Reduced CTGF expression promotes cell growth, migration, and invasion in nasopharyngeal carcinoma
Authors: Xiaoli Yu
Qiangyun Wu
Xiaoming Lyu
Mengyang Zhao
Qiaofen Fu
Ye Song
Weiyi Fang
Huiling Yang
Yajie Zhang
Yan Chen
Yan Zhen
Yanfen Ye
Ying Zhou
Zhen Liu
Hao Wang
Shengni Hua
Chunping Mai
Journal: PLoS ONE (2013)
Discipline: Multidisciplinary
Keywords: CTGF; nasopharyngeal carcinoma; tumor progression; prognosis; cell proliferation
Affiliations: Cancer Research Institute of Basic Medicine School, Southern Medical University, Guangzhou, PR China.

Abstract:  Background: The role of CTGF varies in different types of cancer. The purpose of this study is to investigate the involvement of CTGF in tumor progression and prognosis of human nasopharyngeal carcinoma (NPC). Experimental design: CTGF expression levels were examined in NPC tissues and cells, nasopharynx (NP) tissues, and NP69 cells. The effects and molecular mechanisms of CTGF expression on cell proliferation, migration, invasion, and cell cycle were also explored. Results: NPC cells exhibited decreased mRNA expression of CTGF compared to immortalized human nasopharyngeal epithelial cell line NP69. Similarly, CTGF was observed to be downregulated in NPC compared to normal tissues at mRNA and protein levels. Furthermore, reduced CTGF was negatively associated with the progression of NPC. Knocking down CTGF expression enhanced the colony formation, cell migration, invasion, and G1/S cell cycle transition. Mechanistic analysis revealed that CTGF suppression activated FAK/PI3K/AKT and its downstream signals regulating the cell cycle, epithelial-mesenchymal transition (EMT) and MMPs. Finally, DNA methylation microarray revealed a lack of hypermethylation at the CTGF promoter, suggesting other mechanisms are associated with suppression of CTGF in NPC. Conclusion: Our study demonstrates that reduced expression of CTGF promoted cell proliferation, migration, invasion and cell cycle progression through FAK/PI3K/AKT, EMT and MMP pathways in NPC. 

Retracted: No

PubPeer comments:
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 2020 Expression of Concern. https: journals.plos.org plosone article?id=10.1371 journal.pone.0231520 After publication of this article [1], concerns were raised about the identity of the cell lines used in the study, regions of overlap between some image panels in Fig 4, western blot methodology and representation in Fig 5, and the availability of the underlying data. Four of the eight nasopharyngeal carcinoma (NPC) cell lines used in the qPCR experiment shown in Fig 1B have been reported elsewhere to be possibly contaminated or misidentified (CNE1, CNE2, HONE1, and HNE1, see ICLAC Register of Misidentified Cell Lines (iclac.org databases cross-contaminations ) [2], Cellosaurus [3] and [4u20135]). Of these potentially misidentified cell lines, only HONE1 was used in the further functional studies reported in the article. The corresponding author has provided STR profile reports for the 5-8F cells and the HONE1 cells (S1 File). In addition to HONE1, the follow-up experiments reported in [1] use only one other NPC cell line, 6-10B, which may limit the generalizability of the findings as representative of nasopharyngeal cancer biology. Clarification of the source and provenance of the cell lines used is as follows: 5-8F, 6-10B, and SUNE1 are from stock kept at the Cancer Institute of Southern Medical University since 2003, originally obtained from Cancer Center, Sun Yat-sen University where these cell lines were generated [6, 7]. C666-1 cell line was generated by the Chinese University of Hong Kong [8]. It is from stock kept by the Cancer Institute of Southern Medical University since 2003, originally obtained from Cancer Center of Sun Yat-sen University. CNE1 and CNE2 are from stock kept by the Cancer Institute of Southern Medical University since 1999. HONE1 and HNE1 are from stock kept by the Cancer Institute of Southern Medical University, originally obtained from the Cancer Institute of Hunan Medical University (now the Central Southern University) where these cell lines were generated. NP69 [9] is from stock kept by the Cancer Institute of Southern Medical University, since 2009, originally obtained from Cancer Center, Sun Yat-sen University where the cell line was generated. In Fig 4C, it was noted that the si-CTGF panel in 6-10B cells is the same as Fig 4D si-CTGF panel in 6-10B cells. Additionally, there is a region of overlap between the images in Fig 4C si-CTGF panel in HONE1 cells and Fig 4C si-Ctr in HONE1 cells. The authors confirmed that incorrect image files were used for the following panels: Fig 4A shCTGF-B panel; All panels of Fig 4B; Fig 4C HONE1 cell, si-CTGF panel; Fig 4C HONE1 cell, si-Ctr panel; All panels of Fig 4D. A revised Fig 4 is provided here in which the incorrect image panels are replaced. The underlying data for Fig 4, including the representative image files, other replicate image files, and individual-level datasets for the charts, are provided as S2 File. The authors stated that the errors in selecting the representative image files for Fig 4 do not affect the statistical data collected and reported in the accompanying charts. In Fig 5, the authors clarified that the u03b2-actin panels shown are representative only, and do not show the actual loading controls for all target proteins shown in parts A, B and C, respectively. Actin loading controls were not run on the same gels as target proteins in each experiment. Protein quantification was carried out to ensure equal sample loading, and blots were checked for consistency of actin control bands. Target protein expression levels were compared when actin protein expression was observed to be relatively consistent. The available uncropped western blot images underlying Fig 5 are provided as Supporting Information (S3 File). The underlying data files are no longer available for the immunohistochemistry images in Fig 1D, the western blots in Figs 2A and 3A, the western blots for PI3K, pPI3K, AKT, pAKT, pRb, E2F1, Snail, E-cadherin, Vimentin and u03b2-actin in Fig 5, and the charts in Figs 2B and 3B. The raw data for the NimbleGen DNA methylation microarray experiment reported in Fig 6 were not deposited in a public repository as required by the standards of the field. The dataset was subsequently deposited and is available at https: datadryad.org stash dataset doi:10.5061 dryad.wh70rxwj1. No additional underlying data are available for this article. The following additional issues were noted: The Transient Transfection with siRNAs subsection of the Materials and Methods of [1] states that the siRNA sequences for the gene and the controls are provided in Table 3. However, the control sequences are not provided. The control siRNA sequences were designed and synthesized by Guangzhou RiboBio (RiboBio Inc, China) and are protected under a patent. The antisense strand of the siRNAs targeting CTGF are: 5u2019 UAUGUCUUCAUGCUGGUGC dTdT 3u2019 5u2019 UAAUCAUAGUUGGGUCUGG dTdT 3u2019 5u2019 UUUGGGAGUACGGAUGCAC dTdT 3u2019 These sequences are reported in Table 2 of [1] in 3u2019-5u2019 direction. There is an error in the text legend for Fig 1D. A complete, correct Fig 1 legend is provided here. The PLOS ONE Editors issue this Expression of Concern to alert readers to the concerns about the accuracy of the representation of the western blot data and of the migration and invasion image panels, and the use of cell lines previously reported to be misidentified. ,