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Circular RNA circZFR Promotes Hepatocellular Carcinoma Progression by Regulating miR-375/HMGA2 Axis

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Abstract

Background

Mounting evidence indicates that circular RNAs (circRNAs) have vital roles in human diseases, especially in cancers.

Aims

The aim of this study was to explore the biological functions and underlying mechanism of circRNA zinc finger RNA binding (circZFR) in hepatocellular carcinoma (HCC).

Methods

The expression levels of circZFR, microRNA-375 (miR-375) and high mobility group A2 (HMGA2) were detected by qRT-PCR or western blot assay. Glycolytic metabolism was examined via the measurement of extracellular acidification rate, oxygen consumption rate, glucose uptake, lactate production, and ATP level. MTT assay and flow cytometry were used to assess cell proliferation and cell apoptosis, respectively. The interaction between miR-375 and circZFR or HMGA2 was verified by dual-luciferase reporter and RNA Immunoprecipitation (RIP) assays. The mice xenograft model was established to investigate the role of circZFR in vivo.

Results

CircZFR and HMGA2 were upregulated while miR-375 was downregulated in HCC tissues and cells. CircZFR silence inhibited HCC progression by inhibiting cell proliferation, glycolysis and tumor growth and promoting apoptosis. MiR-375 was a direct target of circZFR and its knockdown reversed the inhibitory effect of circZFR silence on the progression of HCC cells. Moreover, HMGA2 was a downstream target of miR-375, and miR-375 suppressed proliferation and glycolysis and induced apoptosis by targeting HMGA2 in HCC cells. Besides, circZFR acted as a molecular sponge of miR-375 to regulate HMGA2 expression.

Conclusion

Knockdown of circZFR suppressed the progression of HCC by upregulating miR-375 and downregulating HMGA2, providing new insight into the pathogenesis of HCC.

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Authors and Affiliations

  1. Department of Interventional Radiology, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, No. 1 Swan Lake Road, New District of Political Affairs and Culture, Hefei, 230036, Anhui, China

    Rui Xu, Meng Zheng, Xiaohong Pei & Xuebing Ji

  2. Department of Interventional Radiology, The Second People’s Hospital of Hefei, Hefei, 230000, Anhui, China

    Shiwu Yin

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  1. Rui Xu
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Correspondence to Xuebing Ji.

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10620_2020_6805_MOESM1_ESM.tif

Supplementary Fig. 1. Downregulation of circZFR suppressed proliferation and promoted apoptosis by upregulating miR-375 in HCC cells. (A-D) Hep3B and Huh7 cells were divided into four groups: Mock, si-con, si-circZFR-1, and si-circZFR-2. (A and B) MTT assay was utilized to assess cell proliferation. (C and D) Flow cytometry analysis was used to determine apoptosis rate. (E–H) Hep3B and Huh7 cells were divided into four groups: si-con, si-circZFR, si-circZFR + in-miR-con, and si-circZFR + in-miR-375. (E and F) Cell proliferation was assessed by MTT assay. (G and H) Flow cytometry analysis was employed to determine the apoptosis rate. *P < 0.05. (TIFF 2433 kb)

10620_2020_6805_MOESM2_ESM.tif

Supplementary Fig. 2. MiR-375 overexpression inhibited the progression of HCC cells by downregulating HMGA2. Hep3B and Huh7 cells were transfected with miR-con, miR-375, miR-375 + vector, or miR-375 + HMGA2. (A-F) Glucose Uptake Colorimetric Assay kit, Lactate Assay kit and ATP Colorimetric Assay kit were used to measure glucose uptake, lactate production and ATP level, respectively. (G and H) Cell proliferation was examined by MTT assay. (I and J) Cell apoptosis was analyzed using flow cytometry analysis. *P < 0.05. (TIFF 756 kb)

10620_2020_6805_MOESM3_ESM.tif

Supplementary Fig. 3. Silence of circZFR repressed the progression of HCC cells by downregulating HMGA2. Hep3B and Huh7 cells were transfected with si-con, si-circZFR, si-circZFR + vector, or si-circZFR + HMGA2. (A and B) Western blot assay was performed to detect the protein expression of HMGA2. (C-F) ECAR and OCR were measured by Seahorse Bioscience XF96 extracellular flux analyzer. (G-L) Glucose Uptake Colorimetric Assay kit, Lactate Assay kit and ATP Colorimetric Assay kit were used for detecting glucose uptake, lactate production and ATP level, respectively. (M and N) MTT assay was applied to examine cell proliferation. (O and P) Cell apoptosis was analyzed by flow cytometry analysis. *P < 0.05. (TIFF 1687 kb)

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Xu, R., Yin, S., Zheng, M. et al. Circular RNA circZFR Promotes Hepatocellular Carcinoma Progression by Regulating miR-375/HMGA2 Axis. Dig Dis Sci 66, 4361–4373 (2021). https://doi.org/10.1007/s10620-020-06805-2

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