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. 2020 May 14:12:3457-3468.
doi: 10.2147/CMAR.S244024. eCollection 2020.

Circ_0000517 Contributes to Hepatocellular Carcinoma Progression by Upregulating TXNDC5 via Sponging miR-1296-5p

Hongliang Zang  1 , Yuhui Li  1 , Xue Zhang  1 , Guomin Huang  1
Affiliations

Circ_0000517 Contributes to Hepatocellular Carcinoma Progression by Upregulating TXNDC5 via Sponging miR-1296-5p

Hongliang Zang et al. Cancer Manag Res. .

Abstract

Background: Circular RNAs (circRNAs) function as essential regulators in diverse human cancers, including hepatocellular carcinoma (HCC). However, the function of circ_0000517 in HCC was unknown. We aimed to explore the roles and mechanisms of circ_0000517 in HCC.

Materials and methods: The levels of circ_0000517, RPPH1 mRNA and microRNA-1296-5p (miR-1296-5p) were measured using quantitative real-time polymerase chain reaction (qRT-PCR). The characteristics of circ_0000517 were explored by RNase R digestion and actinomycin D assays. Cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8) and colony formation assays. Cell cycle process and cell apoptosis were analyzed by flow cytometry analysis. The function of circ_0000517 in vivo was explored by a murine xenograft model. The association between miR-1296-5p and circ_0000517 or thioredoxin domain containing 5 (TXNDC5) was determined by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. The protein level of TXNDC5 was detected by Western blot assay.

Results: Circ_0000517 was upregulated in HCC tissues and cells. Silencing of circ_0000517 suppressed HCC cell viability and colony formation and promoted cell cycle arrest and apoptosis in vitro and hampered tumor growth in vivo. MiR-1296-5p was a target of circ_0000517 and the effects of circ_0000517 silencing on HCC cell viability, cell cycle, colony formation and apoptosis were abolished by miR-1296-5p inhibition. TXNDC5 functioned as a target gene of miR-1296-5p, and the inhibitory effect of miR-1296-5p on HCC cell progression was rescued by TXNDC5 overexpression. Moreover, circ_0000517 promoted TXNDC5 expression via targeting miR-1296-5p.

Conclusion: Circ_0000517 accelerated HCC progression by upregulating TXNDC5 through sponging miR-1296-5p.

Keywords: HCC; TXNDC5; circ_0000517; miR-1296-5p.

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Conflict of interest statement

The authors declare that they have no conflicts of interest in this work.

Figures

Figure 1
Figure 1
High expression of circ_0000517 was observed in HCC tissues and cells. (A) The expression of circ_0000517 in HCC tissues and normal tissues was measured by qRT-PCR. (B) The expression of circ_0000517 in HCC tissues at different TNM stages (I-III) was determined by qRT-PCR. (C) The expression of circ_0000517 in THLE-2, SNU-387 and Huh7 cells was examined using qRT-PCR. (D) QRT-PCR was performed to determine the level of circ_0000517 and linear RPPH1 mRNA in SNU-387 and Huh7 cells treated with or without RNase R. (E) QRT-PCR assay was conducted to determine the levels of circ_0000517 and linear RPPH1 mRNA in SNU-387 and Huh7 cells after treated with Actinomycin D for 0 h, 4 h, 8 h, 12 h and 24 h. (F) The levels of circ_0000517 and linear RPPH1 mRNA were examined by qRT-PCR after total RNA was reversely transcribed using Random primers or Oligo (dt)18 primers. (G) The structure of circ_0000517 was shown. *P<0.05.
Figure 2
Figure 2
Circ_0000517 silencing hampered HCC cell viability and colony formation and induced cell cycle arrest and apoptosis in vitro and blocked tumor growth in vivo. (A-H) Si-NC or si-circ_0000517 was transfected into SNU-387 and Huh7 cells. (A) The expression of circ_0000517 in SNU-387 and Huh7 cells was detected via qRT-PCR. (B) The viability of SNU-387 and Huh7 cellswas assessed by CCK-8 assay. (C and D) Cell cycle in SNU-387 and Huh7 cells was analyzed by flow cytometry analysis. (E and F) Colony formation numbers of SNU-387 and Huh7 cells were examined by colony formation assay. (G and H) The apoptosis of SNU-387 and Huh7 cells was analyzed by flow cytometry analysis. (I-K) Huh7 cells were transfected with sh-NC or sh-circ_0000517 and then injected into mice. (I) Tumor volume was monitored every 7 days. (J) Tumor weight was examined after 28 days. (K) The level of circ_0000517 in the collected tissues was measured by qRT-PCR. *P<0.05.
Figure 3
Figure 3
MiR-1296-5p was a target of circ_0000517 and the inhibitory effect of circ_0000517 knockdown on HCC cell progression was weakened by miR-1296-5p inhibition. (A and B) The expression level of miR-1296-5p in HCC tissues, cells (SNU-387 and Huh7 cells) and corresponding normal tissues and cells (THLE-2) was examined by qRT-PCR. (C) The potential binding sites between circ_0000517 and miR-1296-5p were predicted by starBase 2.0. (D and E) The levels of circ_0000517 and miR-1296 in Anti-Ago2/Anti-IgG immunoprecipitates in SNU-387 and Huh7 cells were measured by qRT-PCR following RIP assay. (F) The expression levels of circ_0000517 and miR-1296-5p in the nuclear and cytoplasm of SNU-387 and Huh7 cells were examined by qRT-PCR. (G) Dual-luciferase reporter assay was conducted for the luciferase activity in SNU-387 and Huh7 cells transfected with WT-circ_0000517/MUT-circ_0000517 and miRNA NC/miR-1296-5p mimic. (H-M) SNU-387 and Huh7 cells were transfected with si-NC, si-circ_0000517, si-circ_0000517+inhibitor NC and si-circ_0000517+miR-1296-5p inhibitor. (H) The expression of miR-1296-5p in SNU-387 and Huh7 cells was determined using qRT-PCR. (I) CCK-8 assay was adopted for the viability of SNU-387 and Huh7 cells. (J and K) Flow cytometry analysis was employed to analyze cell cycle process in SNU-387 and Huh7 cells. (L) Colony formation assay was performed for cell colony formation ability in SNU-387 and Huh7 cells. (M) Flow cytometry analysis was employed to evaluate the apoptosis SNU-387 and Huh7 cells. *P<0.05.
Figure 4
Figure 4
MiR-1296-5p regulated HCC cell viability, cell cycle process, colony formation and apoptosis by targeting TXNDC5.(A) The complementary sequences between miR-1296-5p and TXNDC5 were presented. (B) The targeting relationship between miR-1296-5p and TXNDC5 was verified by dual-luciferase reporter assay. (C and D) The protein level of TXNDC5 in HCC tissues, cells and corresponding normal tissues and cells was measured using western blot assay. (E) The protein level of TXNDC5 in SNU-387 and Huh7 cells transfected with pc-NC or pc-TXNDC5 was determined through western blot assay. (F-K) SNU-387 and Huh7 cells were transfected with miRNA NC, miR-1296-5p mimic, miR-1296-5p mimic+pc-NC or miR-1296-5p mimic+pc-TXNDC5. (F) The protein level of TXNDC5 in SNU-387 and Huh7 cells was measured by western blot assay. (G) Cell viability in SNU-387 and Huh7 cells was analyzed by CCK-8 assay. (H and I) Cell cycle in SNU-387 and Huh7 cells was analyzed through flow cytometry analysis. (J) Cell colony formation ability was evaluated by colony formation assay. (K) Cell apoptosis in SNU-387 and Huh7 cells was assessed via flow cytometry analysis. *P<0.05.
Figure 5
Figure 5
The suppressive role of circ_0000517 knockdown in HCC cell progression was reversed by TXNDC5 elevation. SNU-387 and Huh7 cells were transfected with si-NC, si-circ_0000517, si-circ_0000517+pc-NC or si-circ_0000517+pc-TXNDC5. (A) The protein level of TXNDC5 in SNU-387 and Huh7 cells was measured by western blot assay. (B) The viability of SNU-387 and Huh7 cells was assessed by CCK-8 assay. (C) The colony formation ability of SNU-387 and Huh7 cells was evaluated by cell colony formation assay. (D-F) Cell cycle and cell apoptosis in SNU-387 and Huh7 cells were analyzed by flow cytometry analysis. *P<0.05.
Figure 6
Figure 6
Circ_0000517 promoted TXNDC5 expression by binding to miR-1296-5p in HCC cells.(A and B) The luciferase reporter vectorWT-TXNDC5-3’UTR together with relevant vectors and miRNA mimics were transfected into SNU-387 and Huh7 cells and then the luciferase activity was determined. (C) SNU-387 and Huh7 cells were transfected with si-NC, si-circ_0000517, si-circ_0000517+inhibitor NC or si-circ_0000517+miR-1296-5p inhibitor and then the protein level of TXNDC5 was measured by western blot assay. *P<0.05.
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