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Abstract

BACKGROUND:

Blood-circulating miRNAs have been reported to act as potential biomarkers in various cancers including non-small cell lung cancer (NSCLC).

OBJECTIVE:

This study was to assess serum miR-98 levels in NSCLC patients and explore its potential prognostic value.

METHODS:

The relative expression levels of miR-98 were detected by quantitative RT-PCR in the sera of 127 NSCLC patients and 60 healthy controls.

RESULTS:

Our results showed that serum miR-98 expression was down-regulated in NSCLC patients compared with healthy controls. Receiver operating characteristic (ROC) curve analysis suggested that serum miR-98 could be used as a potential marker in the diagnosis of NSCLC. In addition, decreased serum miR-98 was positively correlated with worse TNM stage, lymph node metastasis, as well as unfavorable overall survival. Multivariate Cox regression analysis confirmed that serum miR-98 expression was an independent prognostic factor for NSCLC.

CONCLUSIONS:

Therefore, serum miR-98 might be useful as a promising biomarker for prognosis prediction of NSCLC.

Keywords

Non-small cell lung cancer serum miR-98 biomarker diagnosis prognosis

1. Introduction

Non-small cell lung cancer (NSCLC), which accounts for approximately 80%–85% cases of lung cancer patients, is one of the leading fatal malignancy around the world [1, 2]. Although huge progress has been made in the treatment of this deadly disease over the past decades, the 5-year overall survival rate of NSCLC patients remains dismal [3, 4, 5]. Therefore, it is extremely urgent to identify potential novel biomarkers to improve the prognosis of patients with NSCLC.

MicroRNAs (miRNAs) are a subset of small, non-coding RNAs consisting of 19–25 nucleotides. MiRNAs can negatively modulate target genes expression by binding to the 3 $\prime$ -untranslated region (3 $\prime$ -UTR) of target mRNAs, inducing mRNAs degradation or translation suppression [6, 7]. It was widely realized that aberrantly expressed miRNAs involved in the tumor development and progression of many cancer types [8, 9]. Some miRNAs were reported to act as tumor suppressors or oncogenes in NSCLC. For instance, a previous study by Tang et al showed that decreased miR-30a was associated with unfavorable clinical outcome and survival of NSCLC patients [10]. Conversely, Lei and colleagues showed that over-expression of miR-92b markedly promoted NSCLC cell proliferation, invasion and migration through targeting RECK [11].

MiR-98 belongs to the let-7/miR-98 family which is highly evolutionally conserved [12, 13]. Recently, miR-98 had been reported to play critical roles in the development of various cancers such as glioma [14], prostate cancer [15], head and neck squamous cell carcinoma [16] and NSCLC [17, 18]. However, the diagnostic and prognostic significance of serum miR-98 in NSCLC patients was still unknown. This study was to detect serum miR-98 level in NSCLC patients and explore its potential clinical value.

2. Materials and methods

2.1 Patients and sample collection

A total of 127 NSCLC patients prior to any treatment were enrolled in this study. The patient group included 79 men and 48 women aged between 38 to 72 years old (median age $=$ 58 years). Moreover, 60 healthy volunteers included 35 men and 25 women between 41 to 70 years old were recruited as controls (median age $=$ 59 years). All the participants are Han Chinese. About 29.13% NSCLC patients had current cigarette smoking, which was higher than that of healthy controls (18.33%). Tumor stage was determined according to the criteria of the International Union against Cancer (UICC) Staging System. Detailed clinicopathological features of NSCLC patients were listed in Table 1. All NSCLC patients received regular follow-up. Overall survival (OS) was defined as the time from the date of diagnosis to the date of death or last follow-up.

Blood samples were collected from patients with NSCLC and healthy controls early in the morning. Up to 5 ml of fasting venous blood was collected in a serum separator tube from every participant. The blood samples of healthy controls were not mixed with those of tumor patients. All blood samples were centrifuged at 2800 g for 10 min within half an hour after collection. Then the separated supernatant was stored in 1.5-mL tubes at $-$ 80 ${}^{\circ}$ C until use. This study was approved by the Ethics Committee of Daqing Oil Field General Hospital and written informed consent was received from all participants.

2.2 Total RNA isolation and quantitative RT-PCR

Total RNA was extracted from serum samples using a miRVana PARIS Kit (Applied Biosystems, Foster City, CA, USA) in accordance with the manufacturer’s protocols. RNA purity and concentration was determined using a NanoDrop ND-1000 spectrophotometer (NanoDropTechnologies, Wilmington, DE). Reverse transcription reaction was performed using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Subsequently, Quantitative RT-PCR was carried out using the Maxima SYBR Green qPCR Kit (Thermo Scientific, CA, USA) on an ABI 7500 fast real-time PCR system (Applied Biosystems, Foster City, USA) according to the manufacturer’s instructions. The PCR cycling conditions were as follows: 95 ${}^{\circ}$ C for 30 s, followed by 40 amplification cycles of 95 ${}^{\circ}$ C for 5 s and 60 ${}^{\circ}$ C for 34 s. Each sample was independently repeated in triplicate. U6 small nuclear RNA (snRNA) was used for miRNA template normalization. Furthermore, the 2 ${}^{\mbox{--}\Delta\Delta\text{Ct}}$ method was used to calculate the relative expression level of miR-98 in serum. The sequence of primers were shown in below.

miR-98
Forward primer	5 $\prime$ -GGACTGAGGTAGTAAGTTG-3 $\prime$
Reverse primer	5 $\prime$ -CATCAGATGCGTTGCGTA-3 $\prime$
U6
Forward primer	5 $\prime$ -GCTTCGGCAGCACATATACTAAAAT-3 $\prime$
Reverse primer	5 $\prime$ -CGCTTCACGAATTTGCGTGTCAT-3 $\prime$

2.3 Statistical analysis

Statistical analysis was performed using GraphPad Prism v5.0 software (GraphPad Software, Inc., San Diego, CA). The data was not subjected to normal distribution. Therefore, the median value of serum miR-98 in this NSCLC cohort was used as a cut-off point to divide the patients into two groups namely high serum miR-98 group and low serum miR-98 group. The difference in serum miR-98 levels between two groups was compared by Mann-Whitney U test. The Pearson’s chi-squared test was used to evaluate the association between serum miR-98 levels and clinical parameters. Survival curves were constructed using the Kaplan-Meier method and compared by log-rank test. The Cox proportional hazards model was used for multivariate analyses of the independent prognostic factors. Receiver-operating characteristic (ROC) curve was constructed to determine the diagnostic value of serum miR-98 for NSCLC. $P$ value less than 0.05 was considered statistically significant.

Table 1
Association between serum miR-98 expression and clinicopathological characteristics of NSCLC patients

Clinical variables	Number of patients	Serum miR-98 expression
	( $n=$ 127)	Low ( $n=$ 65)	High ( $n=$ 62)	p
Age				0.136
$<$ 60	57	25	32
$\geqslant$ 60	70	40	30
Sex				0.105
Male	79	36	43
Female	48	29	19
Smoking status				0.112
Never/former	90	42	48
Current	37	23	14
TNM stage				$<$ 0.001
I $+$ II	74	23	51
III $+$ IV	53	42	11
Tumor size				0.065
$\leqslant$ 3 cm	51	21	30
$>$ 3 cm	76	44	32
LN metastasis				0.010
No	61	24	37
Yes	66	41	25
Histology				0.347
Adenocarcinoma	54	28	26
Squamous carcinoma	56	31	25
Other types	17	6	11
Differentiation				s0.168
G1 $+$ G2	72	33	39
G3	55	32	23

Figure 1.

Serum miR-98 expression was significantly decreased in patients with NSCLC.

3. Results

3.1 Down-regulation of serum miR-98 in NSCLC patients and its diagnostic value

qRT-PCR was used to detect the levels of serum miR-98 in 127 NSCLC patients and 60 healthy controls. The results showed that serum miR-98 expression was greatly lower in NSCLC patients than that in healthy control ( $p<$ 0.05, Fig. 1). Furthermore, serum miR-98 levels in low clinical stages (stages I and II) patients were significantly higher compared to those in advance clinical stages (stages III and IV) patients. In addition, serum miR-98 levels in patients with lymph node metastasis were significantly lower than those without metastasis (both $p<$ 0.01, Fig. 2).

fAs shown in Fig. 3, ROC curve analysis revealed that serum miR-98 was an accurate indicator for discriminating NSCLC patients from healthy controls. The area under the curve (AUC) was 0.857, and the sensitivity and specificity were 80.3% and 81.7%, respectively.

Figure 2.

Association between the relative miR-98 expression and TNM stage as well as lymph node metastasis of NSCLC patients.

Table 2

Univariate and multivariate analysis for OS in NSCLC patients by Cox regression model

Variables	Univariate analysis			Multivariate analysis
	HR	95% CI	P	HR	95% CI	P
MiR-98 expression	2.77	1.37–4.18	0.018	3.08	1.42–4.72	0.011
LN metastasis	2.29	1.25–3.28	0.031	1.97	0.98–3.23	0.124
TNM stage	3.12	1.47–4.85	0.006	3.57	1.58–5.52	0.003

Figure 3.

ROC curve analysis of the serum miR-98 to distinguish NSCLC patients from healthy controls.

subSerum miR-98 correlated with clinical characteristics in NSCLC patients

All the 127 NSCLC patients were divided into high miR-98 expression group ( $n=$ 62) and low miR-98 expression group ( $n=$ 65) according to the median expression level of serum miR-98. A significant association was observed between serum miR-98 level and lymph node metastasis ( $p=$ 0.010) as well as TNM stage ( $p<$ 0.001). However, no significant correlation was found between serum miR-98 expression and age, sex, smoking status, tumor size, histology or differentiation (all $p>$ 0.05, Table 2).

3.2 Serum miR-98 correlated with prognosis in NSCLC patients

The Kaplan-Meier method and log-rank test demonstrated that the NSCLC patients in low serum miR-98 expression group had shorter OS than those in high serum miR-98 expression group, suggesting that lower serum miR-98 expression was correlated with worse prognosis of NSCLC ( $p=$ 0.021, Fig. 4A). Also, NSCLC patients in the advanced clinical stage ( $p=$ 0.002, Fig. 4B) or with lymph node metastasis ( $p=$ 0.048, Fig. 4C) suffered worse OS compared with the controls respectively.

Figure 4.

Overall survival analysis of clinical variables in NSCLC patients.

Besides, univariate cox regression analysis found out that serum miR-98 expression (HR $=$ 2.77, 95% CI $=$ 1.37–4.18, $p=$ 0.018), lymph node metastasis (HR $=$ 2.29, 95% CI $=$ 1.25–3.28, $p=$ 0.031) and TNM stage (HR $=$ 3.12, 95% CI $=$ 1.47–4.85, $p=$ 0.006) were closely correlated with OS. Moreover, multivariate Cox regression analysis revealed that serum miR-98 expression (HR $=$ 3.08, 95% CI $=$ 1.42–4.72, $p=$ 0.011) and TNM stage (HR $=$ 3.57, 95% CI $=$ 1.58–5.52, $p=$ 0.003) were independent risk factors for NSCLC patients (Table 2).

4. Discussion

In this study, a significant decrease in serum miR-98 expression was detected in NSCLC patients when compared to healthy controls. Moreover, ROC curve analysis suggested that serum miR-98 could effectively discriminate NSCLC patients from healthy controls. Furthermore, a positive correlation was observed between down-regulated serum miR-98 expression and poor clinical features as well as unfavorable OS. Finally, serum miR-98 expression was confirmed to be an independent prognostic factor for NSCLC. These findings indicated that miR-98 might serve as a tumor suppressor in NSCLC. Consistent with previous studies, Ni et al showed that miR-98 was down-regulated in both NSCLC tissues and cells. Ectopic miR-98 expression suppressed the oncogenic potential of NSCLC cells in vitro and in vivo [17]. Similarly, decreased miR-98 expression markedly stimulated NSCLC cell proliferation and invasion by regulating P21-activated protein kinase 1 [18]. Du et al. found that miR-98 could downregulate the expression of tumor suppressor gene FUS1 in lung cancer cell lines. However, they also showed that the expression level of miR-98 was lower in NSCLC cell line compared with normal bronchial epithelial cell line. In addition, no correlation was found between miR-98 and FUS1 expression levels in the lung cancer specimens [19]. Therefore, these data does not indicate miR-98 plays an oncogenic role in NSCLC.

MiR-98 was also found to exhibit a tumor suppressive role in other types of cancers. For instance, miR-98 was significantly reduced in hepatocellular carcinoma and decreased miR-98 levels were associated with unfavorable clinicopathological parameters. Overexpression of miR-98 inhibited hepatocellular carcinoma cell proliferation, migration, invasion and epithelial-mesenchymal transition through down-regulating SALL4 [20]. In esophageal squamous cell carcinoma (ESCC), reduced miR-98 was associated with various clinical features such as pathological grade, tumor stage and lymph node metastasis. In addition, ectopic expression of miR-98 inhibited the migration and invasion capacity of ESCC cells by targeting EZH2 [21]. MiR-98 levels were decreased in both oral squamous cell carcinoma (OSCC) tissues and cell lines. Moreover, upregulation of miR-98 repressed OSCC cell growth and migration via inversely regulating IGF1R [22]. Liu and colleagues showed that miR-98 level was dramatically down-regulated in salivary adenoid cystic carcinoma tissues. In addition, restoration of miR-98 suppressed cell proliferation, migration and invasion in vitro [23]. Enforced miR-98 expression reduced melanoma cell migration in vitro and suppressed metastatic tumor size in vivo by a negative feedback loop with IL-6. Additionally, increased miR-98 expression reduced tumorigenicity and enhanced survival in part by down-regulating IL-6 levels [24]. Up-regulation of miR-98 strongly inhibited glioma cell migration and invasion through degrading I $\kappa$ B kinase expression, suggesting that miR-98 acted as a tumor suppressor in glioma [14].

Interestingly, miR-98 was found to act as an onco-miRNA in breast cancer. Deng et al. revealed that higher miR-98 levels were observed in breast cancer tissues in comparison with cancer free controls. Furthermore, miR-98 was proved to serve as serum marker for discriminating breast cancer patients from healthy controls, indicating that over-expression of miR-98 contributed to carcinogenesis of breast cancer [25]. One possible reason is that the biological function of miR-98 is tissue specific. Further exploration of the hidden molecular mechanisms and downstream targets accounting for the role of miR-98 in tumorigenesis is required.

In conclusion, this study revealed that serum miR-98 expression was greatly down-regulated in patients with NSCLC. Moreover, downregulated miR-98 level was significantly associated with poor clinical outcome of NSCLC. Therefore, our findings demonstrate that serum miR-98 might serve as a promising biomarker for prognosis prediction of NSCLC.

Conflict of interest

None.

References

Gansler

Ganz

P.A.

Grant

Greene

F.L.

Johnstone

Mahoney

Newman

L.A.

W.K.

Thomas

C.R.

, Jr. Thun

M.J.

Vickers

A.J.

Wender

R.C.

and Brawley

O.W.

, Sixty years of CA: a cancer journal for clinicians, CA Cancer J Clin 60(6) (2010), 345–350.

Siegel

R.L.

Miller

K.D.

and Jemal

, Cancer Statistics, CA Cancer J Clin 65 (2015), 5–29.

Y.L.

and Zhou

, Clinical trials and biomarker research on lung cancer in China, Expert Opin Ther Targets 16 (2012), S45–S50.

Ihde

D.C.

and Minna

J.D.

, Non-small cell lung cancer. part II: treatment, Curr Probl Cancer 15(3) (1991), 105–154.

Rosell

and Karachaliou

, Lung cancer: maintenance therapy and precision medicine in NSCLC, Nat Rev Clin Oncol 10(10) (2013), 549–550.

Bartel

D.P.

, MicroRNAs: genomics, biogenesis, mechanism, and function, Cell 116(2) (2004), 281–297.

Di Leva

Briskin

and Croce

C.M.

, MicroRNA in cancer: new hopes for antineoplastic chemotherapy, Ups J Med Sci 117(2) (2012), 202–216.

Schratt

G.M.

Tuebing

Nigh

E.A.

Kane

C.G.

Sabatini

M.E.

Kiebler

and Greenberg

M.E.

, A brain-specific microRNA regulates dendritic spine development, Nature 439(7074) (2006), 283–289.

Qian

Nag

S.A.

Voruganti

Qin

J.J.

Zhang

and Cho

W.C.

, MiRNAs in cancer prevention and treatment and as molecular targets for natural product anticancer agents, Curr Cancer Drug Targets 13(5) (2013), 519–541.

10.

Tang

Liang

Luo

Feng

Huang

Gan

Yang

and Chen

, Downregulation of miR-30a is associated with poor prognosis in lung cancer, Med Sci Monit 21 (2015), 2514–2520.

11.

Lei

Huang

and Gong

, Inhibition of miR-92b suppressed non small cell lung cancer cells growth and motility by targeting RECK, Mol Cell Biochem 387(1–2) (2014), 171–176.

12.

Rizos

Siafakas

Katsantoni

Skourti

Salpeas

Rizos

Tsoporis

J.N.

Kastania

Filippopoulou

Xiros

Margaritis

Parker

T.G.

Papageorgiou

and Zoumpourlis

, Let-7, mir-98 and mir-183 as biomarkers for cancer and schizophrenia, PLoS One 10(4) (2015), e0123522.

13.

Wendler

Keller

Albrecht

Peluso

J.J.

and Wehling

, Involvement of let-7/miR-98 microRNAs in the regulation of progesterone receptor membrane component 1 expression in ovarian cancer cells, Oncol Rep 25(1) (2011), 273–279.

14.

Fan

Y.H.

M.H.

S.G.

M.J.

Xiao

Liao

C.C.

Q.K.

Chai

and Zhu

X.G.

, Overexpression of miR-98 inhibits cell invasion in glioma cell lines via downregulation of IKKε, Eur Rev Med Pharmacol Sci 19(19) (2015), 3593–3604.

15.

Ting

H.J.

Messing

Yasmin-Karim

and Lee

Y.F.

, Identification of microRNA-98 as a therapeutic target inhibiting prostate cancer growth and a biomarker induced by vitamin D, J Biol Chem 288(1) (2013), 1–9.

16.

Hebert

Norris

Scheper

M.A.

Nikitakis

and Sauk

J.J.

, High mobility group A2 is a target for miRNA-98 in head and neck squamous cell carcinoma, Mol Cancer 6 (2007), 5.

17.

Huang

and Wang

, MiR-98 targets ITGB3 to inhibit proliferation, migration, and invasion of non-small-cell lung cancer, Onco Targets Ther 8 (2015), 2689–2697.

18.

Yang

Zhang

and Shi

, MiR-98 inhibits cell proliferation and invasion of non-small cell carcinoma lung cancer by targeting PAK1, Int J Clin Exp Med 8(11) (2015), 20135–20145.

19.

Schageman

J.J.

Subauste

M.C.

Saber

Hammond

S.M.

et al., miR-93, miR-98, and miR-197 regulate expression of tumor suppressor gene FUS1, Mol Cancer Res 7 (2009), 1234–1243.

20.

Zhou

Zou

Liu

Cui

Gao

Yuan

and Cong

, MicroRNA-98 acts as a tumor suppressor in hepatocellular carcinoma via targeting SALL4, Oncotarget 7(45) (2016), 74059–74073.

21.

Huang

S.D.

Yuan

Zhuang

C.W.

B.L.

Gong

D.J.

Wang

S.G.

Zeng

Z.Y.

and Cheng

H.Z.

, MicroRNA-98 and microRNA-214 post-transcriptionally regulate enhancer of zeste homolog 2 and inhibit migration and invasion in human esophageal squamous cell carcinoma, Mol Cancer 11 (2012), 51.

22.

Zhou

Sun

and Wang

, MiR-98 suppresses tumor cell growth and metastasis by targeting IGF1R in oral squamous cell carcinoma, Int J Clin Exp Pathol 8(10) (2015), 12252–12259.

23.

Liu

Zhang

Guo

Yue

and Zhuo

, MiR-98 functions as a tumor suppressor in salivary adenoid cystic carcinomas, Onco Targets Ther 9 (2016), 1777–1786.

24.

X.J.

Qiao

Shi

Liu

Dang

Y.P.

W.J.

Wang

X.G.

and Liu

, MiR-98 suppresses melanoma metastasis through a negative feedback loop with its target gene IL-6, Exp Mol Med 46 (2014), e116.

25.

Deng

Z.Q.

Yin

J.Y.

Tang

Liu

F.Q.

Qian

Lin

Shao

Zhang

and He

, Over-expression of miR-98 in FFPE tissues might serve as a valuable source for biomarker discovery in breast cancer patients, Int J Clin Exp Pathol 7(3) (2014), 1166–1171.

Low serum miR-98 as an unfavorable prognostic biomarker in patients with non-small cell lung cancer

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Materials and methods

2.1 Patients and sample collection

2.2 Total RNA isolation and quantitative RT-PCR

2.3 Statistical analysis

Table 1 Association between serum miR-98 expression and clinicopathological characteristics of NSCLC patients

3.1 Down-regulation of serum miR-98 in NSCLC patients and its diagnostic value

Conflict of interest

References

Table 1
Association between serum miR-98 expression and clinicopathological characteristics of NSCLC patients