Title: Mechanism of action and therapeutic potential of lncRNA MIR497HG/miR-16-5p axis
in breast cancer
Version: 2 Date: 12 Mar 2024
Reviewer's report:
Dear Authors,
Thank you for the opportunity to review this interesting manuscript. The authors emphasise
the role of lncRNA molecules in tumor regulation and thus, suggest that lncRNA molecules,
particularly MIR497HG/miR-16-5p axis, plays a significant role in breast cancer. The authors
tried to elucidate the role of this axis using patient tumor samples, normal controls, and
commercially available cell lines. The authors used RT-qPCR with subsequent statistical
evaluation of the obtained data for research of the molecules in breast cancer. Furthermore,
overexpression plasmid for MIR497HG and luciferase gene reporter assay was used to determine
the relationship between lncRNA MIR497HG and miRNA miR-16-5p. The authors are reporting
the following main findings:
1. Using RT-qPCR, it was observed that MIR497HG expression level was reduced in cancer
tissues relative to the normal tissues;
2. MIR497HG expression (arbitrarily set as nominal groups high/low) was associated with
clinical findings, such as tumor size, lymphocytic metastasis and TNM stage;
3. Using BT549 and Hs578T cell lines, it was observed that upregulation of MIR497HG
expression reduced the relative migration level of both cell lines;
4. Authors guessed a potential relationship between MIR497HG and miR-16-5p that is based
on a bioinformatic analysis;
5. Additionally, the luciferase activity of BT549 or Hs 578T cells was reduced when miR-16-5p
mimic was co-transfected with MIR497HG-WT, whereas MIR497HG-MUT did not influence
the expression as seen from the figures only;
6. Mir-16-5p expression was upregulated in breast cancer tissue specimens;
7. PcDNA3.1-MIR497HG downregulated the miR-16-5p expression in BT549 and Hs578T
cells;
8. Correlation analysis proved negative correlation between MIR497HG and miR-16-5p;
9. MiR-16-5p expression was upregulated in BT549 and Hs 578T cells after co-transfection with
pcDNA3.1-MIR497HG;
10. Cell invasion level increased after transfection of cells with miR-16-5p mimic.
In my opinion the results of the present work are of sufficient novelty to be considered
for publication in BMC Women’s health. However, I have a few major observations and
recommendations the authors may want to consider:
The language of the manuscript needs to be thoroughly checked and word style should
be consistent throughout the manuscript (Hs 578T or Hs578T; down/up-regulated or down/
upregulated; RT-qPCR or RT-PCR; et al. or et al).
Line 69: The aim statement of the present study needs to be improved.
Line 71: The word “cure” should be used cautiously. It would be better to use something gently
that does not imply the cancer could be cured.
Line 76: Collection of patients specimens needs to be updated with detailed inclusion/exclusion
criteria. Also, an ethics statement with approval numbers should be included in this chapter.
Line 85: ATCC accession numbers need to be stated for each cell line. Media and FBS
manufacturers need to be added. Also add the percentage of FBS. If antibiotics were added,
please add the concentration, which and manufacturer. Please add information on how the cell
lines were passaged and which passage did you use for your experiments.
Line 90: Please describe in detail and add manufacturer of the plasmid.
Line 94: Please state the initial concentration of RNA that went into RT reaction. Please add
how was the relative expression calculated (2-ddCT method or equivalent). Please add if both
reference genes were used as geometrical average or were they used independently? Please add
assessment of reference genes for expression stability throughout the experiments. Please add
information about primers or assays used for the detection of lncRNA, miRNA and reference
genes.
Line 101: Please add detailed information such as volume and manufacturers. What is the
meaning of reasonable culture?
Line 108: Please add manufacturer information where applicable.
Line 115: Please add information on how the data was assessed for normal distribution
prior analyses with parametrical tests. Please provide information about what software or
programming language was used for the statistical analyses. If the use of parametrical one-
way ANOVA is justified, please state if homogeneity of variances were a priori assessed and if
Welch’s test was used instead of ANOVA. Please also state if post hoc tests were carried out.
Please add detailed information on how ROC analysis was performed.
Line 129: Please add CI95 for the calculated AUC.
Line 133: Please state how was the expression threshold for low/high expression determined.
Line 134: Chi-square test is used for association determination, not correlation.
Line 152: Please describe in methods what kind of bioinformatics analysis was carried out.
Line 155: In the figure is clearly seen that MIR497HG-MUT was also used. Please add a
description of what was observed when MIR497HG-MUT was transfected.
Line 165: Statement “Moreover, miR-16-5p expression was upregulated in BT549 and Hs 578T
cells after co-transfection with” is superfluous. If cells were transfected with miR-16-5p mimic,
that in turn implies that upregulated expression of miR-16-5p is going to be detected. What
was the exact reason to transect with both, pcDNA3.1-MIR497HG and miR-16-5p mimic? It is
understandable that cell migration and invasion was not counteracted but measuring miR-16-5p
levels only proves that mimic transfection was successful. If the latter is the reason for the
statement, please rephrase.
Line 170: Discussion. Please improve the discussion. Add some more information about
miR-16-5p in terms of its gene targets and how these targets are associated with cancers. This
would greatly improve the manuscript. For the stated limitations, I would advise undertaking
power calculations for statistically significant results in order to back up the limitation statement.
Additionally, the second and third limitations are not clear. Please rephrase.