MicroRNA-1269a promotes the occurrence and progression of osteosarcoma by inhibit-ing TGF-β1 expression
Authors: Y-M Dai
B T Zhang
S-N Yu
Y-Y Miao
Z-L Gao
L Liu
G-F Liu
Journal: European review for medical and pharmacological sciences (2019)
Discipline: PHARMACOLOGY & TOXICOLOGY
Keywords: MicroRNA-1269a; Osteosarcoma; TGF-β1; Metastasis; Prognosis
Affiliations: Departments of Radiology, China-Japan Union Hospital of Jilin University, Changchun, China. .

Abstract:  Objective: MicroRNAs are endogenous, non-coding small RNAs that are capable of regulating biological and pathological processes. Previous studies have shown that microRNA-1269a serves as an oncogene. However, the role of microRNA-1269a in the pathogenesis of osteosarcoma (OS) has not been reported. The aim of this work was to investigate the expression characteristics of microRNA-1269a in OS and to further study its regulatory effects on the malignant progression of OS. Patients and methods: The expression of microRNA-1269a in 61 pairs of OS tissues and para-cancerous tissues was detected by quantitative Real Time-polymerase Chain Reaction (qRT-PCR). Chi-square test was used to analyze the relationship between microRNA-1269a expression and the characteristics of OS patients, including age, sex, clinical stage and distant metastasis. Subsequently, microRNA-1269a expression in OS cell lines was detected as well. After knockdown of microRNA-1269a by constructing relevant small interference RNA, biological performances of MG63 and U2OS cells were accessed by cell counting kit-8 (CCK-8), colony formation and transwell assay. Meanwhile, the protein expressions of key genes in the EMT/Smad pathway were detected by Western blot. Finally, si-TGF-β1 (transforming growth factor-β1) was transfected into OS cells, and cell migration and invasion were detected by transwell assay. Results: MicroRNA-1269a was highly expressed in OS tissues compared with para-cancerous tissues. High expression of microRNA-1269a was positively correlated with young OS patients and high rate of distant metastasis, whereas was not correlated with age, sex and Enneking stage. Kaplan-Meier survival curves showed that high expression of microRNA-1269a was significantly associated with poor prognosis of OS. The knockdown of microRNA-1269a in MG63 and U2OS cells significantly inhibited cell proliferation, migration and invasion. Meanwhile, microRNA-1269a knockdown in OS cells markedly downregulated the expressions of TGF-β1, p-Smad2, p-Smad3, N-cad, Vimentin and MMP9. Furthermore, TGF-β1 knockdown remarkably decreased migratory and invasive abilities of OS cells. Conclusions: MicroRNA-1269a is highly expressed in OS, which is remarkably correlated with tumor stage, distant metastasis and poor prognosis of OS. In addition, microRNA-1269a promotes the malignant progression of OS by regulating TGF-β1 expression. 

Retracted: Yes

PubPeer comments:
 This paper appears to be part of a set of papers (n=170 as of today and growing) that share a very similar layout. Subsets of these papers appear to share similar Kaplan-Meier survival plots, correlation plots, and overlapping photos of wound-healing, transwell assays, etc. Yet, there is little to no overlap between the authors of these papers or their departments and affiliated institutions. The very similar layout and overlapping photos suggests these papers might have all been created by the same entity, perhaps a paper mill. I have been calling this set the  Comb papers, because the Kaplan-Meier plots have a comb-like appearance. For PubPeer user Hoya Camphorifolia, the survival plots look like Lego blocks. This particular paper is part of a subset of four papers within the Comb papers that share very similar correlation plots: * SJ Sun et al., ERMPS (2018), DOI: 10.26355 eurrev_201808_15623 - https: pubpeer.com publications 59A8783B6D785B5491494DC81449A6 * SN Yu et al., ERPMS (2019), DOI: 10.26355 eurrev_201902_16984 - https: pubpeer.com publications 4F6E713824DCE57BD2D6AE0E3FE740 * Tongfei Wang et al., JBUON (2019), PMID: 31128011 - https: pubpeer.com publications 7559D04363B36906338AE8220768A8 * Zi Chen et al., JBUON (2018), PMID: 30610808 - https: pubpeer.com publications 95E6C4CBEC09A7BF5BC5895ED07923 Here is a compilation of the correlation plots from these four different papers. Colored boxes and lines point out some characteristic groups of data points. ![file] , 
 Another characteristic features of this papermill s products is a Table 1 in which the column heads promise to convert the subgroup numbers into percentages, without delivering. ![file] In this case the authors were not consistent about the numbers within the low- and high-miR-1269a expression subgroups. , 
 Dear Dr. Elisabeth M Bik, I am the first author of this article, Shaonan Yu. For some reason, the e-mail registration audit failed, so I used anonymous registration. We are really appreciating your careful check and raising this concern. The work of this experiment was done in 2016. The original data and experimental records are shown in the following figures. The experiment in Figure 4c was completed on August 31, 2017. After that, in order to save time, we once found a company on the internet that helped us make the data into pictures. We have reason to suspect that the company used our data to sell or write papers to others. At the same time, we are repeating our experiments to ensure that there are no problems with the data. Finally, thank you again for your academic supervisionuff01![file] ![file] , 
 *We have reason to suspect that the company used our data to sell or write papers to others.* It would be very helpful if you could name the company, to warn other researchers against using its services. , 
 #3 * ...in order to save time, we once found a company on the internet that helped us make the data into pictures. * I fail to understand why a research lab would need an external company in order to make ordinary graphs and tables. Don t lab members know how to use Excel? , 
 Another question about Fig 4C comes to mind.  Subsequently, we selected 16 pairs from 61 pairs of OS tissues and para-cancerous tissues to explore the correlation between microRNA-1269a and TGF-u03b21. QRT-PCR data indicated that the expression of microRNA-1269a was negatively correlated with TGF-u03b21 expression in OS tissues (Figure 4C). When you have already measured miRNA-1269a and TGF-u03b21 **for all 61 OS samples**, what is the rationale for selecting 32 of them to keep in the spreadsheet you displayed (CT values for 32 wells), and **only 16** samples to plot? What was the criterion for **not** plotting the other 45 samples? , 
 More basic than the points raised before, the provided Ct (Cq) data are inconsistent with the conclusions and suggest additional problems. The first issue is that the data do not indicate that  microRNA-1269a was negatively correlated with TGF-u03b21 expression in OS tissues. Exactly the opposite: miRNA and TGF-beta expression were positively correlated. Lower Cq values mean more abundant target. More abundant miR-1269 went along with more abundant TGF-beta.![file] This plot was drawn by me, and I am not Elisabeth Bik. I think I switched the axes from how they are presented in the paper, but the point is the same: positive correlation, not negative. Second, these Cq values are not believable. Cq below 10 are highly suspect and usually not very quantitative. You would see such low values in most biological samples only for extremely high abundance RNAs like rRNAs. The earlier the  amplification (lower Ct), the less likely this is real signal. Usually, a Cq value below 10 is the result of a threshold that has been placed too low, in the region of machine noise that predominates before  real signal that is the result of amplification. The authors are encouraged to show the amplification curves. Third, even if the values were correct, there are no replicates and no normalization. In qPCR, samples are typically run in triplicate and normalized to one or more internal references. I also see that two groups of subsequent CT values, 3 and 4 for the miR and 11 and 12 for TGF, are identical out to 3 or 4 decimal places. This is curious and may suggest unexpected influences on the data. , 
 #6 A total of 61 samples were collected three times in this experiment. The first 16 samples are in Fig. 4c, the second 21 samples as shown in the first picture, and the third 24 samples, as shown in the second picture below. ![file] Obviously, the linearity of the results of the other two groups is far worse than that of Fig. 4c, and the data in the first picture may be due to the contamination of the experimental medicines which leads to greater deviation of the results. Moreover, as stated by Aconitum Delphiniifolium, miR-1269 and TGF-u03b21 are positively correlated, so our previous conclusions are incorrect. In addition, after the guidance of the seventh floor, we know the reliability of Cq values below 10 is very low, so we also generated these data doubt. In addition, in our PCR result, a single sample is tested only once, which is also the cause of inaccuracy. Based on the above reasons, we will carry out repeated experiments, and in order not to mislead more readers, we will immediately contact the editor of Journal to withdraw or erratum the article. Finally, thank you again for your supervision of scientific research. It is you who made the academic environment better. , 
 #7 Thank you very much for your professional guidance. Our professionalism in this field is indeed seriously insufficient, which has led to many operational and data analysis errors in the experiment. As shown on another reply, in the other 45 samples, the correlation is obviously not as shown in Figure 4c. In addition, as you said, the experimental results of words and the low cq value greatly reduce the accuracy of the experimental results. And we write the positive correlation of the conclusion as negative correlation, which seriously affects the experimental results. We will repeat the experiment and process the article withdrawn or erratum. Finally, thank you again for your careful guidance, so that I know that I need to be more rigorous and serious in the details of the experiment. , 
 Retracted [April 2021] . Since this article has been suspected of research misconduct and the corresponding authors did not respond to our request to prove originality of data and figures, u201cMicroRNA-1269a promotes the occurrence and progression of osteosarcoma by inhibiting TGF-u03b21 expression, by S.-N. Yu, Y.-Y. Miao, B.T. Zhang, Y.-M. Dai, L. Liu, Z.-L. Gao, G.-F. Liu, published in Eur Rev Med Pharmacol Sci 2019; 23 (3): 972-981u2013DOI: 10.26355 eurrev_201902_16984u2013PMID: 30779063u201d has been withdrawn. The Publisher apologizes for any inconvenience this may cause. ,