Korean J Physiol Pharmacol.  2023 Jul;27(4):333-344. 10.4196/kjpp.2023.27.4.333.

Circ-SNX27 sponging miR-375/RPN1 axis contributes to hepatocellular carcinoma progression

• Zheng C
• Liang J
• Yu S
• Xu H
• Dai L
• Xu D

• KMID: 2544135
• DOI: http://doi.org/10.4196/kjpp.2023.27.4.333

Abstract

Hepatocellular carcinoma (HCC) is a prevalent malignant tumor with high fatality. It has yet to be reported whether circ-SNX27 can affect the progression of HCC. This study attempted to analyze circ-SNX27’s precise role and underlying mechanisms in HCC. HCC cell lines and tumor specimens from HCC patients were analyzed using quantitative real-time PCR and Western blotting to quantify the expressions of circ-SNX27, miR-375, and ribophorin I (RPN1). Cell invasion and cell counting kit 8 experiments were conducted for the evaluation of HCC cell invasion and proliferation. Caspase-3 Activity Assay Kit was utilized to gauge the caspase-3 activity. Luciferase reporter and RNA immunoprecipitation assays were executed to ascertain the relationships among miR-375, circ-SNX27, and RPN1. To determine how circ-SNX27 knockdown affects the growth of HCC xenografts in vivo, tumor-bearing mouse models were constructed. Elevated expressions of circ-SNX27 and RPN1 as well as a reduced miR-375 expression were observed among HCC cells and HCC patient tumor specimens. Knocking-down circ-SNX27 in HCC cells abated their proliferative and invasive abilities but raised their caspase-3 activity. Moreover, the poor levels of circ-SNX27 inhibited HCC tumor growth among the mice. Circ-SNX27 enhanced RPN1 by competitively binding with miR-375. Silencing miR-375 in HCC cells promoted their malignant phenotypes. Nonetheless, the promotive effect of miR-375 silencing was reversible via the knockdown of circ-SNX27 or RPN1. This research demonstrated that circ-SNX27 accelerated the progression of HCC by modulating the miR-375/RPN1 axis. This is indicative of circ-SNX27’s potential as a target for the treatment of HCC.

Keyword

circRNA; Hepatocellular carcinoma; miRNA; Proliferation; Sponging activity

Figure

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  Fig. 1 Circ-SNX27 upregulation was observed in hepatocellular carcinoma (HCC) cell lines and tumors. (A) Circ-SNX27 expressions in the normal or tumor samples from HCC patient specimens were quantified via qRT-PCR. (B) Circ-SNX27 expressions in normal liver cells (THLE-2) and HCC cells (Hep 3B, SNU-182, Huh7 and MHCC-97H) were determined by means of qRT-PCR. **p < 0.001 vs. THLE2 group. (C) qRT-PCR analysis of circ-SNX27 expression within the nuclei and cytoplasm of MHCC-97H and Huh7 cells. (D) The circ-SNX27 and linear SNX27 expressions in the MHCC-97H and Huh7 cells after RNase R treatment were measured via qRT-PCR. **p < 0.001 vs. control group. Values are presented as mean ± SD.

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  Fig. 2 Knocking-down circ-SNX27 inhibited the progression of hepatocellular carcinoma (HCC) in vivo and in vitro. MHCC-97H and Huh7 cells were transfected with either si-NC or si-circ-SNX27 (si-circ). (A) The circ-SNX27 expression in the transfected HCC cells was analyzed through qRT-PCR. **p < 0.001 vs. si-NC group. (B) CCK-8 experiment was executed to assess the proliferative ability of these transfected HCC cells. *p < 0.05 vs. sin-NC group. **p < 0.001 vs. si-NC group. (C) Caspase-3 Activity Assay Kit was utilized to detect caspase-3 activity and assess apoptosis among the transfected HCC cells. **p < 0.001 vs. si-NC group. (D) Cell invasion assay was performed to assess the invasion ability of the transfected HCC cells. **p < 0.001 vs. si-NC group by Transwell invasion assays. ×200. (E) Tumor photos, weight, and volume of tumors in mice following the injection of LV-sh-circ-SNX27-infected Huh7 cells or LV-sh-NC-infected Huh7. **p < 0.001 vs. sh-NC group. Values are presented as mean ± SD.

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  Fig. 3 Circ-SNX27 directly targeted miR-375. (A) The circ-SNX27 and miR-375 binding site was predicted by CircInteractome. (B) Luciferase reporter experiment was executed to examine the interaction of circ-SNX27 with miR-375. **p < 0.001 vs. miR-NC group. (C) RIP assay was performed to assess circ-SNX27’s interaction with miR-375. **p < 0.001 vs. Anti-IgG group. (D) The miR-375 expressions in normal and tumoral hepatocellular carcinoma (HCC) tissues were quantified via qRT-PCR. (E) Spearman’s correlation analyzed the association of circ-SNX27 with miR-375. (F) MiR-375 expressions in normal cells (THLE2) and HCC cells (MHCC-97H and Huh7) were determined by means of qRT-PCR. **p < 0.001 vs. THLE2 group. (G) The relative miR-375 expression in MHCC-97H and Huh7 cells following the transfection of si-NC, inhibitor-NC, si-circ-SNX27 (si-circ), miR-375 inhibitor (inhibitor), or si-circ + inhibitor was analyzed by means of qRT-PCR. **p < 0.001 vs. si-NC group; &&p < 0.001 vs. inhibitor-NC group; ##p < 0.001 vs. si-circ + inhibitor group. Values are presented as mean ± SD. WT, wild-type; MUT, mutant; Ago2, Argonaute 2.

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  Fig. 4 Circ-SNX27 knockdown promoted apoptosis and repressed the proliferation and invasion of hepatocellular carcinoma (HCC) cells by regulating the expression of miR-375. MHCC-97H and Huh7 cells were transfected with si-NC, inhibitor-NC, si-circ-SNX27 (si-circ), miR-375 inhibitor (inhibitor), or si-circ + inhibitor. (A) CCK-8 assay was conducted to evaluate the proliferation of the transfected HCC cells. (B) The caspase-3 activity in the transfected HCC cells was examined by means of the caspase-3 activity assay. (C) Cell invasion assay was executed to assess the invasion ability of the transfected HCC cells by transwell invasion assays. ×200. Values are presented as mean ± SD. *p < 0.05 and **p < 0.001 vs. si-NC group; &&p < 0.001 vs. inhibitor-NC group; #p < 0.05 and ##p < 0.001 vs. si-circ + inhibitor group.

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  Fig. 5 Ribophorin I (RPN1) was a miR-375 target gene. (A) The miR-375 and RPN1 binding site was predicted by TargetScan. (B) Luciferase reporter assay was conducted to assess miR-375’s interaction with RPN1. **p < 0.001 vs. miR-NC group. (C) RPN1 expression in normal and tumoral hepatocellular carcinoma (HCC) tissues was quantified by means of qRT-PCR. (D) Spearman’s correlation analyzed the association of miR-375 with RPN1. (E) The RPN1 expressions among normal cells (THLE2) and HCC cells (MHCC-97H and Huh7) were determined via qRT-PCR. **p < 0.001 vs. THLE2 group. (F) The relative protein expressions of RPN1 expression in the MHCC-97H and Huh7 cells transfected with si-NC, inhibitor-NC, si-RPN1, miR-375 inhibitor (inhibitor), or si-RPN1 + inhibitor were quantified via Western blotting. *p < 0.05 and **p < 0.001 vs. si-NC group; &p < 0.01 and &&p < 0.001 vs. inhibitor-NC group; ##p < 0.001 vs. si-RNP1 + inhibitor group. Values are presented as mean ± SD. WT, wild-type; MUT, mutant.

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  Fig. 6 MiR-375 knockdown inhibited apoptosis and stimulated the proliferation and invasion of hepatocellular carcinoma (HCC) cells by modulating ribophorin I (RPN1) expression. MHCC-97H and Huh7 cells were transfected with si-NC, inhibitor-NC, si-RPN1, miR-375 inhibitor (inhibitor), or si-RPN1 + inhibitor. (A) CCK-8 assay was used to evaluate the proliferation of the transfected HCC cells. (B) The caspase-3 activity in the transfected HCC cells was examined by means of the caspase-3 activity assay. (C) Cell invasion assay was executed to assess the invasion ability of the transfected HCC cells by transwell migration assay. ×200. Values are presented as mean ± SD. *p < 0.05 and **p < 0.001 vs. si-NC group; &&p < 0.001 vs. inhibitor-NC group; #p < 0.05 and ##p < 0.001 vs. si-RPN1 + inhibitor group.

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