miRNA-210 promotes the progression of hepatitis B cirrhosis to liver cancer via targeting inhibition of EGR3

Background: This paper was aimed to research the mechanism of miRNA-210 in the progression of hepatitis B cirrhosis to liver cancer. Methods: Health examiners liver tissues (Control group), liver tissues of patients with hepatitis B cirrhosis (Cirrhosis group) and liver cancer tissues of patients induced by hepatitis B virus infection (Liver cancer group) were collected. HL-7702, HepG2 and HepG2.2.15 cells were cultured. HepG2 and HepG2.2.15 cells were transfected by miRNA-210 inhibitor (miRNA-210 Inhibitor group) and its negative control (miRNA-210-NC group). Normal HepG2 and HepG2.2.15 cells were named Blank group. Cells proliferation and apoptosis was analyzed. qRT-PCR technology, Western blot analysis and dual luciferase reporter gene assay were performed. Results: Tissues of Liver cancer group had higher miRNA-210 and lower EGR3 expression than Cirrhosis group (P < 0.05). Increased miRNA-210 and decreased EGR3 expression in HepG2.2.15 cells was presented when compared with those in HepG2 cells (P < 0.05). Compared to Blank group and miRNA-210-NC group, HepG2 and HepG2.2.15 cells of miRNA-210 Inhibitor group had lower A590 value, higher apoptosis rate and EGR3 expression (P < 0.05). EGR3 was directly inhibited by miRNA-210. Conclusions: miRNA-210 might promote the progression of hepatitis B cirrhosis to liver cancer via targeting inhibition of EGR3. HepG2 and HepG2.2.15 cells of Blank group, miRNA–210 Inhibitor group and miRNA–210-NC group were cultured for 72 h and then prepared into cell suspensions with PBS. All cell suspensions were transferred to centrifuge tubes respectively, and subjected to centrifugation for 5 min at 1500 r/min, 4°C. Cells at the bottom of tubes were resuspended by 1 × Binding Buffer to cell suspensions of (0.5–1) × 10 6 cells/mL. These cell suspensions with a volume of 100 μL were transferred to a 5 mL flow tube, and incubated with FITC-labeled annexin V (5 µL) and PI (5 µL) at room temperature in the dark for 15 min. A total of 400 µL 1×Binding Buffer was added into cells and apoptosis was detected using flow cytometry. EGR3 was predicted to be a target gene for miRNA–210 via TargetScan. Thus, Dual luciferase reporter gene assay was performed to further verify the regulatory relationship between EGR3 and miRNA–210. Briefly, HepG2 cells seeded in 24-well plates were transfected at 80–90% confluence. EGR3 wild-type plasmid vectors combined with miRNA– 210 mimics or miRNA–210 negative control was used to co-transfect HepG2 cells. HepG2 cells were also co-transfected by EGR3 mutant plasmid vectors and miRNA–210 mimics or miRNA–210 negative control. In this study, all plasmid vectors were provided from Shanghai GenePharma Co., Ltd. (Shanghai, China). Transfected HepG2 cells were named as mimic + wild group, mimic + mutation group, NC + wild group and NC + mutation group depending on the difference of transfection vectors. HepG2 cells of each group were subjected to dual luciferase activity assay after 48 h culturing. The relative activity of luciferase was presented as the ratio of Fireny Luciferase activity to Renilla Luciferase activity.

HBV carriers in the world 2 . HBV infection would result in damage to liver tissues and eventually lead to chronic hepatitis B as well as cirrhosis and even liver cancer 3 . It was reported that about 1 million patients were died because of liver failure, liver cirrhosis or liver cancer induced by HBV infection every year 4 . In most instances, cirrhosis caused by HBV infection would further develop into liver cancer, which was a manifestation of serious poor prognosis in patients with HBV infection 5 .
MicroRNAs (miRNAs), known as a class of noncoding small single-stranded RNAs consisting of 18-25 nucleotides, were proved to participate in majority of cellular biological functions, such as differentiation, apoptosis, proliferation, and even tumorigenesis, by targeting the regulation of other specific coding genes expression 6-8 . A recent study researched that miR-122-5p, miR-199a-5p, miR-486-5p, miR-193b-5p, miR-206, miR-192-5p, miR-141-3p and miR-26a-5p were differential expressed in cirrhosis as well as liver cancer, which were involved in the conversion of cirrhosis to hepatitis B virusassociated liver cancer 9 . Riazalhosseini et al. 10 demonstrated that miRNA-196 and miRNA-146 were involved in the development of liver cirrhosis infected by HBV to liver cancer. Xie et al. 11 proved that serum miR-101 level could be used as a candidate biomarker to distinguish between HBV-infected liver cancer and HBV-infected cirrhosis. miRNA-210 had been shown to be participated in tumorigenesis and progression of several malignant tumors, including liver cancer 12,13 . However, there was no research to prove whether miRNA-210 was participated in the progression of cirrhosis to liver cancer. Therefore, in this paper, we investigated the expression of miRNA-210 in cirrhosis and liver cancer infected with HBV by in vitro studies in order to explore whether miRNA-210 affected the progression of liver cirrhosis to liver cancer. This study would provide a novel candidate therapeutic target for blocking the development of liver cirrhosis to liver incubator at 37° C, 95% humidity.
HepG2 and HepG2.2.15 cells were digested with 0.25% trypsin and were dispersed in RPMI1640 medium (without FBS) with a density of 1.3 * 10 5 cells/mL. Six-well plates containing 1 mL of cell suspensions per well was placed in the 5% CO 2 incubator at 37° C, 95% humidity. One day later, residual liquid in each well was discarded, and HepG2 and HepG2.2.15 cells were divided into 3 groups: Blank group (cells were recultured by 1 mL serum free RPMI1640 medium), miRNA-210 Inhibitor group (cells were recultured by 1 mL serum free RPMI1640 medium containing 100 nmol/L miRNA-210 inhibitor and 2 μL DharmaFECT 4) and miRNA-210-NC group (cells were recultured by 1 mL serum free RPMI1640 medium containing 100 nmol/L miRNA-210 inhibitor negative control and 2 μL DharmaFECT 4). All plates were returned to the 5% CO 2 incubator for 8 h incubation at 37°C, 95% humidity. Then residual liquid in each well was replaced by 1 mL RPMI1640 medium (10% FBS) and cells were cultured for 2 days at the same conditions. In this study, miRNA-210 inhibitor and miRNA-210 inhibitor negative control were both provided Co., Ltd., China) for 1 h at room temperature. The membrane was washed with TBS buffer 3 times again. ECL kit was used to visualize blots and image was processed by gel imaging analysis system. GAPDH was considered as the internal reference and EGR3 protein relative expression was expressed as the integral optical density ratio of target strip to internal reference strip.
Dual luciferase reporter gene assay EGR3 was predicted to be a target gene for miRNA-210 via TargetScan. Thus, Dual luciferase reporter gene assay was performed to further verify the regulatory relationship between EGR3 and miRNA-210. Briefly, HepG2 cells seeded in 24-well plates were transfected at 80-90% confluence. EGR3 wild-type plasmid vectors combined with miRNA-210 mimics or miRNA-210 negative control was used to co-transfect HepG2 cells. HepG2 cells were also co-transfected by EGR3 mutant plasmid vectors and miRNA-210 mimics or miRNA-210 negative control. In this study, all plasmid vectors were provided from Shanghai GenePharma Co., Ltd. (Shanghai, China). Transfected HepG2 cells were named as mimic + wild group, mimic + mutation group, NC + wild group and NC + mutation group depending on the difference of transfection vectors. HepG2 cells of each group were subjected to dual luciferase activity assay after 48 h culturing. The relative activity of luciferase was presented as the ratio of Fireny Luciferase activity to Renilla Luciferase activity.

Statistical analysis
Data were exhibited in the form of mean ± standard deviation (SD). Statistical analysis was conducted by applying two-tailed Student t-test or one-way ANOVA with P < 0.05 as the threshold.

miRNA-210 was overexpressed in chronic hepatitis B cirrhosis and hepatitis B virus infected liver cancer
miRNA-210 relative expression was determined by qRT-PCR (shown in Figure 1). When compared with relative miRNA-210 expression in liver tissues of Control group, it was significantly overexpressed in Cirrhosis group and Liver cancer group (P < 0.05). Tissues of Liver cancer group had much higher miRNA-210 relative expression when compared with Cirrhosis group (P < 0.05) ( Figure 1A). In addition, HepG2 and HepG2.2.15 cells showed much higher relative miRNA-210 expression than that of HL-7702 cells (P < 0.05).

EGR3 was decreased in chronic hepatitis B cirrhosis and hepatitis B virus infected liver cancer
As shown in Figure 5A, B and C, relative EGR3 mRNA and protein expression in liver tissues of Cirrhosis group and Liver cancer group was both dramatically declined when compared with Control group (P < 0.05). Meanwhile, liver tissues of Liver cancer group showed much lower relative EGR3 mRNA and protein expression than those of Cirrhosis group (P < 0.05). Furthermore, much lower relative EGR3 mRNA and protein expression was observed in HepG2 and HepG2.2.15 cells when compared with that in HL-7702 cells (P < 0.05), and significantly lower relative EGR3 mRNA and protein expression was discovered in HepG2.2.15 cells when compared with HepG2 cells (P < 0.05) ( Figure 5D, E and F). All of the above results revealed that EGR3 was decreased in chronic hepatitis B cirrhosis and hepatitis B virus infected liver cancer.

EGR3 was directly inhibited by miRNA-210
Target Scan prediction indicated that EGR3 bound to miRNA-210 in the 3'-UTR region ( Figure 6A). EGR3 mRNA and protein was lowly expressed in HepG2 and HepG2.2.15 cells of Blank group. However, after miRNA-210 expression was inhibited, HepG2 and HepG2.2.15 cells of miRNA-210 Inhibitor group presented significantly increased EGR3 mRNA and protein expression, which was dramatically higher than that Blank group and miRNA-210-NC group (P < 0.05) ( Figure 6B, C and D). According to dual luciferase reporter gene activity assay, there was no obvious difference in relative fluorescence unit among mimic + mutation group, NC + wild group and NC + mutation group. However, the relative fluorescence unit of mimic + wild group was much lower than that of the other three groups (P < 0.05) ( Figure 6E). All of these results illustrated that miRNA-210 was able to directly suppress EGR3 expression by binding EGR3 at 3'-UTR region.

Discussion
Liver cancer ranked the third leading cause of cancer-related death worldwide, with cirrhosis being the main cause. The evolutionary feature of liver cancer was from intermediate stage progressed to advanced stage and even death in most cases 14 .
Preventing the development of liver cirrhosis to liver cancer was an urgent problem to be solved clinically, which could extremely improve the prognosis of patients. In this article, we discovered that miRNA-210 was extremely highly expressed in HBV-infected liver cancer than that in liver cirrhosis caused by HBV infection, and its down-regulation inhibited proliferation and promoted apoptosis of HepG2 and HepG2.2.15 cells via interfering with EGR3 expression targetedly.
Accumulating data had proved that miRNA-210 was participated in the regulation of multiple tumors. In pancreatic cancer, Amponsah et al. 15  EGR3, one of the important members of EGR family, was a well-known suppressor of tumor initiation and progression in certain cancer events. In head and neck cancer, EGR3 level was significantly reduced when compared with that in adjacent normal tissues, and meanwhile, overexpression of EGR3 in head and neck cancer cells significantly impaired these cells colony forming ability in vitro 20 . Pio et al. 21 declared that EGR3 expression level was closely associated with the recurrence of prostate cancer. Patients with nonrecuttent prostate cancer had relatively higher EGR3 expression, whereas lower EGR3 expression was found in patients with recurrent prostate cancer. Disorder expression of EGR3 expression was also involved in the progression of liver cancer. It was found to be exerted an inhibitor influence in the growth of liver cancer by elevating Fas ligand, and lower EGR3 expression was associated with enhanced proliferation ability and reduced apoptotic ability of liver cancer cells 22 . In fact, little work had been done in researching the link between EGR3 and liver cancer. In this study, we observed that EGR3 was lowly expressed in liver cirrhosis and liver cancer induced by HBV infection. Its expression was also declined in HepG2 and HepG2.2.15 cells, with HepG2.2.15 cells exhibiting much higher EGR3 expression. Our further data also suggested for the first time that EGR3 expression was directly inhibited by miRNA-210. Therefore, we speculated that miRNA-210 might promote the deterioration of liver cirrhosis to liver cancer by inhibiting the expression EGR3 protein. This mechanism proposed from this article might propose a reasonable theoretical basis for the targeted therapy of liver cancer clinically.

Conclusions
In conclusion, we investigated the mechanism of miRNA-210 in influencing liver cancer induced by HBV-infected liver cirrhosis. It could be discovered that miRNA-210 might promote the progression of HBV-infected liver cirrhosis to liver cancer by targeting