LncRNA RNCR3 Promotes the Progression of HCC by Activating the Akt/GSK3β Signaling Pathway

Objective To explore the potential biological roles of long noncoding RNA (lncRNA) RNCR3 in human hepatocellular carcinoma (HCC). Methods First, the expression of RNCR3 was detected by qRT-PCR. Then, in vitro experiments were performed to investigate the effects of RNCR3 on the proliferation, cell cycle, migration, and invasion of HCC cells, while the effects of RNCR3 on HCC tumor growth and metastasis were investigated using in vivo experiments. Finally, western blot was used to study the activation of the Akt/GSK3β signaling pathway. Results RNCR3 was highly expressed in both HCC tissues and cells, and the expression of RNCR3 was closely related to tumor size, tumor number, TNM stage, and overall survival time. In vitro, RNCR3 served as an oncogene to promote cell proliferation, migration, and invasion, and in vivo, RNCR3 promoted the growth and metastasis of HCC tumors. In terms of mechanism, RNCR3 induced the phosphorylation of Akt (thr308 and ser473) and GSK3β (ser9) but decreased the expression of GSK3β, which activated the Akt/GSK3β signaling pathway. Conclusion The high expression of lncRNA RNCR3 in HCC can promote the proliferation, migration, invasion, growth, and metastasis of HCC by activating the NF-κB signaling pathway.


Introduction
Hepatocellular carcinoma (HCC) accounts for nearly 80% of all primary liver cancers, ranking third among cancer-related deaths, and a large number of patients die from HCC every year globally [1,2]. Surgical resection is still the main choice for patients with resectable HCC, but due to the rapid tumor progression and intrahepatic and extrahepatic metastasis, the postoperative tumor recurrence rate is still very high, which leads to a poor prognosis [3]. erefore, preventing the progress and metastasis of hepatocellular carcinoma is the most important task at present, and exploring the molecular mechanism of the occurrence and development of liver cancer is of great significance for finding new therapeutic targets.
Long noncoding RNA (lncRNA) is a kind of noncoding RNAs with no protein-coding ability, and its length exceeds 200 nucleotides [4]. It has been reported that lncRNAs are involved in various cellular processes including cell proliferation, apoptosis, cell cycle, and invasion, and more and more evidence shows that the abnormal expression of lncRNAs that served as oncogenes or tumor suppressor genes is related to the occurrence and development of various cancers [5,6]. In HCC, many lncRNAs play an important role in the occurrence and development of tumors, indicating that lncRNAs can be used as diagnostic markers and therapeutic targets for HCC. For example, the expression of LINC01554 in HCC is significantly downregulated, and its expression has a significant relationship with the tumor size, multiple lesions, TNM stage, tumor recurrence rate, and long-term survival rate of liver cancer patients [7]. It is also reported that LINC00467 can promote the proliferation and invasion of HCC cells through miR-509-3p/PDGFRA, inhibit cell apoptosis, and contribute to Axitinib resistance of hepatocellular carcinoma, which indicates that LINC00467 is a promising biological target for the treatment of liver cancer [8].
Retinal noncoding RNA3 (RNCR3, also known as LINC00599) is a lncRNA transcribed from intergenic regions of the genome and is highly conserved among mammals [9]. Many studies have shown that RNCR3 plays a critical regulatory role in cell proliferation, differentiation, and atherosclerosis [10,11]. In human cancer, RNCR3 as an oncogene promotes the progression of malignant tumors such as colorectal cancer, glioma, and prostate cancer [12][13][14]; however, the biological function and molecular mechanism of RNCR3 in HCC have not been fully elucidated. erefore, this study will investigate the expression, biological effects, and corresponding mechanisms of RNCR3 in HCC.

Tissue Samples and Ethics.
Both HCC cancer tissues and para-cancerous tissues were from patients undergoing surgical treatment in our hospital, and all patients signed informed consent. Before the sample collection, this study was approved by the ethics committee of our hospital. e para-cancerous specimen was taken from the tissue 3 cm away from the edge of liver cancer, after which the collected tissue was immediately placed in a RNA long-term preservation solution (Solarbio, China) and stored at −80°C.

RNA Isolation, Reverse Transcription (RT), and Real-Time Quantitative Polymerase Chain Reaction (qRT-PCR).
Total RNA was extracted from all tissues and cells using TRIzol reagent ( ermo Fisher Scientific, USA), and the concentration of the total RNA was quantified using a NanoDrop 2000 spectrophotometer ( ermo Fisher Scientific). After that, the RNA was reverse transcribed into cDNA using a PrimeScript RT kit (TAKARA, Japan), and the PCR reaction was performed on PikoReal 96 real-time PCR system ( ermo Scientific) using SYBR Green qPCR Master Mix (TAKARA, Japan). GAPDH was used as an internal reference, and the relative expression level of RNCR3 was calculated using the 2 −ΔΔCt method. Primer sequences are shown in Table 1.

CCK8 Experiment.
Cell proliferation ability was measured using the CCK8 kit (Dojindo, Japan). First of all, cells were seeded in 96-well plates at a density of 1 × 10 3 cells/well and cultured for 24 to 72 hours. After that, 10 μL of CCK8 solution was added to each well and incubated at 37°C for an appropriate time, and then, the absorbance at 450 nm was measured with a spectrophotometer.

Cell Cycle Analysis.
First, the cells were washed in PBS and fixed with 75% ethanol at 4°C overnight. en, the cells were stained with PI/RNase staining buffer (BD Biosciences, USA). Finally, the cells were analyzed by flow cytometry (BD Biosciences), and the proportion of G1, S, and G2 phase cells was compared.

Scratch Test.
e scratch test was used to study the effect of RNCR3 on cell migration. First, the transfected cells were seeded in 12-well plates, to be grown to a density of 80-90%, and then, the same width of scratches was scratched with the tip of a sterile 10 μL pipette. After that, the cells were washed with PBS solution to remove the crossed-out cells, and then, the cells were cultured with a serum-free medium for 24 hours. Images were taken using an optical microscope (Olympus, Japan) at 0 hours and 24 hours after scratching.

Transwell Experiment.
e transwell experiment was used to determine the cell invasion ability. First, the transfected cells (5 × 10 4 cells per well) were added to the upper chamber of the transwell chamber (Corning, USA) precoated with matrix gel (BD Biosciences) and cultured in a serum-free medium, and then the DMEM medium containing 10% FBS was added into the lower chamber. After incubating at 37°C for 24 hours, the upper chamber cells were removed with a cotton swab, and the cells that penetrated the membrane were fixed with methanol and stained with 0.1% crystal violet for 5 min. Finally, the stained cells were photographed using an optical microscope (Olympus) and then were counted.

Western Blot Experiment.
Cells were lysed using RIPA buffer (Beyotime, China) to extract proteins. First, the protein samples were electrophoresed by SDS-PAGE, and the proteins were transferred to the PVDF membrane. en, after incubating the membrane with the primary antibody, the secondary antibody was incubated. Finally, an ECL chemiluminescence kit (Beyotime) was used for imaging.

Tumor Formation Experiment in Nude Mice.
Four-week-old BALB/c nude mice used in this study were purchased from Changzhou Cavens Experimental Animal Co., Ltd.
e animal experiments in this study were approved by the Animal Ethics Committee of this hospital. First of all, 2 × 10 7 HepG2 cells stably transfected with sh-NC or shRNCR3 were injected subcutaneously into nude mice. en, the tumor volume was measured and calculated every 5 days using a digital caliper, and the formula was as follows: volume � (length × width 2 )/2. After 20 days, the nude mice were sacrificed and the subcutaneous tumors were excised 2 Evidence-Based Complementary and Alternative Medicine and implanted into new nude mouse livers. Five weeks later, the new nude mice were sacrificed, and lung tissue sections were taken and stained with HE to observe the tumor metastasis.

Statistical Analysis.
All experiments were repeated at least three times, and the data were expressed as mean-± standard deviation. All analyses were performed using SPSS 20.0 software (SPSS, USA). Differences between groups were analyzed using t-test or one-way ANOVA, and the Kaplan-Meier method and Log-rank test were used for survival analysis. P < 0.05 was considered statistically significant.

High Expression and Clinical Significance of RNCR3 in HCC.
First, 46 pairs of HCC tissues and normal liver tissues adjacent to cancer were used to detect the RNCR3 expression by qRT-PCR. e results showed that the expression of RNCR3 in HCC tissues was significantly higher than that in normal tissues (Figures 1(a) and 1(b)). Furthermore, the expression of RNCR3 in the HCC cell line was significantly higher than that of the human normal liver cell line (Figure 1(c)). ese results indicated that RNCR3 was highly expressed in HCC. e expression of lncRNA LINC01554, which has been reported in HCC, was also tested as a positive control, and it was found that the LINC01554 expression was downregulated in HCC tissues (Figure 1(d)). To explore the clinical significance of RNCR3 expression, the expression of RNCR3 was divided into a high-expression group and a low-expression group according to the expression median and compared with clinical factors. It was found that the expression of RNCR3 was closely related to tumor size, tumor number, and TNM stage ( Table 2). rough survival analysis, it was found that patients with the high expression of RNCR3 had a shorter overall survival time (Figure 1(e)).

RNCR3 Overexpression and Knockout Verification In
Vitro. Due to the low expression of RNCR3 in HL-7702 cells and Huh-7 cells and high expression of RNCR3 in HepG2 cells, to study the biological function of RNCR3, the RNCR3 overexpression plasmid was transfected in HL-7702 cells and Huh-7 cells, while RNCR3 shRNAs were transfected in HepG2 cells, and the qRT-PCR test was used to detect for verification.
e results suggest that RNCR3 can be successfully overexpressed in HL-7702 and Huh-7 cells (Figure 2(a) and 2(b)), and shRNCR3#1 had the best silencing efficiency (Figure 2(c)), so this shRNA was selected for subsequent experiments.

RNCR3 Promotes HL-7702 Cell and HCC Cell Proliferation In Vitro.
e overexpression plasmid was transfected in HL-7702 and Huh-7 cells, and RNCR3 shRNA was transfected in HepG2 cells. e CCK8 test was used to detect the changes in cell proliferation ability. e results showed that overexpression of RNCR3 can significantly increase the proliferation capacity of HL-7702 cells and Huh-7 cells (Figure 3(a) and 3(b), but silencing RNCR3 inhibited the proliferation of HepG2 cells (Figure 3(c)). is result suggested that RNCR3 can promote HCC cell proliferation in vitro.

RNCR3 Regulates HL-7702 Cell and HCC Cell Cycle In
Vitro.
e RNCR3 overexpression plasmid was transfected in HL-7702 cells and Huh-7 cells, RNCR3 shRNAs were transfected in HepG2 cells, and flow cytometry was used to detect changes in the cell cycle.
e results showed that overexpression of RNCR3 can significantly increase the percentage of HL-7702 cells and Huh-7 cells in S and G2 phases (Figure 4(a) and 4(b)), while silencing RNCR3 decreased the percentage of HepG2 cells in S and G2 phases (Figure 4(c)). is result indicated that RNCR3 can regulate the HCC cell cycle in vitro.

RNCR3 Promotes HL-7702 Cell and HCC Cell Migration
In Vitro. e RNCR3 overexpression plasmid was transfected in HL-7702 cells and Huh-7 cells, RNCR3 shRNAs were transfected in HepG2 cells, and the changes in cell migration ability were investigated using scratch experiments. e results showed that overexpression of RNCR3 can promote the migration of HL-7702 cells and Huh-7 cells (Figure 5(a) and 5(b)), while silencing RNCR3 inhibited the migration of HepG2 cells ( Figure 5(c)). is result suggested that RNCR3 can promote HCC migration in vitro.

RNCR3 Promotes HL-7702 Cell and HCC Cell Invasion In
Vitro.
e RNCR3 overexpression plasmid was transfected in HL-7702 cells and Huh-7 cells, RNCR3 shRNAs were transfected in HepG2 cells, and the changes in cell migration ability were evaluated using the transwell experiment. e results showed that overexpression of RNCR3 can promote the invasion of HL-7702 cells and Huh-7 cells (Figure 6(a) and 6(b)), while silencing RNCR3 inhibited the invasion of HepG2 cells (Figure 6(c)). is result suggested that RNCR3 could promote the HCC invasion in vitro.

RNCR3 Promotes HCC Growth and Metastasis In Vivo.
HepG2 cells stably transfected with sh-NC or shRNCR3 were injected subcutaneously into nude mice, and the growth rate of subcutaneous tumors was observed in nude mice. e results showed that the growth rate of the tumor was significantly slowed down after silencing RNCR3 (Figure 7(a)). Moreover, after silencing RNCR3, the lung metastasis ability of the tumor was significantly reduced (Figure 7(b)). is result indicated that RNCR3 can promote HCC growth and metastasis in vivo.

RNCR3 Can Activate the Akt/GSK3β Signaling Pathway.
Studies have shown that RNCR3 can activate the Akt/GSK3β signaling pathway in gliomas to exert its cancer-promoting effect [13], so we verified the effect of RNCR3 on the Akt/ GSK3β signaling pathway in HCC. e RNCR3 overexpression plasmid was transfected in HL-7702 cells and Huh-7 cells, RNCR3 shRNAs were transfected in HepG2 cells, and the protein expression changes of the Akt/GSK3β signaling pathway was measured by western blot. e results showed that overexpression of RNCR3 can promote the phosphorylation of Akt ( r308 and Ser473) and GSK3β (Ser9) but reduce the expression of GSK3β (Figure 8(a) and 8(b)). However, silencing RNCR3 inhibited the phosphorylation of Akt ( r308 and Ser473) and GSK3β (Ser9) but upregulated the expression of GSK3β (Figure 8(c)). is result suggested that RNCR3 can activate the Akt/GSK3β signaling pathway in HCC.

Discussion
In the past few years, the lncRNA has been a research hotspot, and its discovery has greatly changed our biological understanding of many complex diseases [15]. To date, HCC still has a poor prognosis and a high mortality rate, and its prevention and treatment have been troublesome for many researchers. erefore, it is of great significance to explore lncRNAs in HCC. is study focuses on the expression and role of lncRNA RNCR3 in HCC. Our results showed that RNCR3 was highly expressed in HCC tissues and cells, and the expression of RNCR3 was closely related to tumor size, tumor number, TNM stage, and overall survival time. It has been reported that the expression of RNCR3 in glioma tissues was increased compared with the corresponding adjacent normal tissues, and the increase in its expression was related to the progression of the tumor and the low survival rate of glioma patients [13]. Moreover, the expression of RNCR3 in prostate cancer was also increased, and its high expression was significantly associated with tumor progression and low survival rate of patients with prostate cancer [14].
ese results indicate that RNCR3 can be used as a potential biomarker in clinical applications.
Our in vitro experiments showed that RNCR3 can be used as an oncogene to promote cell proliferation, migration, and invasion. Further in vivo experiments found that RNCR3 promotes HCC growth and metastasis. In many     Evidence-Based Complementary and Alternative Medicine other tumors, RNCR3 also plays the role of an oncogene; for example, in colorectal cancer, RNCR3 can promote tumor cell proliferation, colony formation, and invasion and inhibit apoptosis [12]. In glioma, RNCR3 can increase the proliferation and invasion ability of glioma cells [13]. In this study, animal experiments were added to confirm that RNCR3 promotes HCC growth and metastasis in vivo; therefore, the biological function of RNCR3 in HCC was clarified both in vitro and in vivo.
In terms of mechanism, our results demonstrated that RNCR3 can promote the phosphorylation of Akt ( r308 and Ser473) and GSK3β (Ser9) but reduce the expression of GSK3β, thereby activating the Akt/GSK3β signaling pathway. AKT, also known as protein kinase B, is a known oncogene whose abnormal overexpression or activation is found in many cancers and is associated with cancer cell proliferation, increased survival rate, invasion, and migration [16]. e activation of Akt depends on the phosphorylation of r308 and Ser473 and the activated Akt can inactivate GSK-3 by phosphorylating a single residue on GSK3β Ser9 [17]. GSK-3 is a versatile serine/threonine kinase that was originally considered to be a regulator of glycogen metabolism. It is now believed that GSK-3 plays various important functions in cell division, proliferation, differentiation, and adhesion [18]. GSK-3 has been shown to regulate cyclin D1 proteolysis and subcellular localization, thereby affecting cell cycle and cell proliferation [19]. Moreover, GSK-3 can also affect the expression of EMT-   related genes, thereby regulating cell migration and invasion [20]. erefore, we believe that RNCR3 can promote the proliferation, migration, invasion, growth, and metastasis of HCC cells by activating the Akt/GSK3β signaling pathway.

Conclusion
Our research shows that lncRNA RNCR3 is highly expressed in HCC, and RNA3 can promote the proliferation, migration, invasion, growth, and metastasis of HCC cells by activating the Akt/GSK3β signaling pathway. erefore, RNCR3 is expected to become a biomarker and therapeutic target for HCC.

Data Availability
All data generated or analyzed during this study are included in this published article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.