MicroRNA-1270 Inhibits Cell Proliferation, Migration, and Invasion via Targeting IRF8 in Osteoblast-like Cell Lines

Round 1

Reviewer 1 Report

1)    The manuscript is well organized, and it represent a good contribution to the field of interest for Osteoporosis. A wider attention to microRNAs and gene regulator as possible biomarkers for the diagnosis of Osteoporosis  represent an added value to approach for treatment                                    2)    The discussion section should be of high quality and comprehensive. Many sentences are redundant  (lines 245-248, line 252,262 line 273,280,283) and revise grammatically the section.                                                              3) I'd like to recommend to publish this manuscript after these small corrections

 

Author Response

Response to Reviewer 1 Comments

 

The discussion section should be of high quality and comprehensive. Many sentences are redundant  (lines 245-248, line 252,262 line 273,280,283) and revise grammatically the section.

Response: Thank you for the comment. We have made changes accordingly.

 

I'd like to recommend to publish this manuscript after these small corrections.

The authors thank the reviewer for their comments and observations.

Reviewer 2 Report

The manuscript of Ramírez-Salazar et al describes the effect of microRNA-1270 on IRF8 mRNA and protein expression and on various characteristics (proliferation, migration and invasion potential) of cell lines derived from human osteosarcomas (U2OS and SaOS-2).

It is a well written and clear article. The Introduction section is appropriate and describes how this research group previously described that miRNA-1270 was upregulated and IRF8 downregulated in monocytes from postmenopausal women. Both the materials and the methods are appropriated for the study and clearly described. The results are clearly exposed, and the figures are adequate (but see the question about Figure 2 below). Perhaps the weakest part of the manuscript is the Discussion section. The authors should describe the possible mechanism(s) through which they believe that osteoclasts-generated miR-1270 may regulate osteoblasts function.

In Figure 2, it appears that the intensities of the bands shown in panels C and D (mainly those from the empty vector transfections) do not correlate with the relative amount of protein shown in the bar graphs. Authors should address this issue.

 

Minor questions

Line 45. Perhaps is more adequate “frequently OP” instead of “frequent OP”.

Line 74. In the last paragraph of the Introduction section, it should be better to indicate the cells in which the studies were carried out.

Line 76. Please specify that the cell lines come from human tissues.

Line 98. Specify which plasmid is the pMir-Target-IRF8-out. Is it the same that the “mutant (mut) IRF8 3’-UTR” (Line 174)? If so, the same name must be used.

Line 144. “cat . no. ab8245), both from Abcam.” instead of “cat . no. ab8245 both from Abcam).”

Line 153. Please specify if the cells used were stably transfected.

Line 154. An incubation time point of 96 h should be added, as it is shown in Figure 3.

Line 177. Which is the miR-1270 inhibitor? It should be shown in Materials and Methods.

Line 178. Delete the period from “Figure. 1” and in other sentences in which a figure is cited.

L179. On the Y axes, “% Normalized luciferase activity” instead of “% Normalized activity luciferase”.

Line 214 (Figure 3). The X axes should show “Time (h)” and not “h”.

Line 214 (Figure 3). Did the authors perform the MTS assay with the cells at time 0 after seeding? (In the Materials and Methods section only 24, 48 and 72 h are specified). Otherwise, the point corresponding to 0 h must be removed from the graphs.

Line 228 (Figure 4). Panel labels should be (as cited in the main text and the figure legend) (A) and (B), not (a) and (b).

Line 264. The hypothesis that osteoclast-generated miRNAs, such as miR-1270, can regulate the gene expression profile in nearby osteoblasts should also be introduced in the Introduction section.

 

Author Response

Response to Reviewer 2 Comments

The manuscript of Ramírez-Salazar et al describes the effect of microRNA-1270 on IRF8 mRNA and protein expression and on various characteristics (proliferation, migration and invasion potential) of cell lines derived from human osteosarcomas (U2OS and SaOS-2).

It is a well written and clear article. The Introduction section is appropriate and describes how this research group previously described that miRNA-1270 was upregulated and IRF8 downregulated in monocytes from postmenopausal women. Both the materials and the methods are appropriated for the study and clearly described. The results are clearly exposed, and the figures are adequate (but see the question about Figure 2 below). Perhaps the weakest part of the manuscript is the Discussion section.

The authors should describe the possible mechanism(s) through which they believe that osteoclasts-generated miR-1270 may regulate osteoblasts function.

Response: We appreciate the reviewer's comment. We have added more information about this point, in lines 59-65.

 

In Figure 2, it appears that the intensities of the bands shown in panels C and D (mainly those from the empty vector transfections) do not correlate with the relative amount of protein shown in the bar graphs. Authors should address this issue.

Response : We appreciate the comment from the reviewer. The reason for this is that we found some differences in the western blots mainly due to the technical variation. These variations are reflected in the error bars for the quantification of protein in the empty vector assays. For the western-blot picture, we are showing the one that looks the better compared to the other pictures that we have.

 

Minor questions

Line 45. Perhaps is more adequate “frequently OP” instead of “frequent OP”.

Response: We have made the changes suggested.

 

Line 74. In the last paragraph of the Introduction section, it should be better to indicate the cells in which the studies were carried out.

Response: We made the change accordingly.

 

Line 76. Please specify that the cell lines come from human tissues.

Response: We have made the changes as requested.

 

Line 98. Specify which plasmid is the pMir-Target-IRF8-out. Is it the same that the “mutant (mut) IRF8 3’-UTR” (Line 174)? If so, the same name must be used.

Response: We have included some sentences to clarify, in lines 98-102.

“The pMir-Target plasmid encompassing the human wild-type (wt) IRF8 3’-untranslated region (pMir-Target-IRF8_3′-UTR wt) was purchased from OriGene Technologies, Inc. (cat. no. SC214035). The mutated version of the binding sequence of miR-1270 on IRF8 3’-UTR (pMir-Target-IRF8_3’-UTR mut) was generated using the QuickChange II site-directed mutagenesis system..”

 

Line 144. “cat . no. ab8245), both from Abcam.” instead of “cat . no. ab8245 both from Abcam).”

Response: We change the sentence accordingly.

 

Line 153. Please specify if the cells used were stably transfected.

Response: We change the sentence accordingly.

 

Line 154. An incubation time point of 96 h should be added, as it is shown in Figure 3.

Response: Thank you for the comment. We apologize for the mistake. We have added the time point as suggested.

 

Line 177. Which is the miR-1270 inhibitor? It should be shown in Materials and Methods.

Response: We appreciate the comment. We have included the information for the inhibitor construction, in lines 108-110.

 

Line 178. Delete the period from “Figure. 1” and in other sentences in which a figure is cited.

Response: We have removed the points accordingly.

 

Line 179. On the Y axes, “% Normalized luciferase activity” instead of “% Normalized activity luciferase”.

Response: We have modified the figure accordingly.

 

Line 214 (Figure 3). The X axes should show “Time (h)” and not “h”.

Response: We have modified the figure accordingly.

 

Line 214 (Figure 3). Did the authors perform the MTS assay with the cells at time 0 after seeding? (In the Materials and Methods section only 24, 48 and 72 h are specified). Otherwise, the point corresponding to 0 h must be removed from the graphs.

Response: Sorry for the mistake. We have included time 0 in the Materials and Methods section.

 

Line 228 (Figure 4). Panel labels should be (as cited in the main text and the figure legend) (A) and (B), not (a) and (b).

Response: We have modified the figure accordingly.

 

Line 264. The hypothesis that osteoclast-generated miRNAs, such as miR-1270, can regulate the gene expression profile in nearby osteoblasts should also be introduced in the Introduction section.

Response: We have included the following paragraph in lines 59-65, in the introduction section, as the reviewer suggested.

“It is well known that osteoclasts can regulate osteoblast activity either by direct interaction or indirectly by the secretion of cytokines [23]. Moreover, some other studies have demonstrated the presence of miRNAs in body fluid, such as serum, and transported in extracellular vesicles or exosomes, unveiling their key function as extracellular signals between cells. However, it is unclear whether the osteoclast-osteoblast communication can be accomplished via other efficient ‘paracrine’ ways [24,25].”

Accordingly, references 23, 24, and 37 were added:

23. Tang, Y.; Wu, X.; Lei, W.; Pang, L.; Wan, C.; Shi, Z.; Zhao, L.; Nagy, T.R.; Peng, X.; Hu, J., et al. TGF-β1–induced migration of bone mesenchymal stem cells couples bone resorption with formation. Nature Medicine 2009, 15, 757-765, doi:10.1038/nm.1979.
24. Li, D.; Liu, J.; Guo, B.; Liang, C.; Dang, L.; Lu, C.; He, X.; Cheung, H.Y.; Xu, L.; Lu, C., et al. Osteoclast-derived exosomal miR-214-3p inhibits osteoblastic bone formation. Nat Commun 2016, 7, 10872, doi:10.1038/ncomms10872.
25. Sun, W.; Zhao, C.; Li, Y.; Wang, L.; Nie, G.; Peng, J.; Wang, A.; Zhang, P.; Tian, W.; Li, Q., et al. Osteoclast-derived microRNA-containing exosomes selectively inhibit osteoblast activity. Cell Discovery 2016, 2, 16015, doi:10.1038/celldisc.2016.15.

 

All reference order was modified and updated.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

General comments:

I) Several statements were insufficiently supported by reference. There was a little bit too much self-citations (6 over 36).

II) Materials and methods lacked information to adequately reproduce the experiments by others

III) It was unclear why SaOS-2, 142 U2OS and 293FT cell lines were selected since there were no justification. The phenotypes of the cells were not checked. It was unclear if they were functional or if they were mineral competent. There was no information if cells were incubated in osteogenic medium to stimulate their mineral function. This was exemplified in the result section lines 180-182, p5 in “By the other hand, the overexpression of miR-1270 in 293FT cells induce an increase in the proliferation (figure 3), this may be is due to the cell lines in not bone-related. However, more experiments will be necessary to analyze the effects of miR-1270 181 in different cell lines.” At this stage it is not possible to extract firm conclusion since it was unclear if cells behaved as bone cells and if they were able to induce mineral formation.

IV) More investigations are needed y to support the findings as stated by the authors, (discussion, lines 257-259, p7) in “However, more 257 studies focused on the study of multiple genetic and regulatory factors are necessaries for 258 the deep understanding of the bone metabolism and its pathological alterations.

 

Minor comments:

1) Abstract, lines 17-18 p1: Delete “Osteoporosis is the most common bone disease affecting elderly people. Diagnosis of this pathology is frequently done once a bone fracture is present. To solve these many microRNAs have been proposed as biomarkers to the improvement of osteoporosis diagnosis and even treatment. ”

2) Abstract, lines 23-14 p1: Specify IRF8 in “By luciferase activity assays and real-time PCR, and western-blot, we evaluate the functional role of IRF8 mediated by miR-1270. ”

3) Introduction, lines 33-34, p1: Add references to support “Osteoporosis (OP) is the most common bone disease; it is representing a public health problem because of the increase in life expectancy.”

4) Introduction, lines 34-35, p1: Add references to support “OP development is close related to genetic and environmental factors.”

5) Introduction, lines 35-36, p1: Add references to support “This pathology is highly prevalent in elderly and post-menopausal women, which suffer from fractures that diminish their quality of life.”

6) Introduction, lines 39-41, p1: Add explanation, and references to support “The study of those well-differentiated cell types it is the key to understand the remodeling process, this has been accomplished by using primary cultures and established cell lines.”

7) Introduction, from line 45, p1 to line 47, p2: Add references to support “Since the bone remodeling process is regulated by osteoclasts and osteoblasts, the crosstalk between those two types of cells is crucial.”

8) Introduction, lines 47-49, p2: Add references to support “It has been probed those mechanisms aim to restore the balance of formation and resorption in the bone represents an effective therapy for post-menopausal osteoporosis”

9) Introduction, lines 49-50, p2: “However, the current treatment protocols are only focused on the inhibition of osteoclast activity (resorption).”

10) Introduction, lines 62-64, and lines 65-68 p2: Justify the use of cell lines, and add references to support“All cell lines were obtained from American Type Culture Collection (ATCC). U2OS and SaOS-2 cell lines were cultured in McCoy’s 5A medium (Cat. M4892, Sigma-Aldrich. San Luis, MO, USA) with 10% and 15% of heat-inactivated fetal bovine serum (FBS), respectively (Cat: S165H, Biowest, MO, USA.).” and “The 293FT cell line was maintained in DMEM 65 medium (Cat. 12100-046, Gibco. Waltham, MA, USA) supplemented with 10% of heat in activated FBS. All cell cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2.”

11) Cell lines and transfections, lines 69-73, p2: Add more information how transfection was performed, specify GFP, how phenotypes of cells were monitored, and it was unclear if cells were mineral competent in “The cell lines were transfected with 10 μg of the plasmid pcDNA6.2-miR-1270 using Lipofectamine 2000 transfection reagent (Cat: 11668019, Invitrogen, Waltham, MA, USA). GFP reporter expression was analyzed 48 hrs. after transfection. Stable transfections were established by adding blasticidin 10 ng/uL (Cat: 15205, Sigma-Aldrich, St. Luis, MO, USA) to the cultures during two weeks before they were analyzed.”

12) Luciferase activity assays, lines 74-76, p2: Add more information, and references to support “The human wild-type IRF8 3′-UTR was amplified and inserted downstream of a fire- 75 fly luciferase gene in vector pMir-Target (OriGene, Rockville, MD, USA).”

13) Luciferase activity assays, lines 77-79, p2: Add more information, and references to support “The miR-1270 76 binding sequence on the 3’-UTR of IRF8 was mutated by using the QuickChange II site- directed mutagenesis system (Cat: 200524, Stratagene, La Jolla, CA, USA) following the manufacturer’s instructions.”

14) Luciferase activity assays, lines 87-88, p2: Independent measurements shall be performed instead of triplicate to obtain valid statistics in “All measurements were performed by triplicate.”

15) Real-time Reverse Transcription-PCR, Title, line 89, p2: Replace PCR by its full name in the title.

16) Real-time Reverse Transcription-PCR, Title, lines 90-91, p2: Add more information to support “Isolation of total RNA from stably transfected and non-transfected cells was per-formed by using TRIzol Reagent (Invitrogen-Life Technologies) accordingly to manufacturer's instructions.”

17) Real-time Reverse Transcription-PCR, Title, line 106, p3: : Independent measurements shall be performed instead of triplicate to obtain valid statistics in “All reactions were performed in triplicate.”

18) Western blot, lines 108-110, p3: Add more information to support “Protein lysates from cells lines were obtained by using RIPA buffer (Cat: 20, Merck Millipore, Burlington, MA, USA.) containing proteinase inhibitors cocktail (Cat: P8340, Sigma Aldrich, St. Luis, MO, USA).”

19) Western blot, lines 110-116, p3: It was unclear if several independent measurements were performed to support “Proteins were resolved in 10% polyacrylamide gels and transferred to PVDF membranes for probing with anti-Irf8 and gapdh antibodies (Cat: ab28696, ab8245. Secondary antibodies anti-rabbit and anti-mouse conjugated to HRP were used (Cat: ab205718, and ab6728, respectively, Abcam, Cambridge, UK.). Chemiluminescence signal was developed using the Amersham ECL Prime Western Blotting System (Cat: RPN2232, Kenilworth, NJ, USA.) and detected in a GelDocTMXR Plus system (Bio‐Rad, Hercules, CA, USA.).”

20) For proliferation analysis, lines 118-121, p3: It was unclear if several independent measurements were performed to support 3x104 transfected and non-transfected cells were seeded in a 24-well plate and incubated for 24, 48, and 72 hours. Cell proliferation assay was carried out using the MTS assay (Promega, Madison, WI, USA). Absorbance was measured at 490 nm.

21) Statistical analysis, lines 131-135, p3: Indicate number of independent measuremts to support “ Mann-Whitney test was for all statistical analyses. Data plotted to represent the mean ± standard deviation as indicated. Statistical analyses were performed by the GraphPad Prism v. 6.0 software (GraphPad Software, La Jolla, CA, USA; www.graphpad.com). P values £ 05 were considered as statistically significant.”

22) MiR-1270 binds to 3’-UTR of IRF8, title, line 141: Replace IRF8 by its full name in the title.

23) MiR-1270 binds to 3’-UTR of IRF8, lines 142-143, p4: There was insufficient information how cell phenotype was monitored, it was unclear if cells were functional, or if they were mineral competent to support “Firs, to determine the bind of miR-1270 to it binding site in 3’-UTR of IRF8, SaOS-2, U2OS and 293FT cells were transitory transfected with miR-1270 and the wild-type or mutated 3’-UTR of IRF8.”

24) IRF8 expression is down-regulated by miR-1270 MiR-1270 binds to 3’-UTR of IRF8, title, line 154: Replace IRF8 by its full name in the title.

25) IRF8 expression is down-regulated by miR-1270 MiR-1270 binds to 3’-UTR of IRF8, title, line 159-163: There was insufficient information how cell phenotype was monitored, it was unclear if cells were functional, or if they were mineral competent to support “To address this, we analyzed the regulation of the expression of IRF8 mediated by miR-1270, we quantified the expression levels of IRF8 in cell culture stably transfected with the microRNA. First, by real-time PCR, we determine that miR-1270 inhibits the levels of mRNA of IRF8 in the osteoblastic cell lines SaOS-2 and U2OS (Figure 2a and b).”

26) miR-1270 inhibits the proliferation, migration, and invasion, lines 176-182, p5 and lines 189-194, p6 : There was insufficient information how cell phenotype was monitored, it was unclear if cells were functional, or if they were mineral competent to support “Stably transfection of miR-1270 in osteoblast-like cells allows to investigate the effects of the microRNA in the proliferation, migration and invasion. We found that miR-1270 induces a low, but statistically significant decrease in proliferation rate in both cell lines (figure 3). By the other hand, the overexpression of miR-1270 in 293FT cells induce an increase in the proliferation (figure 3), this may be is due to the cell lines in not bone-related. However, more experiments will be necessary to analyze the effects of miR-1270 181 in different cell lines.” And in “Furthermore, we evaluate the effects of miR-1270 over the migration and invasion capabilities of the osteoblastic cell lines. We found no statistical difference in the migration in both cell lines (figure 4 a). However, when migration was evaluated, we found that overexpression of miR-1270, inhibits the capability of both cells to migrate through a semi-permeable membrane (figure 4b). These results suggest that miR-1270 can inhibit the proliferation and invasion of osteoblast, interfering in this way with their functions.”

27) Discussion, lines 220-231, p7: Move into the introduction “Osteoporosis is a the most common bone disease which predisposes to fracture risk [16]. Six-teen percent of Mexican women have OP, ant it has been projected that 1 of 12 women over 50 years old will suffer a hip fracture [1,17]. Mexico is facing an increase of the older population; in a short-term future the incidence and prevalence of osteoporosis 223 will increase. An imbalance in osteoclast (resorption) and osteoblast (formation) activity led to the osteoporosis development [18,19]. To prevent this, is very important to maintaining the equilibrium in the processes of bone formation and resorption [16,20]. In this way, the comprehension of the molecular mechanism involved in osteoporosis development, it is very important to design drugs and treatments for this disease. Recently, many reports have highlight to the miRNAs as important modulators in the bone remodeling processes, by regulating the expression of key genes in the osteoclast and osteoblast differentiation [21–23].”

28) Discussion, lines 248-249, p7: Due to repetition, delete “MiR-1270 has been identified in some types of cancer [24,26,33]. However, its role related to bone metabolisms it is unknown.”

29) Conclusion, lines 261-265, p7: Due to repetition, delete “This work is, analyzed by first time the role of miR-1270 in bone-related cells. This microRNA can regulate the expression of IRF8, an important player in the bone remodeling. Besides, we demonstrate that miR-1270 modifies the cell proliferation, migration, and invasion. Although, more investigation are necessaries for a better understanding of the role of this and other microRNAs in the regulation bone remodeling.”

30) References, p8: There was too much self-citations (9, 10, 11,12 35 and 36) as compared to lack of citations of other authors which were missing in the main text.

 

 

 

 

Author Response

We appreciate the comments that have been given to this manuscript.

We address all the observations in the cover letter.

Hope you find the modifications

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Manuscript title:

MicroRNA-1270 inhibits proliferation, migration, and invasion 2 through targeting IRF8 in osteoblast-like cell lines.

The authors summarized that the potentially key role of miR-1270 in the modulation of bone metabolism by regulation of IRF8 expression.

There some major concerns about the style and methods. The reviewer thinks the manuscript can be published after the major revision of below things.

1. All figure: The text in the figures is too small. It must be corrected.
2. Fig4: Scale bar must be added. Also, color image should be necessary.
3. Although there was no significant difference in the migration, a detailed discussion on migration should be added.
4. If this focused on bone remodeling, osteogenic differentiation experiments should also be conducted.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors addressed most of the minor concerns, however there were two general concerns 

General unaddressed concerns:

III) It was unclear why SaOS-2,  U2OS and 293FT cell lines were selected since there were no justification. The phenotypes of the cells were not checked. It was unclear if they were functional or if they were mineral competent. There was no information if cells were incubated in osteogenic medium to stimulate their mineral function. This was exemplified in the result section lines 180-182, p5 in “By the other hand, the overexpression of miR-1270 in 293FT cells induce an increase in the proliferation (figure 3), this may be is due to the cell lines in not bone-related. However, more experiments will be necessary to analyze the effects of miR-1270 181 in different cell lines.” At this stage it is not possible to extract firm conclusion since it was unclear if cells behaved as bone cells and if they were able to induce mineral formation.

Response: We have added information related to the cell lines used. The 293FT cells as a non-related cell line. We have removed the results using this cell line to avoid misunderstandings.

Reviewer's answer: The authors clarified the use of Saos-2 and U2OS cells. However, additional experiments on mineral competent Saos-2 and U2OS cells shall be performed. It was unclear if the ability of microRNA miR-1270 to bind and regulate the expression of Interferon Regulatory Factor IRF8 in mineral competent cells was retained. Mineral competent cells may behave  differently than non mineral competent cells. These experiments have to performed  to validate the concept that the ability of microRNA miR-1270 to bind and regulate the expression of Interferon Regulatory Factor IRF8    in the context of bone metabolism and osteoporosis. 

IV) More investigations are needed y to support the findings as stated by the authors, (discussion, lines 257-259, p7) in “However, more studies focused on the study of multiple genetic and regulatory factors are necessaries for the deep understanding of the bone metabolism and its pathological alterations.

Response: We appreciate the suggestion of the reviewer, however at this moment is too difficult for us to perform the experiments requested. The aim of the current manuscript is to demonstrate the bind and regulation of IRF8 expression mediated by miR-1270. The functional effects of this microRNA will be evaluated soon.

Reviewer's answer: The functional effects of miR-1270 in mineral competent cells shall be completed to strengthen the manuscript.