Long non-coding RNA ANCR promotes progression of NSCLC by inhibiting E-Ca expression
Authors: L Jiang
Z-P Li
Y-X Zhou
Z-H Zhu
T Zhou
W-W Han
J-J Fang
Journal: European review for medical and pharmacological sciences (2020)
Discipline: PHARMACOLOGY & TOXICOLOGY
Keywords: ANCR; NSCLC; E-Ca; qRT-PCR; EZH2
Affiliations: Department of Emergency, Ningbo Medical Center Eastern Hospital, Ningbo, China. .

Abstract:  Objective: This study aimed to investigate whether long-chain non-coding ANCR is involved in the progression of non-small cell LCa (NSCLC) and its possible molecular mechanisms. Patients and methods: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was applied to examine ANCR expression in 48 cases of NSCLC and adjacent normal tissues. In addition, ANCR level in patients of different tumor staging was analyzed. The Kaplan-Meier method was applied to analyze the interplay between ANCR expression and the prognosis of patients with NSCLC. Subsequently, qRT-PCR was performed to detect ANCR level in LCa cell lines. After knocking down ANCR in A549 cells, ANCR and E-Ca mRNA expression were examined by qRT-PCR, while the expression levels of epithelial-mesenchymal transition (EMT)-related proteins were detected by Western blot. At the same time, cell viability and migration ability were analyzed through cell counting kit-8 (CCK-8) and cell wound healing assay, respectively. RNA immunoprecipitation (RIP) test was performed to verify the binding of ANCR to EZH2. After knocking down EZH2 in A549 cells, E-Ca messenger ribonucleic acid (mRNA) expression was detected. Additionally, Chromatin immunoprecipitation (ChIP) assay was performed to detect the binding of EZH2 to the E-Ca promoter region. When E-Ca and ANCR were simultaneously knocked down in A549 cells, Western blot investigation was performed to examine the expression of EMT-related proteins, while CCK-8 and wound healing assays were applied to figure out the changes in cell viability and cell migration capacity. Results: ANCR level was conspicuously higher in NSCLC tissues than that in normal tissues, and that in T3 and T4 tumors was also higher than that in T1 and T2. Meanwhile, ANCR expression in the tissues of patients with lymph node metastasis was conspicuously higher than those without metastasis. Survival analysis revealed that the overall survival of patients with NSCLC with high expression of ANCR was conspicuously lower than patients with low expression of ANCR. The qRT-PCR study verified that ANCR was highly expressed in the LCa cell line A549. After knocking down ANCR in A549 cells, ANCR and E-Ca mRNA levels were found conspicuously decreased, and so were the expression levels of EMT-related proteins, as well as the cell viability and migration ability. The RIP assay result indicated that ANCR can indeed bind to EZH2. E-Ca mRNA expression was elevated after the knockdown of EZH2 in A549 cells. In addition, the result of CHIP test demonstrated that EZH2 could combine with E-Ca. Simultaneous down-regulation of ANCR and E-Ca in A549 cells could reverse the influence of knocking down ANCR alone on cell viability and migration ability. Conclusions: Long-chain non-coding RNA ANCR was highly expressed in NSCLC tissues and could enhance the viability and malignancy of NSCLC cells by inhibiting the expression of E-Ca, thereby promoting the progression of NSCLC. 

Retracted: Yes

PubPeer comments:
 This paper is one of a set of (as of now) five papers that all share unexpected similarities. The papers are from different groups of authors at different hospitals, describing different cancer types. Yet, they share several similar features. * All five papers describe a group of 48 patients * All five papers describe the same set of GAPDH primers. One of them (the RV primer) does not appear to match the GAPDH mRNA sequence. * Figures 1 of all five papers show black bar graphs with error bars and a Kaplan-Meier curve with a blue and a red line * Two papers (XX Ma et al. and JL Xu et al.) share nearly identical positive correlation curves * All five papers show photos of wound healing (scratch) assays with the same background, suggesting they were all taken with the same microscope. Here is the set of papers: 1. J.-L. Xu, T. Hua, J. Ding, Y. Fan, Z.-J. Liu, J.-W. Lian - FOXF2 aggravates the progression of non-small cell lung cancer through targeting lncRNA H19 to downregulate PTEN - Eur Rev Med Pharmacol Sci 2019; 23 (24): 10796-10802 - DOI: 10.26355 eurrev_201912_19782 - Department of Oncology, Xi an Chest Hospital, Xi an, China 2. Q.-Z. Meng, C.-H. Cong, X.-J. Li, F. Zhu, X. Zhao, F.-W. Chen - METTL3 promotes the progression of nasopharyngeal carcinoma through mediating M6A modification of EZH2 - Eur Rev Med Pharmacol Sci 2020; 24 (8): 4328-4336 - DOI: 10.26355 eurrev_202004_21014 - Department of Head and Neck Surgery, Linyi Cancer Hospital, Linyi, China 3. T. Chen, J. Li, M.-H. Zhou, L.-J. Xu, T.-C. Pan - IL-6 stimulates lncRNA ZEB2-AS1 to aggravate the progression of non-small cell lung cancer through activating STAT1 - Eur Rev Med Pharmacol Sci 2020; 24 (7): 3734-3740 - DOI: 10.26355 eurrev_202004_20837 - Department of Cardiovascular Surgery, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 4. T. Zhou, J.-J. Fang, Y.-X. Zhou, Z.-P. Li, L. Jiang, W.-W. Han, Z.-H. Zhu - Long non-coding RNA ANCR promotes progression of NSCLC by inhibiting E-Ca expression - Eur Rev Med Pharmacol Sci 2020; 24 (3): 1250-1257 - DOI: 10.26355 eurrev_202002_20178 - Department of Respiratory Medicine, Zhoushan Hospital of Zhejiang University, Zhoushan, China 5. X.-X. Ma, Z.-G. Cao, S.-L. Zhao - m6A methyltransferase METTL3 promotes the progression of prostate cancer via m6A-modified LEF1 - Eur Rev Med Pharmacol Sci 2020; 24 (7): 3565-3571 - DOI: 10.26355 eurrev_202004_20817 - Department of Urology, Nanjing First Hospital, Nanjing Medical University, Nanjing, China Here is a comparison of the Figures 1 from these papers. The very similar looking correlation curves of XX Ma et al. and JL Xu et al. have been highlighted with red boxes.  Here is a comparison of the wound healing assay photos, with similar background spots highlighted in green and yellow boxes. Contrast enhanced. These appear to be irregularities in the microscope, which by itself is not a problem. But it is unexpected to see the same similarities in photos obtained across different laboratories.   Could the authors please clarify how these photos look so similar? , 
 Withdrawn. https: www.europeanreview.org article 23365 *The article u201cLong non-coding RNA ANCR promotes progression of NSCLC by inhibiting E-Ca expression, by T. Zhou, J.-J. Fang, Y.-X. Zhou, Z.-P. Li, L. Jiang, W.-W. Han, Z.-H. Zhu, published in Eur Rev Med Pharmacol Sci 2020; 24 (3): 1250-1257u2013DOI: 10.26355 eurrev_202002_20178u2013PMID: 32096155u201d has been withdrawn from the authors.* *They stated that u201cduring the last few months, new experimental data have been obtained and analyzed, so we want to rewrite the paper to further test the cell experiment and give more evidence to support our paper. It is difficult to repeat the part of the cell experiment, and the results need to be further improved. In addition, the internal reference pictures in the paper are wrong and need to be re experimented and modified. This needs more detailed studies and maybe a long time, so we want to withdraw our paper and resubmit it when it is readyu201d.* *The Publisher apologizes for any inconvenience this may cause.* , 
 Although already retracted, it might be worth noticing that the survival curve in Figure 1D suggests the patients were followed for 10 years (120 months). Yet, they were recruited from July 2016 to December 2018 and the paper was published in 2020. This time line does not seem to be correct. HT: Hoya Camphorifolia.  , 
 This paper is one of a set of (as of now) five papers that all share unexpected similarities. The papers are from different groups of authors at different hospitals, describing different cancer types. Yet, they share several similar features. * All five papers describe a group of 48 patients * All five papers describe the same set of GAPDH primers. One of them (the Reverse primer) does not appear to match the GAPDH mRNA sequence. * Figures 1 of all five papers show black bar graphs with error bars and a Kaplan-Meier curve with a blue and a red line * Two papers (XX Ma et al. and JL Xu et al.) share nearly identical positive correlation curves * All five papers show photos of wound healing (scratch) assays with the same background, suggesting they were all taken with the same microscope. Here is the set of papers: 1. J.-L. Xu, T. Hua, J. Ding, Y. Fan, Z.-J. Liu, J.-W. Lian - FOXF2 aggravates the progression of non-small cell lung cancer through targeting lncRNA H19 to downregulate PTEN - Eur Rev Med Pharmacol Sci 2019; 23 (24): 10796-10802 - DOI: 10.26355 eurrev_201912_19782 - Department of Oncology, Xi an Chest Hospital, Xi an, China 2. Q.-Z. Meng, C.-H. Cong, X.-J. Li, F. Zhu, X. Zhao, F.-W. Chen - METTL3 promotes the progression of nasopharyngeal carcinoma through mediating M6A modification of EZH2 - Eur Rev Med Pharmacol Sci 2020; 24 (8): 4328-4336 - DOI: 10.26355 eurrev_202004_21014 - Department of Head and Neck Surgery, Linyi Cancer Hospital, Linyi, China 3. T. Chen, J. Li, M.-H. Zhou, L.-J. Xu, T.-C. Pan - IL-6 stimulates lncRNA ZEB2-AS1 to aggravate the progression of non-small cell lung cancer through activating STAT1 - Eur Rev Med Pharmacol Sci 2020; 24 (7): 3734-3740 - DOI: 10.26355 eurrev_202004_20837 - Department of Cardiovascular Surgery, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 4. T. Zhou, J.-J. Fang, Y.-X. Zhou, Z.-P. Li, L. Jiang, W.-W. Han, Z.-H. Zhu - Long non-coding RNA ANCR promotes progression of NSCLC by inhibiting E-Ca expression - Eur Rev Med Pharmacol Sci 2020; 24 (3): 1250-1257 - DOI: 10.26355 eurrev_202002_20178 - Department of Respiratory Medicine, Zhoushan Hospital of Zhejiang University, Zhoushan, China 5. X.-X. Ma, Z.-G. Cao, S.-L. Zhao - m6A methyltransferase METTL3 promotes the progression of prostate cancer via m6A-modified LEF1 - Eur Rev Med Pharmacol Sci 2020; 24 (7): 3565-3571 - DOI: 10.26355 eurrev_202004_20817 - Department of Urology, Nanjing First Hospital, Nanjing Medical University, Nanjing, China Here is a comparison of the Figures 1 from these papers. The very similar looking correlation curves of XX Ma et al. and JL Xu et al. have been highlighted with red boxes.  Here is a comparison of the wound healing assay photos, with similar background spots highlighted in green and yellow boxes. Contrast enhanced. These appear to be irregularities in the microscope, which by itself is not a problem. But it is unexpected to see the same similarities in photos obtained across different laboratories.   Could the authors please clarify how these photos look so similar? , 
 [Retracted] . --------------------------------- The article u201cLong non-coding RNA ANCR promotes progression of NSCLC by inhibiting E-Ca expression, by T. Zhou, J.-J. Fang, Y.-X. Zhou, Z.-P. Li, L. Jiang, W.-W. Han, Z.-H. Zhu, published in Eur Rev Med Pharmacol Sci 2020; 24 (3): 1250-1257u2013DOI: 10.26355 eurrev_202002_20178u2013PMID: 32096155u201d has been withdrawn from the authors. They stated that u201cduring the last few months, new experimental data have been obtained and analyzed, so we want to rewrite the paper to further test the cell experiment and give more evidence to support our paper. It is difficult to repeat the part of the cell experiment, and the results need to be further improved. In addition, the internal reference pictures in the paper are wrong and need to be re experimented and modified. This needs more detailed studies and maybe a long time, so we want to withdraw our paper and resubmit it when it is readyu201d. The Publisher apologizes for any inconvenience this may cause. ,