Retracted Article: MicroRNA-1271 modulates hepatitis B virus replication, cell proliferation and apoptosis in hepatitis B virus-related hepatocellular carcinoma by targeting SIRT1

Abstract

Chronic hepatitis B virus (HBV) infection is a leading cause of hepatocellular carcinoma (HCC). Certain studies have revealed that microRNAs play crucial roles in HBV-related HCC. The aim of this study was to investigate the effects of microRNA-1271 (miR-1271) on HBV replication, cell proliferation and apoptosis in HBV-related HCC. The expression of HBV DNA and miR-1271 was detected by quantitative real time-polymerase chain reaction (qRT-PCR). The mRNA and protein levels of SIRT1 were detected by qRT-PCR and western blot analysis, respectively. HBV replication was assessed by the expression of HBV DNA and the levels of HBsAg and HBeAg. Cell proliferation was assessed by cell counting kit-8 (CCK-8) and 5-bromo-2-deoxyuidine (BrdU) assay, and apoptosis was evaluated by flow cytometry assay, enzyme-linked immunosorbent assay (ELISA) and the activity of caspase-3. The relationship between miR-1271 and SIRT1 was predicated by online software and confirmed by dual-luciferase reporter assay, RNA immunoprecipitation (RIP) and pull-down assay. We first found that the expression of miR-1271 was downregulated and SIRT1 was upregulated in both HBV-related HCC tissues and cells. Overexpression of miR-1271 inhibited HBV replication and cell proliferation whilst promoting apoptosis in HBV-related HCC cells. Subsequently, SIRT1 was identified as a target of miR-1271. Moreover, overexpression of SIRT1 reversed the effects of miR-1271 overexpression on HBV replication, cell proliferation and apoptosis in HBV-related HCC cells. In conclusion, our study demonstrated that miR-1271 inhibited HBV replication and proliferation and promoted apoptosis of HBV-related HCC cells via targeting SIRT1, which might contribute to the diagnosis and therapy of HBV-related HCC.

Fig. 1. Expression of miR-1271 and SIRT1 in HCC tissues and cells. (A) The expression of miR-1271 in HBV-HCC tissues and normal tissues was detected by qRT-PCR. (B) The expression of miR-1271 in Huh7, Huh7-1.3, HepaRG and HepAD38 cells was detected by qRT-PCR. (C) The mRNA level of SIRT1 in HBV-HCC tissues and normal tissues was detected by qRT-PCR. (D and E) The mRNA and protein levels of SIRT1 in Huh7, Huh7-1.3, HepaRG and HepAD38 cells were detected by qRT-PCR and western blot. *P < 0.05.

Fig. 2. MiR-1271 inhibited HBV replication in HBV-related HCC cells. (A) The level of HBV DNA in Huh7, Huh7-1.3, HepaRG and HepAD38 cells was detected by qRT-PCR. (B and C) HBsAg and HBeAg levels were detected from Huh7, Huh7-1.3, HepaRG and HepAD38 cells culture. (D) The levels of miR-1271 in Huh7-1.3 and HepAD38 cells transfected with miR-1271 mimic was detected by qRT-PCR. (E) The level of HBV DNA in Huh7-1.3 and HepAD38 cells transfected with miR-1271 mimic was detected by qRT-PCR. (F and G) HBsAg and HBeAg levels were detected after miR-1271 was overexpressed in Huh7-1.3 and HepAD38 cells. *P < 0.05.

Fig. 3. MiR-1271 suppressed proliferation and promoted apoptosis in HBV-HCC cells. Huh7-1.3 and HepAD38 cells were transfected with miR-1271 mimic or miR-NC for 24 h. (A) Cell viability was assessed by CCK-8 assay. (B) Cell proliferation was measured by BrdU-ELISA assay. (C and D) Apoptosis was assessed by flow cytometry and cell death ELISA detection kit. (E) The activity of caspase-3 was detected to assess apoptosis. *P < 0.05.

Fig. 4. SIRT1 was a direct target of miR-1271. (A) The potential binding site between miR-1271 and SIRT1 was predicted by online software. (B and C) The relative luciferase activities of SIRT1 3′UTR WT and SIRT1 3′UTR MUT in Huh7-1.3 and HepAD38 cells were detected. (D) Enrichment of SIRT1 was detected by RIP assay after transfected with miR-1271 in Huh7-1.3 and HepAD38 cells. (E and F) Pull down assay was performed to confirm the relationship between miR-1271 and SIRT1 in Huh7-1.3 and HepAD38 cells. (G) The protein level of SIRT1 in Huh7-1.3 and HepAD38 cells transfected with miR-1271 was detected by western blot. *P < 0.05.

Fig. 5. Overexpression of SIRT1 reversed effects of miR-1271 on HBV replication in HBV related HCC cells. (A) The protein level of SIRT1 was detected by western blot in Huh7-1.3 and HepAD38 cells transfected with SIRT1. (B) The level of HBV DNA was detected by western blot in Huh7-1.3 and HepAD38 cells transfected with SIRT1. (C and D) HBsAg and HBeAg levels were detected after SIRT1 was overexpressed in Huh7-1.3 and HepAD38 cells. (E) The protein level of SIRT1 was detected by western blot in Huh7-1.3 and HepAD38 cells transfected with miR-1271 or miR-1271 + SIRT1. (F) The level of HBV DNA was detected by western blot in Huh7-1.3 and HepAD38 cells transfected with miR-1271 or miR-1271 + SIRT1. (G and H) HBsAg and HBeAg levels were detected after Huh7-1.3 and HepAD38 cells were transfected with miR-1271 or miR-1271 + SIRT1. *P < 0.05.

Fig. 6. Overexpression of SIRT1 reversed effects of miR-1271 on proliferation and apoptosis of HBV related HCC cells. (A) Cell viability of Huh7-1.3 and HepAD38 cells transfected with SIRT1 was assessed by CCK-8 assay. (B) The proliferation of Huh7-1.3 and HepAD38 cells transfected with SIRT1 was measured by BrdU-ELISA assay. (C and D) Apoptosis of Huh7-1.3 and HepAD38 cells transfected with SIRT1 was assessed by flow cytometry and cell death ELISA detection kit. (E) The activity of caspase-3 was detected in Huh7-1.3 and HepAD38 cells transfected with SIRT1. (F) Cell viability was assessed by CCK-8 assay in Huh7-1.3 and HepAD38 cells transfected with miR-1271 or miR-1271 + SIRT1. (G) Cell proliferation was measured by BrdU-ELISA assay in Huh7-1.3 and HepAD38 cells transfected with miR-1271 or miR-1271 + SIRT1. (H and I) Apoptosis was assessed by flow cytometry and cell death ELISA detection kit in Huh7-1.3 and HepAD38 cells transfected with miR-1271 or miR-1271 + SIRT1. (J) The activity of caspase-3 was detected in Huh7-1.3 and HepAD38 cells transfected with miR-1271 or miR-1271 + SIRT1. *P < 0.05.