ELANE in leukemia progression, the expression level of ELANE, as well as correlation and survival analyses, was performed in patients with leukemia. Gene expression of ELANE on the normal monocytes and myeloid leukemia samples was obtained and compared vi

Original Article

ELANE

is highly expressed in leukemia

patients and predicts poor survival

Yanli Zhao

_{, Lining Si}

_{, Weihua Zhang}

_{, Wei Huang}

_{, Rong Wang}

1_{Department of Medicine, Qinghai University, Xining, Qinghai Province, China;} 2_{Department of Critical-Care} Medi-cine, Affiliated Hospital of Qinghai University, Xining, Qinghai Province, China; 3_{Department of Pathophysiology,} Harbin Medical University, Harbin, Heilongjiang Province, China; 4_{Division of Cellular Therapy, Duke University,} Durham, USA. *_{Equal contributors.}

Received March 18, 2018; Accepted October 11, 2018; Epub April 15, 2019; Published April 30, 2019

Abstract:ELANE (Elastase, Neutrophil Expressed), which has been identified as an oncogene in various tumors, is associated with neutropenia, both cyclic and autosomal dominant forms. However, little is known about ELANE on leukemia progression. To better understand the role of ELANE in leukemia progression, the expression level of ELANE, as well as correlation and survival analyses, was performed in patients with leukemia. Gene expression of ELANE on the normal monocytes and myeloid leukemia samples was obtained and compared via Oncomine. Corre-lation of ELANE and proto-oncogenes includes MYC and TP53 was assessed. The clinical prognostic value of ELANE was found to be elevated using Metzeler Acute myeloid leukemia microarray. As a result, ELANE was found to be highly expressed in the leukemia samples compared to normal monocytes. ELANE was found to be positively corre-lated with MYC expression, whereas it was negatively correcorre-lated with TP53 expression. Additionally, high expression of ELANE was associated with a relatively shorter survival time in leukemia patients, suggesting that ELANE is an oncogene in leukemia development. In conclusion, ELANE acts as a pro-oncogene in leukemia, which provides a potential therapeutic target.

Keywords: ELANE, leukemia, expression, correlation, survival rate

Introduction

Leukemia occurs in 2.3 million people and has

caused 353,500 deaths by 2018 [1]. Four main

types of leukemia - acute lymphoblastic

leuke-mia (ALL), acute myeloid leukeleuke-mia (AML),

chron-ic lymphocytchron-ic leukemia (CLL) and chronchron-ic my-

eloid leukemia (CML) have been identified in

clinic. Epidemic studies revealed that many risk

factors, such as smoking, ionizing radiation,

chemicals, prior chemotherapy, and even Down

syndrome can be ascribed to the

carcinogene-sis and progression of leukemia [2]. However,

the exact cause of leukemia is still unknown.

ELANE

is a serine protein-coding gene that

pro-vides instructions for making a protein called

neutrophil elastase [3]. Previously, numerous

studies have reported roles for the

ELANE

mu-

tation in pathological processes such as in-

flammatory diseases [4] and cancers [5].

Ph-armacological inhibitors such as alpha

2-anti-plasmin and some natural products (curcumin

and EGCG) have been well documented to

counteract and decrease damage caused by

neutrophil elastase [6, 7]. However, whether

ELANE

can be used as a marker for leukemia

diagnostics is still unclear. In this present stu-

dy, to clarify gene expression changes that

are common in cancer tissues or differ among

cancer tissues, we analyzed leukemia patient

samples downloaded from GEO database. Ex-

pression, correlation, and survival analyses of

ELANE

associated with leukemia

were

per-formed and

ELANE

was identified as a

poten-tial therapeutic target against leukemia.

Material and methods

ELANE expression analysis

Figure 1. ELANE mRNA levels in the Stegmaier leukemia microarray. Expres-sion analysis for ELANE was performed using student t-test method via com-paring acute myeloid leukemia (n = 9, group 3) and monocyte samples (n = 3, group 1). P value and fold change were extracted directly without any manual modification. In this figure, p value was 2.34E-5 and the fold change of ELANE in acute myeloid leukemia was 28.425 compared with monocyte samples in Stegmaier leukemia microarray.

using the online open access

Oncomine database (https://

www.oncomine.org/). The

p

value and fold change were

extracted directly from the

Oncomine analysis without

any manual modification.

Microarray correlation

analy-sis

To address the role of

ELANE

in leukemia,

MYC

and

TP53

were used as internal

refer-ences for the Pearson

correla-tion analysis of

ELANE.

Clinical significance of ELANE

in leukemia patients

Overall survival (OS) of

ELA-

NE

in leukemia patients was

accessed using a data set

of patients originally derived

from Metzeler acute myeloid

leukemia microarray as

previ-ously described [8, 9].

Statistical analysis

Gene expression was con-

sidered to be dramatically

changed if the

p

-value was

less than 5%. For survival

analysis, the overall survival

rate was performed using a

log-rank test and considered

to be different if the

p-

value

Figure 2.ELANE mRNA levels in the Coustan-Smith leukemia mi-croarray. Expression analysis for

ELANE was performed using stu-dent t-test method via comparing T-cell childhood acute lympho-blastic leukemia (n = 46, group 2) and CD10 positive CD19-pos-itive hematogone (n = 4, group 1) samples. P value and fold change were extracted directly without any manual modification. In this figure, p value was 4.63E-5 and the fold change of ELANE

was less than 5% based on the one-way ANOVA

with Tukey’s post-test.

mia cell growth and increase the overall surviv-

al time in leukemia patient. As shown in Figures

Figure 3. ELANE mRNA levels in the Coustan-Smith leukemia microarray. Ex-pression analysis for ELANE was performed using student t-test method via comparing B-cell childhood acute lymphoblastic leukemia (n = 238, group 2) and CD10 positive CD19 positive hematogone (n = 4, group 1) samples. P value and fold change were extracted directly without any manual modifica -tion. In this figure, p value was 0.002 and the fold change of ELANE in acute myeloid leukemia was 4.251 compared with monocyte samples in the Steg-maier leukemia microarray.

Figure 4. Correlation analysis of ELANE and TP53 expression in Herold chron-ic lymphocytchron-ic leukemia (n = 44). Correlation analysis was performed using a linear regression method according to the expression of ELANE and TP53 in the Herold chronic lymphocytic leukemia microarray. In this figure, the best-fit vales of the slopes were -0.02377 ± 0.03255 (A. ELANE ID 206871_at vs. TP53 ID 201746_at) and -0.0009251 ± 0.004012 (B. ELANE ID 206871_at

vs.TP53 ID 211300_s_at), which indicated that ELANE was negatively cor-related with TP53 in Herold chronic lymphocytic leukemia microarray.

Results

ELANE mRNA is highly

ex-pressed in patients with

leu-kemia

To compare

ELANE

expres-sion in leukemia patient

sam-ples, the original Series Ma-

trix. txt File(s) of Stegmaier

leukemia [10] and

Coustan-Smith leukemia [11] were

downloaded from Gene Ex-

pression Omnibus (GEO). Ori-

ginal intensity files were per

-formed MAS 5.0

normaliza-tion before further analysis.

As shown in Figure 1, mRNA

level of

ELANE

was highly

expressed in acute myeloid

leukemia samples compar-

ed to the normal monocyte

cells, with a total of 28.428

fold change in Stegmaier leu-

kemia.

Furthermore, mRNA level of

ELANE

was also found to be

significantly increased in T-

and B-cell childhood acute

myeloid leukemia samples

compared to the CD10

_and

CD19

_{hematogone (Figures}

2 and 3).

ELANE is positively correlated

with pro-oncogene MYC

ex-pression and negatively

corre-lated with tumor suppressor

TP53 expression in leukemia

samples

To address the role of

ELANE

in leukemia progression,

cor-relation analysis of

ELANE

4-6, expression of

ELANE

was found to be

consistently negatively correlated with

TP53

expression. Additionally,

ELANE

was positively

correlated with the expression of

MYC

(Figure

5A). In conclusion, our data strongly suggested

that

ELANE

served as an oncogene in leukemia

patients.

suppressor

TP53

, suggesting that

ELANE

may

act as an oncogene in leukemia. Finally, this

prediction was validated by the survival

analy-sis in Metzeler acute myeloid leukemia patients.

TP53

[16] (tumor protein with MW ~53 kDa) is

a tumor suppressor gene found on

chromo-Figure 6. Correlation analysis of ELANE and TP53 expression in Metzeler acute myeloid leukemia (n = 79). In this figure, the best-fit vales of the slopes were -0.005027 ± 0.005507 (A. ELANE ID 206871_at vs.TP53 ID 201746_ at) and -0.0001598 ± 0.001214 (B. ELANE ID 206871_at vs. TP53 ID 201746_at), which indicates that ELANE is negatively correlated with TP53

in Metzeler acute myeloid leukemia microarray.

Figure 7. Overall survival analysis of ELANE in the Metzeler acute myeloid leukemia 2 dataset. Survival analysis was performed using a log-rank test. In this figure, high and low groups of ELANE were defined using the expression of ELANE (ID: 206871_at) in Metzeler acute myeloid leukemia 2 microarray with a cut-off of 2336.28. For high expression of ELANE group (expression >2336.28, red line), the overall survival time was shorter compared with the low expression group (expression ≤2336.28, blue line) with p value of 0.17.

Low expression of ELANE

sug-gested a good prognosis in

leukemia patients

The clinical significance of

ELANE

was further validated

by Metzeler acute myeloid

leukemia patients [8]. Both

of

Figures 7 and 8 indicated

that mRNA level of

ELANE

with a relatively lower expre-

ssion predicted a good

sur-vival in leukemia patients

compared to the high

expres-sion group.

Discussion

Microarrays have enabled re-

searchers to conduct

large-scale quantitative

assess-ments of gene expression,

defining the transcriptome of

a multitude of cellular types

and states [15]. In addition,

gene expression microarrays

provide a snapshot of all the

transcriptional activity in a

biological sample. Collective-

ly, the Gene Expression Om-

nibus (GEO) at the National

Center for Biotechnology In-

formation (NCBI) has emerg-

ed as the leading fully public

repository for gene

expres-sion data. To better address

the role of

ELANE

in leuke-

mia progression, in this

pres-ent study, we delineated the

expression, correlation and

survival of

ELANE

using the

datasets downloaded from

GEO. As a result,

ELANE

was

found to be highly expressed

in leukemia sample. In

addi-tion, mRNA level of

ELANE

some 17 that is involved in DNA repair [17, 18],

cell cycle arrest [19], and apoptosis [20], and

its overexpression is a hallmark of many human

cancers [21].

TP53

drew initial attention from

cancer community because its expression was

frequently detected in acute myeloid leukemia

(AML) patients and closely connected with the

prognosis in leukemia patients [22, 23]. It has

been validated that a higher expression of the

wild-type

TP53

in leukemia may accelerate the

apoptotic process. In our study,

ELANE

was

found to be negatively correlated with

TP53

expression, which indicated that

ELANE was

an

oncogenic gene in leukemia progression.

Unlike

TP53

,

MYC

(c-

MYC

) is a tumor

suppres-sor that codes for a transcription factor. A

mutated version of

MYC

is found in cancers

[24], which finally causes

MYC

to be

constitu-tively expressed. Malfunctions in

MYC

have

also been found in carcinoma of the cervix,

colon, breast, lung and stomach [25].

MYC

is

thus viewed as a promising target for anti-

We would like to thank Gene Expression Om-

nibus (GEO) and Oncomine for making their

data readily available to the scientific commu

-nity. This work was supported by National Na-

tural Science Foundation of China (No. 8106-

0162, No. 81460284, No. 81460051, No. 31-

560039), Natural Science Foundation of Qing-

hai Provincial (No. 2014-ZJ-944Q, No. 2015-

ZJ-744, 2015-ZJ-929Q, 2015-ZJ-731), Constru-

ction of Laboratory of Qinghai Province (No.

2015-ZJ-Y23). The funders had no role in study

design, data collection and analysis, decision

to publish, or preparation of the manuscript.

Disclosure of conflict of interest

None.

Address correspondence to: Rong Wang, Depart- ment of Biochemistry, Medical College of Qinghai University, No. 16 Kunlun Road, Chengxi District, Xining 810001, Qinghai Province, China. Tel: +86-971-6108393; Fax: +86-+86-971-6108393; E-mail: qh- [email protected]

Figure 8. Overall survival analysis of ELANE in the Metzeler acute myeloid leukemia 1 dataset. Survival analysis was performed using a log-rank test. In this figure, high and low groups of ELANE were defined using the expression of ELANE (ID: 206871_at) in Metzeler acute myeloid leukemia 1 microarray with a cut-off of 9607.86. For the high expression of ELANE group (expres-sion >9607.86, red line), the overall survival time was shorter compared with the low expression group (expression ≤9607.86, blue line) with p value of 0.019.

cancer drugs. In our study,

ELANE

was found to be

posi-tively correlated with

MYC

ex-

pression, which further

vali-dated our hypothesis of the

oncogenic role of

ELANE

in

leukemia progression.

Microarray technology is es-

sentially a measurement tool

for measuring expressions of

genes and gene expressions

could be employed as

predic-tors of patient survival. In this

study, the clinical significance

of

ELANE

was further

validat-ed by Metzeler acute myeloid

leukemia patients. Finally, we

discovered that low express-

ion of

ELANE

predicted a good

survival in leukemia patients.

In conclusion, in this study,

the role of

ELANE

in leuke-

mia patients was determin-

ed using the public available

microarray datasets and de-

monstrate that

ELANE

might

be a potential therapeutic

tar-get against leukemia.

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