circ_0003418 Inhibits Tumorigenesis And Cisplatin Chemoresistance Through Wnt/β-Catenin Pathway In Hepatocellular Carcinoma

Abstract

Background: Accumulating evidences indicate that circRNAs play important roles in the progression and chemoresistance of human cancers. The present study is designated for researching the roles of circ_0003418 in hepatocellular carcinoma (HCC).

Methods: We detected the expression profile of circ_0003418 in human HCC tissues and cell lines by quantitative real-time-PCR assays. CCK-8 assay, transwell migration assay, transwell invasion assay and drug-sensitivity analysis were carried out to estimate the effects of circ_0003418 on HCC cells' proliferation, migration, invasion and resistance to cisplatin, respectively. Mouse xenograft model was conducted to monitor the role of circ_0003418 in cisplatin resistance in vivo. Western blotting was performed to explore the changes of the Wnt/β-catenin pathway after knockdown of circ_0003418. The rescue experiment was carried out to explore circ_0003418-activated biological functions through Wnt/β-catenin pathway.

Results: The expression level of circ_0003418 was downregulated in HCC tissues and cell lines, and the level correlated with tumor size, TNM stage and HBsAg level in HCC patients. circ_0003418 knockdown promoted HCC cells' proliferation, migration, and invasion. Additionally, suppression of circ_0003418 enhanced cisplatin resistance of HCC cells in vivo and vitro. Knockdown of circ_0003418 activated the Wnt/β-catenin signalling pathway in HCC cells. The effect of circ-0003418 on sensitivity of HCC cells to cisplatin was reversed after inhibition of Wnt/β-catenin pathway.

Conclusion: circ-0003418 exerts an antitumorigenic role in HCC and advances the sensitivity of HCC cells to cisplatin by restraining the Wnt/β-catenin pathway. Thus, circ-0003418 may represent a novel biomarker and provide us a new strategy for the treatment of HCC.

Keywords: Wnt/β-catenin; circ-0003418; circRNA; cisplatin resistance; hepatocellular carcinoma.

Figure 1

circ_0003418 downregulation in HCC tissues and cells. Notes: (A) The expression profile of circ_0003418 in HCC tissues and adjacent noncancerous tissues were detected by qRT-PCR assays. (B) qRT-PCR was used to analyze the level of circ_0003418 in five HCC cell lines and one normal human hepatocyte cell line. (C and D) The knockdown efficiency of LV3-circ_0003418 on circ_0003418 in Huh-7 and Hep-3B cells was verified via qRT-PCR. ** P<0.01 and *** P<0.001 compared to control group. Abbreviations: HCC, hepatocellular carcinoma; NC, negative control.

Figure 2

circ_0003418 suppresses proliferation, migration, and invasion and promotes apoptosis in HCC cells. Notes: (A and B) CCK-8 assays were performed to measure the effect of silencing circ_0003418 on the proliferation in Huh-7 and Hep-3B cells. (C–F) Effect of silencing circ_0003418 on cell migration (C and D) and invasion (E and F) were analyzed by transwell migration and invasion assays, respectively. * P<0.05, ** P<0.01 and *** P<0.001 compared to control group. Abbreviations: CCK-8, cell counting kit 8; NC, negative control.

Figure 3

circ-0003418 sensitizes HCC cells to cisplatin in vitro. Notes: (A and B) Huh-7 and Hep-3B cells infected with LV3-NC or LV3-circ_0003418 were treated with different doses of cisplatin (1, 2, 4, 8, 16, 32, 64 and 128 mg/L) for 24 hrs, and then cell viability was determined by CCK-8 assays. (C–F) Huh-7 cells were treated with cisplatin (11.39 mg/L) for 24 hrs as well as Hep-3B cells were treated with cisplatin (20.18 mg/L) for 24 hrs, and then cell migration and invasion were detected by transwell migration (C and D) and invasion (E and F) assays, respectively. * P<0.05, ** P<0.01 and *** P<0.001 compared to control group. Abbreviation: NC, negative control.

Figure 4

circ-0003418 enhances sensitivity of HCC cells to cisplatin in vivo. Female BALB/c nude mice were implanted subcutaneously with Huh-7 cells infected with LV3-NC or LV3-circ_0003418. Twelve days later, the LV3-NC tumor-bearing mice were treated with saline or cisplatin (5 mg/kg) by intraperitoneal injection twice a week up to 36 days posttreatment. The mice bearing LV3-circ_0003418 tumors received the same treatment. Notes: (A) Image of the tumors in the nude mice. (B) The tumors growth curve of xenograft model mice. (C and D) The weight and volume of the tumors in the nude mice. * P<0.05, ** P<0.01 and *** P<0.001 compared to control group. Abbreviation: NC, negative control.

Figure 5

Silencing circ_0003418 induces cisplatin resistance of HCC cells through activating the Wnt/β-catenin pathway. Notes: (A and B) Huh-7 cells were treated with cisplatin (11.39 mg/L) for 24 hrs as well as Hep-3B cells were treated with cisplatin (20.18 mg/L) for 24 hrs, and then the protein levels of β-catenin and c-Myc in the Huh-7 and Hep-3B cells were detected by Western blotting. (C and D) The inhibition efficiency of ICG-001 was detected by Western blotting. (E and F) Cell proliferation assay showed that inhibition of Wnt/β-catenin pathway in cells infected with LV3-circ_0003418 inhibited cell proliferation. (G and H) Chemotherapy sensitivity assay showed that inhibition of Wnt/β-catenin pathway in cells infected with LV3-circ_0003418 enhanced sensitivity of HCC cells to cisplatin. * P<0.05, ** P<0.01 and *** P<0.001 compared to control group. Abbreviation: NC, negative control.