Comparison of Hepatocellular Carcinoma miRNA Expression Profiling as Evaluated by Next Generation Sequencing and Microarray

PLoS ONE 9(9): e106314. doi:10.1371/journal.pone.0106314

Yoshiki Murakami1, Toshihito Tanahashi2,3, Rina Okada4, Hidenori Toyoda5, Takashi Kumada5, Masaru Enomoto1, Akihiro Tamori1, Norifumi Kawada1, Y-h Taguchi6, Takeshi Azuma4

1 Department of Hepatology, Osaka City University Graduate School of Medicine, Osaka, Japan2 Department of Medical Pharmaceutics, Kobe Pharmaceutical University, Kobe, Japan, 3 Division of Gastroenterology, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe, Japan4 Division of Gastroenterology, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe, Japan5 Department of Gastroenterology, Ogaki Municipal Hospital, Ogaki, Japan6 Department of Physics, Chuo University, Tokyo 112-8551, Japan

Purpose: How can we measure microRNA expression using MiSeq + miRDeep2?

MiSeq: Desktop sequencerMiRDeep2: easy to use pipeline for miRNA-seq

We would like to measure miRNA expression as easy & precisely as possible ….

(without any complicated or sophisticated or advanced procedures)

How accurate are those outcomes?

Materials:14 HCC (liver cancer) + 6 Normal tissue (same patients)

MiRNA-seq : 14 HCC + 6 Normal tissueMicroarray : 14 HCC

Questions:How coincident between miRNA-seq and microarray?

Can miRNA-seq diagnose HCC vs normal tissue?

Are novel miRNAs detected?

How coincident between miRNA-seq and microarray?

log10(NGS)

log1

0(m

icro

arra

y)Corr. Coef. = 0.6059

Microarray:Agilent microRNA GprocessedSignal + mean=0 + SD=1

miRDeep2:Bowtie + hg19 + miRBase 18 + default settings

Essentially, no normalization, but they are correlated very well.

Te

ch

ni c

al re

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ate

s

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er p

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nt

Independent of technical replicates / patients, miRNA-seq and microarray are well correlated without any sophisticated/complicated/advanced normalizations

Next: Consistency of differential miRNA expression between miRNA-seq and microarray

log10(NGS)

log1

0(m

icro

arra

y)K-177 vs CU-087

Corr. Coef. = 0.5555

Com

pari

sons

bet

wee

n te

chni

cal r

eplic

ates

An

oth

er p

atie

nt

Independent of technical replicates / patients, miRNA-seq and microarray of differential expression were well correlated without any sophisticated/complicated/advanced normalizations

Comparisons between technical replicates of miRNA-seq

0.986

0.984 0.986

Comparisons between technical replicates of miRNA-seq of differential expression

0.913

0.929 0.907

0.926 0.852 0.865

0.834 0.925 0.843 0.899

0.853 0.830 0.843 0.925 0.896

0.918 0.841 0.856 0.922 0.818 0.850

0.830 0.919 0.842 0.845 0.922 0.847 0.899

0.836 0.815 0.914 0.844 0.818 0.922 0.921 0.899

Independent of technical replicates / patients, reproducibilities of miRNA-seq of expression and differential expression were well without any sophisticated/complicated/advanced normalizations

MiRNA-seq expression/differential expression does not require any sophisticated/complicated/advanced normalizations, possibly because of the fact that limited number of miRNAs are generally expressive.

Can miRNA-seq diagnose HCC ?

11 miRNAs were selected using PCA based unsupervised FE

miR-10a-5p, miR-122-5p, miR-146b-5p, miR-148a-3p, miR-192-5p, miR-22-3p, miR-26a-5p, and miR-27b-3p, miR-10b-5p, miR-143-3p, and miR-21-5p

True Tumor ControlPredicted Tumor 12 0 Control 2 6

M features

N sample

Q

＝＋ noise

M' N✕matrix

Q N matrix✕

N（≪M)samples

M'（≪M）✕Q matrix

M features

M features embedded

PC1

PC1

M'

N（≪M)samples

Individual miRNAs are not always expressed between tumor and control

Combinatorial expression is important !

Discovery of Novel miRNAs

Conclusions

※Miseq+miRDeep2 detect miRNA expression and differential miRNA expression coincident with microarray

・Miseq+miRDeep2　give reproducible miRNA expression and differential miRNA expression

・miRNA expression measured by Miseq+miRDeep2 can diagnose between HCC and normal control

・Miseq+miRDeep2 can identify novel miRNAs