Answers to reviewers’ comments
Reviewer no. 1
1. The language must be checked by a native English-speaking colleague.
Answer: We have checked English writing in our revised manuscript (Changes are marked in
green color).
2. The results section completely lacks a solid statistical description of the data; each data must
be statistically analyzed and detailed in the result section.
Answer: We have added the values of P in the result section.
3. The discussion must not repeat the results, but it must explain the data in light of the current
knowledge and the current literature. The novelty of the findings must be highlighted. Current
controversies that the paper may raise or may settle must be described. Here the discussion
only repeats the observation, which is not the aim of a discussion. Therefore the discussion
section must be entirely rewritten.
Answer: We have already rewritten the discussion in our revised manuscript (Changes in
discussion are highlighted in red color). We also have highlighted the novelty of our study as
follows:
1. “These results suggested that miR-181a acted as an oncogene in HCC.”
2. “We firstly identified E2F5 as a direct target gene of miR-181a.”
3. “Our study found a novel mechanism for the growth- promoting effect of HBV.”
4. E2F5 is one target of miR181a, among many, many others. This must be described (based on
analysis, eg. MiRanda). The paper is much too simplistic, assessing that miR181a and E2F5
are so related. E2F5 is regulated not only by miR181a, but also by several other miR's.
Answer: miR-181a can actually control multiple target genes, and E2F5 is just one of them. At
the beginning of this study, we examined the expression of several possible target genes
predicted by MiRanda and Targetscan in miR-181a overexpression SMMC-7721 cells, but
only E2F5 was found to be down-regulated (Fig. S1). Therefore, we choose E2F5 as a further
study. It has been reported that E2F5 can also be regulated by other miR's, such as miR-205,
miR-128-2, miR-106. But none of these miRNAs were proved to be dysregulated by HBV in
miRNA microarray. Therefore, we focus E2F5 as a target gene of miR-181a.
pTARGET
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Fig. S1. qRT-PCR was performed to analyze the expressions of potential target genes of
miR-181a in SMMC-7721cells transfected miR-181a.
5. Implying that your paper suggest relevant data for diagnostic and therapeutic target is ridiculous,
based on such a very primitive report that does not integrate the data with a global view on the
function of other miR's and other targets, and limited to minor animal experiments on mice.
Answer: Thanks for your frank opinion. As you said, we jumped too rapidly to the conclusion.
We have deleted “the conclusion of miR-181a acting as a diagnostic and therapeutic target”
this sentence.
Reviewer no. 2
1. Construction of vectors: instead of #data not published#, the detail construction information
of Ad-HBV and Ad-GFP should be added.
Answer: We have added “HBV 1.3 fold genome was ligated to shuttle vector pAdTrack-TO4,
then pAdTrack-TO4-HBV1.3 was linearized by PmeI and transfected into BJ5183 cells
containing pAd-Easy1 to form a recombinant plasmid. After that the confirmed recombinant
plasmid was linearize by PacI restriction endonuclease and transfected into HEK-293 cells to
generate recombinant adenoviruses. ” to introduce how the Ad-HBV and Ad-GFP were
constructed in the methods section (red words). Also, we added its reference [14. Luo J，Deng
ZL，Luo X，Tang N，Song WX，Chen J，Sharff KA，Luu HH， Haydon RC，Kinzier KW，
Vogeistein B，He TC: A protocol for rapid generation of recombinant adenoviruses using the
AdEasy system. Nat Protoc 2007, 2:1236-1247.].
2. One of the primer pair for microRNA-181a is missing in the quantitative real-time PCR. This
information should be included.
Answer: In this study, we used miRNA Real-Time PCR Assay Kit (CWBIO, China) for the
miR-181a expression assay. A universal reverse primer is provided by this kit.
The protocol in detail is showed in the following Figure. RNA was firstly incubated in the
presence of poly (A) polymerase, MnCl2, and ATP for 1 h at 37 °C, then, using an oligodT
primer harboring a consensus sequence. Reverse transcription was performed on total RNA
using
miRNA
cDNA
Kit
(CWBIO,
China).
Next, the cDNA was amplified by real-time PCR, this reaction contained a miRNA specific
forward primer which was presented in the article and a universal reverse primer
complementary to the consensus sequence of the oligoDT tail.
3. The authors determine up-regulation of microRNA-181a by HBV using HBV stably
expression cell line HepG2.2.15 and adenovirus transduced HBV expression HepG2 cells. My
question is about whether or not this up-regulation is in a dose-dependent manner. If the
authors provide data confirming dose-dependent up-regulation of microRNA-181a by HBV, it
will be more convincing.
Answer: We have tested the impact of different dose of Ad-HBV on miR-181a expression
and found HBV could exactly regulate miR-181a expression in a dose-dependent manner
(Fig. S2). Ad-GFP was used as a control.
A
B
C
Fig. S2 (A) qRT-PCR was performed to analyze the expression of miR-181a in HepG2 cells
treated with different dose of adenovirus. (B and C) ELISA were used to analyze the
expressions of HBeAg and HBsAg in HepG2 cells treated with different dose of adenovirus.
‘－’ stands for the negative control; ‘＋’ stands for the positive control.
4. Up-regulation of microRNA-181a by HBV has been confirmed. Is there any feedback
regulation of HBV replication by microRNA-181a?
Answer: This is a good question. However, there is no study reporting that miR-181a has an
impact on HBV replication so far. We will focus on your interesting idea in our next research
programs.
Reviewer no. 3
1. In recent years, the miR-181 family was found dysregulated in a variety of human cancers and
significantly associated with clinical outcome of cancerous patients. Lots of studies confirmed
that different miR-181 family members inhibit the growth of cancer cells (for example #Shi,
Brain Res, 2008 Oct 21; 1236:185-93), why the authors chose miR-181a as their research
target?
Answer: A study of miRNA microarray by our laboratory has shown that miR-181a was
significantly increased in HBV-related HCC cell line HepG2.2.15 compared with HepG2
(Zhang et al., Word J Gastroenterol. 2011; 17: 3353-3358). Moreover, Liu et al. reported that
the expression of miR-181a was higher in HepG2.2.15 than that in HepG2 cells (Liu et al.,
Medical Journal of Chinese People’s Liberation Army. 2007; 32: 92-95). So we chose
miR-181a as a research target.
Although lots of studies confirmed that different miR-181 family members inhibit the
growth of cancer cells, several studies have reported that miR-181a promoted the growth of
cancer cells, such as gastric cancer and osteosarcoma (Zhang, Tumor Biol 2012; 33:1589-1597;
Zhu, Tumor Biol 2013; 38:401-407).
2. In #Results#, the authors claimed that HBV could up-regulate the activity of miR-181a
promoter. What is the exactly mechanism? Is there a binding relationship?
Answer: It has not been reported that HBV could directly bind DNA to affect genes expression.
But lots of studies showed that HBV regulated genes expression mainly by affecting the
expression of transcription factors. Further studies are needed to investigate whether HBV
up-regulated the activity of miR-181a promoter through affecting a certain transcription factor.
3. In #Results#, the authors find kinds of E2F5 silencing can enhance HCC cell growth, how
about up-regulate the expression of E2F5?
Answer: We have tried many times to construct a plasmid which could express E2F5, but
failed at last. It is very difficult to clone the sequence of E2F5 CDS because the Tm of the two
primers is quite different. Moreover, the function of E2F5 is very clearly defined in Gene
Cards as ‘The E2F family plays a crucial role in the control of cell cycle and action of tumor
suppressor proteins.’ Therefore, we just did experiments of E2F5 silencing.
4. All the expressions were detected in HCC cells, how about in Hepatocytes?
Answer: It is very necessary to detecting all the expressions in Hepatocytes. However, there
are not hepatocyte cell lines in our laboratory currently, and primary hepatocytes are difficult
to isolate from clinic patient, so we can not detect the expressions in Hepatocytes at present.
Reviewer no. 4
1. What is the HBV infectious status in HepG2 and SMMC-7721 cell lines? Are they HBV
positive or negative in the initial screen?
Answer: Both HepG2 and SMMC-7721 are HBV negative cells. We have added “Both the
two cell lines are HBV negative” in the first part of methods section.
2. If miR-181a is indeed important for HBV positive HCC proliferation, can miR-181a inhibitor
attenuate this effect?
Answer: We added the results of MTS and colony formation assay in HepG2.2.15 cells
transfected by miR-181a inhibitor. As shown in a new Fig. 5C and D.
3. In all bar figures, the control and experimental groups should be highlighted with white and
black respectively. Labeling all in groups black can easily create confusion among readers.
Answer: We have highlighted the control and experimental groups with white and black
respectively in all of our Figures. Please see our new Figures.
Last, we would like to express our appreciation to you and the advices for
suggesting how to improve our paper.
Thank you very much!
Yours sincerely,
Hua Tang.