Abstract. Accumulating evidence highlights the fact that long non-coding RNAs (lncRNAs) serve as critical factors in the growth of hepatocellular carcinoma (HCC). The dysregulation of lncRNA KCNQ1 opposite strand/antisense transcript 1 (KCNQ1OT1) has been reported in numerous human benign diseases. 

Accumulating evidence highlights the fact that long non-coding RNAs (lncRNAs) serve as critical factors in the growth of hepatocellular carcinoma (HCC). The dysregulation of lncRNA KCNQ1 opposite strand/antisense transcript 1 (KCNQ1OT1) has been reported in numerous human benign diseases. However, the role of KCNQ1OT1 in human HCC remains poorly understood. In this study, we demonstrated that the expression of KCNQ1OT1 was abnormally increased in HCC tissues. The ectopic high expression of KCNQ1OT1 was associated with liver cirrhosis, a larger tumor size, an advanced TNM stage, and a worse overall survival and tumor-free survival. For the first time, to the best of our knowledge, we report that KCNQ1OT1 knockdown results in a decreased cell viability and colony formation ability, and an increased rate of apoptosis in vitro. The results from our in vivo results demonstrated that KCNQ1OT1 silencing attenuated tumor growth by impairing cell proliferation. Additionally, we found that KCNQ1OT1 exerted its effects partly by relying on the microRNA-504 (miR-504)-mediated regulation of cyclin-dependent kinase 16 (CDK16), in addition to the regulation of the glycogen synthase kinase 3β (GSK3β)/β-catenin/Bcl-2 signaling pathway. The present study revealed the functions and mechanisms of action of lncRNA KCNQ1OT1 regarding its role in promoting the growth of HCC. Thus, lncRNA KCNQ1OT1 may prove to be a potential therapeutic target for human HCC.


Introduction
Hepatocellular carcinoma (HCC) remains a serious health burden worldwide (1). In China, >466,100 new cases and 422,100 HCC-associated deaths were reported in 2015 (2). Clinical experience indicates that patients affected by tumors of a large size usually have a poor prognosis (3). This may be ascribed to two main reasons: The loss of surgical opportunity due to the large tumor size, and fatal complications following a massive hepatectomy. Thus, the control of tumor growth is a critical strategy for improving the prognosis of patients with HCC.
Long non-coding RNAs (lncRNAs) are a class of abundant and largely uncharacterized non-protein-coding RNAs that are >200 nucleotides in length (4). lncRNAs have been demonstrated to regulate cell proliferation in HCC (5). lncRNA TUG1 has been shown to promote HCC growth in vitro and in vivo by epigenetically suppressing Kruppel-like factor 2 (KLF2) transcription (6). lncRNA HOTAIR has been shown to increase cell viability by enhancing glucose transporter isoform 1 (GLUT1)-mediated glycolysis (7). lncRNA KCNQ1 opposite strand/antisense transcript 1 (known as KCNQ1OT1) was initially identified as an aberrantly expressed lncRNA in Beckwith-Wiedemann syndrome, which is an overgrowth disorder usually presenting in the embryonic stages (8). Recently, KCNQ1OT1 was found to be required for the proliferation of cancer cells. In breast cancer, a high KCNQ1OT1 expression has been shown to be associated with a large tumor size (9). Yoshizawa et al (10) found that pyrrole-imidazole polyamide bound to the CCAAT boxes of the KCNQ1OT1 promoter region to silence its expression. Moreover, KCNQ1OT1 silencing by this drug induced the death of Wilms' tumor-derived G401 cells (10). These results indicate that KCNQ1OT1 may be a novel therapeutic target for patients with high intra-tumoral levels of KCNQ1OT1.
In the present study, we confirmed the cell growth-related lncRNA KCNQ1OT1 to be critical for HCC cell growth in vitro and in vivo. The overexpression of KCNQ1OT1 was found to be associated with a large tumor size and a poor prognosis of patients with HCC. Additionally, KCNQ1OT1 was found to function as a competing endogenous RNA sponge for microRNA-504 (miR-504). Thus, cyclin-dependent kinase 16 (CDK16), a downstream target of miR-504, was harbored by KCNQ1OT1 to facilitate its tumor growth-promoting effects.

Materials and methods
Clinical samples and cell lines. A total of 50 pairs of liquid nitrogen-stored HCC tissues and matched tumor-adjacent tissues were collected from 30 male patients and 20 female patients at the Department of Hepatobiliary Surgery, First Affiliated Hospital of Xi'an Jiaotong University (Xi'an, China) between January 2011 and January 2013. Written informed consent was obtained from all patients enrolled in this study. The use of clinical samples was approved by the Ethics Committee of First Affiliated Hospital of Xi'an Jiaotong University. In total, 5 HCC cell lines (Huh-7, SMMC-7721, Hep3B, MHCC97-H and MHCC97-L), the human immortalized normal hepatocyte cell line, LO2, and 293T cells were obtained from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China), and maintained in our laboratory. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) (both from Gibco, Carlsbad, CA, USA) and 1% V/V penicillin/streptomycin (MilliporeSigma, Bedford, MA, USA) in a humidified atmosphere containing 5% CO 2 at 37˚C.
Cancer Genome Atlas dataset. Gene Expression Omnibus (GEO) data for 95 HCC samples and 39 normal liver samples were collected from the dataset entitled Gene Expression Profiles of Human Hepatocellular Carcinoma (GSE45436), and pre-analyzed using R2: Genomics Analysis and Visualization Platform (http://r2.amc.nl).
Cell counting kit-8 (CCK-8) assay. The cancer cells were seeded into a 96-well plate at 2,000/well in quintuplicate, and cell viability was determined by CCK-8 assay (E606335; Sangon, Shanghai, China) at 0, 24, 48 and 72 h. The absorbance of the samples was measured using a model 550 microplate reader (Bio-Rad Laboratories) at a wavelength of 450 nm.
Colony formation assay. A total of 400 cells were seeded into a 6-well plate and maintained in DMEM containing 10% FBS for 2 weeks. Cell colonies were fixed with 20% methanol and stained with 0.1% crystal violet at room temperature for 15 min. The colonies were counted using a ELIspot Bioreader 5000 (BIO-SYS, Karben, Germany). Apo-ONE ® homogeneous caspase-3/7 assay. A total of 1x10 4 cells (100 µl) were seeded into a 96-well plate. This was followed by the thawing and mixing of the Caspase Substrate and Apo-ONE ® Caspase-3/7 Buffer to produce the Apo-ONE ® Caspase-3/7 Reagent according to the manufacturer's instructions (Promega, Madison, WI, USA) and then by the addition of 100 µl reagent to each well. The wells were then incubated at room temperature for 4 h. The fluorescence of each well at 499 nm was measured using a model 550 microplate reader (Bio-Rad Laboratories).
In vivo tumor growth assays. The in vivo experiments were performed after obtaining ethics approval from the Biomedical Ethics Committee of Xi'an Jiaotong University Health Science Center. A total of 12 BALB/cA male nude mice (6 weeks old, weighing 16-18 g) were obtained from the Laboratory Animal Center of Xi'an Jiaotong University Health Science Center, kept in sterilized cages in an appropriate environment (25˚C, 45% humidity, 12:12 light:dark cycles), and fed a regular chow diet with water available ad libitum. The mice were randomly divided into 4 groups as follows: The Huh-7 sh-control group, Huh-7 sh-KCNQ1OT1 group, SMMC-7721 sh-control group and the SMMC-7721 sh-KCNQ1OT1 group. Modified Huh-7 and SMMC-7721 cells were subcutaneously injected into the outside of the right hind limb of the nude mice to establish tumor xenograft models. The tumor diameter was measured using a vernier caliper every 7 days to monitor the tumor growth from the time of the injection. The tumor volume was calculated as follows: (length x width 2 )/2. All mice were kept under the aforementioned conditions. After 4 weeks, the mice were sacrificed by CO 2 euthanasia (the flow rate of CO 2 was 20% displacement/min). The final weights of the mice at the time of sacrifice were 20-23 g. Each mouse only had a single tumor nodule. The maximum diameter of a single tumor measured was 14.9 mm, and the largest single tumor volume was 1,251.31 mm 3 . Paraformaldehyde-fixed paraffin-embedded mouse subcutaneous tumor tissue sections were created as previously described (12).
Immunohistochemistry. Specific Ki-67 rabbit anti-human primary antibodies (#9027) were purchased from Cell Signaling Technology (Danvers, MA, USA) and diluted 1:100 with PBS to label the antigens at 4˚C overnight. Biotinylated goat anti-rabbit secondary antibodies were used to label the combined primary antibodies. Complexes were detected using HRP-streptavidin conjugates and visualized with DAB. In total, 20 high power (x400) fields were randomly selected to calculate the total cell numbers and Ki-67-positive cell numbers. The percentage of Ki-67-positive cells was identified as the Ki-67 index.
Western blot analysis. The cells were lysed with RIPA buffer (HEART Biotech, Xi'an, China) and quantified using a BCA protein assay kit II (#5000002; Bio-Rad Laboratories). Protein samples (40 µg) were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes (Invitrogen/Thermo Fisher Scientific, Inc.) using a Bio-Rad tank blotting system (Bio-Rad Laboratories). The membranes were incubated with the appropriate primary antibodies at a 1:1,000 dilution overnight at 4˚C. Horseradish peroxidase-conjugated secondary antibodies at a 1:2,000 dilution were used to incubate the membranes for 1 h at room temperature, after washing them with TBST 3 times for 10 min. The targeting proteins on the membrane were visualized with ECL reagents (Millipore, Plano, TX, USA). The primary antibodies used in this study were purchased from Cell Signaling Technology, and included those against glycogen synthase kinase 3β (GSK3β) (#5676), GSK3βser9 (#9322), β-catenin (#8480), Bcl-2 (#2872) and GAPDH (#5174). The secondary antibodies used, included the Amersham ECL Donkey Anti-Mouse IgG Horseradish Peroxidase-Linked Species-Specific Whole Antibody (NA931; GE Healthcare Life Sciences, Pittsburgh, PA, USA). The relative band density was determined using ImageJ software (NIH, Bethesda, ML, USA).
Statistical analysis. Continuous data are presented as the means ± SD. Statistical analysis was performed using SPSS version 21.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA). Correlations between KCNQ1OT1 and clinical characteristics were analyzed using the Pearson's Chi-squared test. The differences between 2 groups were analyzed using a Student's t-test. ANOVA was used to compare the data from multiple groups. The Bonferroni method was used as the post hoc test. Survival analysis was performed using a Kaplan-Meier curve. The hazard ratio and confidence interval were determined using the log-rank (Mantel-Cox) test. The respective correlations between KCNQ1OT1 and miR-504, and CDK16 mRNA levels were analyzed by Spearman's correlation analysis. A value of P<0.05 was considered to indicate a statistically significant difference.

Results
Expression of KCNQ1OT1 is increased in HCC tissues and predicts a worse prognosis. The expression of KCNQ1OT1 in human tissues was screened using RT-qPCR. We found that KCNQ1OT1 expression was significantly increased in the HCC tissues compared with in the adjacent non-tumor tissues (Fig. 1A, P<0.001). To confirm the results obtained by our PCR analysis, we further analyzed KCNQ1OT1 expression data available in the GSE dataset (GSE45436). As expected, KCNQ1OT1 expression was also upregulated in those 95 HCC tissues (Fig. 1B, P=0.002). Additionally, in comparison to the HCC tissues obtained from small tumors (<5 cm in diameter), the KCNQ1OT1 levels were significantly higher in the tissues obtained from large tumors (≥5 cm in diameter) (Fig. 1C,  P<0.001). This result indicated that KCNQ1OT1 may be associated with tumor growth in HCC.
Therefore, in order to further determine the clinical and prognostic significance of KNQ1OT1 in HCC, we selected the mean value of KCNQ1OT1 (2.112) as a cut-off value to divide the 50 patients with HCC into a low KCNQ1OT1 expression group (n=22, <2.112) and a high KCNQ1OT1 expression group (n=28, ≥2.112). The correlations between KCNQ1OT1 expression and the clinical characteristics were analyzed using the Chi-squared test (Table I). High expression of KCNQ1OT1 was confirmed to be associated with liver cirrhosis (P=0.027), a large tumor size (>5 cm, P<0.001) and an advanced TNM stage (stages III and IV, P= 0.002). Moreover, patients with HCC and a high KCNQ1OT1 expression experienced a shorter overall survival ( Fig. 1D; hazard ratio, 2.962; 95% confidence interval, 1.440-6.091; P= 0.003) and tumor-free survival ( Fig. 1E; hazard ratio, 2.588; 95% confidence interval, 1.282-5.221; P= 0.008) than patients with a low KCNQ1OT1 expression. These findings thus suggest that KCNQ1OT1 has the potential to predict the outcomes of patients with HCC.

KCNQ1OT1 enhances HCC proliferation and tumorigenicity in vitro and in vivo.
Next, in vitro cell proliferation and apoptosis experiments were carried out to determine the role of KCNQ1OT1 in tumor growth. We first detected KCNQ1OT1 expression in LO2 cells and in 5 HCC cell lines, and found that KCNQ1OT1 expression was upregulated in all the HCC cell lines, apart from the Hep3B and MHCC97-L cell lines ( Fig. 2A, P<0.05, respectively). Two HCC cell lines with high KCNQ1OT1 expression levels, namely Huh-7 and SMMC-7721, were transduced with a KCNQ1OT1-specific shRNA lentivirus. The PCR results revealed that KCNQ1OT1 expression was silenced in these two cell lines following transduction (Fig. 2B, P<0.01, respectively). KCNQ1OT1 knockdown significantly suppressed the viability of the Huh-7 and SMMC-7721 cells (Fig. 2C, P<0.05, respectively). The capacity of the Huh-7 and SMMC-7721 cells for clone formation was also inhibited by KCNQ1OT1 knockdown (Fig. 2D, P<0.05, respectively). The effect of KCNQ1OT1 on cell apoptosis was also examined in this study. We found that KCNQ1OT1 silencing increased the percentage of apoptotic cells (Fig. 2E, P<0.05, respectively), as well as caspase-3/7 activity (Fig. 2F, P<0.05, respectively) in the Huh-7 and SMMC-7721 cells.
The cells in which KCNQ1OT1 was stably knocked down were implanted into nude mice to examine the growth of HCC cells in vivo. Compared with the mice in the control group, the tumors in mice injected with KCNQ1OT1 shRNA-transduced Huh-7 (Fig. 3A, upper panel) and SMMC-7721 (Fig. 3A, lower panel) cells grew at a slower rate during the 4-week observation period (P<0.05, respectively). Subsequently, a classical proliferation marker, Ki-67, was stained in order to measure the cell proliferation in the tumors xenografts. As was expected, the Ki-67-positive percentage of Huh-7 (Fig. 3B, upper panel) and SMMC-7721 (Fig. 3B, lower panel) cells in the tumor tissues derived from cells in which KCNQ1OT1 was knocked down was significantly lower than that in the tumor tissues derived from the negative controls (P<0.05, respectively). These results thus suggest that KCNQ1OT1 promotes HCC growth by promoting cell proliferation and inhibiting apoptosis.
KCNQ1OT1 drives tumor growth in HCC by sponging miR-504. lncRNAs can bind to miRNAs and function as sponges to regulate the expression of certain miRNAs. As shown in Fig. 4A, we identified that the seed region of miR-504 (2-8 bp from 5') has putative binding sites for KCNQ1OT1. We cloned these sequences into reporter vectors; the reporter vector and miR-504 were co-transfected into 293T cells. The luciferase activities were significantly inhibited by the miR-504 mimics (P<0.05, respectively). The expression of miR-504 was upregulated when KCNQ1OT1 was knocked down in the Huh-7 and SMMC-7721 cells (Fig. 4B, P<0.05, respectively). We also found that the overexpression of miR-504 decreased KCNQ1OT1 expression, whereas miR-504 knockdown increased KCNQ1OT1 expression (Fig. 4C, P<0.05, respectively). Furthermore, a significant inverse correlation was demonstrated between the KCNQ1OT1 and miR-504 levels (Fig. 4D, r=-0.337; 95% confidence interval, -0.5680 to -0.05574; P=0.017). In addition, the miR-504 inhibitor was transfected into the Huh-7 and SMMC-7721 cells in which KCNQ1OT1 was knocked down to further examine whether KCNQ1OT1-induced HCC cell proliferation could be ascribed to miR-504 inhibition. Even though KCNQ1OT1 knockdown inhibited the viability and promoted the apoptosis of Huh-7 and SMMC-7721 cells, these effects were markedly abrogated by miR-504 inhibition ( Fig. 4E and F, P<0.05, respectively).

Discussion
lncRNAs have critical biological functions, and KCNQ1OT1 is one of the few well-characterized lncRNAs. In the current study, we investigated the expression, clinical significance and biological functions of KCNQ1OT1 in human HCC. Several new discoveries were made. First, KCNQ1OT1 was found to be upregulated in human HCC tissues. This was consistent with findings that KCNQ1OT1 was overexpressed in a HCC cohort from the GSE dataset. Second, high levels KCNQ1OT1 were found to correlate with liver cirrhosis, an advanced TNM stage and a large tumor size. Tissues from larger-sized tumors (>5 cm in diameter) also had high KCNQ1OT1 expression levels. Third, patients with a high intra-tumoral KCNQ1OT1 expression experienced worse 3-year overall survival and tumor-free survival times. This evidence strongly suggests that KCNQ1OT1 is a prognostic biomarker, and that it functions to promote tumor growth. With this antecedent, we subsequently found that the downregulation of KCNQ1OT1 inhibited cell proliferation and induced apoptosis in vitro, and attenuated subcutaneous tumor growth in vivo.
Consistent with our findings, Ren et al identified a high KCNQ1OT1 expression in lung adenocarcinoma tissues (14). KCNQ1OT1 knockdown suppressed the proliferation of and sensitized A549 cells to paclitaxel treatment. KCNQ1OT1 silencing also exerted tumor suppressive functions in glioma cells by acting as a sponge for miR-370 (15). The ability to function as a competing endogenous RNA (ceRNA) in order to sponge miRNAs is commonly found in lncRNA networks (16). For example, lncRNA-CCAT1 has been reported to sponge let-7 in HCC (17), miR-410 in glioma (18) and miR-181a in nasopharyngeal cancer (19). In this study, we found that KCNQ1OT1 had three fragments containing miR-504 seed region sequences. Upregulated miR-504 was found in cells in which KCNQ1OT1 was knocked down. The inhibition of miR-504 abrogated the changes in cell proliferation and apoptosis induced by KCNQ1OT1 knockdown. In the 50 pairs of HCC tissues, an inverse correlation was also confirmed between KCNQ1OT1 and miR-504 expression. We then used different algorithms to assume that CDK16 is a putative target of miR-504. The in vitro results from the dual-luciferase reporter gene system, RT-qPCR and western blot analyses verified our hypothesis. Moreover, the downregulation of KCNQ1OT1 inhibited CDK16 expression in HCC cells. Clinical analysis also confirmed the inverse correlation between KCNQ1OT1 and CDK16. Clearly, KCNQ1OT1 modulates CDK16 expression by sponging miR-504 in order to regulate cell proliferation in human HCC.
The underlying mechanisms through which KCNQ1OT1 increases HCC proliferation were also explored. CDKs are a family of serine/threonine-protein kinases. CDK16 is a recently recognized, atypical member of the CDK family (20). The activation of CDK16 usually occurs upon binding to membrane-associated cyclin Y (CCNY) (21). CDK16 has been reported to exert its functions by phosphorylating Akt at Ser473 (22), KAP0 at Ser83 (23) and caspase-8 at Ser387 (24). In this study, we found that KCNQ1OT1 silencing downregulated the phosphorylation of GSK3β at Ser9. Ser9 functions as an inhibitory phosphorylating position (25), which indicates that GSK3β phosphorylation was decreased by KCNQ1OT1 knockdown. Therefore, we detected the downregulation of β-catenin in the cells in which KCNQ1OT1 was knocked down. The expression of Bcl-2, an anti-apoptotic gene that can be regulated by β-catenin (26,27), was also suppressed by transfection with KCNQ1OT1 shRNA. Notably, all these changes were attenuated by miR-504 inhibition. Our results suggested that the role of KCNQ1OT1 in the regulation of the GSK3β/β-catenin/Bcl-2 signaling pathway depends on the inhibition of miR-504. Recently, Sunamura et al reported that β-catenin promoted KCNQ1OT1 transcription by directly binding to the KCNQ1OT1 promoter (28), which may explain why miR-504 overexpression decreased KCNQ1OT1 expression in Huh-7 and SMMC-7721 cells.
In conclusion, our data highlight the critical role played by KCNQ1OT1 in the proliferation and apoptosis of HCC cells. Our findings also indicate that lncRNA KCNQ1OT1 is a potential biomarker for HCC post-surgical surveillance, in addition to a molecular therapeutic target.