RETRACTED ARTICLE: Overexpression of lncRNA ANRIL aggravated hydrogen peroxide-disposed injury in PC-12 cells via inhibiting miR-499a/PDCD4 axis-mediated PI3K/Akt/mTOR/p70S6K pathway

Abstract We, the Editors and Publisher of the journal Artificial Cells, Nanomedicine, and Biotechnology, have retracted the following article: Zhiliang Guo, Lanlan Li, Yu Gao, Xiaoyun Zhang and Min Cheng. (2019). Overexpression of lncRNA ANRIL aggravated hydrogen peroxide-disposed injury in PC-12 cells via inhibiting miR-499a/PDCD4 axis-mediated PI3K/Akt/mTOR/p70S6K pathway. Artificial Cells, Nanomedicine, and Biotechnology. 47:1, 2624–2633. DOI: 10.1080/21691401.2019.1629953 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 The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.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
Spinal cord injury (SCI) is a fatal neurological disorder that impacts significant financial burden on health care systems [1,2]. It can lead to severe motor, sensory and autonomic dysfunction [3]. Moreover, traumatic SCI can cause several devastating symptoms, including chronic pain and paralysis [4,5]. Despite great efforts made to improve the functional outcome, current effective treatment for SCI is limited [6]. Therefore, elucidating the key molecular mechanisms mediating SCI will facilitate to improve the functional outcomes of patients with this injury.
Long noncoding RNAs (lncRNAs) are a class of transcripts longer than 200 nucleotides and lack of protein-coding capacity [7]. Increasing evidence has pointed out that lncRNAs exhibit extensive functions in biological processes, such as gene expression regulation [8] and epigenetic control [9]. Moreover, lncRNA has gained more attention because their aberrant expression is implicated in several neurological disorders [10][11][12]. In recent years, several lncRNAs are pointed out to be crucial in the pathophysiology of SCI, such as lncSCIR1 [13], lncRNA H19 [14] and XIST [15]. However, there is limited report about the biological roles of lncRNAs in SCI. Antisense noncoding RNA in the INK4 locus (ANRIL) is a newly discovered lncRNA existing in the chromosome 9p21 region [16]. It is reported that ANRIL is involved in the development of multiple disease, such as cancers [17,18], coronary artery disease [19] and diabetic retinopathy [20]. However, the roles and possible mechanism of ANRIL in SCI has not been clarified.
Oxidative stress-mediated cellular injury is a major cause of neurodegenerative diseases, and PC-12 cells have been applied for model construction in analysing the molecular mechanism of SCI in vitro [21][22][23]. Moreover, SCI is reported as a fearful neurology-related disease and the latent therapeutic strategies for this injury have been grabbed in PC-12 cells [24]. Therefore, we chose hydrogen peroxide (H 2 O 2 ) to produce injury in PC-12 cells, to imitate the process of SCI. H 2 O 2 -disposed injury in PC-12 cells was assessed by detecting cell viability, migration, invasion, apoptosis and autophagy. The abnormal level of ANRIL in H 2 O 2 -disposed PC-12 cells was analysed, then the impacts of ANRIL silencing on H 2 O 2disposed PC-12 cell injury was determined. To further elucidate the downstream mechanism of ANRIL on H 2 O 2 -disposed PC-12 cell injury, the regulatory associations between ANRIL and miR-499a, between miR-499a and PDCD4, as well as PDCD4 and PI3K/Akt/mTOR/p70S6K signals were investigated. Our findings will inform the future direction of treatments for patients with SCI.
For cell disposes, PC-12 cells were seeded in 96-well plates (Sangon Biotech, Shanghai, China) at a final density of 5 Â 10 4 cells/well for 24 h. To construct an oxidative stress model to stimulate SCI, PC-12 cells were then cultured in fresh medium containing 200 lM of H 2 O 2 for the other 24 h. Cells in control group were disposed with the same fresh medium without H 2 O 2 [25].
Cell proliferation assay PC-12 cells at a final density of 1 Â 10 5 cells/well were incubated in triplicate in 60-mm dishes. After plenty of time incubation, PC-12 cells were washed by PBS buffer. The live cell numbers were counted by trypan blue exclusion.

Migration and invasion test
We chose a modified two-chamber migration to evaluate cell migration assay. In brief, PC-12 cells were suspended in 200 lL of serum-free fresh medium. After that, cells were then plated on the upper chamber of 24-well Transwell with an aperture of 8 lm (BD Biosciences, San Jose, CA). The under chamber was padded with 600 lL of complete medium. After incubation at 37 C, traversed cells on the under chamber were fixed with methanol, stained with crystal violet and then counted microscopically. The invaded behaviour of PC-12 cells was detected using a similar protocol as cell migration except that the Transwell chamber was pre-coated with 20 lg Matrigel (Takara Biotech, Kusatsu, Japan). The data are expressed as the average number of cells attached to the under chamber from five randomly chosen fields.

Apoptosis test
We chose flow cytometry to assess cell apoptosis. In brief, cells were fixed in 70% ethanol after they were washed by PBS. The fixed cells were then stained with Annexin V-FITC/PI apoptosis detection kit (Beijing Biosea Biotechnology, Beijing, China). After incubation for 1 h at room temperature darkly, we chose FACS can (Beckman Coulter, Fullerton, CA) for observing the apoptotic cells, and then chose FlowJo software for calculation.

Luciferase reporter test
We amplified the coding sequence of PDCD4 containing the predicted binding site of miR-499a, then we inserted it into a pmirGlO Dual-luciferase miRNA Target Expression Vector (Promega, Madison, WI) to establish the vectors of PDCD4wild-type (PDCD4-wt). The PDCD4-mutated-type (PDCD4-mut) reporter vector carrying the mutated binding site of miR-499a in the PDCD4 was also constructed as NC. These reporter vectors and miR-499a mimics were also transfected into HEK 293T cells, and the luciferase activities of them were analysed by Dual-Luciferase Reporter Test System (Promega, Madison, WI).

Quantitative PCR
We isolated the total RNA from PC-12 cells by Trizol reagent (TaKaRa Biotech, Kusatsu, Japan). The One Step SYBR V R PrimeScript V R PLUS RT-RNA PCR Kit (TaKaRa Biotech, Kusatsu, Japan) and RNA PCR Kit (AMV) Ver.3.0 (TaKaRa Biotech, Kusatsu, Japan) were respectively used for the real-time qPCR analysis for detection of the expression levels of ANRIL and PDCD4. After that, Taqman MicroRNA Reverse Transcription Kit was chosen for determining the levels of miR-499a in PC-12 cells (TaKaRa Biotech, Kusatsu, Japan). Fold changes of gene expression levels were then evaluated by 2 ÀDDCt method as previous described [26]. GAPDH was chosen as internal control for normalizing the levels of them.

Western blot assay
We isolated the total protein from cells by RIPA lysis buffer (Sangon Biotech, Shanghai, China). Purity and concentration of the isolate protein were determined using the BCA TM Protein Assay Kit (Pierce, Appleton, WI). Bio-Rad Bis-Tris Gel system was applied for separating the protein samples after we isolated the total protein from cells. The polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA) were thereby cultured with primary antibodies at 4 C overnight. Afterwards, the membranes were mixed with secondary antibody symboled by horseradish peroxidase for 1 h at room temperature. After washing three times, we chose Bio-Rad ChemiDoc TM XRS system to assess the membranes taking along blots and antibodies, after that, 200 lL Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA) was chosen for mixed with the membranes. We then chose Image Lab TM Software (Bio-Rad, Shanghai, China) to evaluate the protein signals under membranes. Notably, primary antibodies used in this study were totally obtained from Abcam (Cambridge, UK), and prepared in 5% blocking buffer at a dilution of 1:1000 before use.

Statistical analysis
We carried out all experiments independently with three times repeats. The obtained data are displayed as the mean ± standard deviation (SD). For comparison of difference between groups, the p values were calculated using a oneway ANOVA in GraphPad 6.0 statistical software (GraphPad, San Diego, CA). Statistically significance was presented when p < .05.

H 2 O 2 induces injury in PC-12 cells
wThe impacts of H 2 O 2 on PC-12 cell injury were first investigated. Relative to control, H 2 O 2 treatment markedly depressed PC-12 cell viability (p < .05, Figure 1(A)), migration (p < .01, Figure 1(B)) and invasion (p < .01, Figure 1(C)) in PC-12 cells. Moreover, H 2 O 2 treatment resulted in distinct increase on the apoptotic cells (p < .001, Figure 1(D)). Consistent changes in the expressions of apoptotic proteins were also observed that the expression levels of Bax/Bcl-2, cleaved/pro-caspase-3 and cleaved/pro-caspase-9 were markedly increased after H 2 O 2 disposes relative to control (Figure 1(D)). Furthermore, H 2 O 2 treatment resulted in remarkable increases in the expression levels of LC3-II/I and Beclin-1 and obvious decrease in P62 expression (p < .01, Figure 1(E)), indicating that H 2 O 2 treatment increased P12 cell autophagy.

Silencing of ANRIL alleviates H 2 O 2 -disposed injury in PC-12 cells
Notably, H 2 O 2 treatment markedly promoted ANRIL expression in PC-12 cells relative to control (p < .01, Figure 2(A)), indicating the potential association between ANRIL and SCI.

PDCD4 is targeted by miR-499a
As we all know, miRNAs function important roles in disease development via regulating their target genes [29,30]. We further predicted the potential targets of miR-499a according to the target information of TargetScanHuman. As shown in Figure 4(A), PDCD4 was identified as one of the targets of miR-499a (Figure 4(A)). To verify this, luciferase report assay was further performed and the luciferase report test showed us that of luciferase activity of PDCD4-wt was dramatically inhibited by miR-499a mimic (p < .05, Figure 4(B)), confirming that miR-499a could bind to the 3 0 UTR of PDCD4. Furthermore, our results revealed that the levels of PDCD4 in miR-499a mimic group were markedly depressed compared to those in scramble group, while obviously enhanced in miR-499a inhibitor group relative to those in inhibitor NC group (p < .01, Figure 4(C,D)), pointing out that PDCD4 was a target of miR-499a.
ANRIL silencing decreased H 2 O 2 -disposed injury in PC-12 cells possible by activating PI3K/Akt/mTOR/ p70S6K signals The activated Akt/mTOR/p70S6K signal has been shown to be crucial in accelerating the regenerative environment for

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SCI and may function as a promising tactics for its treatment [31]. We further investigated the regulatory association between ANRIL and PI3K/Akt/mTOR/p70S6K signals ( Figure  5(G)). H 2 O 2 dispose markedly decreased the levels of p/t-PI3K, p/t-AKT, p/t-mTOR and p-p70S6K in PC-12 cells, which were remarkably alleviated after silencing of ANRIL (all p < .05). Moreover, inhibition of miR-499a and silencing of ANRIL concurrently markedly changeover the impacts of silencing of

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ANRIL alone on the levels of these PI3K/Akt/mTOR/p70S6K signal-related proteins in H 2 O 2 -disposed PC-12 cells, which were significantly reversed after inhibition of miR-499a, silencing of ANRIL and knockdown of PDCD4 simultaneously. These data deducted that ANRIL silencing might decrease H 2 O 2 -disposed injury in PC-12 cells possible by activating PI3K/Akt/mTOR/p70S6K signals, which was mediated by miR-499a/PDCD4 axis ( Figure 5(H)).

Discussion
SCI is a fatal damage that brings about severe symptoms, and the prevention and treatment of this injury has aroused more attention [32]. In very recent studies, lncRNA SNHG5 has been shown to promote astrocytes and microglia viability in SCI [33]; and lncRNA ZNF667-AS1 impacts potential function in promoting recovery of SCI [34]. Moreover, lncRNA SNGH16 is shown to reduce H 2 O 2 -disposed cell injury in PC-12 cells, providing a new reference for remedying SCI [35]. These data suggest that lncRNAs may be pivotal factors in SCI.
There are plenty of cancer related researches rounding the pivotal roles of ANRIL; nevertheless, the impacts of ANRIL on SCI remain unfully discovered. In our study, we found that H 2 O 2 disposed PC-12 cell injury and promoted ANRIL level. Silencing of ANRIL inhibited H 2 O 2 -disposed PC-12 cell injury through promoting cell viability, migration, invasion and inhibiting apoptosis and autophagy. Also, miR-499a was upregulated after silencing of ANRIL, and inhibition of miR-499a reversed the effects of silencing of ANRIL on H 2 O 2 -disposed PC-12 cell injury. Also, PDCD4 was a target of miR-499a. Furthermore, ANRIL silencing alleviated the H 2 O 2 -disposed injury in PC-12 cells possible by activating PI3K/Akt/ mTOR/p70S6K signals, which was mediated by miR-499a/ PDCD4 axis. ANRIL may provide a promising proposal for spinal cord repair.
Extensive studies have disclosed that lncRNAs are participating in the processes of plenty of diseases on the identity of can function as ceRNAs via sponging miRNAs [27,28], as well as in SCI by targeting miRNAs [36]. Moreover, miRNAs are involved in the biology and process of nerve development and injury repair [37,38]. As for this research, ANRIL showed a negative association with miR-499a, and PDCD4 was a target of miR-499a. miR-499a polymorphism was associated with the progression of several cancers, such as oral squamous cell carcinoma [39], breast cancer [40] and hepatocellular carcinoma [41]. The genetic polymorphism of miR-499 A>G (rs3746444) is also associated with the risk of ischemic stroke in a Chinese people [42]. Besides, PDCD4 was pointed out that its abnormal level was correlated with cell death of myocardium and brain tissue post injuries [43]. PDCD4 was targeted by miRNA-21, and this pattern can

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protect spinal cords against ischemia-reperfusion injury [43]. Moreover, tetramethylpyrazine could enhance functional recovery after contusion SCI by regulating PDCD4 expression [44]. We indicated that inhibition of miR-499a changeovers the impacts of silencing of ANRIL on H 2 O 2 -disposed PC-12 cell injury, and the impacts of inhibition of miR-499a on aggravated H 2 O 2 -disposed PC-12 injury were reversed post knockdown of PDCD4. Although the role of miR-499a and PDCD4 in SCI has not been fully clarified, we speculate that ANRIL may sponge miR-499a to regulate PDCD4 expression, thus playing a significant role in SCI. The PI3K/Akt signal is a major determinant in regulating multiple cellular processes, including SCI [45,46]. It is also reported that the activated PI3K/Akt signal is pivotal in improving recovery from SCI [24]. Also, the activated Akt/ mTOR/p70S6K signals can help to improve motor function

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T E D and the regenerative environment for SCI [31]. Furthermore, PI3K/Akt signal is discovered as a pivotal mediator participating in the transcriptional regulation of some lncRNAs, including ANRIL [47]. In current research, our data showed that ANRIL silencing alleviated the H 2 O 2 -disposed injury in PC-12 cells possibly by activating PI3K/Akt/mTOR/p70S6K signals, which was mediated by miR-499a/PDCD4 axis. We thus speculate that PI3K/Akt/mTOR/p70S6K signals are a latent pivotal downstream mechanism to mediate the role of ANRIL/miR-499a/PDCD4 axis in SCI. In sum, our results indicate that high level of ANRIL may enhance SCI via targeting miR-499a/PDCD4 axis to regulate the briskness of PI3K/Akt/mTOR/p70S6K signals. Our study will lay a new sight in better understanding of the mechanism of SCI and provide a novel perspective for the treatment of this disorder.

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