MicroRNA-337-5p participates in the development and progression of osteosarcoma via ERBB, MAPK and VEGF pathways
Authors: F Zhou
L-F Zhu
Z-G Tian
Z Jin
Y Zhuang
P-C Shen
Journal: European Review for Medical and Pharmacological Sciences (2018)
Discipline: PHARMACOLOGY & TOXICOLOGY
Keywords: microRNA-337-5p; osteosarcoma; prognosis; survival analysis; GEO database
Affiliations: Department of Orthopedics, The First People's Hospital of Wujiang District, Suzhou, China. .

Abstract:  Objective: To investigate the role of microRNA-337-5p in osteosarcoma (OS) and its underlying mechanism. Patients and methods: The microRNA (microRNA-337-5p) that may be related to OS development was screened out by GEO (Gene Expression Omnibus) database. Survival analysis and ROC curve were performed according to microRNA-337-5p expressions in OA patients. Besides, the correlation between microRNA-337-5p expression and clinical parameters was evaluated by Chi-square analysis. Cox regression analysis was performed to detect the relationship between the overall survival and clinical parameters of OA patients. Subsequently, enriched functions and pathways of microRNA-337-5p were predicted by GESA (gene enrichment sets analysis). MicroRNA-337-5p expression was detected in 65 OS tissue samples and 30 normal tissue samples by qRT-PCR (quantitative Real-Time Polymerase Chain Reaction). For in vitro experiments, after microRNA-337-5p mimics or microRNA-337-5p inhibitor was transfected into OS cells, proliferative and invasive abilities were detected by CCK-8 (Cell Counting Kit-8) and transwell assay, respectively. Finally, Western blot was used to explore the underlying mechanism of microRNA-337-5p in regulating OS. Results: MicroRNA-337-5p was overexpressed in serum and tissue samples of OS patients, which was valuable in diagnosing OS. Besides, microRNA-337-5p expression was correlated with the overall survival and necrosis range of OA patients, whereas not correlated with age and sex. GESA indicated that microRNA-337-5p was enriched in ERBB, MAPK, and VEGF pathways. In vitro experiments indicated elevated proliferative and invasive abilities in MG63 and U2OS cells after microRNA-337-5p overexpression. Furthermore, increased expressions of ERBB2, Erk1/2, and VEGF121 were observed in OS cells after microRNA-337-5p overexpression. Conclusions: MicroRNA-337-5p is upregulated in OS tissues, which is an independent prognostic factor in OS. Overexpressed microRNA-337-5p can promote proliferative and invasive abilities of OS cells via activating ERBB, MAPK, and VEGF pathways. 

Retracted: No

PubPeer comments:
  We then collected clinical information of 65 OS patients in GSE39040, including prognosis, age, sex, and their microRNA-337-5p expression. Kaplan-Meier and log-rank were introduced to evaluate the correlation between microRNA-337-5p expression and prognosis of OS patients. Based on the median expression of microRNA-337-5p, OS patients were further assigned to high-expression group and low-expression group. Our data suggested that the overall survival of OS patients in the high-expression group was shorter than those in the low-expression group (p=0.01949, Figure 1D). ![file] Figure 1D could not have been derived from the data for 65 OS patients from GSE39040, because that data repository only provides time until recurrence (or drop-out) - not overall survival. https: www.ncbi.nlm.nih.gov geo query acc.cgi?acc=GSE39040 The authors also collected their own miR-337-5p measurements for *another* 65 patients.  **Sample Collection** Paraffin slices, peripheral blood samples of OS and corresponding control samples were collected at the Orthopedic Department, The Affiliated Jiangyin Hospital of Southeast University Medical College from **July 2012 to June 2017**. OS patients were surgically treated and pathologically diagnosed. All patients did not have other systemic tumors except for OS. Trauma patients during the same period were selected as the control group.   Expression levels of microRNA-337-5p were detected in 65 OS tissues and 30 normal tissues by qRT-PCR. These seem to be the data plotted in Fig 3.  B. Lower overall survival was found in OS patients with higher microRNA-337-5p expression than those with lower expression . ![file] Inquiring minds are wondering how the authors managed to follow the patients survival for over 11 years after surgery, i.e. through a date range **July 2023 to June 2028** , 
 There s another problem here. The sequence of mature miR-337-5p is GAACGGCUUCAUACAGGAGUU. A specialized reverse transcription process would be needed to allow this short RNA to be amplified at all. The  Prime-Script RT Master Mix mentioned in the methods would not be sufficient. Even if a different and appropriate RT strategy had been used, the primers listed as miR-337-5p specific are F: ACACTCCAGCTGGGTTCAGCTCCTATATGAT and R: CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAAAGGCAT. These primers have melting temperatures around 71 and 84 degrees C, respectively, and would not be suitable for standard PCR even if they were specific for something in the human genome. A BLAST search suggests they are not, and I could also not figure out how any part of these sequences would be suitable for amplifying miR-337-5p. , 
 #2 It s always a delight to see somebody paying attention to the methodology and pointing out sequence errors. While there isn t any binding between hsa-miR-337-5p and the primers the author has listed, there is a small level of binding between the primers and hsa-miR-337-3p instead. Although even there, there is a small overlap between the listed reverse primer and the forward primer which isn t ideal. I can t really comment on the difference in melting temperatures, but if the reverse primer was instead a stem-loop primer (as shown in the dissertation, and potentially ripped off from:  HEPATIC CELL DIFFERENTIATION REQUIRES A HNF6 u2013 miR-122 POSITIVE FEEDBACK LOOP by Ilaria LAUDADIO) and used for RT-PCR instead, then perhaps it would be possible. Interested in hearing from the authors their input on this. Were they aware they were targeting the wrong prime end of the mature miR? Did the primers really work in a conventional qRT-PCR? Did they instead use a universal reverse primer as Laudadio did and just forget to list it? , 
 #3 Thank you for noticing the sequence of the 3p arm. It looks like miR-337-5p was identified in a GEO dataset, but miR-337-3p was mistakenly chosen as the target of the qPCR. The primer described as the  reverse primer could potentially work as a stem-loop RT primer for 337-3p, where eight 3 nt would bind to a sequence internal to 3p (bolded sequences below). 1) Mature 337-3p CUCCUAUAUG**AUGCCUUU**CUUC 2)  Reverse (actually stem-loop RT primer): CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAG**AAAGGCAT** It would be unusual to use the RT primer as a reverse primer. Typically, a much shorter sequence is used as a universal reverse primer. As you said, there is also overlap of the forward and reverse primers if this was the case. But perhaps it would work? It would be helpful if the authors could clarify all of this and also indicate which of the other reagents and experiments were for miR-337-5p and which were actually using the miR-337-3p sequence. ,