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lncRNA KCNQ1OT1 knockdown inhibits colorectal cancer cell proliferation, migration and invasiveness via the PI3K/AKT pathway

Colorectal cancer (CRC) is one of the most common primary malignancies worldwide. Numerous studies have demonstrated that long non-coding RNAs (lncRNAs) are considered as crucial regulators of tumor progression. In particular, upregulation of the lncRNA KCNQ1OT1 was reported in various types of malignancy as a promoter of tumor progression. However, the role and underlying mechanism of KCNQ1OT1 in CRC remain unclear. Thus, the present study aimed to investigate the role of KCNQ1OT1 in colorectal cancer through GEPIA, reverse transcription-quantitative PCR (RT-qPCR) and western blot analyses, and cell assays. GEPIA analysis demonstrated that high expression levels of KCNQ1OT1 in CRC tissues predicted a poor prognosis for patients with CRC. KCNQ1OT1 was overexpressed in CRC tissues and cell lines via RT-qPCR analysis. Furthermore, the results from the cell viability assay, colony formation assay, wound healing assay, invasion assay and flow cytometric analysis demonstrated that KCNQ1OT1 knockdown significantly inhibited CRC cell proliferation, migration and invasiveness, and promoted CRC cell apoptosis, leading to cell cycle arrest. Western blot analysis demonstrated that KCNQ1OT1 knockdown inhibited the PI3K/AKT signaling pathway. These results suggest that KCNQ1OT1 may act as an oncogene through the PI3K/AKT signaling pathway in CRC.


Introduction
Colorectal cancer (CRC) is the third most diagnosed cancer worldwide with a high mortality rate, which reported 1.4 million new cases and 693,900 mortalities in 2012 (1). Based on the thorough understanding of the CRC pathological process, immunotherapy and targeted therapy have emerged and have greatly improved the prognosis of patients with CRC (2). However, because the occurrence and development of CRC is a complex process caused by numerous factors, the prognosis of patients with advanced-stage CRC remains unsatisfactory (3). It is therefore crucial to identify the underlying mechanisms of CRC tumorigenesis, in order to develop novel therapeutic strategies.
The human genome consists of 2% genes coding for proteins and several non-coding RNAs (ncRNAs) (4). Long non-coding (lnc)RNAs are a type of ncRNAs that are defined as transcripts with lengths >200 nucleotides, and that are not translated into proteins (5,6). Although lncRNAs were previously considered as transcriptional noise, previous studies have reported that lncRNAs serve crucial roles in the regulation of biological processes involved in numerous diseases, including various types of cancer (6,7). Previous studies have reported that the lncRNA KCNQ1OT1 is involved in the progression of multiple tumors, including tongue cancer, hepatocellular carcinoma, and breast cancer (8)(9)(10). However, the underlying mechanisms of KCNQ1OT1 in CRC development and metastasis remain unknown.
The present study investigated KCNQ1OT1 expression in CRC tissues and its association with the prognosis of patients with CRC. Furthermore, the role of KCNQ1OT1 in CRC cell proliferation, migratory and invasive abilities, apoptosis and the cell cycle was also analyzed. The underlying mechanisms of KCNQ1OT1 in CRC cells were subsequently investigated, which may help to provide a new therapeutic and prognostic method for colorectal cancer.  (12). Following submission of an analysis request, GEPIA can provide the visual image results for users (12). The gene symbol KCNQ1OT1 was submitted on GEPIA for differential expression analysis between data from CRC and normal tissues, and provided results consisting of box plots, stage plots and survival analysis.

Materials and methods
Cell culture. The human CRC cell lines RKO, SW620, colo320, HCT116 and LoVo, and the normal colon epithelial cell line NCM460 were obtained from the Southern Medical university Corporation. All cells were maintained in RPMI-1640 (HyClone; GE Healthcare Life Sciences) containing 10% FBS (Serana Europe GmbH) and placed at 37˚C in a humidified incubator containing 5% CO 2 . PI3K inhibitor was purchased from Selleck Chemicals. The stably transfected SW620 and RKO cells were treated with 10 µM PI3K inhibitor (LY294002) for 24 h (13) to suppress the PI3K/AKT signaling pathway when the density of cells reached 50%.
RNA extraction and reverse transcription-quantitative (RT-q) PCR. Total RNA was extracted from the primary tissues and cell lines using TRIzol™ reagent (Takara Bio, Inc.), and reverse transcribed into cDNA with the PrimeScript RT Kit (Takara Bio, Inc.) according to the manufacturers' instructions. RT-qPCR reactions were performed as follows: 37˚C for 15 min, followed by 85˚C for 5 sec. qPCR was performed using the TB Green premix (Takara Bio, Inc.) in a CFx96 real-time PCR detection system (Bio-Rad Laboratories, Inc.). The sequences of the primers (Sangon Biotech, Co., Ltd.) were as follows: KCNQ1OT1 forward, 5'-CAA GCA GCC AGA AGG ATG AGA AGG-3' and reverse 5'-GGT CAG CAC CAG AAG GCA GAA TG-3'; and GAPDH forward, 5'-TGC ACC ACC AAC TGC TTA GC-3' and reverse 5'-GGC ATG CAC TGT GGT CAT GAG-3'. The relative expression level of KCNQ1OT1 normalized to endogenous control and expressed as 2 -ΔΔCq (14).
Cell viability assay. The stably transfected SW620 and RKO cells were seeded into 96-well plates at the density of 2x10 3 cells per well. Cell viability was assessed every 24 h for 5 days using the Cell Counting-Kit 8 reagent (CCK-8; Beijing Transgen Biotech Co., Ltd.). Briefly, cells were incubated with 10 ul CCK-8 solution for 2 h and absorbance was read at 450 nm on a microplate reader.
Colony formation assay. The stably transfected cells were seeded in 6-well plates at a density of 2x10 3 cells/well and cultured at 37˚C for 12-14 days. Once the colonies were visible, cells were washed twice with PBS, and colonies were fixed with 5% paraformaldehyde for 20 min at room temperature and stained with 0.1% crystal violet (1 mg/ml) for 20 min. The stained colonies were counted, and the colony formation rate was calculated using ImageJ 8.0 software (National Institutes of Health).
Wound healing assay. The stably transfected cells were seeded into 6-well plates at a density of 5x10 5 cells/well and cultured until they had reached 90% confluence. The complete medium was replaced with serum-free medium and the cell monolayer was scratched with a sterile 100-µl pipette tip. After further culture for 48 h, the wound gap was observed under an inverted light microscope (magnification x100). The extent of wound healing was calculated using the ImageJ 8.0 software. The wound healing percentage was calculated using the following formula: (Scratch healing area)/(original scratch area) x100%.
Invasion assays. The stably transfected cells were suspended in serum-free medium at a density of 2x10 5 cells/200 µl and seeded into the Matrigel-coated upper chamber (8-µm pore size) of Transwell inserts in 24-well plates at 37˚C for 1 h. the lower chamber was filled with 600 µl complete medium. Following 48 h of culture, cells remaining on the upper surface of the filter were swabbed off using cotton wool, and cells that had invaded the lower surface were fixed with 5% paraformaldehyde for 20 min at room temperature and stained with Giemsa for 10 min. Cells were observed under an inverted light microscope (magnification x200), and the number of cells per field was calculated using ImageJ 8.0 software.
Cell cycle and apoptosis analysis. The stably transfected SW620 and RKO cells were trypsinized (without EDTA) and washed with PBS. For cell cycle analysis, 10 5 cells/100 µl cells were fixed with 70% ethanol at 4˚C for 12 h and resuspended in staining buffer containing 450 µl propidium iodide (PI) and 50 µl RNaseA in the dark for 30 min at room temperature (cat. no. KGA512; Nanjing KeyGen Biotech Co., Ltd.) For apoptosis analysis, the Annexin V APC/7AAD apoptosis kit (cat. no. KGF004; Nanjing KeyGen Biotech Co., Ltd.) was used to stain for early and late apoptotic cells according to the manufacturers' instructions. Subsequently, stained cells were analyzed using a BD FACS-Verse flow cytometer (BD Biosciences). Cell cycle distribution was obtained using ModFit 3.2 software (Verity Software House, Inc.), and the apoptosis rate was calculated using FlowJo 7.6.1 software (FlowJo LLC).
Statistical analysis. All statistical analyses were performed using GraphPad Prism 7.0 (GraphPad Software, Inc.). Data were presented as the means ± standard deviation of at least three independent experiments. Differences between two groups were assessed using the t-test, whereas one-way ANOVA, followed by Newman-Keuls post-hoc test were used to compare differences between multiple groups (>2). P<0.05 was considered to indicate a statistically significant difference.

KCNQ1OT1 expression is upregulated in CRC tissues and is associated with disease progression and poor survival in patients with CRC.
Previous studies have reported that KCNQ1OT1 expression is upregulated in various types of tumor, including hepatocellular carcinoma, non-small cell lung cancer and tongue cancer (8,9,15), and that this is correlated with poor prognosis in patients. In the present study, GEPIA was used to predict KCNQ1OT1 expression in CRC. The results from box plot analysis in GEPIA demonstrated that KCNQ1OT1 expression was significantly higher in tumor tissues compared with normal tissues in patients with COAD and READ (P<0.05; Fig. 1A). The results from stage plot analysis in GEPIA demonstrated that KCNQ1OT1 expression increased with tumor stage progression in patients with CRC (P= 0.0287; Fig. 1B). Furthermore, results from survival analysis in GEPIA demonstrated that high KCNQ1OT1 expression in CRC tissues was associated with lower overall and disease-free survival times (Fig. 1C and D). These predicted results suggest that KCNQ1OT1 expression may be upregulated in CRC tissues and may be associated with poor prognosis in patients with CRC.
Subsequently, 28 pairs of CRC and adjacent normal tissues were collected from patients with CRC, and KCNQ1OT1 expression was examined by RT-qPCR. The results demonstrated that the KCNQ1OT1 expression level was significantly higher in CRC tissues compared with adjacent normal tissues (P= 0.0142; Fig 1E). Furthermore, KCNQ1OT1 expression level was significantly higher in CRC tissues obtained from large tumors (≥5 cm) compared with those obtained from small tumors (<5 cm; P= 0.0302; Fig. 1F). In addition, the KCNQ1OT1 expression level in CRC tissues obtained from patients with advanced stage CRC (IIIB+IIIC+IV) was significantly increased compared with those obtained from patients with limited stage disease (I+II+IIIA; P= 0.0372; Fig. 1G). These findings indicate that KCNQ1OT1 may be associated with tumor growth and metastasis in CRC.

KCNQ1OT1 knockdown inhibits CRC cell proliferation in vitro.
To investigate the role of KCNQ1OT1 in CRC growth, in vitro experiments were performed. Firstly, KCNQ1OT1 expression was assessed by RT-qPCR in five CRC cell lines and the normal NCM460 intestinal epithelial cell line. The results demonstrate that KCNQ1OT1 expression level was upregulated in four out of five CRC cell lines compared with the normal cell line ( Fig. 2A). SW620 and RKO cells were subsequently selected to construct stably transfected cell lines using two KCNQ1OT1-specifc shRNAs lentivirus or negative control lentivirus. The results from RT-qPCR demonstrate that KCNQ1OT1 expression level was efficiently knocked down in the two cell lines following transfection (Fig. 2B). Subsequently, cell viability and colony formation assays were performed to investigate the effect of KCNQ1OT1 on CRC cell proliferation in vitro. The results demonstrate that KCNQ1OT1 knockdown significantly decreased SW620 and RKO cell viability (Fig. 2C and D). Furthermore, the results of the colony formation assay demonstrated that KCNQ1OT1 knockdown significantly inhibited clone formation capacity in SW620 and RKO cells (Fig. 2E). These findings confirm that KCNQ1OT1 knockdown inhibits CRC cell proliferation.

KCNQ1OT1 knockdown stimulates CRC cell apoptosis and cell cycle arrest.
A previous study reported that abnormal cell cycle and apoptosis are associated with cancer growth (16). To better understand the underlying mechanism of KCNQ1OT1 in CRC cell proliferation, apoptosis and cell cycle distribution were evaluated in the present study. The results demonstrated that the apoptotic rates of KCNQ1OT1 knockdown SW620 and RKO cells were significantly increased compared with the control cells ( Fig. 3A and B). Furthermore, as presented in Fig. 4A and B, the expression of the pro-apoptotic protein BAx and of the anti-apoptotic protein BCL-2, in KCNQ1OT1 knockdown CRC cells was significantly increased and decreased, respectively, compared with control group. The results from western blotting are therefore consistent with results from apoptosis detection by flow cytometry. In addition, the results from cell cycle analysis demonstrated that KCNQ1OT1 knockdown significantly increased the percentage of cells in the G 0 /G 1 phase and decreased the percentage of cells in the S phase in SW620 and RKO cell lines, compared with control cells (Fig. 3C and D). These findings suggest that downregulation of KCNQ1OT1 in CRC cells may induce apoptosis and arrest the cell cycle at the G 0 /G 1 phase.

KCNQ1OT1 knockdown inhibits CRC cell migratory and invasive abilities.
In order to investigate the biological function of KCNQ1OT1 in CRC cell invasiveness, a Transwell assay was performed. The results demonstrate that the invasive ability of KCNQ1OT1 knockdown cells was significantly decreased compared with control cells (Fig. 5A). Similarly, the results from the wound healing assay demonstrated that silencing KCNQ1OT1 in SW620 or RKO cells significantly decreased the scratch healing ability (Fig. 5B). These results indicate that KCNQ1OT1 knockdown may decrease CRC cell invasion in vitro.
KCNQ1OT1 knockdown inhibits the activation of the PI3K/AKT signaling pathway. Previous studies have reported that the PI3K/AKT pathway is one of the most commonly altered pathways in human cancer that is associated with the regulation of tumor growth and metastasis (17,18). In order to identify the underlying mechanism of KCNQ1OT1 in CRC progression, the PI3K/AKT pathway was investigated. To do so, the expression of proteins from the PI3K/AKT pathway was analyzed by western blotting in CRC cells transfected with sh-KCNQ1OT1 or sh-nc. The results demonstrate that KCNQ1OT1 knockdown inactivates the PI3K/AKT pathway, as evidenced by the decreased expression of p-PI3K and p-AKT in SW620 and RKO cell lines ( Fig. 6A and B). In addition, following CRC cell treatment with a PI3K inhibitor, the level of p-PI3K and p-AKT was decreased compared with non-treated cells (Fig. 6C and D). The inhibition experiment was successful, confirming that KCNQ1OT1 inhibited the proliferation and invasion of colorectal cancer by inhibiting the PI3K signaling pathway. These data suggest that KCNQ1OT1 knockdown may inhibit PI3K/AKT signaling pathway activation.

Discussion
Numerous studies have demonstrated that lncRNAs serve crucial roles in malignant tumor progression, including CRC (5,19). For example, Chen et al (20) reported that lncRNA XIST is significantly overexpressed in tumors from patients with CRC and is associated with poor overall survival. Furthermore, it was demonstrated that xIST can regulate CRC progression and metastasis by competing with the micro-RNA miR-200b for the regulation of zinc finger E-box binding homeobox 1 expression (20). In addition, Han et al (21) reported that lncRNA CRNDE could promote chemoresistance and CRC cell proliferation by regulating the expression levels of miR-181a-5p and the activity of the Wnt/β-catenin signaling pathway. A previous study reported that β-catenin could directly target KCNQ1OT1 to promote CRC development (22). However, the role and underlying mechanism of KCNQ1OT1 in CRC remains unclear.
The present study demonstrated that KCNQ1OT1 is highly expressed in CRC tissues compared with normal tissues, and that high KCNQ1OT1 expression in CRC tissues is associated with poor survival according to GEPIA predictions. These results were similar to those observed in 28 CRC tissue samples, in which KCNQ1OT1 was upregulated compared with adjacent normal tissues. Furthermore, KCNQ1OT1 expression was significantly associated with tumor size (<5 cm vs. ≥5 cm) and clinical stage (limited vs. advanced). These results suggest that high KCNQ1OT1 expression may be associated with the malignant progression of CRC. Previous research demonstrates that KCNQ1OT1 overexpression serves a crucial role in various types of tumor. For example, it was reported that KCNQ1OT1 is upregulated in chemoresistant tongue squamous cell carcinoma tissues compared with chemosensitive tissues, and that high KNCQ1OT1 expression is positively correlated with poor prognosis in patients (8). Another study demonstrated that KCNQ1OT1 is highly expressed and promotes tumor growth and metastasis in cholangiocarcinoma via miR-140-5p/SRY-box transcription factor 4 axis (23). It was demonstrated that KCNQ1OT1 is upregulated in patients with hepatocellular carcinoma (9), breast cancer (10), non-small-cell lung cancer (15) and glioma (24), and that it promotes malignant tumor progression. The results from these studies indicate that KCNQ1OT1 may serve an oncogenic role in various types of cancer.
The present study demonstrated that KCNQ1OT1 knockdown inhibited the proliferation and migratory and invasive abilities of CRC cells, which was consistent with previous studies on cholangiocarcinoma and hepatocellular carcinoma (9,23). Numerous studies have reported that the cell cycle and apoptosis are associated with cancer cell proliferation and tumor growth (16,25,26). Subsequently, KCNQ1OT1 was knocked down in the present study and the cell cycle and apoptosis were evaluated. The results demonstrated that KCNQ1OT1 knockdown promoted apoptosis and cell cycle arrest in CRC cells. These findings indicate that KCNQ1OT1 may regulate CRC cell proliferation, migratory and invasive abilities, cell cycle and apoptosis in order to promote tumor growth and metastasis.
The PI3K/AKT signaling pathway serves an essential role in the growth and metastasis of multiple tumors. For example, lncRNA ABHD11-AS1 promotes tumor progression by regulating PI3K/AKT signaling in papillary thyroid carcinoma (27). In addition, lncRNA SNHG7 facilitates CRC cell proliferation and metastasis via the PI3K/AKT/mTOR signaling pathway, and by regulating GALNT7 expression via miR-34a sponging (28). Following PI3K/AKT pathway inactivation, G 1 /S transition, proliferation and migratory and invasive abilities of cancer cells are inhibited, whereas apoptosis is stimulated (17,28). The results from the present study demonstrate that KCNQ1OT1 knockdown decreases the protein expression of p-PI3K and p-AKT. These findings suggest that CRC cell proliferation and metastasis may be regulated by KCNQ1OT1 via the PI3K/AKT signaling pathway.
In conclusion, the present study demonstrated that KCNQ1OT1 expression was significantly increased in CRC cells and tissues compared with normal cells and tissues. Furthermore, KCNQ1OT1 knockdown inhibited CRC cell proliferation and migratory and invasive ability, promoted CRC cell apoptosis and arrested the cell cycle via PI3K/AKT signal pathway inactivation. These findings indicate that KCNQ1OT1 may be considered as a potential prognostic marker and therapeutic target for CRC. However, the study had several limitations. For example, the effect of KCNQ1OT1 overexpression in CRC cells in vitro or its function in vivo, were not investigated. Future studies will further explore the underlying mechanisms of KCNQ1OT1 in CRC, in particular, its potential role as a competitive endogenous RNA.