Long noncoding RNA PCAT-14 induces proliferation and invasion by hepatocellular carcinoma cells by inducing methylation of miR-372

Long non-coding RNAs (lncRNAs) regulate oncogenesis by inducing methylation of CpG islands to silence target genes. Here we show that the lncRNA PCAT-14 is overexpressed in patients with hepatocellular carcinoma (HCC), and is associated with a poor prognosis after surgery. Our results demonstrate that PCAT-14 promotes proliferation, invasion, and cell cycle arrest in HCC cells. In addition, PCAT-14 inhibits miR-372 expression by inducing methylation of the miR-372 promoter. Simultaneously, miR-372 eliminates the effects of PCAT-14 on proliferation, invasion, and cell cycle in HCC cells. Moreover, PCAT-14 regulates expression of ATAD2 and activation of the Hedgehog pathway via miR-372. These findings indicate that PCAT-14 plays an important role in HCC, and may serve as a novel prognostic factor and therapeutic target.


INTRODUCTION
Hepatocellular carcinoma (HCC) is one of the most prevalent tumor types with the highest mortality rate [1]. The occurrence of HCC is increasing, especially in Asia and Africa [2]. Many HCC patients do not have any symptoms until an advanced stage [3]. Even though HCC prognosis has improved thanks to the development of effective surgical techniques and diagnostic methods over recent years, long-term prognosis is still unsatisfactory largely due to the high recurrence (50-70% at 5 years) [4,5]. Thus, there is an urgent need to identify valuable diagnostic and prognostic biomarkers to improve clinical outcomes and develop effective individual therapeutic strategies for patients with HCC.
LncRNA prostate cancer-associated transcripts (PCATs) were originally identified as biomarkers for prostate cancer [14]. They include PCAT-1, PCAT-6, PCAT-14, and PCAT-29, which play critical roles in the progression of several cancer types [15][16][17]. Increased expression of PCAT-1 has been associated with advanced clinical parameters and poor overall survival of HCC patients [18]. Upregulation of PCAT-1 contributes to HCC cell proliferation, migration, and apoptosis [19]. PCAT-14 is located on chromosome 22, GRCh38.p7. An integrative analysis revealed that PCAT-14 is the most prevalent lncRNA that is aberrantly expressed in prostate cancer patients [20], and predicts a poor outcome [21]. However, the expression and function of PCAT-14 in HCC have not been studied.
In this study, we have analyzed the expression and function of PCAT-14 in HCC. Our results demonstrate that the PCAT-14 expression is associated with HCC metastasis, tumor size, and TNM stage, suggesting that PCAT-14 may be involved in the tumorigenesis and progression of HCC. In addition, Kaplan-Meier and Cox regression analysis shows that high PCAT-14 expression correlates with poor OS, indicating that it could serve as

Research Paper
an independent prognostic factor for overall survival in HCC. Our results demonstrate that PCAT-14 promotes cellular invasion and proliferation, and inhibits miR-372 expression by inducing methylation of CpG islands in the miR-372 promoter. In addition, PCAT-14 regulates ATAD2 expression and activation of Hedgehog pathway in HCC cells, depending on miR-372. Together, these results indicate that PCAT-14 plays a critical role in HCC, and may serve as a candidate target for new HCC therapies.

Clinical significance of PCAT-14 expression in HCC
According to the results of in situ hybridization, the association between PCAT-14 expression and clinicopathological factors of the 120 HCC patients was analyzed. The expression of PCAT-14 was significantly higher in HCC tissues with advanced TNM stage compared with those with early TNM stage (P = 0.021). In addition, the increased PCAT-14 expression was associated with tumor metastasis (P = 0.022) and larger tumor size (P = 0.006, Table 1). The OS was higher in HCC patients with lower PCAT-14 expression than in those with higher PCAT-14 expression (P < 0.01, Figure 1I). In addition, a multivariate analysis using the Cox model indicated that PCAT-14 expression, metastasis, and AFP status were independent, poor prognostic factors (Table 2).

Expression of miR-372 negatively correlates with PCAT-14 expression in HCC
In this study, we used the same HCC samples for qRT-PCR and in situ hybridization as we have previously used for miR-372 analysis [22]. Analysis of HCC samples from 120 patients revealed a negative correlation between PCAT-14 and miR-372 ( Figure 1A-1F). There was a significant negative association between PCAT-14 and miR-372 analyzed in the same specimens, using the Spearman Rank C test (Table 3, P < 0.01). Consistent with the results of in situ hybridization, miR-372 expression also negatively correlated with PCAT-14 expression by using linear regression analysis of 37 clinical HCC samples analyzed by qRT-PCR (R 2 = 0.504, P < 0.01, Figure 5A-5B).

PCAT-14 inhibits miR-372 expression in HCC by inducing methylation of CpG islands in miR-372 promoter
To investigate the relationship between PCAT-14 and miR-372, we evaluated the expression of miR-372 in three HCC cell lines (SMMC7721, HepG2, and HCCLM3) transfected with either pcDNA-PCAT-14 or si-PCAT-14. As shown in Figure 5C, the expression of miR-372 was inhibited in SMMC7721 cells when PCAT-14 was overexpressed (P < 0.01). In contrast, PCAT-14 silencing promoted miR-372 expression in HepG2 and HCCLM3 cells ( Figure 5D, 5E, P < 0.01). We have previously demonstrated that aberrant DNA methylation of CpG islands upstream of the miR-372 promoter induces epigenetic silencing of miR-372 in HCC cells [22]. To explore the mechanisms of the negative miR-372 regulation by PCAT-14, we analyzed the levels of three active DNA methyltransferases (DNMT1, DNMT3a, and DNMT3b) in HCC cells by qRT-PCR when PCAT-14 was aberrantly expressed. The expression of DNMT1, DNMT3a, and DNMT3b was increased when PCAT-14 was overexpressed ( Figure 5F, P < 0.01). An opposite result was observed when PCAT-14 was knocked-down ( Figure 5G, P < 0.01), suggesting that PCAT-14 might regulate miR-372 methylation. In addition, MSP analysis showed that PCAT-14 overexpression promotes the methylation of miR-372 CpG islands in SMMC7721

DISCUSSION
LncRNAs can regulate protein-coding genes, transcription, and post-transcription, and play important roles in biological processes [25,26]. Many functions of lncRNAs in biological processes have been proposed, including cardiovascular diseases, inflammatory responses, and cancers [27,28]. These roles are mediated by epigenetic regulation, and transcriptional and posttranscriptional regulation [29]. A number of lncRNAs have been identified in HCC, such as H19, HOTAIR, MALAT1, and MEG3 [30,31].
PCAT-14 was first reported in prostate cancer. Down-regulation of PCAT-14 in prostate cancer has been associated with an increased probability of metastatic progression and mortality across multiple independent datasets and ethnicities [20]. In-vitro data confirmed that low PCAT-14 expression increases migration of prostate cancer cells, while overexpression of PCAT-14 reduces their growth, migration, and invasion [20,21]. The above studies suggested that PCAT-14 suppresses development of prostate cancer. However, the function and regulation of PCAT-14 in HCC remained unknown.
In this study, we found a negative correlation between PCAT-14 and miR-372 in HCC tissues. We have previously shown that aberrantly high DNA methylation in the miR-372 gene promoter induces epigenetic silencing of miR-372 in HCC and that miR-372 inhibits cancer cell proliferation and invasion. Thus, we speculated that PCAT-14 might regulate the expression of miR-372, thus regulating HCC cell invasion and proliferation. It has been reported that lncRNAs could adjust the methylation status of target genes, such as LncRNA PVT1 regulates prostate cancer cell growth by inducing methylation of miR-146a, LncRNA DBCCR1-003 regulates methylation of DBCCR1      elevated miR-372 expression. To explore the mechanisms of the negative regulation of miR-372 by PCAT-14, we analyzed the levels of three active DNA methyltransferases (DNMT1, DNMT3a, and DNMT3b) in HCC cells aberrantly expressing PCAT-14. The results demonstrated that PCAT-14 could up-regulate the expression of DNMT1, DNMT3a, and DNMT3b, suggesting that PCAT-14 might regulate miR-372 methylation. In addition, MSP analysis provided evidence that PCAT-14 overexpression promotes the methylation of miR-372 CpG islands in SMMC7721 cells, and PCAT-14 suppression inhibits the CpG islands methylation in HepG2 and HCCLM3 cells. Together, these findings indicate that in HCC cells, PCAT-14 inhibits the miR-372 expression through inducing the methylation of CpG islands in its promoter. Furthermore, our results show that miR-372 overexpression or downregulation can eliminate the effect of PCAT-14 on cell proliferation, invasion, and cell cycle in SMMC7721 and HepG2 cells, suggesting that PCAT-14 regulates the HCC carcinogenesis depending on miR-372.
The ATPase family AAA domain-containing protein 2, ATAD2, is highly expressed in several types of tumors, such as breast cancer, lung cancer, and large B-cell lymphoma [35][36][37][38][39][40]. ATAD2 is one of the target genes of miR-372, and regulates the Hh pathway to influence HCC cell proliferation and metastasis [23,24]. To investigate the role of PCAT-14 in ATAD2 and Hh signaling, we altered the expression of PCAT-14 in SMMC7721 and HepG2 cells, and evaluated gene and protein levels of ATAD2 and Hh pathway key proteins (PTCH-1, SMO, and Gli2). Results showed that PCAT-14 could promote the expression of ATAD2, PTCH-1, SMO, and Gli2, while miR-372 could inhibit them, suggesting that PCAT-14 regulates the ATAD2 expression and Hedgehog pathway via miR-372.
In summary, here we demonstrate the function of PCAT-14 in HCC carcinogenesis by providing the following experimental evidence (Figure 8). 1) HCC tissues and cells express high levels of PCAT-14, which promotes HCC cell proliferation and invasion. 2) PCAT-14 inhibits miR-372 expression by inducing the methylation of CpG islands in the miR-372 promoter. 3) PCAT-14 regulates HCC cell growth and invasion depending on miR-372. 4) ATAD2 is one of the target genes of miR-372, and regulates the Hh pathway to influence HCC proliferation and metastasis. 5) PCAT-14 regulates ATAD2 expression and Hedgehog pathway via miR-372. Together, these findings indicate that PCAT-14 plays an important role in HCC carcinogenesis and may provide a new target for HCC detection and treatment. Future studies should determine the precise mechanisms of the aberrant expression of PCAT-14 in HCC.

Patient tissue samples and liver cancer cell lines
HCC tissue slice samples were obtained from 120 patients (51 males and 69 females) diagnosed with HCC who had undergone a routine hepatic resection in the First Affiliated Hospital of China Medical University between January 2007 and January 2009 [22]. The follow-up period for survivors was 5 years. None of the patients had received preoperative radiotherapy or chemotherapy prior to surgical resection. A total of 37 paired fresh specimens, including both tumor tissues and the corresponding paired noncancerous parenchyma, were snap-frozen in liquid nitrogen and stored at −70°C immediately after resection until processing. Histological diagnosis and differentiation were evaluated independently by three pathologists according to the WHO classification system [41]. The project protocol was approved by the Institutional Ethics Committee of China Medical University prior to the initiation of the study. All patients provided written informed consent for the use of the tumor tissues for clinical research. The liver cancer cell lines Huh7, HCCLM3, HepG2, SMMC7721, PLC5, and QGY7701 and the normal liver cell line LO2 were obtained from Shanghai Cell Bank (Shanghai, China).

RNA preparation and quantitative real-time PCR
Total RNA was extracted from approximately 100 mg of the 37 paired tissue samples and liver cancer cell lines using TRIzol reagent ( Invitrogen Company, USA) according to the manufacturer's instructions. The PCAT-14 primers were purchased from Takara Company (Japan). The GADPH gene was used as a reference control for PCAT-14. The relative gene expression was expressed as ΔCt = Ct gene -Ct reference and the fold change in gene expression was calculated using the 2 −ΔΔCt method [42].

Western blot
Cells were washed with ice-cold PBS and lysed by RIPA (Beyotime, China) containing protease inhibitors (Beyotime, China). Protein lysates were separated in 10 % SDS-PAGE and transferred onto PVDF membrane (Millipore, USA). The membranes were blocked by 5 % skim milk in TBST buffer, and incubated with primary antibodies for ATAD2, PTCH1, SMO, and Gli2 (Abcam, UK) overnight at 4°C. PVDF membranes were washed in TBST and incubated with horseradish peroxidase-conjugated secondary antibodies (ProteinTech Group, USA). Antibody against GAPDH (Cell Signaling Technology, USA) was used as an internal control. Proteins were visualized using ECL Western blotting substrate (Pierce, USA).
After 48 h, cells were trypsinized, fixed with 70% ethanol at 4°C, and washed with PBS. RNase A (100 μL) was added, and the mixture was incubated in a 37°C water bath for 30 min. Next, 400 μL of PI staining solution was added and samples were incubated at 4°C in the dark for 30 min; a computer was then used to detect and record the red fluorescence upon excitation at a wavelength of 488 nm.

MTT assay
After transfection, 5000 cells/well were seeded in 96-well plates in media containing 10 % FBS and incubated for 0, 24 h, 48 h, 72 h. On the indicated days, 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-diphenytetrazoliumromide (MTT) (KyeGEN BioTECH, Nanjing, China) was added into each well according to the manufacturer's instructions, and the cells were incubated for 4 h at 37°C. The supernatants were then removed and 150 mL of DMSO (Sigma-Aldrich, Germany) was added to each well to dissolve the formazan crystals. Absorbance levels were measured at the wavelength of 490 nm using an automatic microplate reader (Gene, HK). The data derived from triplicate samples are presented as mean ± s.d.

Colony formation assay
After transfection, 500 cells were counted and seeded in 6-well plates. The plates were incubated for 10 days, and the cells were fixed by 4 % paraformaldehyde and stained using 0.1 % crystal violet. Colonies were counted only if they included 50 cells at least. Triplicate independent experiments were performed. www.impactjournals.com/oncotarget Cell invasion assay SMMC-7721 or HepG2 cells were transfected with pcDNA-lincRNA-PCAT-14 or si-lincRNA-PCAT-14 for 48 h, and seeded onto a synthetic basement membrane in the inset of a 24-well culture plate. In the invasion assay, polycarbonate filters coated with 50 μL Matrigel (1:9, BD Bioscience) were placed in a Transwell chamber (Costar). After incubation, filters were fixed and stained with 0.1% crystal violet solution. Non-invading cells were removed using a cotton swab, and invading cells on the underside of the filter were counted with an inverted microscope.

Tumorigenicity experiments in nude mice
Eighty male nude mice weighing 18 to 20 g, provided by Shanghai Laboratory Animal Center (Chinese Academy of Science, China), were bred under aseptic conditions; the animals were housed in an area with a constant humidity of 60-70% and a room temperature of 18-20°C. Animal maintenance, husbandry and experimental procedures were performed in accordance with the rules of China Medical University for the Use of Experimental Animals and approved by the Medical Animal Care and Use Committee of China Medical University (Shenyang, China). Mice were separated into four groups: pcDNA-lincRNA-PCAT-14 VS pcDNA-NC; si-lincRNA-PCAT-14 VS si-NC. The corresponding cell groups were administered by a subcutaneous injection (0.1 ml of a solution containing 1 × 10 4 cells/ml). Mice were examined every 5 days, and sacrificed 28 days after the initial subcutaneous injection. The tumors were resected and weighed.