Yonsei Med J.  2018 Jun;59(4):511-518. 10.3349/ymj.2018.59.4.511.

miR-215 Enhances HCV Replication by Targeting TRIM22 and Inactivating NF-κB Signaling

Affiliations
  • 1Department of Infectious Disease, Huaihe Hospital of Henan University, Kaifeng, China. hezhenshenccn@163.com

Abstract

PURPOSE
Hepatitis C virus (HCV) infection is a major cause of liver disease. Several miRNAs have been found to be associated with HCV infection. This study aimed to investigate the functional roles and possible molecular mechanisms of miR-215 in HCV replication.
MATERIALS AND METHODS
The expression levels of miR-215 and TRIM22 were detected by quantitative real-time PCR (qRT-PCR) and western blot analysis in Con1b subgenomic genotype 1b HCV replicon cells (Con1b cells) and JFH1 full genome infecting Huh7.5.1 cells (Huh7.5.1 cells). HCV RNA levels were measured by qRT-PCR. The protein levels of NS3, NS5A, p65 subunit of NF-κB (p65), and phosphorylated p65 (p-p65) were determined by western blot analysis. The relationship between miR-215 and TRIM22 were explored by target prediction and luciferase reporter analysis.
RESULTS
miR-215 overexpression enhanced HCV replication in Con1b cells, while miR-215 knockdown suppressed HCV replication in Huh7.5.1 cells. TRIM22 was confirmed to be a direct target of miR-215. TRIM22 upregulation resulted in a decline in HCV replication, while TRIM22 inhibition led to enhancement of HCV replication. Additionally, exogenous expression of TRIM22 reversed the facilitating effect of miR-215 on HCV replication, while TRIM22 downregulation counteracted the inhibitory effect of miR-215 knockdown on HCV replication. Furthermore, miR-215 targeted TRIM22 to block the NF-κB pathway, and exerted a positively regulatory role on HCV replication.
CONCLUSION
miR-215 facilitated HCV replication via inactivation of the NF-κB pathway by inhibiting TRIM22, providing a novel potential target for HCV infection.

Keyword

miR-215; tripartite motif 22; hepatitis C virus; NF-κB

MeSH Terms

Blotting, Western
Down-Regulation
Genome
Genotype
Hepacivirus
Liver Diseases
Luciferases
MicroRNAs
Real-Time Polymerase Chain Reaction
Replicon
RNA
Up-Regulation
Luciferases
MicroRNAs
RNA

Figure

  • Fig. 1 miR-215 promotes HCV replication in Con1b and Huh7.5.1 cells. Con1b cells transfected with miR-con or miR-215 and Huh7.5.1 cells introduced with anti-miR-con or anti-miR-215 were incubated for 48 h. (A and B) qRT-PCR analysis was performed to detect miR-215 expression. (C and D) The levels of HCV RNA were measured by qRT-PCR. (E and F) The expression levels of HCV NS5A and NS3 proteins were determined by western blot analysis.*p<0.05. HCV, hepatitis C virus; qRT-PCR, quantitative real-time PCR.

  • Fig. 2 miR-215 directly targets TRIM22. Con1b cells were transfected with miR-con or miR-215, and Huh7.5.1 cells were introduced with anti-miR-con or anti-miR-215. (A) WT and MUT binding sites of miR-215 in TRIM22 3'UTR are shown. (B and C) The relative luciferase activities were measured in Con1b and Huh7.5.1 cells transfected with WT-TRIM22-3'UTR or MUT-TRIM22-3'UTR. The mRNA (D and E) and protein (F and G) levels of TRIM22 were evaluated by qRT-PCR and western blot in Con1b and Huh7.5.1 cells. *p<0.05. WT, wild-type; MUT, mutant; 3'UTR, 3'-untranslated region; qRT-PCR, quantitative real-time PCR.

  • Fig. 3 TRIM22 inhibits HCV replication. Con1b cells were transfected with Vector or TRIM22, followed by detection of TRIM22 mRNA (A) and protein (B), HCV RNA level (C), as well as expressions of NS5A and NS3 proteins (D). Huh7.5.1 cells were introduced with si-con or si-TRIM22, followed by determination of TRIM22 mRNA (E) and protein (F), HCV RNA level (G), as well as expressions of NS5A and NS3 proteins (H). *p<0.05. HCV, hepatitis C virus.

  • Fig. 4 miR-215 promotes HCV replication by targeting TRIM22. (A and B) Con1b cells were transfected with either miR-215 alone or in combination with TRIM22, after which the levels of HCV RNA and NS5A and NS3 proteins were assessed. (C and D) Huh7.5.1 cells were transfected with anti-miR-215 or cotransfected with anti-miR-215 and si-TRIM22, and then the levels of HCV RNA and NS5A and NS3 proteins were measured. *p<0.05. HCV, hepatitis C virus.

  • Fig. 5 miR-215/TRIM22 modulates HCV replication by affecting NF-κB signaling. (A) The protein levels of p65 and phosphorylated p65 were determined by western blot in Con1b cells transfected with either miR-215 alone or combined with TRIM22. The activity of p65 was determined by calculating p-p65/p65 ratio. (B and C) The levels of HCV RNA and NS5A and NS3 proteins were determined in Con1b cells treated with NC or TNF-α. (D) The protein levels of p65 subunit of NF-κB (p65) and phosphorylated p65 (p-p65) were measured by western blot in Huh7.5.1 cells transfected with anti-miR-215 or cotransfected with anti-miR-215 and si-TRIM22. The activity of p65 was determined by calculating p-p65/p65 ratio. (E and F) The levels of HCV RNA and NS5A and NS3 proteins were analyzed in Huh7.5.1 cells treated with NC or PDTC. *p<0.05. HCV, hepatitis C virus; PDTC, pyrrolidinedithiocarbamate.


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