Discovery of miRNA-939 as Pos5ranscrip8onal Regulator of hiNOS in Human Hepatocytes: A Cri8cal Review

Lim Yi Fei M15606

Background

Guo et al., in 2012, claimed to have demonstrated the role of microRNA (miRNA), specifically

miRNA-939, in the direct regulation of the human inducible Nitric Oxide Synthase (hiNOS) genes in

human hepatocytes (HC)1. hiNOS genes codes for Nitric Oxide (NO) synthase, which in turn

catalyzes the reaction below to form NO2:

Unlike its isoforms, hiNOS, when induced, results in sustained release of NO in large quantities,

which, in the case of cytokine induction, contributes to the livers extraordinary regeneration

abilities3. Regulation of hiNOS gene expression is paramount to prevent liver inflammation3, and

yet the mechanism is not well understood today.

Guo et al. thus proposed miRNA as the main post-transcriptional regulatory mechanism after

observing the relationship between hiNOS mRNA with its protein. They then proceeded to verify

their hypothesis by locating miRNA response elements (MREs) in hiNOS mRNA 3 Untranslated

Region (3UTR) - a region commonly associated with miRNA binding - followed by the identification

of miRNA candidate miR-939 as a translational repressor. Lastly, a detailed investigation of the

repression mechanism involving both MRE and miR-939 was carried out.

In short, Guos research began as an attempt to identify a particular miRNA, if any, that have a

direct influence on hiNOS gene expression, and concluded with the characterisation of miR-939 as

a mediator for hiNOS mRNA translation blockade in HC.

Major Findings

MRE Properties of hiNOS mRNA 3UTR

By inserting hiNOS 3UTR into its counterpart in a reporter gene and observing its results, Guo et

al. concluded that the region causes post-transcriptional blockade of protein expression. 1

Additional experiments show that the blockade is dependent on the orientation of the hiNOS

3UTR in the mRNA, and its effect could be observed both during basal transcription and when

induced by cytokine.

After miR-939 was determined as the best candidate as a repressor, mutagenesis was carried out

on the 3UTR region, whereby the integrity of two of the five suspected miRNA binding sites (as

determined from gene sequencing), located +99bp and +112bp from the start of the 3UTR, were

shown to be crucial for miR-939 to exert its ability as translational repressor.

Properties of miR-939

Introduction of exogenous miR-939, one of the five miRNAs complementary to suspected miRNA

binding sites to a hepatocyte culture led to reduced NO synthesis, and hence the proposal of

miR-939 as the main translational repressor.

It was later suggested that miR-939 binds to hiNOS mRNA with high specificity, as indicated by

Ago co-immunoprecipitation and the unsuccessful inhibition of non-specific antisense miRNA.

Western blot and RT-qPCR implied that miR-939 confers repression by translational blockade

instead of mRNA degradation, as mRNA levels remain high despite low protein expression. As

miR-939 levels were shown to increase following cytokine and LPS induction (both in vitro

and in vivo), it was predicted that miR-939 functions to inhibit hiNOS gene expression following

induction of hiNOS gene, a theory that was later reinforced by the observation of increase in NO

synthesis after inhibition of miR-939 in cytokine-induced hepatocytes.

Conclusion

From the findings above, Guo et al. postulates that as cytokines stimulate the transcription of

hiNOS gene, it simultaneously induces miR-939 expression, which binds to both miRNA

binding sites in hiNOS 3UTR, leading to translational inhibition. They believe that this

contributes to the bodys regulation of NO synthesis in order to prevent hiNOS over-expression.

Comments

In general, the research was well done, whereby many aspects of the miR-939 regulatory

mechanism, from identifying the binding site to understanding how miR-939 causes repression of

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gene expression, was investigated in a comprehensive manner, as the limitation of each

experiment were mostly addressed by subsequent experiments. However, certain flaws remain,

which we will now go through:

Validity of Conclusions

In an attempt to show specificity for miR-939 binding, Guo et al. performed antisense miRNA

inhibition on the candidate, and found that specific inhibitors managed to increase NO synthesis in

presence of miR-939 in a dose dependent manner. I feel that the results are insufficient to show

specificity for miR-939 binding as claimed by Guo et al., as specific inhibitors merely

incapacitates a proportion of miR-939 - the remaining miR-939 could very well bind to other miRNA

binding sites. The method was never meant to demonstrate the specificity of miRNA binding in the

first place - other research draw their conclusions from investigation of miRNA:mRNA interactions

instead4,5.

Another set of experiments, aimed to demonstrate that miR-939 is functioning in the co-

immunoprecipitated complex, showed high amount of miRNA/Ago complex when the miR-939 was

transfected into HCs, which they believe implies the specificity of miR-939 binding to hiNOS mRNA

in vivo. I feel that the data merely shows that in the presence of miR-939, more miRNA/Ago

complex were formed, and that does not necessarily correlate to the direct binding of miR-939 to

hiNOS mRNA, especially in the complex in vivo environment of HCs. Previous research has shown

that as many as 16% of the miRNAs are prone to alteration by nuclear RNA editing6, thus the

conclusion should not be made so hastily.

Completeness of Research

The results of the experiment were not sufficiently interpreted - the section was merely a laundry

list of findings arranged in chronological order instead of by area of focus. The mechanisms of

miR-939 regulation were not well elaborated upon in the discussion, which were littered by

irrelevant literature reviews.

Also, certain experiments did not provide interpretation of the results in context of the research.

When investigating the effects of exogenous miRNA-939 on Cytokine-induced NO synthesis, for

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example, no explanation were provided regarding the dose-dependency of miR-939; in other

research, this was supplemented with reference to the reaction pathways7.

The significance of the findings were not sufficiently expanded in the discussions, as there were no

indication of application of their findings, resulting in failure to add depth to their results. The team

also did not identify future work for their project, whereby much more could be done for LPS-

induced HCs, which is another major source of induction.

In short, the research is good, but presentation could be done in a more coherent manner.

(1058 words)

Biblography

1. Guo Z, Shao L, Zheng L, Du Q, Li P, John B, Geller D. 2012. miRNA-939 regulates human inducible nitric oxide synthase posttranscriptional gene expression in human hepatocytes. Proceedings of the National Academy of Sciences 109:5826-5831.

2. Alderton W, Cooper C, Knowles R. 2001. Nitric oxide synthases: structure, function and inhibition. Biochem. J. 357:593.

3. Clemens M. 1999. Nitric oxide in liver injury. Hepatology 30:1-5.

4. Idtdna.com. 2015. Inhibiting miRNAs using Antisense Oligonucleotides.

5. Doench J. 2004. Specificity of microRNA target selection in translational repression. Genes & Development 18:504-511.

6. Kawahara Y, Megraw M, Kreider E, Iizasa H, Valente L, Hatzigeorgiou A, Nishikura K. 2008. Frequency and fate of microRNA editing in human brain. Nucleic Acids Research 36:5270-5280.

7. Shu J, Xia Z, Li L, Liang E, Slipek N, Shen D, Foo J, Subramanian S, Steer C. 2012. Dose-dependent differential mRNA target selection and regulation by let-7a-7f and miR-17-92 cluster microRNAs. RNA Biology 9:1275-1287.

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