Tuberculosis 93 S1 (2013) S47–S50

1. Introduction

The ability of Mycobacterium tuberculosis to survive inside host

macrophages and tubercle granulomas constitutes the major

virulence attributes of this species. Whereas the macrophages are

capable of generating a variety of antimicrobial responses such

as anti-bacterial peptides, hydrolases and toxic reactive oxygen

and nitrogen intermediates,1 granulomas are deprived of oxygen

and nutrients.2 Although how M. tuberculosis is able to survive

in these environments still remains a mystery, accumulating

evidence indicates that M. tuberculosis is capable of modulating

cellular processes such as cytokine responses of macrophages,

MHC class II expression and antigen presentation in phagocytes,

phagosomal processing by MHC class I pathway, phagolysosome

biogenesis and apoptosis of macrophages/dendritic cells.3

Currently, many of the above cellular processes of eukaryotic

cells are under the control of microRNAs (miRNAs)4,5 and it is

possible that M. tuberculosis alters the expression of miRNAs to

modulate the function of macrophages for its favor.

miRNAs are small non-coding RNAs ranging from 19 to 24

nucleotides in length and are encoded by eukaryotic and some

viral genomes.4 They are processed from hairpin structures by

the sequential action of two RNAse type III nucleases, namely

Drosha and Dicer.6 Mature miRNAs bind with complementary

sequences in the 3´ untranslated region of target protein coding

mRNAs and repress their translation post-transcriptionally or

degrade the mRNA, causing an effect more or less similar to that

of gene silencing by RNAi.7,8 It is estimated that miRNAs regulate

approximately 1/3 of the protein coding genes in human and

miRNAs play critical roles in human cancer, cardiovascular and

neurodegenerative diseases.9-11 They are now considered, due to

their importance in disease pathogenesis, as possible therapeutic

targets for diseases.12,13

Recently, attempts have been made to determine the effects of

mycobacterial infection on the expression of miRNAs of the host.

Interestingly, some of the studies were conducted in samples

directly derived from patients infected with mycobacteria. Liu

et al14 analyzed the expression of miRNAs in peripheral blood

mononuclear cells (PBMCs) of TB patients and healthy controls

and reported that PBMCs from TB patients have elevated levels

of several miRNAs which include miR-500, miR-144 and miR-

452. In the skin biopsy of leprosy patients, the levels of miR-21

are elevated.15 While M. tuberculosis infection of PBMCs derived

macrophages down regulates the expression of miR-155,16

infection of mouse bone marrow derived macrophages (BMDMs)

upregulates miR-155.17 Murine T cells infected with BCG and

human monocyte derived macrophages infected with M. avium

also display differential expression of several miRNAs.18,19 Further,

mycobacterial component lipomannan (LM) of M. tuberculosis

and M. smegmatis has been reported to induce miR-125a and

miR-155, respectively, in human macrophages.16

Although some of the studies reported above have used

macrophages to determine the miRNAs affected by mycobacterial

K E Y W O R D S

Macrophage

M. tuberculosis

Virulence

Infection

miRNA

Differential expression

A B S T R A C T

MicroRNAs (miRNAs) are small non-coding RNAs which post-transcriptionally regulate a wide range of

biological processes that include cellular differentiation, development, immunity and apoptosis. There

is a growing body of evidences that bacteria modulate immune responses by altering the expression

of host miRNAs. Since macrophages are immune cells associated with innate and adaptive immunity,

we investigated whether Mycobacterium tuberculosis infection affects miRNAs of macrophages.

THP-1 macrophages infected with virulent (H37Rv) and avirulent (H37Ra) strains of M. tuberculosis

were analyzed for changes in miRNAs’ expression using microarray. This revealed that nine miRNA

genes (miR-30a, miR-30e, miR-155, miR-1275, miR-3665, miR-3178, miR-4484, miR-4668-5p and

miR-4497) were differentially expressed between THP-1cells infected with M. tuberculosis H37Rv and

M. tuberculosis H37Ra strains. Additional characterization of these genes is likely to provide insights

into their role in the pathogenesis of tuberculosis.

© 2013 Elsevier Ltd. All rights reserved.

Differential expression of miRNAs by macrophages infected with virulent and avirulent Mycobacterium tuberculosis

Kishore Dasa,b, Sankaralingam Saikolappana,b, Subramanian Dhandayuthapani*a,b

aDepartment of Microbiology and Immunology and Regional Academic Health Center, University of Texas Health Science Center at San Antonio, Edinburg, TX 78541, USAbCenter of Excellence in Infectious Diseases, Paul L. Foster School of Medicine, Texas Tech Health Sciences Center, 5001 El Paso Drive, El Paso, TX 79905, USA

* Corresponding author at: Center of Excellence in Infectious Diseases, Paul L.

Foster School of Medicine, Texas Tech Health Sciences Center, 5001 El Paso Drive,

El Paso, TX 79905. Tel.: Tel.: 915 215-4239; Fax: 915 215-1271

E-mail address: s.dhandayuthapani@ttuhsc.edu (S. Dhandayuthapani).

1472-9792/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.

Contents lists available at ScienceDirect

Tuberculosis

j our na l homepage: h t tp : / / in t l .e lsev ie rhea l th .com/ jour na ls / tube

S48 K. Das et al. / Tuberculosis 93 S1 (2013) S47–S50

infections, none of these studies analyzed miRNAs responding

specifically to virulent and avirulent strains of M. tuberculosis.

Identification of such miRNAs will not only help understand

their role in pathogenesis but also provide targets for potential

therapy. Therefore, we conducted experiments in this study

to analyze the expression of miRNAs in THP-1 macrophages

infected with virulent and avirulent strains of M. tuberculosis. We

report here that nine miRNA genes are differentially regulated by

virulent and avirulent strains of M. tuberculosis in THP-1 cells.

2. Materials and Methods

2.1 Bacterial strains and culture

M. tuberculosis H37Rv (27294) and H37Ra (25177) strains

are from ATCC. They were grown in Middlebrook 7H9 broth

containing oleic acid-albumin-dextrose-catalase (OADC) (10%)

and Tween80 (0.05%) at 37°C. They were washed with phosphate

buffered saline (PBS) and passed through a syringe, to disrupt the

clumps, before infecting THP-1 cells.

2.2 Cell culture and infection

THP-1 cell line was grown in RPMI medium containing 10%

fetal bovine serum (FBS) at 37ºC with 5% CO2.

Adherent THP-1

cells (3x106 cells), differentiated into macrophages by treating

with 100 nM PMA (Phorbol-12-myristate-13-acetate) for 72 h,

were infected with M. tuberculosis H37Rv and H37Ra strains,

separately, at an MOI of 10 (10 bacteria: 1 cell). Uninfected cells

which received only phosphate buffered saline (PBS) served

as controls. After 4-6 h incubation at 37ºC, non-phagocytosed

bacteria were washed off using PBS. Cells were replenished with

fresh RPMI and incubated for 24 h at 37ºC with 5%CO2. After 24 h,

cells were harvested and RNA was isolated.

2.3 RNA isolation

RNA from macrophages was isolated using PureZol (BioRad,

Hercules, CA) reagent following manufacturer’s protocol. Quanti-

tation of RNA was done using a Nanodrop (Thermo Scientific).

2.4 miRNA microarray

Microarray hybridization and data analyses were performed

by commercial provider ‘LC Sciences’, Houston, TX, (www.

lcsciences.com). The hybridization of microarray was performed

as follows. Two g total RNA samples were 3’-extended with a

poly(A) tail using poly(A) polymerase. An oligonucleotide tag was

then ligated to the poly(A) tail for later fluorescent dye staining.

Hybridization was performed overnight on a Paraflo microfluidic

chip using a micro-circulation pump (Atactic Technologies).20

On the microfluidic chip, each detection probe consisted of a

chemically modified nucleotide coding segment complementary

to target microRNA (from miRBase, http://microrna.sanger.

ac.uk/sequences/). The detection probes were made by in situ

synthesis using PGR (photogenerated reagent) chemistry. The

hybridization melting temperatures were balanced by chemical

modifications of the detection probes. For hybridization, 100 L

6x SSPE buffer (0.90 M NaCl, 60 mM Na2HPO

4, 6 mM EDTA, pH 6.8)

containing 25% formamide was used. After RNA hybridization,

tag-conjugating Cy3 was circulated through the microfluidic

chip for dye staining. Fluorescence images were collected using

a laser scanner (GenePix 4000B, Molecular Device) and digitized

using Array-Pro image analysis software (Media Cybernetics).

Data were analyzed by first subtracting the background and then

normalizing the signals using a LOWESS filter (Locally-weighted

Regression).21 miRNA expression data between two groups were

subjected to t-Test and more than two groups were subjected to

ANOVA.

3. Results and Discussion

Figure 1 shows the clusters of differentially expressed

miRNAs. A total of 41 miRNA genes were differentially expressed

between the uninfected control, M.tuberculosis H37Rv and

M.tuberculosis H37Ra infected cells at p value >0.01. This included

seven under expressed genes (values below 500) and they were

not considered for further analysis. The expression data for the

remaining 34 miRNA genes are shown in Table 1. Among these,

15 and 19 miRNAs were up- and down regulated, respectively,

in THP-1 cells infected with M. tuberculosis H37Rv strain when

Figure 1. Diagram showing clustering of differentially expressed miRNAs.

Each row represents a miRNA and each column represents a sample. The color

scale shown at the top illustrates the relative expression of miRNA. Red color

represents high expression and green color represents low expression. MT1, MT2

and MT3 are RNA samples from uninfected, Mtb H37Rv infected, Mtb H37Ra

infected THP-1 cells, respectively.

K. Das et al. / Tuberculosis 93 S1 (2013) S47–S50 S49

compared to uninfected cells. A similar up- and down regulation

of miRNAs were also noticed between THP-1 cells uninfected

and infected with M. tuberculosis H37Ra strain. However, it was

noticed that a cluster of genes showed differential expression

between THP-1 cells infected with M. tuberculosis H37Rv and

H37Ra strains. This included five genes (miR-4668-5p, miR-30e,

miR-1275, miR-30a and miR-3178) that showed more than

one fold elevated expression in cells infected with H37Rv than

H37Ra, one gene (miR-4484) that showed more than one fold

elevated expression in cells infected with H37Ra than H37Rv,

and 3 genes (miR-155, miR-3665 and miR-4497) that showed

more than one fold reduced expression in cells infected with

M. tuberculosis H37Rv than M. tuberculosis H37Ra. Pubmed

search for the function of these miRNAs provided only limited

information. While miR-30e is activated by -catenin,22 miR-30a

inhibits epithelial to mesenchyme transition.23 miR-1275 seems

to be associated with liver metastases of cancer24 and miR-155

targets a negative regulator associated with TNF- production.16

Other miRNAs have not been studied in detail so far and this

prevents us to predict any potential roles for the differentially

expressed miRNAs.

It was difficult to compare the results of the present study

directly with previously published studies on M. tuberculosis

because each study had used a different cell/tissue type like

PBMCs,14 PBMCs derived macrophages,16 BMDMs,17 RAW264.7

cell line,17 human serum25 and sputum.26 However, a single

miRNA that is reported to be affected due to M. tuberculosis

infection in most of these studies, including this study which

used THP-1 cell line, is miR-155. But this miRNA, contrary to our

results which showed slight down regulation of expression, has

been found to be upregulated in all these studies, although the

extent of upregulation varies with different cell types/tissues.

Specifically, M. tuberculosis components lipomannan and ESAT-6

have been shown to induce miR-155 in PBMCs and BMDMs,

respectively.16 This is in sharp contrast to the observation made

with the intracellular pathogen F. tularensis which suppresses

the induction of miR-155. It seems that miR-155 is induced

through TLR and NOD like receptors mediated pathways and

any discrepancy in the induction of this miRNA by different

bacterial pathogens may indicate the differences in the ligands

for the above receptors. What is interesting, however, is that the

induction of miR-155 by M. tuberculosis seems to enhance its

survival in BMDMs.17

The virulent M. tuberculosis strain H37Rv and avirulent H37Ra

used in this study have the same ancestral parent, H37, a clinical

isolate from a pulmonary TB patient at the Trudeau Sanatorium.

Table1Data obtained for the differentially expressed miRNA genes.

No Infection Mtb H37Rv infected Fold change from Mtb H37Ra Infected Fold change from

No miRNA MT1a MT2a uninfected MT3a uninfected

1 hsa-miR-4668-5p 100±3 4290±922 43.0 170±31 1.7

2 hsa-miR-30e 448±0 2148±266 4.8 1038±30 2.3

3 hsa-miR-106b 337±24 693±18 2.0 460±24 1.36

4 hsa-miR-27a 2355±210 5247±612 2.2 5256±211 2.23

5 hsa-miR-4484 253±50 5185±406 20.5 5537±199 21.88

6 hsa-miR-4532 180±38 546±32 3.0 571±12 3.17

7 hsa-miR-1246 8857±908 19463±1264 2.19 18370±755 2.07

8 hsa-miR-21 15001±717 27577±2868 1.83 23126±1521 1.54

9 hsa-miR-1275 150±18 680±36 4.53 427±21 2.84

10 hsa-miR-19b 1132±58 3077±202 2.71 2231±77 1.97

11 hsa-miR-106a 5105±314 7607±140 1.49 6801±169 1.33

12 hsa-miR-29a 5686±304 10576±241 1.86 8872±11 1.56

13 hsa-miR-30a 188±19 1010±126 5.37 641±57 3.40

14 hsa-miR-3178 494±58 3351±243 6.78 2576±172 5.21

15 hsa-miR-762 176±7 564±41 3.20 423±37 2.40

16 hsa-miR-155 4395±500 2446±122 0.55 4510±201 1.02

17 hsa-miR-3665 7719±276 6526±5 0.85 8422±317 1.09

18 hsa-miR-4497 4109±54 2961±1 0.72 4514±51 1.09

19 hsa-miR-1280 831±6 289±62 0.34 448±16 0.54

20 hsa-miR-151-5p 1606±59 565±81 0.35 709±62 0.44

21 hsa-miR-151b 1011±72 450±65 0.45 570±41 0.56

22 hsa-miR-1307 276±27 164±4 0.59 191±5 0.69

23 hsa-miR-3141 1696±49 841±1 0.49 999±109 0.59

24 hsa-miR-4298 1408±40 735±9 0.52 957±53 0.68

25 hsa-miR-4739 842±3 759±28 0.90 674±9 0.88

26 hsa-miR-4281 1443±107 894±54 0.62 750±8 0.89

28 hsa-miR-191 9545±431 7059±149 0.74 7040±176 0.74

29 hsa-miR-193a-5p 1539±49 662±32 0.43 712±4 0.46

30 hsa-miR-361-5p 1703±12 787±104 0.46 913±43 0.53

31 hsa-miR-92b 3885±86 1922±308 0.49 1912±12 0.49

32 hsa-miR-92a 13673±531 8645±661 0.63 8011±143 0.58

33 hsa-miR-222 20731±585 15388±912 0.74 14999±182 0.72

34 hsa-miR-93 12376±102 1394±126 0.58 1344±31 0.56

a Values are Mean±SD. MT1, MT2 and MT3 are RNA samples from uninfected, Mtb H37Rv infected, Mtb H37Ra infected THP-1 cells, respectively.

S50 K. Das et al. / Tuberculosis 93 S1 (2013) S47–S50

They were initially differentiated based on the differences in

cord factor (TDM)27 production and ability to multiply within

macrophages.27,28 However, recent developments in ‘omics’

(genomics, proteomics, bioinformatics) have highlighted several

additional differences between them. In particular, the H37Ra

genome appears to be 8445 bp larger than H37Rv, despite the fact

it has 21 deletions.29 In addition, the most significant difference

between the two strains is the deficiency of H37Ra to translocate

the secretory and immunogenic proteins ESAT-6 and CFP-10 of

ESX-1 locus.30 ESAT-6 and CFP-10 are major virulence factors of

M. tuberculosis and some of the effects of these proteins in hosts

are suppression of proinflammatory responses,31 necrosis,32

apoptosis,33 membrano-lysis34 and cytolysis.35,36 Whether these

molecules have any roles in the observed alterations in the

expression of miRNAs between THP-1 cells infected with H37Rv

and H37Ra remains an important area of investigation.

In summary, we have identified nine miRNAs showing

differential expression in THP-1 cells infected with M. tuberculosis

H37Rv and H37Ra strains. Additional characterization of these

genes is necessary to better understand their role in tuberculosis

pathogenesis.

Acknowledgement

This study was partly supported by NIH/NIAID. This agency

has no role in the following: study design, data collection,

analysis and interpretation of data, writing of the manuscript

and in the decision to submit the manuscript for publication.

Funding

NIH/NIAID Grant R21AI089346.

Competing Interests

None declared

Ethical Approval

Not required

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