OVERALL CONSIDERATIONS
MicroRNA control of osteoblast differentiation remains poorly understood and there is a paucity of compelling in vivo data of how this critical class of post-transcriptional epigenetic regulators controls cellular pathways that contribute to osteogenesis. This study by the Delany group, which is one of the leading miRNA groups in the bone field, follows up on her previous paper in JBC (Smith SS et al., J Biol Chem. 2016;291(41):21717-21728) showing that miR433-3p controls glucocorticoid signaling. The present work provides compelling data for the novel concept that miR-433-3p also controls several other osteogenic pathways required for bone formation in vivo and osteoblast differentiation in vitro to provide an updated integrated model for miR433 function in osteoblasts (see Fig. 7).

In all its component parts, this paper reflects what an outstanding good miRNA paper should include. It establishes that a single miRNA can inhibit several critical pathways in vitro and in vivo, that selectively affect one biological process (osteogenesis) but not another (adipogenesis) and providing sufficient proof that this miRNA regulates bone formation in vivo. This work includes an important translational component by showing the in vivo efficacy of miR-433-3p antagonists, modified versions of which could potentially be developed as RNA therapeutics.

As such, this paper represents the perfect example that raises the standard for the ballooning number of low-quality microRNA papers that frequently offer very unrealistic ‘one miRNA-one gene-one biological effect’ concepts. These papers typically follow a prescribed template of, one, an arbitrary miRNA picked for no apparent reason, two, this miRNA has a singular arbitrary mRNA target that fits a pre-conceived mechanistic notion, and, three, supposedly affects osteoblastogenesis by a cursory examination of very select biomarkers for this biological process. In this sense, this paper provides an outstanding example of what a miRNA paper in JBMR should look like.

Beyond this profuse praise, there are really no concerns with the overall design, methodology, technical execution, data, statistical rigor, interpretation or visual presentation for the miR-433-3p study presented here. This paper provides a minimal standard for the miRNA studies in the bone field to follow. This study deserves to be recommended for an editorial commentary with the encouragement for future authors and reviewers to appreciate what a solid miRNA paper should look like.

CRITIQUE
It is fully understood that laboratory limitations due to COVID alter perspectives of what can be achieved during revisions of papers. The points below reflect due diligence as reviewer. The authors are encouraged to include what they can in good faith and otherwise acknowledge limitations and other ideas in the Discussion.

MAJOR POINTS
1) The paper, excellent as it is, would have reached its fullest potential, if it also had included a broader examination of transcriptome level changes upon miR-433-3p modulation (e.g., straight-forward RNA seq analysis). Such data would have clarified whether the model in Figure 7 is comprehensive or still requires further expansion. If these data are available, then inclusion would make this paper truly a future candidate for a citation classic for its comprehensiveness. Otherwise, the authors may wish to acknowledge the known unknowns of the present work in the Discussion.

2) Figures 1 and 2 (in vitro osteogenic differentiation of BMSCs with or without miR-433-3p decoy) deserve a slightly broader analysis of other osteogenic markers and a few other representative miRNAs that have different temporal expression patterns and/or that should not be affected by the decoy. Similarly, the adipogenic story could be deepened with a broader analysis of a few select Adipogenic mRNAs and miRNA markers.

3) Figure 4 ought to be complemented with a few representative western blots showing that representative pathways are activated or repressed at the protein and phosphor-protein level.

4) Figures 5 and 6 could benefit from basic biomechanical analysis.

5) The reference listing is comprehensive with greater than 100 cited papers (n=107). Yet, many of these papers are very peripheral to the present work and would be more appropriate for the next concise review that the Delany group could write. Yet, as comprehensive as this reference listing is, it is rather disappointing that key pioneering papers from other leading groups on miRNAs in the bone field are not included. For example, the top most cited references in the categories “miRNA & bone & osteoblast” appear to be excluded from this paper. Rather than diffusing the discussion and interpretation by inclusion of miRNA concepts in a bewildering number of biological contexts (e.g., cancer, angiogenesis, non-skeletal aspects of development), the authors could trim this unfocused excess. The latter would provide space for one or two dozen overlooked papers from multiple colleagues in the bone community. These other prior papers together with the major overall achievements of the Delany group provide the solid foundation for a true understanding of this exciting class of molecules in osteoblast differentiation and bone formation. If this paper would be commented on by an accompanying editorial, then it would be misfortunate that it is flawed by an unbalanced presentation of the experimental art in the bone field prior to this work.

MINOR POINTS
6) Appropriate gene symbols should be provided throughout this paper (e.g., AP=ALPL, OC=BLGAP p38alpha=MAPK14).

This paper provides in vivo evidence for miRNA function in bone formation. Excellent papers like this represent only a very small but very important fraction of the available literature on miRNA papers which is remains heavily biased for less informative in vitro studies. The authors have adequately addressed the comments of the reviewers and this high quality paper will be of much interest to the readership of JBMR.