miR-425-5p Acts as a Molecular Marker and Promoted Proliferation, Migration by Targeting RNF11 in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most common and dangerous malignant tumors in China, which causes a large number of deaths every year. MicroRNAs (miRNAs) dysfunction contributes to the malignant progression of tumors. The aim of our study was to investigate the relationship between the biological role of miR-425-5p and malignant progression of HCC. Our results showed that miR-425-5p expression was significantly upregulated in HCC tissues and closely related to the poor prognosis of HCC patients. The knockdown of miR-425-5p inhibited cell proliferation and migration. Further, we identified RNF11 as the downstream target gene of miR-425-5p. In addition, the rescue experiments showed that the upregulation of RNF11 could rescue the inhibitory effect of miR-425-5p on HCC. In general, miR-425-5p as an oncogene promotes the malignant development of HCC via RNF11 and serves as a molecular target for predicting the prognosis of HCC patients.


Introduction
Hepatocellular carcinoma (HCC) is the third most common malignant tumor with mortality after gastric cancer and esophageal cancer [1,2]. Like other cancers, the onset of HCC is a complex process with multiple factors and multiple steps, which is affected by both environmental and genetic factors. Epidemiological and experimental research data have shown that HBV and HCV infection, aflatoxin B1, alcohol, liver cirrhosis, and sex hormones are related to the onset of HCC, and HBV and HCV infection are the most important factors [3,4]. The existing research results indicate that the X protein of HBV plays an important role in the occurrence and development of liver cancer. X protein is a multifunctional protein encoded by the HBV genome. Its functions include tyrosine phosphokinase activity, p53 protein binding activity, transcription activation, Src kinase activation, and DNA excision repair system inhibition [5]. In addition, the activation or amplification of Ras and Myc genes plays an important role in the proliferation of HCC cells [6]. Ras-GTPase and its downstream mitogen-activated protein kinase effector pathways positively regulate the cell cycle in a variety of cells [6,7]. Wnt signaling pathway and TGFβ/Smads signaling pathway are signal transduction chains with extensive functions. Mutations in this pathway exist in HCC cells, which promote the malignant growth of liver cancer [8]. However, the biological characteristics of HCC are that the disease is concealed and the prognosis is poor, which brings a heavy burden to the individual and society [9,10]. Therefore, it is very important to clarify the molecular mechanism of the occurrence and development of HCC and find effective target genes for molecular therapy.
The role of microRNAs (miRNAs) in tumorigenesis and development has been recognized by scientists. A large number of studies have found the expression and dysfunction of miRNAs in multiple malignant tumors including gastric cancer, colon cancer, and bladder cancer [11][12][13]. In oral carcinoma, miR-31 causes the stability of mitochondria and membrane potential through SIRT3 and promotes the occurrence of oxidative stress, resulting in an increase in glycolysis in tumor cells, ultimately promoting the malignant development of tumors [14]. A study also found that miR-29c-3p promotes the malignant development of HCC by regulating the methylation of LATS1 caused by DNMT3B and inhibiting the anticancer function of the Hippo signaling pathway [15]. In addition, the miRNA-140-5p regulation of TGFBRI and IGF1R expressions results in the abnormal expression of downstream target gene SMAD2/3 and p-AKT and causes the malignant development of Wilms' tumor [16]. Recently, a study has found that the high expression of miR-425-5p can promote the malignant development of gastric cancer by regulating the expression of CYLD [17]. However, the biological role of miR-425-5p in HCC is still unclear, and its role in HCC deserves our in-depth study.
RNF11 gene is a key member of the E3 ubiquitin ligase family [18]. Protein is precisely balanced by de novo synthesis, posttranslational modification, and degradation. 80% of the proteins in cells are destroyed by ubiquitin of the ubiquitin enzyme system [19]. RNF11 has a dominant position in the ubiquitination signal pathway. The function of RNF11 is very powerful, covers a wide range, and is very important to the body [20]. In the promoter region of mouse RNF11, three conserved ETS1 transcription factor binding sites were found. Among them, ETS1 is produced by many cells and is necessary for embryonic development, blood vessel formation, hormone production, lymphocyte production, and acti-vation of immune cells, and ETS1 is activated by the action site of RNF11 bound to the ETS1 site and regulate transcription, so that RNF11 will work together with ETS1 at the transcription level of bone cells to play a regulatory role [21,22]. RNF11 can increase the TGF-β signal starting from the receptor level. RNF11 can directly connect with SMAD4, and SMAD4 is a common Smad that interacts with TGF-β. RNF11 can enhance the transcriptional activity of SMAD4 and SMAD2-SMAD4 transcriptional complexes, and then play an important role in regulating the pathway [23]. However, there are few studies on RNF11 in HCC, and its specific role is still unknown.
In this study, we detected the expression of miR-425-5p in HCC samples and analyzed the relationship between miR-425-5p and poor prognosis of patients in HCC. In addition, the biological effects of miR-425-5p on HCC cells were confirmed by in vivo and in vitro. Important, we further analyzed the downstream target genes of miR-425-5p and its role in the malignant progression of HCC. Therefore, we aim at demonstrating whether miR-425-5p contributes to the diagnosis and treatment of HCC.

Western Blot.
Total protein was isolated from HCC tissue and cell lines by protein extraction buffer (RIPA lysis buffer) and use BCA protein assay kit (Beyotime, Haimen, China) to determine the extracted protein concentration. The total protein was resolved by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Then, block the membranes for 1 hour at 37°C with 5% nonfat powdered milk. The membranes were probed at 4°C overnight with anti-RNF11 (1 : 1000, ab154831, Abcam) and anti-GAPDH (1 : 5000, ab181602, Abcam). Next, the membranes were incubated with the appropriate secondary antibodies. Finally, the chemiluminescence detection system was used to analyze protein expression.

Cell-Counting
Kit-8 Assay. The CCK8 assay was used to determine the cell viability in the HCC cells. Transfected cells are seeded on a 96-well plate; cells were processed for the CCK8 assay according to the manufacturer's instructions.
2.6. Wound Healing Assay. Cells were inoculated on a 6-well plate. The cell monolayer was scratched with a pipette tip (1 ml) to generate 3 scratch wounds after the cells were full, and suspended cells were washed off with PBS. The distance between the wound sides was measured with a microscope. 2.7. 5-Ethynyl-2 ′ -Deoxyuridine (EdU) Assay. The transfected cells were inoculated into a 96-well plate. Cells were immobilized with 4% polyformaldehyde and nucleus perforation with 0.5% Triton X-100 solution. According to the manufacturer's instructions, cells were incubated with EdU (50 μM), 1 × ApolloR reaction cocktail (100 μl), and 1 × Hoechst 33342 (100 μl) for 30 min. Cell proliferation was analyzed using the mean number cells.
2.10. Statistical Analysis. All data were expressed as mean ± SD and analyzed using SPSS 22.0 software (SPSS Inc., Chicago, IL, USA) or GraphPad Prism version 7.0 (CA, USA). The differences were analyzed by one-way analysis of variance, followed by the Newman-Keuls test, were used to evaluate the differences between groups, and repeated measures analysis of variance. The relationship between miR-425-5p and RNF11 expressions was evaluated using Spearman's correlation analysis. The Kaplan-Meier and log-rank tests were used to assess RFS and DSS. The clinical prognostic significance of miR-425-5p was calculated by univariate and multivariate Cox regression analysis.

Results
3.1. miR-425-5p Was Upregulated in HCC Tissues. To explore the role of miR-425-5p, RT-qPCR was performed to evaluate the expression of miR-425-5p in HCC. The results showed that miR-425-5p was significantly upregulated in HCC tissues compared with paired adjacent tissue (Figure 1(a)). Moreover, the expression of miR-425-5p in metastatic HCC is higher than that in nonmetastatic HCC (Figure 1(b)). To further explore the role of miR-425-5p in HCC, HepG2 and Huh-7 cells were transfected with pre-miR-425-5p to increase the expression of miR-425-5p, and miR-425-5p inhibitor to knockdown the expression of miR-425-5p (Figures 1(c) and 1(d)).

Reducing the Expression of miR-425-5p Inhibits Cell
Proliferation and Migration in HCC. We used cell biology experiments to examine the role of miR-425-5p in HCC. CCK-8 assays showed that the decreased expression of miR-425-5p significantly inhibits the proliferation in HepG2 and Huh-7 cells (Figures 3(a) and 3(b)). Wound healing assays revealed that the decreased expression of miR-425-5p suppressed the migration of HepG2 and Huh-7 cells (Figures 3(c) and 3(d)). Moreover, HepG2 and Huh-7 cells incorporating EdU in the miR-425-5p downexpression group was less than the control group (Figures 3(e) and 3(f)).

RNF11
Was Downregulated in HCC Tissue. Based on the above results, we further studied the expression of RNF11 in HCC. RT-qPCR results showed that RNF11 was downregu-lated in HCC tissues than that in paired adjacent tissue ( Figure 5(a)). Importantly, miR-425-5p expression levels were inversely correlated with RNF11 ( Figure 5(b)).

Restoration of RNF11 Rescued the miR-425-5p-Mediated
Effect on Proliferation and Migration of HCC. To explore whether miR-425-5p plays a biological role via RNF11 in HCC, we restored the expression of RNF11 by using RNF11 plasmids in HepG2 and Huh-7 cells, which overexpress miR-425-5p (Figure 6(a)). CCK-8 assays showed that the increased expression of RNF11 alleviates the promotion of miR-425-5p on the proliferation in HepG2 and Huh-7 cells (Figure 6(b)). Moreover, wound healing assays revealed that the increased expression of RNF11 alleviates the promotion of miR-425-5p on the migration in HepG2 and Huh-7  (Figures 6(c) and 6(d)). After increasing the expression of RNF11, the ability of HepG2 and Huh-7 cells to absorb EDU was limited (Figures 6(e) and 6(f)).

Discussion
HCC is one of the most common malignant tumors in China [24]. The mortality rate ranks third among malignant tumors of the digestive system. About 110,000 people die of HCC every year in China, accounting for 45% of the world's HCC deaths [25]. At present, many oncogenes are known to induce or promote the metastatic potential of HCC cells, such as Ras, Myc, Raf, Fos, EGFR, and c-Met, while KAI1 and PTEN can inhibit the metastasis and recurrence of cancer [26,27]. Recent research found that microRNA (miRNA) is a kind of endogenous noncoding RNA with regulatory function found in eukaryotes. The main biological function of mature miRNA is to downregulate gene expression by inhibiting mRNA translation and promoting mRNA degradation [28]. However, the current research on microRNAs in HCC mainly focuses on the microRNAs with high expression in cancer tissues, such as miR-21-5p, miR-107, and miR-92a, or the microRNAs with low expression in advanced liver cancer, such as miR-122, miR-7, and miR-29a [29,30]. Whether the change of miR-425-5p expression is related to the malignant behavior of HCC cells and whether the downstream molecules play an important role in the occurrence and development of HCC need further study. The purpose of this study is to explore the molecular mechanism of malignant development of HCC from the perspective of miR-425-  BioMed Research International 5p and to provide a scientific basis for the treatment of patients with HCC. The role of miRNA tumors has been widely recognized. As a functional microRNA, miR-425-5p plays different roles in tumors [31][32][33]. Recently, a study reported that miR-425-5p targeted genes are involved in the EGFR tyrosine kinase inhibitor resistance pathway, and miR-425-5p might act as an oncogene to participate in the pathogenesis of KRASmutated CRC [34]. miR-425-5p as a tumor suppressor gene is low expressed in prostate cancer and can affect the sensitiv-ity of prostate cancer to cisplatin through the action of GSK3β and Wnt/β-catenin signaling pathways [35]. It has been found that long noncoding RNA LINC-PINT regulates laryngeal carcinoma cell stemness and chemoresistance by targeting miR-425-5p in laryngeal tumors [36]. Our results are also consistent with previous researches; miR-425-5p is highly expressed in HCC and is closely related to the poor prognosis of HCC patients. The inhibition of miR-425-5p expression can significantly control the malignant development of HCC via RNF11. These findings, combined with 7 BioMed Research International our existing results, suggest that miR-425-5p might play different roles in different tumor microenvironments. This is a very interesting phenomenon; we will explore the mechanism that may cause the above arrears in the next study.
RNF11 is a member of the ubiquitin ligase family and capable of modulating protein function through ubiquitination and perhaps through sumoylation, which may play an important role in tumor formation [37,38]. RNF11 is abnormally expressed in breast cancer and can promote the malig-nant development of breast cancer [39]. In addition, RNF11 is activated by ETS1 and plays an important role in the growth of bone cells [40]. Moreover, RNF11 can directly enhance TGFb signaling through a direct association with SMAD4 and promote the malignant development of tumor cells [18]. Ubiquitination is activated by ubiquitination activator E1 in the form of ATP, and the activated ubiquitin protein is transported to ubiquitination polymerase E2, and then interacts with special E3 ubiquitin linker, resulting in protein    ubiquitination. Importantly, RNF11 can interact with E2 binding enzyme and E3 ubiquitin ligase to promote protein ubiquitination [41]. In addition, RNF11 enhances the signal transduction of TGF-β at the receptor level. RNF11 directly connects with SMAD4. SMAD4 is a common SMAD that interacts with TGF-β. RNF11 can enhance the transcriptional activity of SMAD4 and SMAD2-SMAD4 transcriptional complexes, thus playing an important role in regulating the pathway [42]. The research results also showed that RNF11 could regulate the single phosphorylation of ESP-15 to degrade AMSH and regulate the ubiquitination of ESP-15, thus promoting the degradation of EGFR [20]. According to our research results and previous literature reports, we hypothesized that miR-425-5p affects the ubiquitination modification by regulating the expression of RNF11, which weakens the binding of RNF11 to E2 binding enzyme and E3 ubiquitination ligase, and then affects the malignant biological behavior of HCC by regulating TGF-β and EGFR signaling pathway. In the present work, we demonstrated that the high expression of miR-425-5p leads to the dysfunction of RNF11, and promoting the development of HCC. Therefore, through clinical samples and biological function experiments, we have determined that RNF11 as a new target for miR-425-5p to regulate tumorigenesis and development in HCC.

Conclusion
In general, miR-425-5p is highly expressed in HCC and is associated with poor prognosis. Reducing the expression of miR-425-5p can inhibit the proliferation and migration of HCC. Moreover, it is verified that RNF11 is a new downstream target of miR-425-5p. These findings will provide a new target for the diagnosis and treatment of HCC and provide a scientific basis for the further study of miR-425-5p in HCC.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.