP9, Bcl2, and Bax expression in resected tumor tissues. *p < 0.05, **p < 0.01