The human CRC cell lines SW480, SW620, DLD-1, HT29, HCT116, Ls-174T and LOVO were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All CRC cell lines were cultured in RPMI 1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences, Logan, UT, USA) at 37°C in a humidified incubator containing 5% CO₂.

Fresh colorectal tumor tissue samples and adjacent non-tumor tissues were obtained from patients who were diagnosed with primary CRC from January to June in 2010. Elective surgery was carried out on these patients at Nanfang Hospital, Southern Medical University (Guangzhou, China). In total, 14 cases of fresh CRC tissue and adjacent non-tumor tissues were freshly frozen in liquid nitrogen and stored at −80°C until further use. The mean age of the patients was 60.1±12.4 years; 5 of the 14 patients (35.7%) were female and the remaining 9 were male (64.3%). According to the tumor size, lymph node and metastasis classification system (11), among the patients the following was observed: 2 patients (14.3%) were T stage 2 (T2), 9 patients (64.3%) were T3 and 3 patients (21.4%) were T4; 8 patients (57.1%) had lymph-node metastasis and 6 patients (42.9%) did not; 2 patients (14.3%) developed a distant metastasis while the remaining patients did not. The use of tissues for this study has been approved by the Ethics Committee of Nanfang Hospital, Southern Medical University. Prior to using these clinical materials for research purposes, all the patients signed an informed consent.

Total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was synthesized using the PrimeScript RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China). qPCR was performed with the SYBR Premix Ex Taq kit (Takara Biotechnology Co., Ltd.) on an ABI 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). 3-step qPCR was performed with the following thermocycling conditions: 95°C for 10 min, followed by 40 cycles of amplification (95°C for 5 sec, 60°C for 20 sec and 72°C for 34 sec) and then a dissociation stage profile (95°C for 15 sec, 60°C for 1 min and 95°C for 15 sec). GAPDH and U6 were used as an endogenous controls. Fold changes were calculated through the relative quantification 2^−ΔΔCq method (12). Primer sequences are listed in Table I. To account for technical variability, the assay was performed in triplicate for each sample.

Nuclear and cytosolic RNA isolation was performed as described previously (13), with some modifications. Briefly, cells were rinsed twice with ice-cold PBS centrifuged at 111.8 × g at 4°C for 5 min. Cell pellets were resuspended by gentle pipetting in 150 µl 0.1% NP-40 (dissolved in water), and incubated on ice for 5 min, followed by centrifugation at 5,000 × g at 4°C for 1 min. The resulting lysate was the cytosolic fraction of cells, which was added in TRIzol reagent for cytosolic RNA extraction. Following gentle rinsing and centrifugation at 111.8 × g at 4°C for 5 min twice, the cell pellets were resuspended by gentle pipetting in 75 µl 0.1% NP-40 and incubated on ice for 5 min. TRIzol reagent was then added to the lysate for nuclear RNA purification.

HCT116 and HT29 cells were seeded into six-well plates. The sequence of the small interfering RNA (siRNA) to knockdown HNF1A-AS1 was: 5′-CACCUGCAUUCAAACUCGGACUGUU-3′. The target sequence of siRNA-HNF1A-AS1 was: 5′-CACCTGCATTCAAACTCGGACTGTT-3′. The sequence of the negative control siRNA was: 5′-UUGUACUACACAAAAGUACUG-3′. HNF1A-AS1 siRNA and negative control siRNA were transfected (0.1 nmol siRNA/well) into HCT116 and HT29 cells using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Cells were harvested 48 h post-transfection for RT-qPCR and functional experiments.

Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc., Rockville, MD, USA) was used in order to evaluate the rate of cell proliferation, according to manufacturer's instructions. Briefly, log-phase cells were trypsinized into a single-cell suspension and plated into 96-well plates at a density of 1,000 cells/well. CCK-8 solution was added to each well. After 2 h, the absorbance of each well was measured at 450 nm using a microplate reader (Enspire 2300 Multimode plate reader; PerkinElmer, Inc., Waltham, MA, USA).

Experiments were performed based on those previously described (14). The cells were plated in 6-well plates at 2×10² cells/well and maintained in RPMI 1640 containing 10% FBS for 2 weeks. After 2 weeks, the cells were washed twice with PBS, fixed with methanol and stained with Giemsa. The number of colonies with diameter >150 µm was counted using Image Pro Plus v6.0 (Media Cybernetics, Inc., Rockville, MD, USA); whole plates were counted and 3 replicate plates were performed per sample. A total of 3 independent experiments were performed.

Cell motility was measured using a wound healing assay by measuring the movement of cells in a scraped, acellular area created by a 10 µl pipette tube. Wound closure was observed at 0 and 48 h. Photographs were captured in order to assess the level of migration in each group of transfected cells. The migration was quantified by counting the total number of cells that migrated toward the original wound field. A total of 5 random fields were measured per plate, 3 plates per sample, and 3 independent experiments were performed using Image Pro Plus v6.0 (Media Cybernetics Corporation, USA) for analysis.

For invasion assay, Matrigel-coated chambers (BD Biosciences, San Jose, CA, USA) containing 8-µm pores were used. Cells were seeded in the upper chambers (coated in Matrigel) at 1×10⁵ concentration in serum-free medium. The lower chamber of the transwells was filled with culture media containing 10% FBS as a chemoattractant. After incubation at 37°C for 48 h, non-invaded cells on the top of the transwells were scraped off with a cotton swab. The successfully invaded cells at the bottom of the transwells were fixed with methanol and stained with Giemsa for 15 min, prior to counting under a light microscope. A total of 5 random fields were measured per plate, 3 plates per samples, and 3 independent experiments were performed.

Cells were seeded at a density of 1×10⁶ cells/well in six-well plates. After 48 h, cells were washed with PBS and then treated with a Cell Cycle Staining kit [MultiSciences (Lianke) Biotech Co., Ltd., Hangzhou, China] for 30 min at 4°C in the dark. The cell cycle profiles were analyzed using a Elite ESP flow cytometer (Beckman Coulter, Inc., Brea, CA, USA) at 488 nm, and data were analyzed with the CellQuest software v3.1 (BD Biosciences).

All statistical analyses were performed using the SPSS v16.0 statistical software package (SPSS, Inc., Chicago, IL, USA). Data were presented as mean ± standard error of the mean from at least three independent experiments. The differences between variables were assessed using the following statistical tests: t-test, factorial analysis, χ²-test, Fisher's exact test, or one-way analysis of variance. Multiple comparisons between groups were performed using the Student-Newman-Keuls post hoc test. P<0.05 was considered to indicate a statistically significant difference.

As a first step towards understanding the role of lncRNA HNF1A-AS1 in CRC, the expression of HNF1A-AS1 was examined in CRC tissues. RT-qPCR was performed to detect the expression of HNF1A-AS1 in a total of 14 paired clinical CRC tissues and adjacent non-tumor tissues. The results revealed that HNF1A-AS1 expression levels in tumors were significantly higher compared with the paired normal tissues (Fig. 1A). Next, the association between HNF1A-AS1 expression and the lymph-node metastasis status of the CRC samples was examined. The results revealed that upregulation of HNF1A-AS1 was significantly associated with lymph-node metastasis of CRC (Fig. 1B). Taken together, these results suggested that HNF1A-AS1 might have an important role in CRC pathogenesis and progression.

Since HNF1A-AS1 was highly expressed in CRC tissues, the role of HNF1A-AS1 was further examined in CRC cell lines. RT-qPCR was used to examine the expression levels of HNF1A-AS1 in seven CRC cell lines (SW480, SW620, DLD-1, HT29, HCT116, Ls-174T and LOVO). The results demonstrated that the HCT116 and HT29 cell lines displayed the highest expression levels of HNF1A-AS1 (Fig. 1C). In order to determine the subcellular localization of HNF1A-AS1, a nuclear/cytosolic fractionation was performed on HT116 and HT29 cells, followed by RT-qPCR. The differential enrichment of GAPDH and U6 RNAs were used as fractionation indicators for the cytosolic and nuclear extracts respectively. The results revealed markedly higher levels of HNF1A-AS1 in the nucleus compared with the cytosol in both cell lines tested (Fig. 1D), which indicated that HNF1A-AS1 was mainly localized in the nuclear fraction of CRC cells.

To further investigate the impact of HNF1A-AS1 on CRC cell biological behaviors, specific siRNA was employed to knockdown HNF1A-AS1 expression in CRC cells. According to endogenous HNF1A-AS1 expression in the CRC cell lines mentioned above, the HCT116 and HT29 cell lines were selected for HNF1A-AS1 knockdown by RNA interference. RT-qPCR analysis revealed that HNF1A-AS1 expression was downregulated by 62.13% in HCT116 cells and by 52.18% in HT29 cells following specific siRNA transfection compared with negative control siRNA transfection (Fig. 1E). Growth curves determined by CCK-8 assay indicated that cell proliferation was reduced following HNF1A-AS1 knockdown in HCT116 and HT29 cells (Fig. 2A and B). Notably, the results from the colony formation assay also demonstrated that decreased HNF1A-AS1 expression resulted in a decreased colony formation ability in HCT116 and HT29 cells (Fig. 2C).

The effect of HNF1A-AS1 on cell migration and invasion was next assessed in vitro. The migration ability of HCT116 and HT29 cells was determined by wound healing assay. The results demonstrated that knockdown of HNF1A-AS1 significantly inhibited HCT116 and HT29 cell migration compared with the negative control cells (Fig. 2D). Furthermore, Matrigel invasion assay revealed that knockdown of HNF1A-AS1 inhibited the invasion ability of HCT116 cells by 74.62% and of HT29 cells by 57.23% compared with the negative control cells (Fig. 2E). These results indicated that the downregulation of HNF1A-AS1 expression effectively reduced both migration and invasion of CRC cells in vitro.

To further examine the role of HNF1A-AS1 in CRC, the relationship between HNF1A-AS1 knockdown and cell cycle progression was assessed. Cell cycle phase distribution was evaluated in HCT116 and HT29 cells by flow cytometry. The results demonstrated that knockdown of HNF1A-AS1 resulted in a significant increase of the % of cells at G0/G1 phase, but a significant decrease in the % of cells in the S phase of the cell cycle (Fig. 3).