RETRACTED ARTICLE: Long non-coding XIAP-AS1 regulates cell proliferation, invasion and cell cycle in colon cancer

Abstract We, the Editors and Publisher of the journal Artificial Cells, Nanomedicine, and Biotechnology, have retracted the following article:  Xiaohong Lu, Yuanjie Yu & Shiyun Tan (2019) Long non-coding XIAP-AS1 regulates cell proliferation, invasion and cell cycle in colon cancer. Artificial Cells, Nanomedicine, and Biotechnology, 47:1, 767–775, DOI: https://doi.org/10.1080/21691401.2019.1577880 It has come to our attention that the full authorship list and affiliations for this manuscript were changed after the article was submitted. We have contacted the author for an explanation, but we have not received a satisfactory explanation. As determining authorship is core to the integrity of published work, we are therefore retracting the article. The authors listed in this publication have been informed and do not agree with the retraction. 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  The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.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 s the third most common type of malignancy in the population, colon cancer affects millions of people worldwide [1]. The morbidity of colon cancer is rapidly increasing in eastern Asian countries including China [2]. About 50% of patients with colon cancer will present with metastasis either at the time of diagnosis or develop distant relapses after therapy [3]. Current primary treatments of colon cancer include surgery, chemotherapy, and radiotherapy [4]. Chemical drugs targeting epidermal growth factor receptor (EGFR) and KRAS have been reported to significantly prolong the survival of CRC patients [5]. A progressive accumulation of genetic changes during the development and progression of colon cancer results in tumour transformation from the normal colonic mucosa [6]. A deeper understanding of the intracellular and biological mechanisms in the pathogenesis of colon cancer is beneficial for exploring novel therapeutic approaches and agents.
Rapid development of genome sequencing results in the identification of non-coding RNA (ncRNA), which have similar properties to mRNA but are not translated into proteins [7]. However, these molecules have important biological functions. Long non-coding RNAs (lncRNAs) are a class of nonprotein coding transcripts over 200 nucleotides (nt) in length [8]. LncRNAs have been reported to regulate gene expression at the epigenetic level, transcriptional level, and post-transcriptional level through interacting with other molecules such as proteins, RNAs, and DNAs [9]. LncRNAs have been shown to be intensively involved in the pathogenesis of human diseases. Notably, lncRNAs have been identified as an important regulator of cancer progression and metastasis and play key roles in the physiological and pathological processes of various types of cancer [10]. The physiological roles of lncRNAs in colon cancer development have attracted considerable attention [11]. LncRNAs such as H19, CCAT1, CCAT2, and TUG1 are involved in the initiation and progression of colon cancer and regulate the biological properties of cancer cells [3]. The protein X-linked inhibitor of apoptosis (XIAP) acts as an important regulator and one of the most promising targets for a variety of cancers [12]. XIAP-AS1 is a novel lncRNA complementary to the XIAP transcript. It has been reported that XIAP-AS1 regulates apoptosis in gastric cancer cells by interacting with Sp1 [13].
In this study, we analyzed the expression of lncRNA XIAP-AS1 in normal and CRC tissues by real-time PCR analysis. Our results show that lncRNA XIAP-AS1 expression was significantly increased in CRC tissues. Next, using CCK-8 and flow cytometric analysis assays, we found that lncRNA XIAP-AS1 promotes CRC cell growth by regulating the cell cycle as well as cell migration and invasion. These results suggest that lncRNA XIAP-AS1 might act as an oncogene during CRC progression and may also provide a potential target for the treatment of CRC.

Patients and tissue samples
A total of 75 cancer tissue samples and corresponding adjacent normal tissues were obtained from stages I/II and III/IV (according to the seventh version of the American Joint Committee on Cancer staging system) patients who underwent surgery for colon cancer without preoperative chemotherapy or radiotherapy. This study was approved by the Medical Ethics Committee of Sun Yat-Sen University. Experiments were guided by the World Medical Association Declaration of Helsinki Ethical Principles for Medical Research Involving Human Subjects. We confirmed that all methods were performed in accordance with the relevant guidelines and regulations of the Medical Ethics Committee of Sun Yat-Sen University. Tumour specimens and corresponding adjacent normal tissues were collected and stored in liquid nitrogen until use.

Cell-cycle analysis
Cell cycle was evaluated by flow cytometric analysis. After the necessary transfection, cells were fixed with 70% ethanol and stored at 4 C overnight. After washing three times with PBS, cells were incubated with 50 mg/ml PI with 100 mg/ml of RNase A in 0.5% Triton-X 100 in darkness at 37 C for 30 min. Signals were analyzed using a FACS Calibur Cytometer (BD Biosciences, Franklin Lakes, NJ).

Cell viability assay
After the indicated transfection, the cell viability was determined using a cell counting kit-8 (CCK-8) assay. Briefly, cells were seeded into 96-well plates at the density of 5 Â 10 3 cells 768 X. LU ET AL.

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per well and transfected with siRNA-XIAP-AS1 or siRNAscramble. The CCK-8 reagent was added and incubated with the cells for 1 h. Absorbance measured at 450 nm was used to index cell proliferation.

Cell invasion assay
Matrigel with Transwell inserts (8.0 mm pore size with polyethylene terephthalate membrane) was used to determine cell invasion. Briefly, 10 mg of Matrigel (BD Biosciences, Franklin Lakes, NJ) was added to pre-coat filters. After the necessary transfection, cells (10 5 ) in serum-free medium were seeded into the upper chamber and incubated for 24 h. Cells that invaded to the lower side of the membrane were fixed with methanol and stained with 0.1% crystal violet.

Statistical analysis
Statistical analysis was carried out using SPSS (version 12.0) software (SPSS, Chicago, IL). Experimental data are expressed as means ± standard error (SE) using two-way analysis of variance (ANOVA). p values less than .05 were considered statistically significant.

Results
To investigate the role of lncRNA XIAP-AS1 in CRC progression, we first examined its expression in CRC. Samples were obtained from 75 tissue specimens and an equal number of adjacent normal tissues. Quantitative PCR showed that the level of XIAP-AS1 was significantly increased in primary CRC tumour tissues as compared to adjacent normal tissues (Figure 1(A)). This result suggests that lncRNA XIAP-AS1 may play a functional role in the progression of CRC. To address whether XIAP-AS1 expression is correlated with the clinicopathological development of colon cancer, its expression was determined in different stages of CRC. As expected, XIAP-AS1 expression was higher in TMN stage IV samples than in TMN stage I/II/III tissue samples (Figure 1(B)). In addition, we examined the effects of XIAP-AS1 on disease outcomes in colon cancer patients using Kaplan-Meier survival analysis. We found that the relapse-free survival rate was significantly lower in patients with high expression of XIAP-AS1 than in those with low XIAP-AS1 expression (Figure 1(C)).
To investigate how XIAP-AS1 regulates CRC progression, we first examined its expression in several CRC cell lines. Interestingly, we found that expression of XIAP-AS1 in the colon cancer cell line SW480 (Figure 2(A)) and LoVo cells (Figure 2(B)) was significantly higher than that in normal colonic mucosa epithelial cells (NCM460). The transcriptional factor p63 has been reported to regulate the expression of lncRNAs in colon cancer cells [14]. The expression of p63 was silenced by transfection with p63-siRNA in SW480 and LoVo cells. The results in Figure 2(C,D) indicate that knockdown of p63 increased the expression of XIAP-AS1 in both cell lines, whereas p63 overexpression produced the opposite result ( Figure 2(E,F)). Furthermore, the effects of XIAP-AS1 on cell proliferation of colon cancer cells were assessed. Cell proliferation assay (CCK-8) results indicate that XIAP-AS1 knockdown significantly suppressed the growth of SW480 (Figure 3(A)) and LoVo cells (Figure 3(B)), whereas p63 overexpression produced the opposite result (Figure 3(C,D)). To investigate how XIAP-AS1 suppresses CRC cell growth, the effects of XIAP-AS1 on the CRC cell cycle were also examined. As shown in Figure 4(A,B), the cell cycles of both SW480 and LoVo cells, which were transfected with XIAP-AS1-siRNA, were arrested in the G0/G1 phase, whereas p63 overexpression produced the opposite result (Figure 4(C,D)). Additionally, we determined the effects of XIAP-AS1 on the expression of cell cyclerelated proteins. As expected, XIAP-AS1 overexpression significantly reduced the expression of cyclin D1, cyclin E, and c-Myc in both SW480 ( Figure 5(A)) and LoVo cells ( Figure  5(B)). However, cyclin A levels remained consistent. As we know, b-catenin is a crucial signalling factor regulating oncogenesis. Cyclin D1 and c-Myc are important target genes of the Wnt/b-catenin pathway. Therefore, we measured the expression of b-catenin. As expected, the results in

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silencing of XIAP-AS1 significantly increased the expression of ZO-1 and E-cadherin, but decreased the expression of N-cadherin and vimentin in both SW480 (Figure 7(A)) and LoVo cells (Figure 7(B)). These results indicate that XIAP-AS1 might regulate colon cancer cell metastasis via EMT. STAT3 has been considered as an important transcriptional factor which plays an essential role in gene transcription and cell fate determination. Here, we found that XIAP-AS1 knockdown reduced the phosphorylation of STAT3 in both SW480 (Figure 8(A)) and LoVo cells (Figure 8(B)), suggesting a possible molecular mechanism.

Discussion
Colon cancer is the third most diagnosed malignancy in the world, therefore, posing to a serious threat to human health. The incidence is increasing in young adults, especially in developing countries [15]. In past decades, efforts have been made to study the pathological mechanisms as a means to reduce the mortality of colon cancer. However, the prognosis of this disease is still poor due to late clinical diagnosis of the malignancy [16]. The accumulation of genetic alterations such as NRAS, KRAS, and BRAF has been associated with the initiation and progression of colon cancer [17]. These biological markers are in use for treatment decisions and patient stratification. However, there are still some insufficiencies. LncRNAs have recently become a novel molecular star and have been shown to be involved in a variety of human diseases, especially in cancers. Importantly, lncRNAs have been considered as novel and significant biological biomarkers for early diagnosis and prognosis of colon cancer [11]. To date, the biological functions of lncRNAs such as TUG1, CCAT2, and UCA1 in colon cancer have been reported [18]. Therefore, identifying novel biomarkers for the diagnosis and prognosis of diseases may be of clinical significance. In this study, we examined lncRNA XIAP-AS1 expression in CRC tissues by quantitative PCR. The results show that XIAP-AS1 expression was increased in CRC tissues, and a high expression level of XIAP-AS1 in colon cancer patients was positively correlated with TNM stage. The cumulative survival rate was significantly higher in patients with low XIAP-AS1 expression than in those with high XIAP-AS1 expression. These findings suggest that lncRNA XIAP-AS1 might be a novel diagnostic biomarker for CRC.
Aberrant cell proliferation and cell-cycle progression have been found in the majority of human malignancies, including colon cancer [19]. Cell-cycle progression is regulated by a series of checkpoint proteins. Elevated expressions of cyclin D1, cyclin E, and c-Myc have been reported in colon cancer [20]. Modification of the cell cycle has been considered as an important therapeutic strategy for colon cancer treatment [20]. The current study demonstrates that silencing of XIAP-AS1 causes G0/G1 phase cell-cycle arrest via downregulation of cyclin D1, cyclin E, and c-Myc levels, which appears to be the underlying mechanism in colon cancer cell growth inhibition. The expression of cell-cycle-related proteins is regulated by the Wnt-signalling pathway. Stabilization and subsequent nuclear translocation of b-catenin are critical steps in activating downstream signalling and expression of cell cycle proteins. b-Catenin activates the transcription of various genes involved in cell proliferation, migration, and metastasis [21]. Hyperactivation of Wnt-b-catenin signalling and increased expression of b-catenin have been considered as characteristic features of colon cancer development. Here, we found that XIAP-AS1 knockdown significantly suppressed CRC cell growth and arrested the cell cycle at the G0/G1 phase. Furthermore, cell-cycle-related genes, including cyclin D, cyclin A, and cyclin E, are also regulated by XIAP-AS1 silencing. Additionally, our findings indicate that XIAP-AS1 knockdown significantly reduced the expression level of b-catenin in colon cancer cells, implying that XIAP-AS1 promoted cell growth and invasion by potentiating the Wnt/b-catenin pathway.
As a critical mechanism of tumour metastasis [22], EMT has been shown to be involved in the aggressive tumour biology of a variety of cancers, including colon cancer [23]. During the process of EMT, cells lose their cellular epithelial features and gain mesenchymal properties with increased migration and invasion abilities, which can invade and migrate through the body [24]. Epithelial markers such as Ecadherin and ZO-1 and mesenchymal markers such as vimentin and N-cadherin have been widely used to index EMT [25]. Indeed, decreased levels of E-cadherin and ZO-1 in tumour cells have been associated with liver metastasis of colon cancer [26]. Meanwhile, increased expression of vimentin and Ncadherin has been found in colon cancer cells [27]. A recent study reported that another lncRNA ATB promotes the progression of colon cancer and predicts poor prognosis by

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repressing the expression of E-cadherin [28]. In the current study, our results indicate that silencing of XIAP-AS1 decreased the expression of E-cadherin (E-cad) and ZO-1 but increased the expression of vimentin and N-cadherin, suggesting that XIAP-AS1 might have an important potential role in facilitating EMT colon carcinoma. Agents or therapeutic approaches targeting XIAP-AS1 might be beneficial for the blockage of EMT and metastasis of colon cancer. The pathological mechanism of colon cancer is complicated and needs to be elucidated. A variety of risk factors have been linked with the pathogenesis of colon cancer, including genetics, ageing, obesity, and a personal history of inflammatory bowel disease [29]. The biological function of XIAP-AS1 in vivo is rarely reported. In an in vivo mouse xenograft gastric cancer model, tumour cell proliferation was inhibited by XIAP-AS1 knockdown in response to tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) administration [13]. It should be noted that a major limitation of this study is that all the findings here are based on in vitro cell culture model experiments. More future in vivo studies are necessary to confirm the physiological function of XIAP-AS1 on colon cancer.
In summary, our findings indicate that XIAP-AS1 promoted cell growth and invasion by facilitating the Wnt/b-catenin pathway and EMT. Based on these observations, we speculate that XIAP-AS1 plays an oncogenic role in colon cancer progression and serves as a potential target for cancer prevention and treatment.

Disclosure statement
None of the authors of this work need to disclose has any conflicts of interest.