MicroRNA-98-5p Inhibits Tumorigenesis of Hepatitis B Virus-Related Hepatocellular Carcinoma by Targeting NF-κB-Inducing Kinase

Abstract

Purpose: MicroRNAs play key regulatory roles in the tumorigenesis of hepatitis B virus-related hepatocellular carcinoma (HBV-HCC). This study aimed to explore the regulatory effects of microRNA-98-5p (miR-98-5p) on the proliferation, migration, invasion, and apoptosis of HBV-HCC cells, as well as the underlying mechanisms involving nuclear factor-κB-inducing kinase (NIK).

Materials and methods: The expressions of miR-98-5p and NIK in HBV-HCC tissues and cells, and the level of HBV DNA in HBV-HCC cells were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The proliferation, migration, invasion, and apoptosis of HBV-HCC cells were analyzed by cell counting kit-8, wound healing, transwell, and flow cytometry assay, respectively. The targeting relationship between miR-98-5p and NIK was predicted by StarBase3.0 and verified by dual-luciferase reporter assay. HBV-HCC xenograft tumor model was constructed in mice to observe the tumor growth in vivo.

Results: The expression of miR-98-5p was declined in HBV-HCC tissues and cells. Overexpression of miR-98-5p markedly reduced the level of HBV DNA; inhibited the proliferation, migration, and invasion; and promoted the apoptosis of HBV-HCC cells. NIK was a target of miR-98-5p. Overexpression of miR-98-5p markedly decreased the protein expression of NIK in MHCC97H-HBV cells. NIK reversed the tumor-suppressing effect of miR-98-5p on HBV-HCC cells. Furthermore, overexpression of miR-98-5p significantly inhibited the xenograft tumor growth and decreased the expression of NIK in mice.

Conclusion: MiR-98-5p inhibits the secretion of HBV, proliferation, migration, and invasion of HBV-HCC cells by targeting NIK.

Keywords: MicroRNA-98-5p; hepatitis B virus-related hepatocellular carcinoma; invasion; nuclear factor-κB-inducing kinase; proliferation.

Fig. 1. The expression of microRNA-98-5p (miR-98-5p) was down-regulated in hepatocellular carcinoma-related hepatitis B virus (HBV-HCC) tissues and cells. (A) Relative expression of miR-98-5p in adjacent normal tissues and HBV-HCC tissues was detected by qRT-PCR (p<0.001). (B) Relative expression of miR-98-5p in LO2, MHCC97H, MHCC97H-HBV, Huh7, and Huh7-1.3 cells was detected by qRT-PCR. ^***p<0.001 vs. LO2; ^†p<0.05 vs. MHCC97H; ^‡p<0.05 vs. Huh7. qRT-PCR, quantitative real-time polymerase chain reaction.

Fig. 2. MiR-98-5p inhibited the secretion of HBV. (A) Relative expression of miR-98-5p in MHCC97H-HBV and Huh7-1.3 cells was detected by qRT-PCR. (B) The level of HBV DNA in MHCC97H-HBV and Huh7-1.3 cells was detected by qRT-PCR. (C) The ratio of HBeAg secretion in MHCC97H-HBV and Huh7-1.3 cells. (D) The ratio of HBsAg secretion in MHCC97H-HBV and Huh7-1.3 cells. ^**p<0.01, ^***p<0.001 vs. Mock and mimics-NC. MiR-98-5p, microRNA-98-5p; HBV, hepatitis B virus; qRT-PCR, quantitative real-time polymerase chain reaction.

Fig. 3. MiR-98-5p inhibited the proliferation, migration, and invasion of HBV-HCC cells. (A) Proliferation of MHCC97H-HBV and Huh7-1.3 cells was assessed by cell counting kit-8 (CCK-8) assay. (B) Apoptosis of MHCC97H-HBV and Huh7-1.3 cells was assessed by flow cytometry. (C) Migration of MHCC97H-HBV and Huh7-1.3 cells was assessed by wound healing assay. (D) Invasion of MHCC97H-HBV and Huh7-1.3 cells was assessed by transwell assay. ^*p<0.05, ^**p<0.01, ^***p<0.001 vs. Mock and mimics-NC. MiR-98-5p, microRNA-98-5p; HBV-HCC, hepatitis B virus-related hepatocellular carcinoma.

Fig. 4. NIK was a direct target of miR-98-5p. (A) Relative mRNA expression of NIK in normal liver tissues and HBV-HCC tissues was detected by qRT-PCR (p<0.001). (B) The mRNA expression of NIK was negatively correlated with that of miR-98-5p (p=0.0008). (C) Relative mRNA expression of NIK in LO2, MHCC97H, MHCC97H-HBV, Huh7, and Huh7-1.3 cells was detected by qRT-PCR (^***p<0.001 vs. LO2; ^†††p<0.001 vs. HepG2; ^‡‡‡p<0.001 vs. Huh7). (D) Schematic diagram of the target site between miR-98-5p and NIK. (E) Luciferase renilla/firefly activity of HEK-293T cells transfected with NIK Wt/Mut and miR-98-5p mimics/mimics-NC (^***p<0.001 vs. mimics-NC). (F) Relative protein expression of NIK in MHCC97H-HBV and Huh7-1.3 cells was detected by western blot (^***p<0.001 vs. Mock and mimics-NC). NIK, nuclear factor-κB-inducing kinase; MiR-98-5p, microRNA-98-5p; HBV-HCC, hepatitis B virus-related hepatocellular carcinoma; qRT-PCR, quantitative real-time polymerase chain reaction.

Fig. 5. NIK eliminated the effect of miR-98-5p on HBV-HCC cells. (A) Relative mRNA expression of NIK in MHCC97H-HBV cells was detected by qRT-PCR (^***p<0.001 vs. Mock and pcDNA3.1-NC). (B) The level of HBV DNA in MHCC97H-HBV cells was detected by qRT-PCR. (C) Secretion ratio of HBeAg in MHCC97H-HBV cells. (D) Secretion ratio of HBsAg in MHCC97H-HBV cells. (E) Proliferation of MHCC97H-HBV cells was assessed by cell counting kit-8 (CCK-8) assay. (F) Apoptosis of MHCC97H-HBV was assessed by flow cytometry. (G) Migration of MHCC97H-HBV cells was assessed by wound healing assay. (H) Invasion of MHCC97H-HBV cells was assessed by transwell assay. ^*p<0.05, ^**p<0.01, ^***p<0.001 vs. mimics-NC+pcDNA-NC; ^†p<0.05, ^††p<0.01 vs. miR-98-5p mimics+pcDNA-NC; ^‡p<0.05, ^‡‡p<0.01 vs. mimics-NC+pcDNA-NIK. NIK, nuclear factor-κB-inducing kinase; MiR-98-5p, microRNA-98-5p; HBV-HCC, hepatitis B virus-related hepatocellular carcinoma; qRT-PCR, quantitative real-time polymerase chain reaction.

Fig. 6. MiR-98-5p inhibited xenograft tumor growth in mice. (A) Tumor volume. (B) Tumor weight. (C) Relative expression of miR-98-5p in xenograft tumor was detected by qRT-PCR. (D) Relative mRNA expression of NIK in xenograft tumor was detected by qRT-PCR. ^**p<0.01, ^***p<0.001 vs. Mock and mimics-NC. NIK, nuclear factor-κB-inducing kinase; MiR-98-5p, microRNA-98-5p; qRT-PCR, quantitative real-time polymerase chain reaction.