MicroRNA-520c-3p negatively regulates EMT by targeting IL-8 to suppress the invasion and migration of breast cancer
Authors: Han-Jing Zhou
Jian Qin
Tao Zhang
Yi Luo
Cui-Ping Tang
Journal: Oncology Reports (2017)
Discipline: CLINICAL MEDICINE
Keywords: Interleukin-8; miR-520c-3p; EMT; breast cancer cells; 3'-UTR
Affiliations: Department of Oncology, The First Affiliated Hospital of Chongqing Medical University, Yuzhong, Chongqing 400016, P.R. China.

Abstract:  Interleukin-8 (IL-8), which is secreted by cancer cells undergoing epithelial-mesenchymal transition (EMT), can promote EMT in adjacent epithelial-like cells. MicroRNAs (miRNAs/miRs) can affect the expression of target genes via binding to their 3'-untranslated regions (3'-UTRs), which may subsequently affect the biological behaviors of cancer cells. In our previous study, miR-520c-3p was predicted to directly target the 3'-UTR of IL-8. Therefore, the present study was carried out to investigate whether miR-520c-3p can interact with the IL-8 gene and regulate the EMT of breast cancer cells. Web-based prediction algorithms were used to identify miRNAs that potentially target the IL-8 transcript. Luciferase reporter assays were used to confirm the targeting of IL-8 by miR-520c-3p. Reverse transcription-quantitative PCR and western blot analyses were used to examine the levels of IL-8 and EMT-related genes in breast cancer cells. The functional impact of miR-520c-3p on EMT phenotype was evaluated using Transwell and wound-healing assays, and rescue experiments were conducted by overexpressing IL-8 to determine its effect on cell properties. miR-520c-3p was predicted by all three databases, which strongly suggested its interaction with the 3'-UTR of IL-8. The relative Renilla luciferase activity of luciferase reporter construct containing the wild-type 3'-UTR of IL-8 was markedly decreased by miR-520c-3p transfection when compared with scrambled miRNA control transfection (P&lt;0.001). In addition, compared with the scrambled miRNA control transfection, the overexpression of miR-520c-3p significantly reduced the expression of IL-8, and resulted in increased E-cadherin and decreased vimentin and fibronectin levels in MCF-7 and T47D cells (all P&lt;0.001). Introduction of miR-520c-3p inhibited the invasion and migration of MCF-7 and T47D cells (all P&lt;0.001). By contrast, the rescue of IL-8 expression led to the recovery of EMT-related protein expression patterns and cell motility and invasion capabilities. In conclusion, aberrant miR-520c-3p expression may lead to reduced IL-8 expression and promote the mesenchymal phenotype in breast cancer cells, thereby increasing invasive growth. 

Retracted: No

PubPeer comments:
 I contend that the paper does not support its conclusion that:  ...contemporary injection of carbon dioxide in the atmosphere as a result of combustion of fossil fuels is not important for the greenhouse effect. The majority of the paper analyzes how CO2 behaves independent of its source, arriving at this value:  ...the change of the global temperature as a result at doubling of the concentration of atmospheric CO2 molecules [is] (0.4 + - 0.1) K... Only one sentence of the analysis addresses anthropogenic CO2 sources from fossil fuel combustion, in section 6, page 9:  Because anthropogenic fluxes of carbon dioxide in the atmosphere resulted from combustion of fossil fuels is about 5%, the contribution of the human activity of ECS (the temperature change as a result of doubling of the atmospheric carbon dioxide amount) is 0.02 K, i.e. injections of carbon dioxide in the atmosphere as a result of combustion of fossil fuels is not important for the greenhouse effect. The 5% figure is uncited and the paper does not provide supporting data nor confidence level for it. Nevertheless, by calculating 0.4K x 0.05 = 0.02K, the paper implicitly asserts that: **Fossil fuel combustion is about 5% responsible for an increase in atmospheric CO2 concentration.** This assertion requires supporting evidence, which the paper does not provide. Other research contradicts it: u201cWith a very high level of confidence, the increase in CO2 emissions from fossil fuel burning and those arising from land use change are the dominant cause of the observed increase in atmospheric CO2 concentration.u201d [1] The paper s 5% figure may refer to an approximation of total anthropogenic CO2 sources divided by total global CO2 sources. Therefore, the implied assertion might be: **If anthropogenic CO2 from fossil fuel combustion is about 5% of global CO2 sources, then it is also about 5% responsible for an increase in atmospheric CO2 concentration.** This argument is not valid because it does not consider if any of the CO2 source types increased more than others, which could skew the proportion of responsibility for increased atmospheric concentrations. For example, suppose if anthropogenic CO2 sources had recently increased while other CO2 sources remained relatively flat, then anthropogenic CO2 s contribution to the increase in atmospheric CO2 could be much closer to 100%, even if it only comprised 5% of total sources. The paper does not consider that if humans didn t burn any fossil fuels, whether or not the atmospheric CO2 would still have hypothetically doubled. Research shows that anthropogenic CO2 emissions from fossil fuel combustion have recently increased: u201cSince 1970, cumulative CO2 emissions from fossil fuel combustion, cement production and flaring have tripled...u201d [2] u201cFossil-fuel-related CO2 emissions reached 32 (u00b12.7) GtCO2 yr, in 2010, and grew further by about 3% between 2010 and 2011, and by about 1 to 2% between 2011 and 2012. CO2 remains the major anthropogenic GHG, accounting for 76% of total anthropogenic GHG emissions in 2010.u201d [2] This data would need to be compared to changes in other CO2 sources in order to ascertain the percentage of responsibility per source. Regarding the u201cimportanceu201d of fossil fuel combustion on greenhouse effect, the paper does not specify what the threshold to be considered important is for a CO2 source contribution to atmospheric CO2 concentrations or resulting temperature change. Even if fossil fuel s contribution to temperature increases could be demonstrated to be a comparatively lower percentage, this is not enough to conclude that it is not important. Smaller amounts can sometimes have significant effects, and it must be demonstrated rather than assumed that a proportionally lower contribution does not have significance. In conclusion, properly calculating the contribution and importance of fossil fuel combustion on the greenhouse effect requires significantly more data and analysis than this paper provides, and therefore its conclusion regarding fossil fuel combustion is not supported by this paper. That analysis seems out of scope for this paper which is predominantly focused on how CO2 behaves independent of source. I kindly request the author s response with any relevant clarifications or corrections. REFERENCES [1] IPCC, 2013: Climate Change 2013: The Physical Science Basis. Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change [Stocker, T.F., D. Qin, G.-K. Plattner, M. Tignor, S.K. Allen, J. Boschung, A. Nauels, Y. Xia, V. Bex and P.M. Midgley (eds.)]. Cambridge University Press, Cambridge, United Kingdom and New York, NY, USA, 1535 pp. [2] IPCC, 2014: Climate Change 2014: Synthesis Report. Contribution of Working Groups I, II and III to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change [Core Writing Team, R.K. Pachauri and L.A. Meyer (eds.)]. IPCC, Geneva, Switzerland, 151 pp. , 
 #1 In reality, the evaluations I made differ from the climatology ones because I account for overlapping of spectra of CO2 molecules with those due to water molecules and water drops which constitute the clouds. In other words, an increase of carbon dioxide concentration in the atmosphere causes and increase of the radiative flux due to CO2 molecules and simultaneously this leads to a decrease of the flux from water molecules and clouds. The total change of the radiative flux according to spectroscopic data of the HITRAN database is less that the radiative flux due to CO2 molecules approximately in six times. This fact does not take into account in climatology codes. , 
 #1 In reality, the evaluations I made differ from the climatology ones because I account for overlapping of spectra of CO2 molecules with those due to water molecules and water drops which constitute the clouds. In other words, an increase of carbon dioxide concentration in the atmosphere causes and increase of the radiative flux due to CO2 molecules and simultaneously this leads to a decrease of the flux from water molecules and clouds. The total change of the radiative flux according to spectroscopic data of the HITRAN database is less that the radiative flux due to CO2 molecules approximately in six times. This fact does not take into account in climatology codes. , 
 Thank you for replying. I understand your comment as a summary and defense of the diligent scientific work you have done to produce this paper and its findings. I do not dispute your comment nor the portions of the paper that it refers to. However, your comment does not address the concern I have detailed, which is specifically regarding the responsibility of human activity and combustion of fossil fuel. I will summarize the concern here: **The paper asserts that the contribution of anthropogenic fossil fuel combustion to increased atmospheric CO2, and its corresponding temperature increase, is about 5%, but does not provide any support for that figure. Therefore the resulting conclusion, that fossil fuel combustion is not important for the greenhouse effect, is not supported for the same reason.** How did you arrive at the low percentage of fossil fuel combustion s responsibility for increased atmospheric CO2? Where does the 5% figure come from? , 
 #2 Mr. Smirnov, Why did you not address my concern in your response? You ve commented about CO2 in general, but my concern is about the anthropogenic percentage of CO2. There is a lot of science compiled by the IPCC indicating that human activity is responsible for most or nearly all of the increased atmospheric CO2, i.e. if doubling atmospheric CO2 causes 0.4K temperature change, then CO2 from human activity is responsible for most or nearly all of that 0.4K temperature change. (Note that  the contribution of the human activity of ECS is not only from fossil fuel combustion as the paper equates; it must also include cement and steel production, land use change, etc.) - Asserting that human activity is only 5% responsible is contentious and demands supporting evidence. Your paper has not provided any. - What caused the other 95% of the increased atmospheric CO2? Your paper does not offer any explanation. - You say your evaluations differ from the climatology ones, but you do not say what evaluations you made that support this low estimate of human responsibility. I sincerely ask that you address this concern. , 
 #2 Mr. Smirnov, Will you acknowledge that 5% of total CO2 sources does not imply 5% responsibility for an ***increase*** in atmospheric CO2 concentration? , 
 There is a comment from M. Lino da Silva and J. Vargas at the Institute for Plasma Research and Nuclear Fusion (IPFN), University of Lisbon, Portugal. https: arxiv.org abs 2002.10601 Abstract: Recently, Smirnov published a paper ... which dismisses the role of increasing concentrations of anthropogenic CO2 on global warming of planet Earth. We show that these conclusions are the consequence of two flaws in Smirnov s theoretical model which neglect the effects of the increased concentrations of CO2 on the absorption of Earth s blackbody radiation in the 12-15u03bcm region. The influence of doubling the concentration of CO2 in the atmosphere on the surface temperature is not u0394T=0.02K, or even u0394T=0.4K if only one of the two mistakes in Smirnov s analysis is corrected. The correct value lies within u0394T=1.1-1.3K as outlined by other authors analysis using simplified, yet more theoretically consistent models. Page 6: ...one may not simply claim that the contribution from the combustion of fossil fuels to the Equilibrium Climate Sensitivity (ECS) is 5% since this simply neglects the CO2 sink values from photosynthesis (-450Gt yr) and from the Ocean (-294Gt yr). The key effect of anthropogenic pumping of CO2 into the atmosphere is to disrupt the balance between source and sink terms of CO2, and the aforementioned percentage is irrelevant since in a first approach (excluding any feedback mechanisms), doubling halving the natural source sink terms would significantly change the assumption by Smirnov (eq. 6.20) without actually changing the net growth of CO2 in the atmosphere (4Gt yr), and hence the actual temperature increases. Instead, any doubling of CO2 in the atmosphere has by all evidence to be assumed as the effect of anthropogenic sources since firstly, concentrations of CO2 have increased from about 300ppm in 1950, when fossil fuel burning became the major anthropogenic source of atmospheric CO2u00a7, up to 410ppm today, a higher than 1 3 increase [11, 12], and secondly, only about 57% of human-emitted CO2 is removed by the biosphere and oceans [13, 14, 15]. Therefore the correct value to use for the temperature increase would rather be the one from eq. 6.19 if this wasnu2019t also wrong (although much closer to the right value). , 
 Prof. Smirnov is a well known climate denialer, you don t need to go further than to read the conclusions of a very recent paper of his:  *The climate problem (e.g., [149u2013154]) is much more complex and various than the considered problem of the greenhouse effect of the atmosphere. This allows the media to disseminate unreliable conclusions about the evolution of Earthu2019s climate in the interests of European financial groups.* Source: Smirnov,  Infrared Radiation in the Energetics of the Atmosphere , ISSN 0018-151X, High Temperature, 2019, Vol. 57, No. 4, pp. 573u2013595. u00a9 Pleiades Publishing, Ltd., 2019. The main flawed argument (among others) is that anthropogenic CO2 only accounts for 5% of global warming. Actually the 5% hoax has been around for a long while, going as far back as the 70 s and 80 s where EXXON MOBIL used these kind of arguments in newspapers (at the time it was about 3-4%, see for example http: www.realclimate.org images Exxon_NYT_ausriss-600x429.jpg The whole discussion on this very common hoax is discussed at large in this lik: http: www.realclimate.org index.php archives 2018 01 the-global-co2-rise-the-facts-exxon-and-the-favorite-denial-tricks We did debunk this hoax in our comment to Smirnov article, although this is very trivial to do (you just need to check the evolution for the isotopic content of CO2 in the atmosphere) In layman terms, we may see this as an analogy to a large water vessel where you have a syphon that evacuates 95litre min and a faucet that adds 95litre min. Then you connect a hose to the vessel which adds 5litre minute and the vessel starts overflowing. You cannot claim that your responsibility is solely 5% can you? More like 100% But what is more troubling in this whole situation is that such a work made it through a journal such as JPhys D which deals with Plasma physics and not climate change. This article should be plain and simply withdrawn, however JPhysD instead declined to publish my comment to his article, which is why I published it in arXiv instead. This is the most embarrassing part of this whole situation, and you may find a more detailed account of this complete disgrace in this post: https: pubpeer.com publications 3914CE7735C54567189C6DC7E28FF6#2 ,