RETRACTED ARTICLE: Upregulation of long non-coding RNA OGFRP1 facilitates endometrial cancer through regulating miR-124-3p/SIRT1 axis and activating PI3K/AKT/GSK-3β pathway

Abstract We, the Editors and Publisher of the journal Artificial Cells, Nanomedicine, and Biotechnology, have retracted the following article: Yuqiong Lv, Shaorong Chen, Jingjing Wu, Ruyin Lin, Limei Zhou, Guimin Chen, Huiqing Chen and Yumin Ke. (2019). Upregulation of long non-coding RNA OGFRP1 facilitates endometrial cancer by regulating miR-124-3p/SIRT1 axis and by activating PI3K/AKT/GSK-3b pathway. Artificial Cells, Nanomedicine, and Biotechnology. 47:1, 2083–2090, DOI: 10.1080/21691401.2019.1617727 Since publication, concerns have been raised about the integrity of the data in the article. When approached for an explanation, the authors checked their data and confirmed there are fundamental errors present. Therefore, they have agreed to the retraction of this article. The authors apologise for this oversight. 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 The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.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
Endometrial cancer remains to be a lethal gynaecologic malignancy with a tremendous increase in the incidence over recent years [1]. Although the survival rate is high if patients were diagnosed at an early stage, the percentage of patients who are diagnosed at a relative advanced stage is still high, which is about 30% [2]. The prognosis remains poor in patients at the advanced stage or with a high risk of recurrence [3]. Moreover, the molecular mechanisms of endometrial cancer have been poorly illustrated.
Long non-coding RNAs (lncRNAs), some non-coding RNAs with over 200 nucleotides in length, is widely pointed out as pivotal players in many biology of various diseases [4][5][6]. Aberrant expression of lncRNAs has been widely discovered in many cancers and is considered as a character in cancer [7][8][9]. Several lncRNAs, including HOTAIR, BANCR, and FER1L4, have been identified to be crucial in the progression of endometrial cancer [10][11][12]. OGFRP1, a newly reported lncRNA, has been shown to induce autophagy and growth inhibition in human coronary artery endothelial cells [13]. Moreover, dysregulation of OGFRP1 was demonstrated to be pivotal in the biology of non-small cell lung cancer (NSCIC) [14] and hepatocellular carcinoma [15]. However, OGFRP1 in endometrial cancer still remain incomplete reported. In addition, Dong et al. pointed out that miR-124 was lowly expressed in tumour tissues of endometrial cancer [16]. However, there was no study focusing on investigating the regulatory pattern between OGFRP1 and miR-124 in endometrial cancer.
During this research, we elucidated the function and possible mechanism of OGFRP1 in endometrial cancer. We determined the expression of OGFRP1 in endometrial cancer and assessed the effect of OGFRP1 dysregulation on the malignant behaviour of endometrial cancer cells. Further, we analyzed the regulatory relationships between OGFRP1 and miR-124-3p, and those between miR-124-3p and sirtuin1 (SIRT1). Moreover, the interaction between OGFRP1 dysregulation and activation of PI3K/AKT/GSK-3b pathway was also revealed. All of these data may offer a theoretical basis for designing novel strategies for illustrating the biology of endometrial cancer.

Patients
Between April 2015 and July 2018, 48 patients diagnosed with endometrial cancer were recruited. Table 1 shows the characteristics of endometrial cancer patients. Forty-eight tumour tissues from endometrial cancer patients and an equal number of matched normal endometrial tissues were collected during an initial hysterectomy. After extraction, the collected tissues were immediately frozen in liquid nitrogen and then stored at À80 C for total RNA extraction.

Cell lines
The human endometrial cancer cell lines HHUA, KLE, Ishikawa, and ECC-1 and normal endometrium (NE) cells were acquired from the authoritative organization of American Type Culture Collection (ATCC). These cells were cultured in medium named as Eagle's Minimum Essential Medium (Gibco, Darmstadt, Germany), which were mixed with 15% foetal bovine serum (FBS), 100 U/mL of penicillin (Gibco), and 100 lg/mL of streptomycin (Gibco) by incubating at 37 C in a humidified atmosphere with 5% CO 2 .

qRT-PCR
We isolated the total RNA from cells using TRIzol reagent (Life Technologies, Gaithersburg, MD). Briefly, the RNA quantity and purity were determined by a SmartSpec Plus spectrophotometer (BioRad, Hercules, CA). qPCR was performed to amplify the target genes using a qRT-PCR kit (GoTaq 2-Step RT-qPCR, Promega, Madison, WI) in an Mx3005P qPCR System (Stratagene, La Jolla, CA). To detect the expression of different miRNAs, qPCR was performed using miScript II RT Kit (Takara, Mountain View, CA), miScript SYBR Green PCR Kit (Takara, Mountain View, CA), and miScript Primer Assays in Mx3005P qPCR System (Stratagene, La Jolla, CA). GAPDH and U6 were used as the internal controls for RNA and miRNA expression analyses, respectively. Relative quantification of gene expression was performed by the 2 ÀDDCt method.

Cell viability assay
Approximately 24 h post-transfection, the cells (10 4 cells/well) were cultured into 96-well plates for the subsequent analysis using MTT assay. Briefly, MTT (0.5 mg/mL; Sigma, St. Louis, MO) was added in the 96-well plates equipped with cells for a 4 h incubation at 37 C. After taking away the cultured medium, dimethyl sulphoxide (DMSO, 100 lL; Sigma, St. Louis, MO) was added in for melting the precipitated formazan. After shaking for 15 min, cell viability was measured via reading the absorbance at 570 nm (A570) using a spectrophotometer (lQuant universal microplate, BioRad, Hercules, CA).

Cell apoptosis assay
Approximately 24 h post-transfection, the cultured cells were harvested and washed by PBS buffer. The cells were stained with fluorescein isothiocyanate-labelled annexin V and propidium iodide following the protocol recommended for Annexin V-FITC Kit (Sangon Biotech., Shanghai, China). The percentage of apoptotic cells was assessed using a flow cytometry (FACSCalibur, BioRad, Hercules, CA).

Cell migration and invasion assays
Transwell assay was used for the assessment of cell biological processes of migration and invasion. For invasion, the transwell chambers (8 lm pore size; Corning co. Ltd., Maine, NY) were pre-coated with Matrigel (Becton-Dickinson, Franklin Lakes, NJ). Briefly, after a 24-h incubation, cells were cultured in serum-free RPMI 1640 medium for 24 h. Subsequently, the cells were seeded in the upper chamber and RPMI 1640 medium mixed with 10% FBS was added in the lower chamber. After incubating for another 48 h, transwell chambers were fixed with methanol and stained with Giemsa. Cell migration and invasion were analyzed by counting the migrated and invaded cells under a microscope (IX83, Olympus Corporation, Tokyo, Japan).

Luciferase reporter assay
We used PCR analysis to amplify the sequences of binding site between 3 0 -UTR of SIRT1 mRNA and miR-124-3p and then inserted the obtained sequences into pGL3 vector (Promega, Madison, WI) to construct the wild type (WT) luciferase reporter vector pGL3-SIRT1. Moreover, Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) was used to synthesis the mutated luciferase reporter vector pGL3-SIRT1-MUT with point mutations in the seed sequence. Cells at a density of 1 Â 10 6 per well were co-transfected with the following vectors, including 50 pmoL of miR-124-3p inhibitor (or control miRNA), 1 lg of pGL3-SIRT1-WT/pGL3-SIRT1-MUT, and 1 lg of

R E T R A C T E D
Renilla luciferase expression construct pRL-TK (Promega, Madison, WI), using Lipofectamine 3000 reagent following the manufacturer's protocol. The luciferase activity of Renilla group was used as a control. Approximately a 48 h incubation, luciferase activity in each group was assessed using the dual-luciferase reporter assay system (Promega, Madison, WI).

Western blot
Cells were harvested and lysed with the purchased cell lysis buffer (Beyotime, Haimen, China). After centrifugation, the total protein was extracted. The protein samples (50 lg/lane) were subjected to 12% SDS-polyacrylamide gel electrophoresis and then we transferred the obtained protein band signals onto PVDF membranes (Millipore, Billerica, MA). Then, we performed the immunoblotting by incubating the membranes with the primary antibodies (1:1000 dilutions) and subsequently with the recommended secondary antibodies (1:5000). The obtained protein signals were then observed by an Imaging Analysis System (Odyssey Infrared, LI-COR, Lincoln, NE). The primary antibodies including b-actin, Bax, Bcl-2, pro-and cleaved caspase-3, pro-and cleaved caspase-9, SIRT1, N-cadherin, E-cadherin, vimentin, snail, PI3K, p-PI3K, AKT, p-AKT, GSK-3b, and p-GSK-3b were obtained from Abcam (Cambridge, UK). b-Actin was used as the internal control.

Statistical analysis
All experiments in this research were conducted three times independently. Data were expressed as the type of mean-± standard deviation (SD). Statistical analyses were evaluated using SPSS 20.0 (IBM, Armonk, NY). The significant differences among groups were evaluated by two-tailed or one-way ANOVA followed by Turkey's post hoc analysis. p < .05 reveals a statistically significance.

OGFRP1 upregulates in endometrial cancer
The mRNA level of OGFRP1 in endometrial cancer tissues was significantly more than in non-tumour tissues (p < .01, Figure 1(A)). Furthermore, OGFRP1 was highly expressed in endometrial cancer cells (HHUA, KLE, Ishikawa, and ECC-1) compared with NE cells (p < .01, Figure 1(B)). We selected the Ishikawa cells for further experiments because OGFRP1 expression was the highest among the four endometrial cancer cell lines (Figure 1(B)).

OGFRP1 suppression inhibits the biological performance of the Ishikawa cells
The expression of OGFRP1 was suppressed by transfecting the Ishikawa cells with si-OGFRP1 to explore the role of OGFRP1 in endometrial cancer development. OGFRP1 expression in si-OGFRP1#1 or si-OGFRP1#2 was markedly less than that in si-NC (p < .05, Figure 1(C)). si-OGFRP1#2 was selected for the subsequent experiments due to higher transfection efficiency compared with si-OGFRP1#1. We further evaluated the influences of OGFRP1 down-regulation on the Ishikawa cell biological processes including viability, apoptosis, migration and invasion. As shown in Figure 1(D), OGFRP1 suppression markedly reduced the viability of the Ishikawa cells (p < .05). The percentage of cell apoptosis was significantly increased after OGFRP1 suppression (p < .001, Figure 1(E)). Besides, we analyzed the level of apoptotic proteins including caspase-3, caspase-9, Bcl-2, and Bax. OGFRP1 down-regulation promoted the levels of pro-apoptotic proteins including cleaved caspase-3, cleaved caspase-9, and Bax, while it suppressed the anti-apoptotic protein Bcl-2 (Figure 1(E)). Furthermore, OGFRP1 down-regulation markedly decreased the Ishikawa cell migration and invasion (p < .01, Figure 1(F,G)). The levels of EMT-related proteins including Ecadherin, N-cadherin, vimentin, and snail were also examined. OGFRP1 suppression promoted E-cadherin expression; however, it restrained the level of N-cadherin, snail, and vimentin ( Figure 1(H)), which indicates that OGFRP1 downregulation inhibited EMT in the Ishikawa cells.

OGFRP1 suppression inhibits the malignant behaviour of the Ishikawa cells by negatively regulating miR-124-3p
Previous evidences pointed out that the abnormal expression of OGFRP1 is associated with the progression of NSCLC by sponging miR-124-3p [14]. We, therefore, investigated the association between OGFRP1 and miR-124-3p in the Ishikawa cells. miR-124-3p in si-OGFRP1#2 was markedly higher compared with si-NC (p < .001, Figure 2(A)). Then, we measured the mRNA level of miR-124-3p to ensure this result. Interestingly, miR-124-3p was markedly decreased in endometrial cancer tissues and cells in comparison to the normal tissues and cells (p < .01, Figure 2(B,C)). To further explore the pivotal role of OGFRP1 in endometrial cancer development via miR-124-3p, the expression of miR-124-3p was upor down-regulated in the Ishikawa cells (p < .001, Figure 2(D)). The combined effects of OGFRP1 downregulation and miR-124-3p inhibition were examined subsequently. All of these findings implied that the inhibition of miR-124-3p obviously reversed the influences of OGFRP1 down-regulation on the biological performances of the Ishikawa cells including viability, apoptosis, migration and invasion, as well as the expression of apoptotic proteins and EMT-markers (p < .05, Figure 2(E-I)).

SIRT1 is a functional target gene of miR-124-3p
Several potential targets of miR-124-3p were identified using TargetScan software, and the interaction pattern between miR-124-3p and SIRT1 was examined to elucidate the possible mechanism of miR-124-3p. The predicted binding sequence between miR-124-3p and SIRT1 is shown in Figure 3(A). Luciferase experiment showed that SIRT1 3'UTR was bounded by miR-124-3p (p < .05, Figure 3(B)), which indicates that SIRT1 is a functional target of miR-124-3p. Besides, ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 2085

R E T R A C T E D
at mRNA and protein levels, the SIRT1 expression in the miR-124-3p mimic group was markedly lower compared with the mimic control group, while its expression in miR-124-3p inhibitor group was markedly higher compared with the inhibitor NC group (p < .01, Figure 3(C,D)), which indicates the negative association between miR-124-3p and SIRT1.

R E T R A C T E D
miR-124-3p regulates endometrial cancer growth and metastasis by targeting SIRT1 To further verify the possible influences of miR-124-3p in endometrial cancer development was adjusted by targeting SIRT1, SIRT1 was successfully suppressed in the Ishikawa cells (p < .001, Figure 4(A)). The combined influences of miR-124-3p inhibition and SIRT1 suppression were then investigated. The suppression of miR-124-3p in the Ishikawa cells increased cell viability (p < .05, Figure 4(B)). Meanwhile, it inhibited cell apoptosis by increasing BCL-2 level but decreasing the expression of Bax, cleaved caspase-3, and cleaved-caspase-9 (Figure 4(C)). Cell migration and invasion were promoted by inducing EMT through decreasing E-cadherin but increasing the expression of N-cadherin, snail, and vimentin (p < .05, Figure 4(D,F)). Further, miR-124-3p inhibition and SIRT1 suppression at the same time reversed the effects of the suppression of miR-124-3p on the Ishikawa cell viability, apoptosis, invasion and migration (p < .05, Figure 4(B-F)), which indicates that miR-124-3p regulated the malignant behaviour of the Ishikawa cells by targeting SIRT1.
OGFRP1 suppression inhibits the activation of the PI3K/ AKT/GSK-3b pathway in the Ishikawa cells via miR-124-3p/SIRT1 axis PI3K/AKT/mTOR pathway was reported to be activated by the up-stream mediators in endometrial cancer pathogenesis [17]. A recent study has shown that lncRNA DLEU1 enhances the progression of endometrial cancer through activation of PI3K/AKT/GSK-3b pathway [18]. Moreover, OGFRP1 downregulation was shown to inhibit hepatocellular carcinoma by regulating AKT/mTOR and Wnt/b-catenin signals [19]. We investigated whether activation of PI3K/AKT/GSK-3b pathway is a key mechanism that mediates the role of OGFRP1 in endometrial cancer. Our experiments discovered that the suppression of OGFRP1 significantly decreased the protein levels of p/t-PI3K, p/t-AKT, and p/t-GSK-3b in the Ishikawa cells, which was neutralized by the inhibition of OGFRP1 and miR-124-3p at the same time (Figure 4(G)). Additionally, the concurrent inhibition of OGFRP1, miR-124-3p, and SIRT1 remarkably decreased the expression levels of these proteins (Figure 4(H)). These findings indicate that the activation of PI3K/AKT/GSK-3b pathway may be a subsequent mechanism mediating the role of OGFRP1/miR-124-3p/SIRT1 axis in the Ishikawa cells (Figure 4(I)).

Discussion
Emerging evidence points out that lncRNAs are pivotal players in endometrial carcinogenesis [20]. Understanding the crucial lncRNAs involved in endometrial cancer may facilitate designing effective therapeutic strategies. This research discovered that OGFRP1 was up-regulated in endometrial cancer tissues and cells, and down-regulation of OGFRP1 inhibited the malignant behaviour including metastasis and apoptosis of endometrial cancer cells. Consistent with previous findings [15,21], our results also imply that OGFRP1 may serve as an oncogene in endometrial cancer and play important roles in tumour progression. Increasing studies have suggested the pivotal roles of lncRNAs in various kinds of diseases, and lncRNAs act as endogenous miRNA sponges and promote these diseases [22][23][24]. In line with the previous findings that OGFRP1 could sponge miR-124-3p in NSCLC cells [21], OGFRP1 negatively regulated miR-124-3p expression and downregulation of OGFRP1 inhibited the malignant behaviour of the Ishikawa cells by negatively regulating miR-124-3p in this experiment. Accumulating evidence indicates that miR-124-3p undertakes a tumour suppressor and is associated with the progression of various cancers, such as females-related cancers including breast cancer [25] and cervical cancer [26], bladder cancer [27], and gastric cancer [28]. This research showed that miR-124-3p expression was dramatically decreased in endometrial cancer tissues and cells. Take into the key role of miR-124-3p in other cancers consideration, we confirmed that miR-124-3p may play a tumour suppressive role in endometrial cancer, and upregulation of OGFRP1 may promote endometrial cancer by inhibiting miR-124-3p. SIRT1, a NAD-dependent histone deacetylase, is reported to play a dual role, including modifying histone protein via the following deacetylation manners, including deacetylation of lysine residues K26 on histone H1, lysine residues K9 on histone H3, and lysine residues K16 on histone H4, acting both as an oncogene and as a tumour suppressor [29]. Previous studies have confirmed that dysregulation of SIRT1 is associated with the malignant phenotypes of various cancers, such as prostate cancer [30], gastric cancer [31], and cervical cancer [32]. Moreover, it has been reported that SIRT1 can contribute to endometrial tumour growth by promoting lipogenesis [33] and is involved in regulating progestin resistance in endometrial cancer [34]. On the other point, Brennan et al pointed out that the down-regulated miR-124 was produced by the SIRT1-mediated histone deacetylase [35]. In this study, SIRT1 was determined as a functional target of miR-124-3p, and SIRT1 suppression could changeover the influences of miR-124-3p inhibitor on the Ishikawa cell biological processes including viability, apoptosis, migration, and invasion. Therefore, we speculate that miR-124-3p may regulate the cell proliferation and metastasis of endometrial cancer by SIRT1.
Furthermore, PI3K/AKT/GSK-3b pathway, a pivotal signal transduction pathway, regulates cell proliferation, apoptosis, and migration [36]. Aberrant activation of this pathway occurs in many malignant tumours including endometrial cancer [17,37]. In addition, the downregulation of OGFRP1 has been revealed to inhibit hepatocellular carcinoma via the regulation of AKT/mTOR and Wnt/b-catenin signals [19]. Koga et al. pointed out that the PI3K/AKT/GSK-3b signals is crucial to SIRT1 induction by stress reaction of endoplasmic reticulum and consequently regulates hepatocellular injury [38]. In this study, suppression of OGFRP1 restrained the activation of PI3K/AKT/GSK-3b signals in the Ishikawa cells, which was neutralized after inhibition of miR-124-3p, and further the concurrent inhibition of OGFRP1 inhibited this pathway. We thus speculate that the activation of PI3K/AKT/GSK-3b

R E T R
A C T E D pathway may be a subsequent mechanism that mediates the role of OGFRP1/miR-124-3p/SIRT1 axis in endometrial cancer. Taken together, our experiments revealed that the upregulation of OGFRP1 may promote the development of endometrial cancer through regulation of miR-124-3p/SIRT1 axis and by activating PI3K/AKT/GSK-3b pathway. OGFRP1 may serve as a marker for the diagnosis and treatment of endometrial cancer.

Disclosure statement
No potential conflict of interest was reported by the authors.