Regulation of miR-146a by RelA/NFkB and p53 in STHdh(Q111)/Hdh(Q111) cells, a cell model of Huntington's disease
Authors: Jayeeta Ghose
Nihar R Jana
Mithun Sinha
Eashita Das
Nitai P Bhattacharyya
Journal: PloS one (2011)
Discipline: Multidisciplinary
Keywords: Huntington's disease; miRNAs; transcription factors; mutant HTT; p53
Affiliations: Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata, West Bengal, India.

Abstract:  Huntington's disease (HD) is caused by the expansion of N-terminal polymorphic poly Q stretch of the protein huntingtin (HTT). Deregulated microRNAs and loss of function of transcription factors recruited to mutant HTT aggregates could cause characteristic transcriptional deregulation associated with HD. We observed earlier that expressions of miR-125b, miR-146a and miR-150 are decreased in STHdh(Q111)/Hdh(Q111) cells, a model for HD in comparison to those of wild type STHdh(Q7)/Hdh(Q7) cells. In the present manuscript, we show by luciferase reporter assays and real time PCR that decreased miR-146a expression in STHdh(Q111)/Hdh(Q111) cells is due to decreased expression and activity of p65 subunit of NFkB (RelA/NFkB). By reporter luciferase assay, RT-PCR and western blot analysis, we also show that both miR-150 and miR-125b target p53. This partially explains the up regulation of p53 observed in HD. Elevated p53 interacts with RelA/NFkB, reduces its expression and activity and decreases the expression of miR-146a, while knocking down p53 increases RelA/NFkB and miR-146a expressions. We also demonstrate that expression of p53 is increased and levels of RelA/NFkB, miR-146a, miR-150 and miR-125b are decreased in striatum of R6/2 mice, a mouse model of HD and in cell models of HD. In a cell model, this effect could be reversed by exogenous expression of chaperone like proteins HYPK and Hsp70. We conclude that (i) miR-125b and miR-150 target p53, which in turn regulates RelA/NFkB and miR-146a expressions; (ii) reduced miR-125b and miR-150 expressions, increased p53 level and decreased RelA/NFkB and miR-146a expressions originate from mutant HTT (iii) p53 directly or indirectly regulates the expression of miR-146a. Our observation of interplay between transcription factors and miRNAs using HD cell model provides an important platform upon which further work is to be done to establish if such regulation plays any role in HD pathogenesis. 

Retracted: No

PubPeer comments:
 I became interested in the far-reaching influence of a 2018 in-vitro paper about Daidzein, [Hua et al (2018)] . Google Scholar informs us that it has been cited **71** times. Many of these are review papers, but we also find a surprising number of papers using it in  Apoptosis Assay paragraphs to illustrate the use of flow-cytometry and Annexin V PI staining to quantify cell apoptosis. Indeed, Hua et al did apply the method, but they were not pioneers, nor is their explanation superior to many others. Alternatively, the paper is cited as a precedent for a phytochemical (daidzein) inhibiting cancer proliferation by interrupting the cell cycle. Given all the earlier precedents, the choice again invites some explanation. It is almost as if all these papers were prepared by a third-party service (a papermill ), following a template from one paper to the next, without checking its contents. Consistent with this notion, the papers came to my attention by recycling the same figures (despite a lack of authors in common). Please excuse the length and list-based nature of this comment. Feel free to skip to the end. There will not be a test. - [Jia et al (2019)] .  The slides were covered with a covers lip and examined with a fluorescent microscope. Annexin V PI staining of the OVACAR3 cells was performed as described previously [17]. - [Luo et al (2019)] .  Many plant-derived molecules have been reported to interfere with the **ROS** level in the cancer cells [13]. ... The slides were then covered with a covers lip and examined with a fluorescent microscope. Then 4u2019,6-diamidino-2-phenylindole (DAPI) and annexin V propidium iodide (PI) staining was performed as described previously [13]. - [Zeng et al (2019)] .  The slides were then covered with a covers lip and examined with a fluorescent microscope (BDBiosciences, San Jose, CA, USA). The annexin V PI staining was performed as described previously [8]. - [Wu et al (2019)] *[retracted]*,  The slides were covered with a coverslip and examined with a fluorescent microscope (BD Biosciences, San Jose, CA, USA). Annexin V PI staining was performed as described previously [13]. - [Yang, Chen Wang (2019)] .  The slides were covered with cover slips and examined with a fluorescent microscope. Annexin V PI staining of the SW-948 cells was performed as described previously 16 . - [Yang et al (2019)] .  The slides were covered with cover slips and examined with a fuorescent microscope. DAPI and annexin V propidium iodide (PI) staining were performed as described previously [14] *[14 is a misprint for 13]*. - [Huang et al (2019)] .  The slides were covered with cover slip and examined under fluorescence microscope. Annexin V propidium iodide (PI) staining of the Icariin-treated HeLa cells was performed as described previously [18]. - [Liu et al (2019)] .  The distribution of the breast cancer SK-BR-3 cells in different cycle phases was performed by flow cytometry after PI staining, following the method reported by Hua et al [11]. ...After overnight incubation at 4u00b0C, the cells were subjected to PI staining and flow cytometry. - [Liu et al (2019)] .  After covering the glass slides with cover slips, the cells were analyzed under a fluorescent microscope (Nikon Instruments, Inc., NY, USA). For determining percentage of apoptotic cells, Annexin V PI staining assay using a FACSCalibur flow cytometry (BD Biosciences, NY, USA) was carried out using a previously reported method [12] - [Yin et al (2019)] .  The slides were covered with covers slips and examined with a fluorescent microscope. Annexin V PI staining of the SW-948 cells was performed as previously described [13] . - [Zhang et al (2019)] .  The slides were cover-slipped and examined with a fluorescent microscope. Annexin V PI staining of the SW48 cells was performed as described previously [11] . - [Ma et al (2019)] .  The slides were cover-slipped and examined under a fluorescent microscope. Annexin V PI staining was performed as described previously [15]. - [Li et al (2019)] .  The slides were cover-slipped and assessed by fluorescent microscopy. ROS estimation was performed as described previously [13]. - [Zhang et al (2019)] .  The slides were covered with a cover slip and examined with a fluorescent microscope. Annexin V PI staining was performed as previously described [12]. - [Wu et al (2020)] .  The slides were covered with a covers lip and examined with a fluorescent microscope. Annexin V PI staining of the AGS cells was performed as described previously [15]. - [Yu et al (2020)] .  The slides were cover-slipped and examined with a fluorescent microscope. Annexin V PI staining was performed as described previously [9]. - [Yin et al (2020)] .  The slides were cover-slipped and examined with a fluorescent microscope. Annexin V PI staining was performed as described previously [10]. - [Wei et al (2019)] .  The slides were covered with cover slips and examined with a fluorescent microscope. Annexin V PI staining of the OVACAR-3 cells was performed as described previously [11]. - [Wen et al (2020)] .  The glass slides laden with cell cultures were then covered with cover slips and then using a fluorescence microscope (BioTek Instruments, Inc., Winooski, VT, USA,) the cells were analysed. Quantification of apoptosis was done with annexin V PI staining assay which was performed as reported already (Hua et al., 2018). -------------------------------------------------------------- Cell-cycle arrest: - [Li Han (2019)] .  Cell cycle arrest is another important mechanism by which anticancer molecules exert their effects [16] . - [Mao et al (2019)] .  The arrest of the cancer cells at G2 M phase prevents the cancer cells from entering mitosis and as such the molecules that trigger the cell cycle arrest prevent the proliferation and tumourigenesis of cancer cells (Hua et al., 2018). - [Si et al (2019)] .  Previous studies have shown that depletion of cyclin A and B1 and upregulation of p27 and p21 results in the G2 M arrest of the cells during cell division [21]. - [Zhou et al (2019)] .  The arrest of cancer cells at G2 M phase prevents the cancer cells from entering mitosis, so the molecules that trigger cell cycle arrest prevent the proliferation and tumorigenesis of cancer cells [20]. - [Tan et al (2019)] .  Cell cycle arrest caused by several anticancer agents is an essential mechanism in the growth inhibition of cancer cells [19] . - [Wei et al (2019)] .  Furthermore, arrest of the cells at different phases of the cell cycle is an important route by which anticancer agents halt the growth of cancer cells [18]. - [Zeng et al (2019)] .  Besides autophagy, cell cycle arrest is another mechanism by which anticancer agents exert their anticancer effects [19] . -------------------------------------------------- Another list of papers cite Hua et al (2018) as precedents for methods which those authors did not in fact apply. - [Zhang et al (2019)] .  For determination of the ROS and mitochondrial membrane potential levels, the A549 cells were treated with 0, 9, 18, and 36 u00b5M concentrations of murrayanine for 24 hours and then the ROS and mitochondrial membrane potential levels in the A549 cells were determined, as described previously [11] *(in fact Hua et al did not measure or even mention ROS or mitochondrial membrane potentials)*. - [Guo et al (2019)] .  Plant-derived molecules have been reported to affect ROS levels in the cancer cells [15] . - [Xu et al (2019)] . Hua et al appear in the References but not in the text at all. - [Du et al (2019)] .  **Osteosarcoma** causes significant mortality world over and the treatments available are not efficient and are associated with a number of side-effects [11]. - [Yu et al (2019)] .  Several of the natural products have been reported to induce apoptosis and autophagy through production of ROS [18]. - [Liu et al (2019)] .  The colony formation assay was carried out as described previously [15] *[Hua et al did not perform colony-formation assays]* - [Zhu et al (2020)] .  Estimation of ROS was measured according to a method previously described [8]. - [Du et al (2020)] . Hua et al appear in the References but not in the text at all. ----------------------------------------------------------- The point of all this is that the present paper appears to have been constructed with the same template, as it includes the same stereotyped Apoptosis Assay paragraph with the same choice of Hua et al as a precedent. I hope the authors will forgive me for speculating that it is perhaps the work of the same third-party company.  The slides were covered with a cover slip and examined with a fluorescence microscope. Annexin V PI staining of the A549 cells was performed as described previously [10]. The reference is to  10. Hua F, Li CH, Chen XG, Liu XP. Daidzein exerts anticancer activity towards SKOV3 human ovarian cancer cells by inducing apoptosis and cell cycle arrest, and inhibiting the Raf MEK ERK cascade. Int J Mol Med 2018;41:3485-92. , 
 *[left]* Figure 3.  Overexpression of miR-133 inhibits the migration and invasion of lung cancer cells. Transwell assay showing A: percent migration and B: invasion in NC or miR-133 mimics transfected A549 cells. The Figure shows that miR-133 overexpression causes decrease in migration and invasion of A549 cells. *[right]* Figure 6A from [Liu et al (2018)] .  Carnosic acid inhibited the invasion of KBM-7 cells. A: Determination of cell invasion by Boyden chamber assay . ![file] , 
 The paper has undergone a stealth revision or correction, replacing Figure 3. The version currently provided on the journal website was created modified on 24 January 2021. I am impressed by the fast turn-around time at this journal, where the authors can obtain replacement non-copied Figures and the editors can insert them into the paper within one day of Comment #1. ![file] Interested readers can still consult the original version (created 11 June 2019), via the copy [archived at ResearchGate] . There they will discover that Fig 2 has also been replaced, suggesting that the original was also untrustworthy. ![file] Just one last question about the new Fig 2A... the Treatment and Control cell-count histograms appear to be identical for horizontal-axis values greater than 120. Can the authors think of a reason for this? ![file] ,