RETRACTED ARTICLE: Silencing hsa_circ_PVT1 (circPVT1) suppresses the growth and metastasis of glioblastoma multiforme cells by up-regulation of miR-199a-5p

Abstract We, the Editors and Publisher of the journal Artificial Cells, Nanomedicine, and Biotechnology, have retracted the following article: Guonan Chi, Fuwei Yang, Donghui Xu & Weiming Liu (2020) Silencing hsa_circ_PVT1 (circPVT1) suppresses the growth and metastasis of glioblastoma multiforme cells by up-regulation of miR-199a-5p. Artificial Cells, Nanomedicine, and Biotechnology, 48:1, 188–196, DOI: 10.1080/21691401.2019.1699825 Since publication, concerns have been raised about the integrity of the data in the article. We reached out to the authors requesting that they supply information that would confirm the article’s integrity. The authors provided several files in response to our queries, but they were unable to provide the original data in a format which satisfies our requirements to confirm the authenticity of some types of data. In particular, they were unable to provide full western blots, where the molecular weight markers are indicated. In addition, the authors did not have a record of the full blot, before cutting it into strips. When checking their data the authors also confirmed there are fundamental errors present. Therefore, they have agreed to the retraction of this article. 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 The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.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
Glioblastoma multiforme (GBM), also known as glioblastoma, one of the most prevailing primary brain tumours in adults and it is one of the most deadly and aggressive cancers [1]. Multiple chromosomal and genetic abnormalities are involved with GBM, leading to rapid infiltration, invasion and destruction of adjacent tissues, especially in spinal cord [2]. Low-grade GBM is generally related with neurological dysfunction [3]. Because of the increasing rate of recrudesce, infiltrative rate and limited treatment means, the prognosis of high-grade GBM is still poor [4]. Despite multiple developments in medical and surgical treatments, GBM is still a deadly disease with a high mortality rate [5]. In addition, radiotherapy for spinal GBM is often accompanied by worse outcomes [6]. Therefore, finding novel molecules participated in tumour progression is vital to improve the survival rate of GBM. Circular RNA (circRNA) is an innovative race of RNA and is widely discovered in eukaryotes [7]. Accumulating evidence suggested that various circRNAs had cell-type specific expressions and were involved in a good deal of cancers [8]. CircRNAs could act as a sponge of diverse microRNAs (miRNAs) and play an essential part in the pathological development of GBM [9]. For instance, up-regulating circTTBK-2 in GMB was proved to be the miR-217 sponge and improved proliferation, migration and invasion [10]. Another study indicated that circ_0046701 remarkably overexpressed in GMB and exerted as a sponge of miR-142-3p to develop tumorgenesis [11]. Furthermore, circRNA could also be targeted by miRNA, and miR-7 sponge CDR1-AS can be deteriorated by miR-671-5p in GMB [12]. Interestingly, several studies illustrated the function of circPVT1 in many cancers. For instance, circPVT1 facilitated growth and migration via sponging miR-125b and mobilising E2F2 pathway in non-small cell lung cancer (NSCLC) [13]. However, the function of circPVT1 on GBM remains unclear.
MiRNAs as a category of small noncoding RNA acts a critical factor of gene expression by promoting mRNA degradation or preventing the translation of target mRNA [14,15]. MiRNA has been investigated in a variety of human diseases, and plays a vital part in cell proliferation, metastasis and EMT process [15]. In spite of a large amount of researches were reported on the aspects of miRNAs, little is known about the complex and various biological effects of miRNA. Numerous literatures demonstrated that miRNAs participated in the biological progresses of GBM. For instance, miR-124 prevented growth and potentiated chemosensitivity via targeting AURKA in GBM [16]. Another study indicated that up-regulating miR-338-5p inhibited proliferation, migration, invasion and improved apoptosis in GBM cells [17]. Furthermore, miR-199a-5p was investigated to play a variety of roles in cancers. Liu et al elucidated miR-199a-5p and let-7c suppressed hepatocellular carcinoma cells metastasis capacity in [18]. In the current research, our purpose was to explore circPVT1 and miR-199a-5p function and mechanism in U591 and U251 cells.

Clinical specimens
Clinical human GBM tissues and normal adjacent tissues (n ¼ 25) were attained from China-Japan Union Hospital of Jilin University (Changchun, China). The whole patients received no therapy before receiving surgery. The agreement of every patient was gotten. Morethan, this work gained the support of the Medical Ethics Committee of China-Japan Union Hospital of Jilin University.
Cell counting kit-8 (CCK-8) assay U539 and U251 cells were hatched in a 96-well plate at the concentration of 5 Â 10 3 cells/well. After culturing, the complete culture medium was removed and added with media including 10 lL CCK-8 (Dojindo Laboratories, Tokyo, Japan), and then the cells were contained for 1 h. The absorbance was measured at 450 nm by applying a Microplate Reader (Thermo Fisher Scientific, Waltham, MA, USA).

Apoptosis assay
After transfection, the rate of U539 and U251 cells apoptosis was assessed applying flow cytometry (Beckman Coulter, Atlanta, GA, USA) in line with the direction of Annexin V-FITC/PI apoptosis detection kit (Beijing Biosea Biotechnology, Beijing, China).

Migration assay
Migration capacity was evaluated utilising transwell chambers (Millipore, Billerica, MA, USA). The 200 lL cell serum-free suspension was placed into the upper layer of chamber. 600 lL complete growth media was added to the lower layer of chamber. After cultivating for 48 h, the migrated cells were fixed with 4% paraformaldehyde (Beyotime) as well as stained with crystal violet reagent for 15 min. A microscope was conducted to assess the number of migrated cells.

Quantitative reverse transcription PCR (qRT-PCR)
The RNA of U539 and U251 cells was separated according to producer's protocols of Trizol purchased from Invitrogen (San Diego, CA, USA). The cDNA of RNA was synthesised through applying the MultiscribeRTkit (Biosystems, Barcelona, Spain). CircPVT1 and miR-199a-5p expression was detected utilising QuantiNova SYBR Green PCR Kit (Qiagen, Hilden, Germany). The relative expression was estimated applying the 2À ᭝᭝ Ct method.

Statistical analysis
Each experimental result was repeated three times. All results of various experiments were represented as the mean ± SD and computed by SPSS 19.0 statistical software (Chicago, IL, USA) with a one-way analysis of variance (ANOVA) or a paired t-test. A p-value of <.05 was appraised statistically significant.

CircPVT1 was highly expressed in GBM tissues
To explore the abnormal expression of circPVT1 in GBM tissues, qRT-PCR was adapted to quantify circPVT1. As the results displayed in Figure 1, circPVT1 levels were dramatically raised in GBM tissues (p < .001).

CircPVT1 expression was silenced by vector transfection in U539 and U251 cells
The qRT-PCR was performed to examine if it is successful for si-circPVT1 transfection. The results suggested that circPVT1 expression was remarkably reduced in U539 and U251 cells through transferring si-circPVT1. (p < .001, Figure 2).
Silencing circPVT1 suppressed viability, migration, and improved apoptosis in U539 and U251 cells For identifying the function circPVT1, the proliferation and migration of U539 and U251 cells transferred by si-circPVT1 were detected. As the evidence showed in Figure 3(A), viability was notably reduced in U539 and U251 cells when circPVT1 was silenced in U539 and U251 cells (p < .01).

Silencing circPVT1 repressed EGF-induced EMT in U539 and U251 cells
In next study, the impact of circPVT1 on was explored in GBM. Firstly, the expression of N-cadherin, Vimentin and Zeb1 was observably escalated in U539 cells by EGF, and Ecadherin expression was remarkably decreased by EGF (p < .001, Figure 4(A-B)). Subsequently, silencing circPVT1 declined N-cadherin, Vimentin and Zeb1 expression as well as elevated E-cadherin expression in U539 cells (p < .05 or p < .01, Figure 4(A-B)). Consistently, the expression of abovementioned proteins showed the same trend in U251 cells as in U539 cells (p < .05, p < .01 or p < .001, Figure 4(C-D)).

Discussion
GBM is listed as the most prevalent type of cancer in China, and its incidence is rising every year, and will remain to advance in the coming years [19]. So far, the prognosis of GBM is still very poor. Early diagnosis and prognosis of GMB require the identification of sensitive bookmakers [20]. Nevertheless, the molecular biological mechanisms of GBM

R E T R A C T E D
are complex and not yet been entirely disentangled, making it difficult to screen for key biomarkers. Thus, understanding the molecular mechanism of GBM is critical to improving the anti-cancer strategies of patients with GBM. Lately, accumulating evidence indicated that circRNAs acted an essential part in the development of GBM. Additionally, abnormal regulation of circRNAs can be observed in cancer tissues, indicating that circRNAs can be a prognostic marker and possible target for GBM therapy [11,21]. Currently, researches have illustrated that circPVT1 as a newly explored circRNA is involved in lung cancer, oral squamous cell carcinoma (OSCC), and osteosarcoma [13,22,23]. For instance, two studies have reported that there is the high expression of circPVT1 in patients suffering from NSCLC and NSCLC lines [13,24]. It was confirmed that reducing circPVT1 expression could weaken cell proliferation and invasion as well as facilitate apoptosis in NSCLC cells [13,24]. The report of OSCC discovered a decision similar to NSCLC. The expression of circPVT1 in OSCC cells and specimens was elevated, and cells growth was alleviated via decreasing circPVT1 expression [23]. Our experimental results were similar to above results of NSCLC and OSCC cancers. We found the levels of circPVT1 expression in GBM tissue were outstandingly increased, as well as silencing circPVT1 decreased viability and migration capacity, while promoted apoptosis in both U539 and U251 cells. More than that, silencing circPVT1 contributed to alleviating EGF-induced EMT in both cells. In this study, the function of circPVT1 was explored in GBM for the first time.
Up to now, miRNAs have been revealed that they are involved in a large amount of biological processes, including tumour formation and inflammatory responses in many human diseases [25][26][27]. It is well known that miR-199a-5p has been studied to take part in the progress of cancer [28]. In glioma cells, there are the lower levels of miR-199a-5p than in normal cells, as well as glioma cells proliferation and invasion were retarded when overexpressing miR-199a-5p [29,30]. Our results were similar to the results of previous studies, we discovered overexpressing miR-199a-5p attenuated U539 and U251 cells proliferation and migration.
In addition, a number of researches have expounded that circPVT1 could effectively sponge miR-125b, miR-145 and miR-497 to promote cell growth and metastasis in colorectal cancer and NSCLC [13,24,31]. Hence, we verified the

R E T R
A C T E D correlation of circPVT1 and miR-199a-5p as well as the impact of circPVT1 and miR-199a-5p on human glioma cell lines. Our results demonstrated that silencing circPVT1 decreased viability and migration and promoted apoptosis by aggrandising miR-199a-5p in U539 and U251 cells. Furthermore, silencing circPVT1 restrained EGF-induced EMT via miR-199a-5p augment. YAP is a proline-rich phosphoprotein and encoded by YAP1 gene. The activation of YAP1 may be associated with stem cells, organ sized and cancer. With the increase of YAP1 expression, the invasiveness of cancer cells is enhanced. In Orr's study, YAP1 was transfected with shRNA plasmid into U87, U373 and HSR-GBMI cells, the data indicated that the knockdown of YAP1 remarkably declined GBM cells growth [32]. And the down-regulation of YAP1 may provide a new approach to treat GBM [32]. PI3K/AKT pathway is a generally uncontrolled pathway in cancer; 86% of GBM clinical specimens indicate the changes of the receptor tyrosine kinases/ PI3K pathway [33,34]. In addition, the sustained activation of AKT1 endothelial cells has been shown to induce the formation of structurally abnormal blood vessels, thereby regenerating tumour vascular abnormalities [33]. PI3K/AKT pathway is a complicated central regulator of a quantity of basic cellular processes that mediates all phenotypes that conduce to human cancer progression, including GBM [35]. The AKT pathway was proved to be mobilised in EC tumours in the previous study [36]. Growth factor signalling, intracellular metabolism, and extracellular signal molecules regulated the growth and proliferation of malignant GBM via PI3K/AKT pathway [37,38]. Moreover, PI3K/AKT pathway was considered to be a key regulator of growth and proliferation in GBM cells [39]. Consistent with previous studies, our data implied that silencing circPVT1 repressed YAP1 and PI3K/AKT pathways. Additionally, these two pathways were partially activated via impededing miR-199a-5p, which displayed that circPVT1 exerted its protective effect on YAP1 and PI3K/AKT pathways by up-regulating miR-199a-5p. Although our study made major breakthroughs in the molecular mechanism of GBM and provided a good theoretical basis for the treatment of GBM, it was mainly limited to in vitro experiments and lacked clinical application.

Conclusion
On the whole, we first investigated the function and interrelation of circPVT1 and miR-199a-5p in GBM. The results indicated a protective effect of circPVT1 on U539 and U251 cells in vitro. Firstly, silencing circPVT1 declined viability, migration and EGF-induced EMT, as well as facilitated apoptosis in U539 and U251 cells. Subsequently, silencing circPVT1 could raise the level of miR-199a-5p in both cells, as well as silencing circPVT1 reduced cells viability, migration, EGF-induced EMT and accelerated apoptosis via promoting miR-199a-5p expression. Moreover, further researches indicated that silencing circPVT1 repressed YAP1 and PI3K/AKT pathway via up-regulating miR-199a-5p. The evidence uncovered an innovative molecular mechanism of the impacts of circPVT1 and miR-199a-5p on GBM, proving an advanced target for clinical therapy.