MicroRNA‑1271‑5p alleviates the malignant development of hepatitis B virus‑mediated liver cancer via binding to AQP5

Abstract

Hepatitis B virus (HBV) is a leading cause of liver‑related cancer. Progress has been made on the study of microRNA (miRNA or miR) function in HBV‑related liver cancer. Hence, the objective of the present study was to determine the role and functional mechanism of miR‑1271‑5p in HBV‑associated liver cancer. miR‑1271‑5p and aquaporin 5 (AQP5) expression at the mRNA level were measured by reverse transcription‑quantitative PCR (RT‑qPCR). The levels of hepatitis B e‑antigen (HBeAg), hepatitis B surface antigen (HBsAg) and HBV DNA were assessed by ELISA or qPCR. Cell viability, apoptosis, migration and invasion were detected by Cell Counting Kit‑8, flow cytometry or Transwell assay. The interaction of miR‑1271‑5p and AQP5 was predicted by TargetScan, and verified by dual‑luciferase reporter assay and RNA binding protein immunoprecipitation assay. The protein levels of AQP5, Bax, Bcl‑2, cleaved‑caspase-3 and proliferating cell nuclear antigen were quantified by western blot analysis. Nude mouse tumorigenicity assay was conducted to examine the role of miR‑1271‑5p in vivo. miR‑1271‑5p was downregulated, while AQP5 was upregulated in HBV‑related liver cancer cells and tissues. Overexpression of miR‑1271‑5p or AQP5 knockdown inhibited the levels of HBeAg, HBsAg and HBV DNA, blocked cell viability, migration and invasion, and induced apoptosis. AQP5 was confirmed to be a direct target of miR‑1271‑5p, and miR‑1271‑5p exerted its role through targeting AQP5. Overexpression of miR‑1271‑5p impeded tumor growth in vivo by weakening the expression of AQP5. In conclusion, miR‑1271‑5p blocked the progression of HBV‑induced liver cancer by competitively targeting AQP5.

Figure 1.

Expression of miR-1271-5p and AQP5. (A) The expression of miR-1271-5p in HBV-related HCC tissues and adjacent normal tissues was measured by RT-qPCR. (B) Expression of AQP5 at the mRNA level was also measured by RT-qPCR. (C) A negative correlation existed between the expression of miR-1271-5p and AQP5. *P<0.05. miR, microRNA; AQP5, aquaporin 5; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; RT-qPCR, reverse transcription-quantitative PCR.

Figure 2.

Effects of miR-1271-5p overexpression on the levels of HBeAg, HBsAg and HBV DNA. (A) The expression of miR-1271-5p in HepG2.2.15 and Huh7-1.3 transfected with miR-1271-5p mimic and miR-NC was measured by RT-qPCR. The levels of (B) HBeAg, (C) HBsAg and (D) HBV DNA were distinguished by ELISA or quantitative PCR. *P<0.05. miR, microRNA; NC, negative control; HBV, hepatitis B virus; RT-qPCR, reverse transcription-quantitative PCR; HBeAg, hepatitis B e-antigen; HBsAg, hepatitis B surface antigen.

Figure 3.

Effects of miR-1271-5p overexpression on cell viability, apoptosis, migration and invasion. (A) Cell viability was detected by Cell Counting Kit-8 assay. (B) Cell apoptosis was assessed by flow cytometry. Apoptosis-related proteins, including Bax, Bcl-2 and cleaved-caspase-3, were quantified by western blotting in (C) HepG2.2.15 and (D) Huh7-1.3 cells. Transwell assay was performed to monitor cell (E) migration and (F) invasion (magnification, ×100). *P<0.05. miR, microRNA; NC, negative control; C-caspase-3, cleaved-caspase-3.

Figure 4.

Effects of AQP5 knockdown on the levels of HBeAg, HBsAg and HBV DNA, cell viability, apoptosis, migration and invasion. (A) The expression of AQP5 in HepG2.2.15 and Huh7-1.3 cells transfected with si-AQP5 or scramble was measured by western blotting. The levels of (B) HBeAg, (C) HBsAg and (D) HBV DNA were assessed by ELISA or quantitative PCR. (E) Cell viability was detected by Cell Counting Kit-8 assay. (F) Cell apoptosis was assessed by flow cytometry. Bax, Bcl-2 and cleaved-caspase-3 were quantified by western blotting in (G) HepG2.2.15 and (H) Huh7-1.3 cells. Transwell assay verified cell (I) migration and (J) invasion. *P<0.05. AQP5, aquaporin 5; HBV, hepatitis B virus; si, small interference.

Figure 5.

AQP5 is a target of miR-1271-5p. (A) The bioinformatics tool TargetScan was used to analyze the binding sites between miR-1271-5p and AQP5 3′ UTR. (B) Dual-luciferase reporter assay was used to verify the association between them. (C) Efficiency of miR-1271-5p inhibitor was assessed via RT-qPCR. (D) Dual-luciferase reporter assay was performed in cells transfected with AQP5-wt or AQP5-mut and anti-miR-1271-5p or anti-miR-NC. (E) RIP assay was performed to further confirm the association. (F) Influence of miR-1271-5p overexpression or knockdown on the expression of AQP5 at the protein level. *P<0.05. AQP5, aquaporin 5; miR, microRNA; UTR, untranslated region; wt, wild-type; mut, mutant; NC, negative control; RIP, RNA binding protein immunoprecipitation.

Figure 6.

miR-1271-5p regulates the levels of HBeAg, HBsAg and HBV DNA, as well as cell viability, apoptosis, migration and invasion, by binding to AQP5. HepG2.2.15 and Huh7-1.3 cells were transfected with miR-1271-5p, miR-NC, miR-1271-5p + AQP5 or miR-1271-5p + vector. (A) The protein level of AQP5 was detected by western blotting. (B) HBeAg, (C) HBsAg and (D) HBV DNA levels were detected by ELISA or quantitative PCR. (E) Cell viability was assessed by Cell Counting Kit-8 assay. (F) Apoptosis was examined by flow cytometry. Bax, Bcl-2 and cleaved-caspase-3 were quantified by western blotting in (G) HepG2.2.15 and (H) Huh7-1.3 cells. (I) Migration and (J) invasion were determined by Transwell assay. *P<0.05. miR, microRNA; HBV, hepatitis B virus; AQP5, aquaporin 5; NC, negative control; C-caspase-3, cleaved-caspase-3.

Figure 7.

Effect of miR-1271-5p overexpression on tumor growth in vivo. (A) Tumor volume was recorded once a week. (B) Tumor weight after 5 weeks. (C) Expression of miR-1271-5p in tumor tissues was measured by reverse transcription-quantitative PCR. (D) Expression of AQP5 at the protein level in tumor tissues was determined by western blotting. (E) Cleaved-caspase-3 and PCNA were quantified by western blotting. (F) Levels of HBeAg and HBsAg were assessed by ELISA. (G) The level of HBV DNA was examined using quantitative PCR. *P<0.05. miR, microRNA; AQP5, aquaporin 5; PCNA, proliferating cell nuclear antigen; HBV, hepatitis B virus; NC, negative control; C-caspase-3, cleaved-caspase-3.