MALAT1 promotes gastric adenocarcinoma through the MALAT1/miR-181a-5p/AKT3 axis

Gastric adenocarcinoma, which originates from the gastric mucosal epithelium, has the highest incidence among various malignant tumours in China. As a crucial long non-coding RNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been suggested to play an important role in many tumours. Here, we aimed to investigate the role and underlying mechanism of MALAT1 in gastric adenocarcinoma. Quantitative reverse transcription polymerase chain reaction was applied to determine the expression levels of MALAT1 in serum and cell lines. A CCK-8 assay and a clonogenic survival assay were used to examine cell proliferation and apoptosis. The protein level of RAC-γ serine/threonine-specific protein kinase (AKT3) was determined by western blot. Our results showed that MALAT1 was highly expressed in the serum of patients with gastric adenocarcinoma and in cell lines. Downregulating MALAT1 inhibited proliferation and promoted apoptosis of MGC-803 cells. In addition, MALAT1 directly targeted and decreased the expression of miR-181a-5p, which in turn upregulated the expression of AKT3. Further, overexpressing miR-181a-5p or directly inhibiting the AKT pathway with the inhibitor ipatasertib exhibited similar effects to MALAT1 knockdown. Our research proposes a novel mechanism where the role of MALAT1 is dependent on the MALAT1/miR-181a-5p/AKT3 axis. MALAT1 competes with AKT3 for miR-181a-5p binding, thereby upregulating the AKT3 protein level and ultimately promoting the growth of gastric adenocarcinoma.


Introduction Yes
Is the length of the paper justified?Yes Should the paper be seen by a specialist statistical reviewer?No

Is it clear how to make all supporting data available? No
Is the supplementary material necessary; and if so is it adequate and clear?Not Applicable

Do you have any ethical concerns with this paper? No
Comments to the Author Major comments: 1.In the results section, the authors mentioned that MALAT1 is highly expressed in gastric adenocarcinoma patients.Yet they only showed that serum MALAT1 level is higher in gastric adenocarcinoma patients compared to healthy control.Why didn't they compare the expression in tumor and adjacent normal tissue as well?
2. The authors should also show the clinical parameter between the two groups of samples, such as age, gender, and use statistical analysis to determine if the serum level of MALAT1 was affected by these parameters.
3. The authors should include more cell-lines of gastric normal and adenocarcinoma cell-line for comparing their MALAT1 levels.
4. More details are needed for the following parts: What bioinformatic methods did the authors use to predict the possible binding targets between MALAT1 and miR-181a-5p/miR-181a-5p and AKT3?How were MALAT1 and miR-181a-5p level knocked down?Did the authors use GAPDH as normalization for lncRNA and miRNA?Why? 5.The expression of miR-181a-5p was negatively correlated with the expression of MALAT1, hence the R2 value should be negative.

6.
No evidence showing direct interaction between MALAT1 and miR-181a-5p/miR-181a-5p and AKT3.Why was luciferase reporter assay not performed?I think the luciferase reporter assay is important to draw the conclusion "Our data further indicates that MALAT1 competitively binds to miR-181a, making miR-181a unable to bind to AKT3 mRNA, thereby up-regulating AKT3 protein levels and ultimately promoting tumor growth".
7. The authors had used miR-181, miR-181a and miR-181a-5p in the manuscript.If they are talking about miR-181a-5p in all occasions, miR-181a-5p should be used consistently.8. Ipatasertib is an inhibitor of Akt pathway, but not specific for Akt3.I think siRNA against Akt3 should be used instead of Ipatasertib.9. To show that MALAT1 promoted gastric adenocarcinoma a through MALAT1-miR-181a-AKT3 axis, the functional effect of MALAT1 overexpression with or without transfection of miR-181a-5p or Akt3 siRNA will be a more direct evidence.We are writing to inform you that the Editor has reached a decision on your manuscript RSOB-19-0095 entitled "MALAT1 promotes gastric adenocarcinoma through MALAT1-miR-181a-AKT3 axis", submitted to Open Biology.
As you will see from the reviewers' comments below, there are a number of criticisms that prevent us from accepting your manuscript at this stage.The reviewers suggest, however, that a revised version could be acceptable, if you are able to address their concerns.If you think that you can deal satisfactorily with the reviewer's suggestions, we would be pleased to consider a revised manuscript.
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Sincerely, The Open Biology Team mailto: openbiology@royalsociety.org Reviewer(s)' Comments to Author(s): Referee: 1 Comments to the Author(s) This paper is well written and I suggest acceptance.

Referee: 2
Comments to the Author(s) This is an interesting study with novel findings.The regulatory role of MALAT1 in gastric adenocarcinoma was well elucidated.First, the authors found that MALAT1 was highly expressed in the serum of gastric adenocarcinoma patients and cell lines.Second, downregulating MALAT1 was able to inhibit the proliferation and promoted apoptosis in MGC-803 cells.Third, MALAT1 was found to directly target miR-181a-5p and decreased the expression of miR-181a-5p, therefore upregulate the expression of AKT3.Last, overexpressing miR-181a-5p or directly inhibiting the AKT pathway with an inhibitor Ipatasertib exhibited similar effects as MALAT1 knockdown.Based on these findings, the authors conclude that MALAT1 competes with AKT3 for miR-181a binding, thereby up-regulating AKT3 protein level and ultimately promoting the growth of gastric adenocarcinoma.These results deserve publication, giving my following concerns could be addressed.1.The rational to study MALAT1 is not cleary explained.Need more background information, especially in the tumor field.2. It is better not to include the results and the significance in the introduction section (the last para.in this section).3. Any informed consents from the participants?This needs to be clarified.4. The time point to conduct the CCK-8 assay needs to be indicated.5. Figure legends should be carefully revised.There are some unidentified characters, and the presentation is poor.6.Why I can not see any error bars in some set of the data in figure 3 and 6? 7. It is preferred to re-arrange the subfigures in Figure 5, especially for the WB image.It looks too small in this figure.

Referee: 3
Comments to the Author(s) Major comments: 1.In the results section, the authors mentioned that MALAT1 is highly expressed in gastric adenocarcinoma patients.Yet they only showed that serum MALAT1 level is higher in gastric adenocarcinoma patients compared to healthy control.Why didn't they compare the expression in tumor and adjacent normal tissue as well?
2. The authors should also show the clinical parameter between the two groups of samples, such as age, gender, and use statistical analysis to determine if the serum level of MALAT1 was affected by these parameters.
3. The authors should include more cell-lines of gastric normal and adenocarcinoma cell-line for comparing their MALAT1 levels.
4. More details are needed for the following parts: What bioinformatic methods did the authors use to predict the possible binding targets between MALAT1 and miR-181a-5p/miR-181a-5p and AKT3?How were MALAT1 and miR-181a-5p level knocked down?Did the authors use GAPDH as normalization for lncRNA and miRNA?Why? 5.The expression of miR-181a-5p was negatively correlated with the expression of MALAT1, hence the R2 value should be negative.

6.
No evidence showing direct interaction between MALAT1 and miR-181a-5p/miR-181a-5p and AKT3.Why was luciferase reporter assay not performed?I think the luciferase reporter assay is important to draw the conclusion "Our data further indicates that MALAT1 competitively binds to miR-181a, making miR-181a unable to bind to AKT3 mRNA, thereby up-regulating AKT3 protein levels and ultimately promoting tumor growth".
8. Ipatasertib is an inhibitor of Akt pathway, but not specific for Akt3.I think siRNA against Akt3 should be used instead of Ipatasertib.9. To show that MALAT1 promoted gastric adenocarcinoma a through MALAT1-miR-181a-AKT3 axis, the functional effect of MALAT1 overexpression with or without transfection of miR-181a-5p or Akt3 siRNA will be a more direct evidence.
10.Some words in figure legend are not displayed appropriately.Figure legend of figure 6 should be A and B.11.There are some minor grammatical errors.Decision letter

Scientific importance: Is the manuscript an original and important contribution to its field? Good General interest: Is the paper of sufficient general interest? Good Quality of the paper: Is the overall quality of the paper suitable? Good It is a condition of publication that authors make their supporting data, code and materials available -either as supplementary material or hosted in an external repository. Please rate, if applicable, the supporting data on the following criteria.
10.Some words in figure legend are not displayed appropriately.Figure legend of figure 6 should be A and B.11.There are some minor grammatical errors.Author's Response to Decision Letter for (RSOB-19-0095.R0)

you have any ethical concerns with this paper? No Comments to the Author
The authors had addressed the comments raised from previous review process, I suggest accept the study in current version.