Circ_0008039 supports breast cancer cell proliferation, migration, invasion, and glycolysis by regulating the miR-140-3p/SKA2 axis

Abstract

Circular RNAs (circRNAs) have been shown to modulate gene expression and participate in the development of multiple malignancies. The purpose of this study was to investigate the role of circ_0008039 in breast cancer (BC). The expression of circ_0008039, miR-140-3p, and spindle and kinetochore-associated protein 2 (SKA2) was detected by qRT-PCR. Cell viability, colony formation, migration, and invasion were evaluated using methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, colony formation assay, and transwell assay, respectively. Glucose consumption and lactate production were measured using commercial kits. Protein levels of hexokinase II (HK2) and SKA2 were determined by western blot. The interaction between miR-140-3p and circ_0008039 or SKA2 was verified by dual-luciferase reporter assay. Finally, a mouse xenograft model was established to investigate the roles of circ_0008039 in BC in vivo. We found that circ_0008039 and SKA2 were upregulated in BC tissues and cells, while miR-140-3p was downregulated. Knockdown of circ_0008039 suppressed BC cell proliferation, migration, invasion, and glycolysis. Moreover, miR-140-3p could bind to circ_0008039 and its inhibition reversed the inhibitory effect of circ_0008039 interference on proliferation, migration, invasion, and glycolysis in BC cells. SKA2 was verified as a direct target of miR-140-3p and its overexpression partially inhibited the suppressive effect of miR-140-3p restoration in BC cells. Additionally, circ_0008039 positively regulated SKA2 expression by sponging miR-140-3p. Consistently, silencing circ_0008039 restrained tumor growth via increasing miR-140-3p and decreasing SKA2. In conclusion, circ_0008039 downregulation suppressed BC cell proliferation, migration, invasion, and glycolysis partially through regulating the miR-140-3p/SKA2 axis, providing an important theoretical basis for treatment of BC.

Keywords: SKA2; breast cancer; circ_0008039; miR-140-3p.

Fig. 1

Validation and characteristics of circ_0008039 in BC cells. (A) The diagram shows that circ_0008039 is generated from exons 2–4 of the PRKAR1B gene. (B) The qRT‐PCR assay was used to determine the subcellular location of circ_0008039 in MB231 cells. (C) The levels of circ_0008039 and PRKAR1B mRNA were detected after reverse transcription with random or oligo (dT)₁₈ primers via qRT‐PCR. (D and E) After treatment with actinomycin D (D) and RNase R (E), circ_0008039 and PRKAR1B mRNA expression levels were examined using qRT‐PCR in MB231 cells. The data are shown as the means ± SD of 3 independent experiments. Statistical analysis was conducted using Student’s t‐test. *P < 0.05.

Fig. 2

The expression of circ_0008039 was enhanced in BC tissues and cells. (A) The expression of circ_0008039 was detected in 51 pairs of BC tissues and adjacent normal tissues. (B) Circ_0008039 level was measured in patients with pathological stages I + II (n = 24) and pathological stage III + IV (n = 27). (C) Circ_0008039 level was detected in BC cells (BT20, BT549, MB231, and MB468) and MCF10A cells. The data are shown as the means ± SD of 3 independent experiments. Statistical analysis was conducted using Student’s t‐test or ANOVA. *P < 0.05.

Fig. 3

Deficiency of circ_0008039 restrained BC cell proliferation, migration, invasion, and glycolysis. MB231 and MB468 cells were transfected with si‐NC or si‐circ_0008039. (A) The knockdown efficiency of circ_0008039 was examined in MB231 and MB468 cells by qRT‐PCR. (B) Cell viability was evaluated by MTT assay. (C) Colony formation assay was applied to detect colony survival rate. (D and E) Transwell assay was applied for detecting cell migration and invasion. (F and G) Glucose consumption and lactate production were evaluated using commercial kits. (H) HK2 protein abundance was analyzed by western blot assay. The data are shown as the means ± SD of 3 independent experiments. Statistical analysis was conducted using Student’s t‐test. *P < 0.05.

Fig. 4

Circ_0008039 acted as a sponge of miR‐140‐3p. (A) The putative binding sites between circ_0008039 and miR‐140‐3p were predicted by circular RNA interactome. (B) The interaction between circ_0008039 and miR‐140‐3p in MB231 and MB468 cells was verified using dual‐luciferase luciferase report assay. (C) MiR‐140‐3p level was measured in BC tissues (n = 51) and adjacent normal tissues (n = 51). (D) The association between miR‐140‐3p level and circ_0008039 abundance was examined in BC tissues. (E) The expression of miR‐140‐3p was examined in BC cells (MB231 and MB468) and normal human breast epithelial cells (MCF10A). (F) The level of miR‐140‐3p was examined in MB231 and MB468 cells with transfection of si‐NC or si‐circ_0008039. The data are shown as the means ± SD of 3 independent experiments. Statistical analysis was conducted using Student’s t‐test. *P < 0.05.

Fig. 5

Circ_0008039 exerted its biological functions via sponging miR‐140‐3p in BC cells. (A) Inhibition efficiency of miR‐140‐3p was assessed via qRT‐PCR in MB231 and MB468 cells. (B‐F) MB231 and MB468 cells were transfected with si‐NC, si‐ circ_0008039, si‐circ_0008039 + anti‐miR‐NC, or si‐circ_0008039 + anti‐miR‐140‐3p. (B) The level of miR‐140‐3p was evaluated using qRT‐PCR. (C–F) Cell viability (C), colony formation ability (D), migration ability (E), and invasion ability (F) were determined in MB231 and MB468 cells. (G) ECAR was measured by using Seahorse XF Glycolysis Stress Test kit. OM, oligomycin; 2‐DG, glucose analog 2‐deoxyglucose. (H) Glucose consumption or lactate production was measured using commercial kits. (I) Western blot was conducted to analyze HK2 protein level. The data are shown as the means ± SD of 3 independent experiments. Statistical analysis was conducted using Student’s t‐test or ANOVA. *P < 0.05.

Fig. 6

SKA2 directly interacted with miR‐140‐3p in BC cells. (A) The putative targeting sites between miR‐140‐3p and SKA2 were provided by starbase. (B) Relative luciferase activity was detected in MB231 and MB468 cells with cotransfection of WT‐SKA2 or MUT‐SKA2 and miR‐140‐3p or miR‐NC. (C) QRT‐PCR was used for measuring the mRNA expression of SKA2. (D) The correlation between SKA2 and miR‐140‐3p expression was tested in BC tissues. (E and F) The protein level of SKA2 was examined via western blot in BC tissues, BC cells, and their corresponding controls. (G) MiR‐140‐3p expression was examined in MB231 and MB468 cells after transfection with miR‐NC or miR‐140‐3p (H) Western blot was carried out for determination of SKA2 protein level in MB231 and MB468 cells with transfection of miR‐NC or miR‐140‐3p. The data are shown as the means ± SD of 3 independent experiments. Statistical analysis was conducted using Student’s t‐test or ANOVA. *P < 0.05.

Fig. 7

Overexpression of SKA2 weakened the anticancer role of miR‐140‐3p restoration in BC cells. MB231 and MB468 cells were transfected with miR‐NC, miR‐140‐3p, miR‐140‐3p + vector, or miR‐140‐3p + SKA2. (A) Western blot assay determined the protein level of SKA2 in MB231 and MB468 cells. (B–E) Cell viability (B), cell colony‐forming ability (C), migration (D), and invasion (E) were detected in MB231 and MB468 cells. (F) ECAR was measured by using Seahorse XF Glycolysis Stress Test kit. OM, oligomycin; 2‐DG, glucose analog 2‐deoxyglucose. (G) Glucose consumption and lactate production were determined using commercial kits. (H) Western blot analysis measured HK2 protein expression. The data are shown as the means ± SD of 3 independent experiments. Statistical analysis was conducted using Student’s t‐test or ANOVA. *P < 0.05.

Fig. 8

SKA2 expression was modulated by miR‐140‐3p and circ_0008039 in BC cells. (A) SKA2 protein abundance was analyzed by western blot analysis in MB231 and MB468 cells after transfection with miR‐NC, miR‐140‐3p, miR‐140‐3p + vector, or miR‐140‐3p + circ_0008039. The data are shown as the means ± SD of 3 independent experiments. Statistical analysis was conducted using ANOVA. *P < 0.05.

Fig. 9

Silence of circ_0008039 suppressed tumor growth via modulating miR‐140‐5p/SKA2 expression. (A and B) The MB468 cells transfected with sh‐circ_0008039 or sh‐NC were inoculated into nude mice, and tumor volume and weight were measured. (C and D) Circ_0008039 and miR‐140‐3p expression levels were examined by qRT‐PCR in resected tumor tissues. (E) SKA2 protein abundance was detected using western blot analysis in resected tumor masses. The data are shown as the means ± SD of 3 independent experiments. Statistical analysis was conducted using Student’s t‐test. *P < 0.05.