Serum miR-375 Levels Are Closely Related to Disease Progression from HBV Infection to HBV-Related Hepatocellular Carcinoma

Abstract

Background: There is an urgent need to identify ideal serological biomarkers that not only are closely related to disease progression from hepatitis B virus (HBV) infection to hepatocellular carcinoma (HCC) but also have high specificity and sensitivity. We conducted this study to analyze whether miR-375 has a potential value in the early prediction of the progression from HBV-related hepatitis or cirrhosis to HCC.

Methods: A total of 177 participants were enrolled. Receiver operating characteristic (ROC) curve was used to evaluate the predictive capability of selected miR-375 for HBV-HCC. We upregulated the miR-375 expression in HepG2, HepG2.2.15, and HepAD38 cells to determine its effect on cellular proliferation and migration, in vitro using Cell Counting Kit-8 (CCK-8) assays.

Results: Serum miR-375 levels decreased in order from healthy controls to chronic hepatitis B (CHB) without cirrhosis, followed by cirrhosis, and finally, HBV-HCC patients. miR-375 levels were significantly lower in HBeAg-positive and HBV DNA-positive patients than negative (P < 0.05) and significantly lower in patients with elevated alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) than normal levels (P < 0.05). miR-375 might be a biomarker for HBV-HCC, with a high area under the curve (AUC) of 0.838 (95% confidence interval (CI) 0.780 to 0.897; sensitivity: 73.9%; specificity: 93.0%). The AUC (0.768 vs. 0.584) and sensitivity (93.8% vs. 75.0%) for miR-375 were higher than those for AFP. The overexpression of miR-375 noticeably inhibited proliferation and migration in HepG2, HepG2.2.15, and HepAD38, especially in HepG2.2.15 and HepAD38, which are stably infected with HBV.

Conclusions: Serum miR-375 levels are closely related to disease progression from HBV-related hepatitis or cirrhosis to HCC.

Figure 1

The differential serum levels of miR-375 in the HCC, cirrhosis, CHB, and healthy control groups. The expression of serum miR-375 decreased in order from healthy controls to patients with chronic hepatitis B infection (CHB) without cirrhosis, followed by patients with CHB with cirrhosis (cirrhosis), and finally, patients with HBV-HCC. Serum miR-375 levels were significantly higher in healthy controls than in cirrhosis patients (^∗∗P < 0.01). Serum miR-375 levels were significantly higher in healthy controls than in HBV-HCC patients (^∗∗∗P < 0.001). Serum miR-375 levels were significantly higher in patients with CHB than in patients with HBV-HCC (^∗∗P < 0.01).

Figure 2

ROC curve analysis of the diagnostic value of serum miR-375 for HCC. (a) AUC estimation for the value of serum miR-375 in identifying HCC patients within the whole cohort (AUC = 0.838, sensitivity = 73.9%, and specificity = 93.0%). (b) AUC estimation of the value of serum miR-375 in distinguishing HCC patients from HBV patients (AUC = 0.768, sensitivity = 93.8%, and specificity = 63.9%). AUC estimation of the value of AFP in distinguishing HCC patients from HBV patients (AUC = 0.584, sensitivity = 75.0%, and specificity = 65.5%).

Figure 3

The expression of miR-375 in HepG2, HepG2.2.15, and HepAD38 cell lines. The expression of miR-375 was clearly lower in HepG2.2.15 cells and HepAD38 cells than that in HepG2 cells, respectively. ^∗P < 0.05.

Figure 4

The effect of miR-375 on the proliferation of HCC cells. We performed the CCK-8 assay in HepG2 cells, HepG2.2.15 cells, and HepAD38 cells at 24 h, 48 h, and 72 h after transfecting the cells with miR-375 mimics. HepG2.2.15 and HepAD38 cells were positive for HBsAg. However, HepG2 cells were negative for HBsAg. The proliferation rates of miR-375-transfected cells were lower than those of nontransfected cells (transfected vs. nontransfected HepG2.2.15 cells, transfected vs. nontransfected HepAD38 cells, and transfected vs. nontransfected HepG2 cells). The proliferation rates of HepG2.2.15 cells and HepAD38 cells were lower than those of HepG2 cells (transfected HepG2.2.15 cells vs. transfected HepG2 cells, transfected HepAD38 cells vs. transfected HepG2 cells, and nontransfected HepG2.2.15 cells vs. nontransfected HepG2 cells).