RETRACTED ARTICLE: The biological function of long noncoding RNA FAL1 in oesophageal carcinoma cells

Abstract We, the Editors and Publisher of the journal Artificial Cells, Nanomedicine, and Biotechnology, have retracted the following article: Zhiguo Yang, Dongyan Liu, Dan Wu, Fang Liu & Cuiping Liu (2019) The biological function of long noncoding RNA FAL1 in oesophageal carcinoma cells. Artificial Cells, Nanomedicine, and Biotechnology, 47:1, 896–903, DOI: 10.1080/21691401.2019.1573738 It has come to our attention that the full authorship list and affiliations for this manuscript, including the study site and ethics committee, were changed after the article was submitted. We have contacted the corresponding author for an explanation, but we have not received a satisfactory explanation. As determining authorship and the location of where the research was conducted is core to the integrity of published work, we are therefore retracting the article. The corresponding author listed in this publication has been informed. We have been informed in our decision-making by our policy on publishing ethics and integrity and the COPE guidelines on retractions. The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.


Introduction
Oesophageal cancer is one of the most prevalent cancer type and a leading lethal cancer worldwide [1]. Oesophageal squamous cell carcinoma (OSCC) and oesophageal adenocarcinoma (EAC) are the two main types of oesophageal cancers [2]. OSCC is the predominant form of oesophageal cancer in China with a high incident rate [3]. Radiochemotherapy and surgical resection have been used to treat OSCC in the past decades, however, the prognosis remains disappointing due to the late diagnosis and rapid progression of the disease [4]. Clinical studies have shown that hundreds of gene expression changes have been associated with disease metastasis and patient survival during the progression of oesophageal cancer [5]. Additionally, in vitro and animal studies also demonstrate that the progressive accumulation of genetic changes plays a causal role in regulating cell proliferation, metastasis and rapid progression [6]. Therefore, a better understanding of the genetic and molecular mechanism and identification of new biomarkers and therapeutic targets of OSCC development are beneficial for the disease diagnosis and treatment.
The human genome contains a huge number of non-coding transcripts, long non-coding RNAs (lncRNAs) refer to RNA transcripts with larger than 200 nucleotides (nts), but lack of protein-coding capacity [7]. LncRNAs participate in plenty of fundamental biological processes, such as inflammation, cell proliferation, immune response and cancer [8]. Aberrant expression of lncRNAs has been associated with the development and pathological progression of oesophageal cancers [9]. The lncRNA focally amplified lncRNA on chromosome 1 (FAL1) has displayed striking oncogenic activity in multiple functional experiments. Increased expression of FAL1 has been found in several types of cancers, including papillary thyroid cancer (PTC). Upregulation of FAL1 promoted the expression of cell cycle-related proteins such as the E2F transcription factors 1 and 2, and cyclin D1 [10]. Increased expression and activity of FAL1 result in the repression of p21 expression in different human cancers such as ovarian cancers [11,12]. However, little information regarding the biological roles of FAL1 in oesophageal cancer has been reported before. In the present study, we evaluated FAL1 expression in oesophageal cancer and investigated the underlying molecular mechanisms.

Clinical specimens
Experimental protocol in the current study was approved by the ethics committee of Heze municipal hospital. All the participants have signed written informed consent. All of the 88 paired tumour and the corresponding non-tumour tissue samples were provided by the second affiliated hospital of Heze municipal hospital. Samples were immediately frozen in liquid nitrogen and stored at À80 C after resection. All of the clinical characteristics of the patients have been recorded and analyzed. Six OSCC cell lines (EC109, Eca9706, KYSE450, KYSE150, TE-1 and TE-2) and the human normal oesophageal epithelium cell line HEEpiC were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were maintained in RPMI-1640 medium (Hyclone, USA) supplemented with 10% foetal bovine serum (FBS) [13]. To knock down the expression of FAL1, TE-1 and Eca9706 cells were transfected with either siRNAs targeting FAL1 (siFAL1) or scrambled negative controls (NS) for 24 h using Lipofectamine 2000 (Life technologies, USA). To overexpress FAL1, TE-1 and Eca9706 cells were transducted with lentiviral-FAL1 (LV-FAL1) or empty vector (LV-vector) using polybrene (Life technologies, USA).

Cell cycle analysis
The cell cycle of TE-1 and Eca9706 cells was analyzed using flow cytometric analysis [14]. After necessary transfection, cells were harvested and washed twice with PBS, followed by fixation with 70% ethanol in a cold room for 24 h. Then cells were stained with propidium iodide using the cell cycle and apoptosis analysis kit (Beyotime, China). Cell cycle distribution was then analyzed on a FACS Calibur Cytometer (BD Biosciences, USA).

Cell invasion assays
Patterns of cell invasion were determined by matrigel with transwell inserts with an 8.0 mm pore size polyethylene tetraphthalate membrane. Cells were seeded in the upper chamber containing 200 ll of serum-free DMEM. The lower chambers contained RPMI1640 supplemented with 10% FBS. 24 h later, cells that invaded into the lower side of the membrane were fixed with methanol, stained with 0.1% crystal violet, and photographed with a digital microscope.

Cell proliferation assay
After necessary transfection or transduction, a commercial cell counting kit-8 (CCK-8) was used to determine cell proliferation of TE-1 and Eca9706 cells. Briefly, TE-1 and Eca9706 cells were seeded in 96-well plates at the density of 3 Â 10 3 per well and incubated for 1, 2 and 3 days. The CCK-8 reagent was added and incubated for 1 h. OD value was recorded at 450 nm.

Real-time polymerase chain reaction (PCR)
Total RNA was extracted from TE-1 and Eca9706 cells using the Trizol reagent (Life technologies, USA). Abundance of intracellular RNA was determined by using a NanoDrop apparatus. Intracellular RNA was reverse transcribed into cDNA with the iScript cDNA synthesis kit (Bio-Rad, USA).
Expression levels of target genes were analyzed with realtime PCR on an ABI7500 real-time PCR system with the SYBR Green qPCR Master Mix (Thermo fisher scientific, USA). Expressions of target gene were normalized to GAPDH using the 2 ÀDDCt method [15].

Western blot analysis
Protein was extracted from TE-1 and Eca9706 cells using RIPA buffer (Bio-Rad, USA) containing the cocktail protease inhibitor. Protein concentration was determined by a BCA assay. Extracted proteins were denatured by boiling at 100 C for 5 min. Samples were run on an electrophoresis using 4-12% Bis-Tris gels (Novex, USA) and blotted onto polyvinyldiene fluoride (PVDF) membranes using the iBlot transfer system (Life technologies, USA). Membranes were then blocked with blocking solution (Roche Applied Science, USA) [16]. Blots were sequentially incubated with primary antibodies and horseradish peroxidase (HRP)-linked anti-rabbit (7074) or antimouse (7076) secondary antibodies (Cell Signaling Technology, USA). Blots were detected using chemiluminescence (Sigma-aldrich, USA). The following antibodies were used in this study: Rabbit monoclonal antibody (mAb) against cyclin D1 (1: 2000

Statistical analysis
Experimental results are expressed as mean ± SEM. Statistical analysis was performed using student t-test or one-way or two-way analysis of variance (ANOVA) where appropriate. pvalues of less than .05 was considered statistically significant.

Results
Firstly, we detected the level of FAL1 in 88-paired OSCC tissues and adjacent normal tissues by real-time PCR. Results in Figure 1(A) indicate that FAL1 expression was significantly higher in OSCC tissues than adjacent normal tissues. We then investigated the relationship between FAL1 expression and the clinic-pathological progression of OSCC patients. We found that the expression of FAL1 in patients with advanced TNM stage (III/IV) was significantly higher than the levels of which in patients with local TNM stage (I/II) (Figure 1(B)). Additionally, the expression of FAL1 in patients with positive lymphy node metastasis was significantly higher than those in patients with negative lymph node metastasis ( Figure 1(C)). The influence of FAL1 expression on clinical outcomes in OSCC patients was analyzed using the Kaplan-Meier analysis. Results in Figure 2 indicate that the survival time for patients with low FAL1 expression was significantly longer than patients with high FAL1 expression. These results suggested that high FAL1 expression predicted a poor prognosis in OSCC patients. We next compared the expression level of FAL1 in six OSCC cell lines (EC109, Eca9706, KYSE450, KYSE150, TE-1 and TE-2) and human normal oesophageal epithelium cell line HEEpiCs by real-time PCR analysis. Results in Figure 3 indicate the results.
OSCC cells displayed a significantly higher FAL1 expression status, especially in TE-1 and Eca9706 cell lines (Figure 3). To assess the biological functions of FAL1 in OSCC, the expression of FAL1 was knocked down by transfection with FAL1 siRNA in both TE-1 and Eca9706 cell lines. Cell growth was evaluated using the CCK-8 assay. Results in Figure 4(A) and Figure 4(B) indicate that silencing of FAL1 resulted in a significant decrease in cell growth relative to negative control at day 4 in both cell lines, respectively. In contrast, overexpression of FAL1 in TE-1 and Eca9706 cell lines significantly promoted cell growth ( Figure 5(A,B)).
We then set out to investigate the effects of FAL1 in the cell cycle. Cells were synchronized at G0/G1 stage by serum starvation (SS) for 24 h. The arrested cells were re-stimulated to enter S phase [17]. Cell cycle was monitored by flow cytometric analysis. Importantly, we found that serum starvationcaused arrest at G0/G1 stage resulted in a significant decrease in the expression of FAL1. As expected, re-enter of S stage restored the reduction of FAL1 caused by serum starvation in both the TE-1 and Eca9706 cells (Figure 6 (A, B)). These results suggested that FAL1 might play an important role in regulating the cell cycle. Results in Figure 7(A,B) indicate that knockdown of FAL1 resulted in a significant arrest of cells at G0/G1 phase and a significant decrease in S phase in both the TE-1 and Eca9706 cells. In contrast, overexpression of FAL1 caused a significant increase of cells in S-phase but a decrease of cells in G0/G1 phase in both TE-1 ( Figure  7(C)) and Eca9706 cells (Figure 7(D)). In addition, the roles of FAL1 on cell cycle protein expressions were then evaluated. Similarly, western blot analysis showed that knockdown of FAL1 significantly decreased the expression of cycline D1, cyclin E and c-Myc in both the TE-1 (Figure 8(A)) and Eca9706 cells (Figure 8(B)). In contrast, overexpression of FAL1 increased the expression of these cell cycle proteins in both the TE-1 (Figure 8(C)) and Eca9706 cells (Figure 8(D)).
We then investigated the effects of FAL1 on invasiveness of OSCC cells. Knockdown of FAL1 significantly reduced the

R E T R A C T E D
invasion of TE-1 (Figure 9(A)) and Eca9706 cells (Figure 9(B)).
In contrast, overexpression of FAL1 stimulated the invasion of TE-1 (Figure 9(C)) and Eca9706 cells (Figure 9(D)). Epithelialmesenchymal transition (EMT) is the remarkable presentation of cell invasion and tumour metastasis. In the physiological process of EMT, cells sacrifice their cell epithelial properties, acquire mesenchymal capacities, and enhance the ability of migration and invasion. Decreased level of the epithelial marker ZO-1 and increased expression of the mesenchymal marker N-cadherin have been associated with tumour metastasis. Therefore, the expressions of EMT markers ZO-1 and Ncadherin were evaluated. Western blot analysis displayed that knockdown of FAL1 obviously elevated the levels of ZO-1, but reduced the levels of N-cadherin in both TE-1 ( Figure 10(A)) and Eca9706 cells (Figure 10(B)). In contrast, overexpression of FAL1 reduced the levels of ZO-1, but increased the levels of N-cadherin in both TE-1 (Figure 10(C)) and Eca9706 cells (Figure 10(D)). These findings suggest that FAL1 might have an impact on OSCC cells metastasis by regulating EMT. The level of PTEN and its downstream AKT activity have been associated with EMT. Therefore, we investigated the effects of FAL1 on the expression of PTEN and ATK phophorylation. Interestingly, we found that knockdown of FAL1 increased the expression of PTEN and reduced the phosphorylation of AKT in both TE-1 (Figure 11(A)) and Eca9706 cells (Figure 11(B)). However, the total level of AKT remains consistent. In contrast, overexpression of FAL1 reduced the expression of PTEN and increased the phosphorylation of AKT in both TE-1 (Figure 11(C)) and Eca9706 cells (Figure 11(D)). Our results were consistent with a previous study showing that FAL1 may promote tumourigenesis and progression of NSCLC through the PTEN/AKT pathway [18].

Discussion
Oesophageal cancer is one of the \most common and fatal malignancies in the world [19]. OSCC is the major subtype of oesophageal cancer, arised from oesophageal epithelial cells [20]. However, the aetiology has not been clearly elucidated. A better understanding of the molecular and genetic basis of OSCC development and progression is beneficial for exploring efficient therapeutic strategy. Additionally, successful identification of novel biomarkers and therapeutic targets are important for improving OSCC diagnosis and treatment. In the past decades, lncRNAs have attracted more and more attention and have been associated with a variety of important cancer phenotypes. Aberrant expression of lncRNAs has been found in various cancers [21]. Multiple lines of evidence have shown that lncRNAs regulate the proliferation, metastasis, invasion, migration and apoptosis in human cancer cells [22]. It is noted that the importance of lncRNAs in OSCC carcinogenesis has been gradually recognized. However, only a small percentage of functional lncRNAs have been well characterized in the pathological progression of OSCC carcinogenesis [23]. In the current study, we analyzed the expression level of lncRNA FAL1 and explored its clinical significance in OSCC.

R E T R A C T E D
Oncogenic properties of FAL1 have been reported in previous studies [24]. However, little information regarding the effects of FAL1 in the pathological progression in OSCC has been reported. Here, we compared FAL1 expression in an independent cohort of OSCC tissues and normal tissues. We found that the expression of FAL1 was higher in OSCC tissues. Clinical analysis demonstrated that FAL1 expression was negatively correlated with a cumulative survival rate in patients with OSCC. OSCC patients with higher FAL1 expression tend to have advanced TNM stage. We also found that FAL1 expression is elevated in OSCC cell lines. Knocking down of FAL1 inhibited cell proliferation of OSCC cells but overexpression of FAL1 promoted cell proliferation of OSCC cells. Consistently, inhibition of FAL1 caused retardation of cell proliferation rates and cellular senescence in different types of tumour cell lines [25]. Another important finding in the current study is that knocking down of FAL1 caused an arrest of cells at G0/G1 phase and a significant decrease in S phase in both the TE-1 and Eca9706 OSCC cell lines. Similarly, it has been recently shown that knocking down of FAL1 expression resulted in cell cycle arrest in both A2780 and MCF7 cell lines in ovarian tumours [12]. Previous studies have proved that numerous lncRNAs are associated with cell cycle arrest [26]. For example, the lncRNA CCAT2 has been shown to affect tumour growth by regulating cell cycle arrest [27]. FAL1 dramatically promoted malignant transformation in primary epithelial cells by combining with other well-known oncogenes, such as RAS and MYC [28]. Here, for the first time, we report that FAL1 could accelerate the invasion activity of OSCC cell lines. EMT is an essential mechanism of tumour metastasis in OSCC [20]. During the process of EMT, epithelial cells temporarily lose their own characteristics and develop characteristics of interstitial cells, which can invade and migrate through the body. Expression of the epithelial markers such as E-cadherin and ZO-1 is reduced. In contrast, mesenchymal markers, such as Vimentin and N-cadherin are increased [29]. In the present study, we confrimed that FAL1 could accelerate EMT of OSCC cell lines by regulating the expression of ZO-1 and N-cadherin in both TE-1 and Eca9706 cells.
To sum up, our current study indicates that elevation of FAL1 is associated with OSCC progression. Our findings provide new insights into the biological functions of lncRNAs in the pathogenesis of OSCC and implicate that FAL1 might be a novel biomarker and a therapeutic target for OSCC.

Disclosure statement
Authors of this article declare they don't have any conflict interest that needs to be disclosed.