RNF185-AS1 Promotes Hepatocellular Carcinoma Progression Through Targeting miR-221-5p/integrin β5 Axis

Background: Recently, long noncoding RNAs (lncRNAs) have been reported to play important role in pathogenesis of various cancers. However, the function of RNF185-AS1 in hepatocellular carcinoma (HCC) metastasis has not been well investigated. The present study aims to explore the role and mechanism of RNF185-AS1 in hepatocellular carcinoma metastasis. Methods: The RNF185-AS1 expression in HCC cells and tissues was measured by quantitative real ‐ time polymerase chain reaction (qRT-PCR). The functional effects of RNF185-AS1 on tumor cell proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) were assessed by Cell Counting Kit-8 (CCK8) assay, colony formation assay, transwell assay and Western blot. The luciferase reporters assay, RNA-binding protein immunoprecipitation assay, qRT-PCR and Western blot were performed to explore and conrm the interaction between RNF185-AS1 and miR-221-5p and integrin β5. The role of RNF185-AS1 in tumor progression was explored through in vivo experiments. Results: RNF185-AS1 was highly expressed in HCC tissues and cell lines. High levels of RNF185-AS1 was correlated with advanced TNM stage, distant metastasis and a poorer overall survival rate. RNF185-AS1 knockdown inhibited cell proliferation, migration, invasion and EMT. Additionally, RNF185-AS1 acted as a sponge for miR-221-5p and integrin β5 was identied as a target gene of miR-221-5p. Rescue assays showed that miR-221-5p inhibitor or integrin β5 overexpression rescued the function of RNF185-AS1 knockdown on cell proliferation, migration, invasion and EMT. Moreover, we found that RNF185-AS1 knockdown inhibited tumor metastases in xenograft tumor mouse model. Conclusion: Our ndings demonstrated that RNF185-AS1 promoted cell EMT and migration by regulating miR-221-5p/integrin


Background
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors. Although the progress in the treatment of HCC has been made, high rate of metastasis and tumor recurrence is the main reason for the poor prognosis of HCC patients [1]. Therefore, it is crucial to explore the molecular mechanisms in HCC recurrence and metastasis, so as to improve the treatment for HCC.

Subcellular fractionation assay
The cytoplasmic and nuclear extracts were extracted from HCC cells with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scienti c, Waltham, MA). The distribution of RNF185-AS1 in cytoplasm or nucleus was analyzed by qRT-PCR analysis. The expression of U6 in nucleus, GAPDH in cytoplasm was used as control.

Colony formation assay
HepG2 and Huh-7 cells (1×10 3 cells per well) were plated into 6 well plates after 48 h transfection and cultured with fresh growth medium every 3 days. Cell were cultured for 21 days and were stained with 0.5% crystal violet.

Cell migration and invasion assays
For analysis of cell migration and invasion, 2.0×10 4 cells were seeded in a transwell chamber (8-μm pore size; EMD Millipore, Billerica, MA, USA) with 200 μL serum-free DMEM. A volume of 500 μL of the medium containing 10% FBS was added to the lower chamber. The 24-well chambers were then incubated at 37°C for 24h, the cells on the lower surface of the membrane were xed in 4% paraformaldehyde for 30 minutes and stained with 0.5% crystal violet. The transmembrane cells were counted under six random microscope elds.

Western blot analysis
Total proteins were prepared using RIPA lysis buffer (Beyotime, China). Proteins were quanti ed by BCA assay. Equal amounts of protein were separated by 10% SDS-PAGE and electrotransferred onto polyvinylidene di uoride membranes (Bio-Rad, USA). After being blocked using 5% non-fat milk for 1 h at room temperature, the primary rabbit anti-human antibodies against E-cadherin (ab1416, Abcam, Cambridge, MA), Vimentin (ab92547, Abcam), integrin β5 (ab184312, Abcam) and GAPDH (ab9485, Abcam) were supplemented overnight at 4℃. Then the members were washed with tris-buffered saline Tween 20 for three times and probed with horse radish peroxidase-conjugated secondary antibodies (ab205718, Abcam) for 1 h at 37°C. The bands were visualized using a ChemicDocXRS system (Bio-Rad, USA).

Luciferase reporter assay
For luciferase reporter assay, the predicted sequences of RNF185-AS1 or integrin β5 were inserted into pGL3 vector (Promega, Madison, WI, USA). Afterward, these vectors were co-transfected with miR-221-5p mimics and its corresponding control into Huh7 and HepG2 cells. 48 hours later, luciferase reporter assay system (Promega) was performed to measure the luciferase activities.
RNA-binding protein immunoprecipitation (RIP) assay RIP was carried out by using the EZMagna RIP kit (Millipore, Bedford, MA, USA) according to the manufacturer's instructions. In brief, cells were lysed and the lysates were incubated with magnetic beads conjugated with human anti-Ago2 antibody (ab57113, Abcam) or control anti-IgG (ab131368, Abcam). For the tumor metastasis model, 1×10 6 Huh7 cells transfected with si-NC or si-RNF185-AS1 were injected into mouse tail veins (n = 6 for each group). The mice were killed at the indicated time. Lungs were surgically retrieved from mice. Micrometastatic tumors in the lungs were counted. Tissues were embedded in para n, sectioned, stained with H&E.

Immunohistochemistry (IHC)
Tissue sections were prepared and subjected to immunohistochemical analysis. Anti-human Ki67 (ab15580 Abcam), ITGB5 (ab15459, Abcam), E-cadherin and Vimentin antibody was used as primary antibodies. HRP-conjugated secondary Ab was used as secondary antibody. Images were obtained using an Olympus-BX51 microscope.

Statistical analysis
All data of multiple experiments are presented as the mean ± standard deviation. SPSS 21.0 software (SPSS, Chicago, IL) was adopted for statistical analysis. The Kaplan-Meier analysis and log-rank test were used to calculate the overall survival. The one-way ANOVA or student's t test was adopted for the differences among groups. The value of p <0 .05 was considered to be signi cant.

RNF185-AS1 is upregulated in in HCC
GEPIA database was utilized to select the upregulated lncRNA in HCC, higher expression of RNF185-AS1 was found in HCC tissues than that in normal nontumor tissues (Fig. 1a,b). The expression of RNF185-AS1 in HCC patients serum and HCC cell lines was measured by qRT-PCR. We found that RNF185-AS1 expression was increased in the serum of HCC patients compared with health controls (Fig. 1c). qRT-PCR results showed that RNF185-AS1 was up-regulated in HCC cell lines when compared with human normal liver cell line HL7702 (Fig. 1d). Kaplan-Meier analysis showed that patients with high levels of RNF185-AS1 had a lower survival rate than those with low levels (Fig. 1e). Further analysis indicated that high levels of RNF185-AS1 were positively correlated with advanced TNM stage, distant metastasis (Fig. 1f, g), suggesting that RNF185-AS1 may promote HCC progression and metastasis.

RNF185-AS1 knockdown inhibits HCC progression in vivo
Finally, we investigated the roles of RNF185-AS1 in vivo by xenograft experiments. We found that tumors from the si-RNF185-AS1 group showed smaller tumor weight than those from the control group (Fig. 6ac). IHC assay showed that Ki67 and ITGB5 expression was signi cantly decreased in the group of si-RNF185-AS1 compared with controls (Fig. 6d). The expression of E-cadherin increased while the expression of vimentin decreased obviously in the si-RNF185-AS1 group (Fig. 6d). The miR-221-5p level in xenograft tumors was signi cantly increased in the si-RNF185-AS1 group (Fig. 6e). In lung metastasis models, the mice injected with Huh7 cells which transfected with si-RNF185-AS1 exhibited less metastatic nodules in the lungs than those injected with the control cells (Fig. 6f). These results suggested that RNF185-AS1 promote HCC cell growth and metastasis in vivo.

Discussion
Mounting evidences have shown that abnormal expression of lncRNAs is involved in the progression of HCC [9][10][11]. In the present study, we identi ed the oncogenic roles of RNF185-AS1 in HCC. We found that RNF185-AS1 expression was overexpressed in HCC and positively correlated with TNM stage, metastasis and low survival rate. We demonstrated that RNF185-AS1 knockdown suppressed the HCC cell proliferation, migration, invasion, EMT in vitro. Furthermore, we found that RNF185-AS1 knockdown inhibited HCC cell growth and metastasis in vivo. We further identi ed that RNF185-AS1 acts as a ceRNA for miR-221-5p and upregulated integrin β5 expression.
IGTB5 is a member of integrin family and has been reported to regulate diverse biological functions in cancers, including proliferation, EMT, migration, invasion and angiogenesis. It has been reported that ITGB5 cooperated with endothelial growth factor receptor 2(VEGF2) to enhance cell survival [23]. Previous studies have shown that integrin β5 ITGB5 promotes cancer cell migration, invasion and contributes to the transforming growth factor β (TGF-β)-induced EMT [24][25]. Studies have revealed that ITGB5 facilitates tumor angiogenesis [26,27]. A recent study reported that miR-185-ITGB5-β-catenin pathway plays an important role in HCC tumorigenesis [28]. In our study, we found that the expression of ITGB5 was increased in HCC. We demonstrated the direct interaction between ITGB5 and miR-221-5p in HCC. We also revealed that ITGB5 expression was inhibited by miR-221-5p and upregulated by RNF185-AS1. Restoration of ITGB5 signi cantly rescued the proliferation, migration, invasion and EMT of HCC cells transfected with si-RNF185-AS1.

Conclusion
Our study provided evidence that RNF185-AS1 promotes HCC progression through miR-221-5p/ITGB5. Our results indicated a novel mechanism of RNF185-AS1 in developments of HCC, which plays a critical role in HCC progression and may be an oncogene in HCC cells.

Declarations Acknowledgements
None.

Funding
This work was supported by the following funds: National Natural Science Foundation of China (No. 81502222) Author contributions JH Zhu and JY Yang conceived and designed the experiments; CM Huang, K Li and RF Huang performed the experiments; RF Huang and CM Huang analyzed the data; CM Huang wrote the paper. All authors contributed to data analysis, drafting or revising the article, gave nal approval of the version to be published, and agree to be accountable for all aspects of the work.

Availability of data and materials
The datasets used during this research are available from the corresponding author upon reasonable request.

Ethics approval and consent to participate
The present study was approved by the Ethics Committee of Wuhan No.1 Hospital. The research has been carried out in accordance with the World Medical Association Declaration of Helsinki. All patients provided written informed consent.

Consent for publication
Not applicable. RNF185-AS1 promoted HCC cell proliferation, migration, invasion and EMT a RNF185-AS1 expression was decreased in Huh7 and HepG2 cells transfected with si-RNF185-AS1. b Cell proliferation was measured in Huh7 and HepG2 cells by CCK8 assay. c Colony formation assays were performed after RNF185-AS1 knockdown. d, e Transwell migration and invasion assay were performed after RNF185-AS1 knockdown. f Expressions of E-cadherin and Vimentin were detected by western blot in RNF185-AS1 knockdown Huh7 and HepG2 cells. * p<0.05. Figure 3 RNF185-AS1 was a sponge for miR-221-5p in HCC. a Subcellular fractionation assays was used to con rm that RNF185-AS1 located mainly in the cytoplasm. b The result of starBase predicted that miR- Figure 5 RNF185-AS1 promoted HCC progression through miR-221-5p/ITGΒ5 axis Huh7 cells were transfected with si-RNF185-AS1, miR-221-5p inhibitor + si-RNF185-AS1 or si-NC+control inhibitor. a, b CCK8 and colony formation assay were performed. c Migration and invasion assay was performed. d EMT markers of cells were detected by western blot. Huh7 cells were transfected with si-RNF185-AS1, si-RNF185-AS1+ ITGΒ5-pcDNA3.1 or si-NC+pcDNA3.1. e, f CCK8 and colony formation assay were performed. g Migration and invasion assay was performed. h Expressions of E-cadherin and Vimentin were measured by western blot.