Circular RNA circBNC2 facilitates glycolysis and stemness of hepatocellular carcinoma through the miR-217/high mobility group AT-hook 2 (HMGA2) axis

Abstract

Hepatocellular cancer (HCC) accounts for approximately 90% of primary liver carcinoma and is a significant health threat worldwide. Circular RNA basonuclin 2 (circBNC2) is implicated with the progression of several cancers. However, its roles in carcinogenesis and glycolysis are still unclear in HCC. In this study, the levels of circBNC2 and high mobility group AT-hook 2 (HMGA2) were highly expressed, while these of miR-217 were poorly expressed in HCC tissues and cells. Upregulation of circBNC2 was related to poor prognosis and tumor node metastasis (TNM) stage. Knockdown of circBNC2 inhibited the HCC progression. Moreover, knockdown of circBNC2 suppressed the levels of Ras, ERK1/2, PCNA, HK2, and OCT4. Notably, circBNC2 functioned as a molecular sponge of microRNA 217 (miR-217) to upregulate the HMGA2 expression. The inhibitory effects of the circBNC2 silence on the growth and stemness of HCC cells, and levels of PCNA, HK2 and OCT4 were aggravated by the miR-217 overexpression, but neutralized by the HMGA2 overexpression. Besides, silencing of circBNC2 blocked the tumor growth through upregulating the expression of miR-217 and downregulating the levels of HMGA2, PCNA2, HK2 and OCT4 in vivo. Thus, the current data confirmed that circBNC2 sponged miR-217 to upregulate the HMGA2 level, thereby contributing to the HCC glycolysis and progression. These findings might present novel insight into the pathogenesis and treatment of HCC.

Keywords: Glycolysis; HMGA2; Hepatocellular carcinoma; circBNC2; miR-217.

Fig. 1

CircBNC2 was highly expressed in HCC tissues and cells. (A) Compared with the adjacent normal tissues (n = 6), circBNC2 was upregulated in HCC tumor tissues (n = 6) according to GSE164803 database. **p < 0.01. (B) The expression of circBNC2 in our cohort of 54 pairs of HCC tumor tissues and adjacent normal tissues was detected by RT-qPCR. ***p < 0.001. (C) HCC patients (n = 54) were divided into circBNC2 high expression group and low expression based on the median value of circBNC2, and the survival curve of HCC patients in two groups was assessed by log-rank test. (D) The correlation between CircBNC2 expression and recurrence-free survival of HCC patients. (E) CircBNC2 expression in HCC cell lines (Huh7, HCCLM3 and Hep3B) and a normal liver cell line (L02) were assessed by RT-qPCR. ***p < 0.001 vs. L02. (F) Expression of circBNC2 and GAPDH in Huh7 cell treated with RNase R were assessed by RT-qPCR. ***p < 0.001 vs. Mock.

Fig. 2

Function of circBNC2 on proliferation, glycolysis and stemness of HCC cells. (A) The knockdown efficiency of si-circBNC2#1, si-circBNC2#2 and si-circBNC2#3 in Huh7 and Hep3B cells was detected by qRT-PCR. After Huh7 and Hep3B cells transfected with si-NC or si-circBNC2, the proliferation, relative glucose uptake, relative lactate production, relative ATP level, sphere formation, and levels of PCNA, HK2 and OCT4 protein were assessed by CCK-8 assay (B), EdU assay(C-D), Glucose Assay Kit (E), Lactate Assay Kit (F), ATP Assay Kit (G), sphere-forming assay(H), Western blot assay (I), respectively. The original images of gels can be found in Supplementary material 2. **p < 0.01, ***p < 0.001 vs. si-NC.

Fig. 3

Knockdown of circBNC2 induced HCC cell S phase arrest and apoptosis. (A) The cell cycle of Huh7 and Hep3B cells was determined by the flow cytometry. (B) The cell apoptosis of Huh7 and Hep3B cells was analyzed by the flow cytometry. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. si-NC.

Fig. 4

CircBNC2 increased HMGA2 expression by absorbing miR-217. (A) The potential binding sites between circBNC2 and miR-217 was predicted by circinteractome. (B) The luciferase activity in Huh7 cells co-transfected with circBNC2-WT/circBNC2-MUT and miR-217/miR-NC was examined by dual-luciferase reporter assay. ***p < 0.001 vs. miR-NC. (C) After Huh7 cells transfected with si-NC or si-circBNC2, miR-217 expression was determined by RT-qPCR assay. ***p < 0.001 vs. si-NC. (D) The level of circBNC2 in Huh7 cells after RNA pull-down assay using Bio-miR-NC probe or Bio-miR-217 probe was determined by RT-qPCR. ***p < 0.001 vs. Bio-miR-NC. (E) MiR-217 expression in HCC tumor tissues (n = 54) and adjacent normal tissues (n = 54) was detected by RT-qPCR. ***p < 0.001. (F) MiR-217 expression in HCC cell lines (Huh7, HCCLM3 and Hep3B) and a normal liver cell line (L02) were assessed by RT-qPCR. ***p < 0.001 vs. L02. (G) The potential binding sites between miR-217 and HMGA2 was predicted by starBase. (H) After co-transfected with HMGA2-WT/HMGA2-MUT and miR-217/miR-NC, the luciferase activity in Huh7 cells was examined. ***p < 0.001 vs. miR-NC. (I) After Huh7 cells transfected with miR-NC or miR-217 vector, HMGA2 protein expression was detected by Western blot assay. The original images of gels can be found in Supplementary material 2. ***p < 0.001 vs. miR-NC. (J) After Huh7 cells transfected with miR-217 inhibitor or NC inhibitor, miR-217 expression was detected by RT-qPCR assay. ***p < 0.001 vs. NC inhibitor. (K–L) After Huh7 cells transfected with si-NC, si-BNC2 or si-circBNC2+miR-217 inhibitor, the levels of PCNA, HK2 and OCT4 in Huh7 cells were assessed by Western blot assay. The original images of gels can be found in Supplementary material 2. ***p < 0.001. (M) HMGA2 expression in HCC tumor tissues (n = 54) and adjacent normal tissues (n = 54) was detected by RT-qPCR. ***p < 0.001. (N) HMGA2 expression in HCC cell lines (Huh7, HCCLM3 and Hep3B) and normal liver cell line (L02) was assessed by RT-qPCR. ***p < 0.001 vs. L02. (O–Q) The correlations between circBNC2 and miR-217 and HMGA2 were analyzed in HCC tissues.

Fig. 5

CircBNC2/miR-217/HMGA2 regulated HCC cell proliferation and glycolysis . (A) After transfection with the empty vector or HMGA2 vector, HMGA2 protein expression was detected by Western blot. The original images of gels can be found in Supplementary material 2. ***p < 0.001 vs. vector. After Huh7 cells transfected with si-NC, si-circBNC2, si-circBNC2+miR-217, or si-circBNC2+HMGA2, the proliferation, relative glucose uptake, relative lactate production, relative ATP level, sphere formation, and levels of PCNA, HK2 and OCT4 protein were assessed by CCK-8 assay (B), EdU assay (C and D), Glucose Assay Kit (E), Lactate Assay Kit (F), ATP Assay Kit (G), respectively. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 6

CircBNC2/miR-217/HMGA2 regulated HCC cell stemness and cycle. (A) After Huh7 cells transfected with si-NC, si-circBNC2, si-circBNC2+miR-217, or si-circBNC2+HMGA2, the sphere formation, and levels of PCNA, HK2 and OCT4 protein were assessed by sphere-forming assay(A) and Western blot assay (B), respectively. The original images of gels can be found in Supplementary material 2. (C) The cell cycle was determined by the flow cytometry. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 7

CircBNC2 inhibition reduced HCC growth in vivo. (A) The growth curve of xenograft tumor in the sh-circBNC2 and sh-NC groups. Mice weight (B) and tumor weight (C) were evaluated in the sh-circBNC2 and sh-NC groups. (D) The expression of circBNC2 and miR-217 in the sh-circBNC2 and sh-NC groups were examined by qRT-PCR. The serum levels of ALT (E) and AST (F) in the sh-circBNC2 and sh-NC groups were examined by ELISA. (G) Representative H&E stain images of tumor foci of liver sample and positive staining rates of HMGA2, PCNA, HK2 and OCT4 in the sh-circBNC2 and sh-NC groups were examined by immunohistochemistry. N = 6 per group. **p < 0.01, ***p < 0.001 vs. sh-NC.

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