Sage Journals: Discover world-class research

Abstract

BACKGROUND:

Circulating microRNAs (miRNAs) are emerging as novel biomarkers for various types of cancer including pancreatic cancer (PC).

OBJECTIVE:

We aimed to explore the diagnostic and prognostic significance of serum miR-373 in PC.

METHODS:

In the current study, we recruited a total of 103 PC patients, 30 patients with benign pancreatic tumor, 20 patients with chronic pancreatitis and 50 healthy volunteers. Total RNA was isolated from all the blood samples, and relative miR-373 expression levels were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

RESULTS:

Our findings demonstrated that serum miR-373 expression was greatly down-regulated in PC patients. The area under the receiver-operating characteristic (ROC) curve (AUC) for serum miR-373 was 0.852 for discriminating PC patients from normal control subjects. In addition, a positive correlation was observed between reduced serum miR-373 level and several clinical parameters, including TNM stage, lymph node metastasis and distant metastasis. Moreover, PC patients with lower serum miR-373 level had shorter 5 year overall survival. Finally, serum miR-373 was proved to be an independent predictor for PC.

CONCLUSIONS:

Taken together, serum miR-373 might serve as a promising biomarker for the early detection and prognosis prediction of PC.

Keywords

Pancreatic cancer serum miR-373 diagnosis prognosis biomarker

Table 1
Correlation between serum miR-373 expression with clinical characteristics in 103 PC patients

Variables Patients ( $n=$ 103) MiR-373 levels $P$ -value

High ( $n=$ 50) Low ( $n=$ 53)

Age 0.4029

$\leqslant$ 65 35 19 16

$>$ 65 68 31 37

Gender 0.3983

Men 62 28 34

Women 41 22 19

Location 0.3450

Pancreas head 76 39 37

Pancreas body/tail 27 11 16

Distant metastasis 0.0182*

Negative 78 43 35

Positive 25 7 18

Lymph node metastasis 0.0394*

Negative 39 24 15

Positive 64 26 38

CA19-9 0.4724

$<$ 37 U/mL 47 21 26

$\geqslant$ 37 U/mL 56 29 27

CEA 0.4027

$<$ 4.3 $\mu$ g/ml 33 18 15

$\geqslant$ 4.3 $\mu$ g/ml 70 32 38

TNM stage 0.0017**

I–IIA 34 24 10

IIB–IV 69 26 43

Variables	Patients ( $n=$ 103)	MiR-373 levels	$P$ -value
		High ( $n=$ 50)	Low ( $n=$ 53)
Age				0.4029
$\leqslant$ 65	35	19	16
$>$ 65	68	31	37
Gender				0.3983
Men	62	28	34
Women	41	22	19
Location				0.3450
Pancreas head	76	39	37
Pancreas body/tail	27	11	16
Distant metastasis				0.0182*
Negative	78	43	35
Positive	25	7	18
Lymph node metastasis				0.0394*
Negative	39	24	15
Positive	64	26	38
CA19-9				0.4724
$<$ 37 U/mL	47	21	26
$\geqslant$ 37 U/mL	56	29	27
CEA				0.4027
$<$ 4.3 $\mu$ g/ml	33	18	15
$\geqslant$ 4.3 $\mu$ g/ml	70	32	38
TNM stage				0.0017**
I–IIA	34	24	10
IIB–IV	69	26	43

1. Introduction

Pancreatic cancer (PC) is one of the most common aggressive malignancies and the fourth cause of cancer related death around the world. In China, the incidence and mortality rates of PC have steadily increased from 2000–2011, making PC a pubic health problem [1, 2]. Although medical and surgical managements for PC have been greatly improved over the past decades, the 5-year survival rate of PC remains dismal. Most PC patients are diagnosed at advanced clinical stage as they usually have no symptoms until the cancer has already spread to distant organs [3, 4, 5]. Therefore, it is extremely urgent to identify effective biomarkers for early diagnosis and prognosis prediction of this fatal disease.

MicroRNAs (miRNAs) are a class of small (19–25 nucleotides in length), non-coding RNAs which regulate gene expression by binding to the 3’-untranslated region (3’-UTR) of target messenger RNA (mRNA), inducing mRNA decay or translational repression [6, 7]. Altered miRNAs expression has been found to play crucial roles in the initiation and progression of PC. For example, overexpression of miR-744 was associated with poor survival of PC patients. Moreover, inhibition of miR-744 repressed the stem cell-like phenotype of cancer cells in vitro and the tumorigenesis in vivo, indicating that miR-744 played an oncogenetic role in PC [8]. In contrast, Yu et al. showed that miR-127 expression was significantly down-regulated in both PC cell lines and tumors. In addition, enhanced miR-127 expression suppressed the tumorigenicity of cancer cells both in vitro and in vivo by targeting Bcl-2-associated athanogene 5 [9].

Recently a growing number of circulating miRNAs have been reported to used as potential biomarkers for diagnosis of PC [10, 11, 12]. In this study, we focused on microRNA-373 (miR-373), which was found to be involved in the carcinogenesis of various tumor types, including PC [15, 16, 17]. However, the serum miR-373 expression pattern and its potential clinical value in PC have not yet been elucidated. Thus, this study aimed to investigate the diagnostic and prognostic value of serum miR-373 in PC.

2. Materials and methods

2.1 Study population and sample collection

A total of 103 PC patients, twenty patients with chronic pancreatitis and thirty patients with benign pancreatic tumor were enrolled from Department of Hepatobiliary Surgery, the General Hospital of Ningxia Medical University. Tumor stages were determined according to the Union for International Cancer Control classification. The PC patients’ features were list in Table 1. Fifty healthy volunteers, who were proved not to have any pancreatic disease or other cancerous disease, were recruited as controls.

Fasting venous blood (6 mL) was taken from each participant before surgery. All the blood samples were centrifuged at 3500 rpm for 10 min within one hour after collection. Then, the separated supernatant was divided into aliquots and stored at $-$ 80 ${}^{\circ}$ C until further processing.

2.2 RNA isolation and RT-qPCR

Total RNA was isolated from serum samples using a mirVana PARIS kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. Reverse transcription was performed using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, San Diego, CA). PCR reaction for quantification of miR-373 was carried out in duplicate using Maxima SYBR Green qPCR Kit (Thermo Scientific, CA, USA), and processed using an Applied Biosystems 7000 Sequence Detection System (Applied Biosystems, Foster City, USA). Relative miR-373 expression in serum was normalized with respect to RNU6, and calculated using the 2 ${}^{-\Delta\Delta\text{Ct}}$ method.

2.3 Statistical analysis

All statistical analyses were performed by using GraphPad Prism 6.02 (GraphPad Software Inc., San Diego, California, USA) and SPSS (version 15.0, SPSS Inc., Chicago, IL, USA) software. The significance of serum miR-373 expression was determined by one way ANOVA. Categorical variables were compared using Chi-square test. Overall survival (OS) curves were constructed using the Kaplan-Meier method plus log-rank tests. Cox’s proportional hazard regression test was performed to evaluate multivariate hazard ratios for prognosis. Receiver operating characteristic (ROC) curve was generated, and the area under the ROC curve (AUC) was computed to assess the diagnostic value of serum miR-373 for PC. Differences were considered significant when $P$ -values $<$ 0.05.

Figure 1.

Serum miR-373 expression was significantly down-regulated in PC patients.

Figure 2.

Patients with lower miR-373 level experienced more frequent distant metastasis and lymph node metastasis.

Figure 3.

High miR-373 expression was closely correlated with lower TNM stage.

2.4 Ethics statement

This study were approved by Fudan University Shanghai Cancer Center and the General Hospital of Ningxia Medical University, and written informed consent was provided by each participant. All serum samples were handled and made anonymous according to the ethical and legal standards.

3. Results

3.1 Expression level and ROC curve of serum miR-373 in PC patients

RT-qPCR was used to detect relative serum miR-373 levels in all the participants. Our results showed that miR-373 expression was dramatically down-regulated in PC patients (all $p<$ 0.01, Fig. 1). As shown in Fig. 2, a significant decrease in serum miR-373 level was observed in patients ( $n=$ 25) with distant metastasis compared to those ( $n=$ 78) without metastasis ( $p<$ 0.05). Moreover, serum miR-373 expression in patients ( $n=$ 64) with lymph node metastasis was significantly lower than those ( $n=$ 39) without metastasis ( $p<$ 0.05). Furthermore, serum miR-373 level in stage I–IIA patients ( $n=$ 34) was higher than in stage IIB–IV patients ( $n=$ 69, $p<$ 0.01, Fig. 3).

Table 2
Multivariate analysis for overall survival of PC patients using the Cox proportional hazard model

Variables Hazard risk 95% CI $P$ value

Lymph node metastasis Positive vs Negative 3.45 1.24–5.68 0.046

Distant metastasis Positive vs Negative 4.84 1.82–7.92 0.029

TNM stage IIB–IV vs I–IIA 5.46 2.53–8.69 0.005

Serum miR-373 Low vs High 5.21 2.12–8.42 0.014

Variables		Hazard risk	95% CI	$P$ value
Lymph node metastasis	Positive vs Negative	3.45	1.24–5.68	0.046
Distant metastasis	Positive vs Negative	4.84	1.82–7.92	0.029
TNM stage	IIB–IV vs I–IIA	5.46	2.53–8.69	0.005
Serum miR-373	Low vs High	5.21	2.12–8.42	0.014

Next, ROC curve was used to determine the potential significance of serum miR-373 for the early diagnosis of PC. The results revealed that serum miR-373 level could differentiate PC patients from healthy controls with an AUC value of 0.852, and the sensitivity and specificity were 80.6% and 84.3%, respectively (Fig. 4).

Figure 4.

Serum miR-373 yielded AUC value of 0.852 with 80.6% sensitivity and 84.3% specificity in distinguishing PC patients from normal subjects.

3.2 Correlation between serum miR-373 expression and clinical factors in PC patients

As shown in Table 1, all 103 PC patients were classified into two groups according to the median value of serum miR-373 level. The high serum miR-373 expression group had 50 cases while the low expression group had 53 people. Low serum miR-373 expression was remarkably correlated with lymph node metastasis ( $p=$ 0.0394), distant metastasis ( $p=$ 0.0182), TNM stage ( $p=$ 0.0017) in PC patients. However, it was not associated with age ( $p=$ 0.4029), gender ( $p=$ 0.3983), location ( $p=$ 0.3450), carbohydrate antigen 19-9 (CA19-9, $p=$ 0.4724), and carcinoembryonic antigen (CEA, $p=$ 0.4027).

3.3 Potential utility of serum miR-373 as a prognostic factor for patients with PC

In 103 PC patients, 72 underwent curative surgical resection. The Kaplan-Meier analysis showed that patients in low serum miR-373 expression group suffered worse OS than those in high expression group ( $p=$ 0.034, Fig. 5(A)). For seventy-two PC patients with pancreatectomy, high serum miR-373 level was associated with longer OS ( $p=$ 0.013, Fig. 5(B)).

Figure 5.

A. Overall survival curve of all PC patients, low miR-373 expression patients had worse prognosis. B. Overall survival curve of PC patients with pancreatectomy, low miR-373 expression patients had worse prognosis.

Subsequently, multivariate analysis for overall survival was performed to evaluate the influence of serum miR-373 level and clinicopathological variables. The data revealed that distant metastasis (hazard risk $=$ 4.84, 95% CI $=$ 1.82–7.92, $p=$ 0.029), serum miR-373 level (hazard risk $=$ 5.21, 95% CI $=$ 2.12–8.42, $p=$ 0.014), lymph node metastasis (hazard risk $=$ 3.45, 95% CI $=$ 1.24–5.68, $p=$ 0.046) and TNM stage (hazard risk $=$ 5.46, 95% CI $=$ 2.53–8.69, $p=$ 0.005) were independent prognostic indicators for PC (Table 2).

4. Discussion

In the present study, we found that serum miR-373 level was significantly decreased in PC patients. Additionally, a positive association was observed between down-regulation of serum miR-373 and aggressive clinical features. ROC curve analysis revealed that serum miR-373 could help discriminate PC patients from normal subjects. Also, PC patients with lower miR-373 expression had shorter OS. Finally, serum miR-373 expression was confirmed to be significant prognostic factor for PC. Collectively, these findings indicated that miR-373 might exhibit a tumor suppressive role in PC.

Previous study had reported the tumor suppressive role of miR-373 expression in PC. They showed that miR-373 was markedly under-expressed in cancer cell lines, formalin-fixed paraffin-embedded and microdissected frozen cancer tissues. Moreover, reexpression of miR-373 greatly restrained invasiveness of cancer cells in vitro and repressed peritoneal dissemination in vivo [14]. Besides PC, miR-373 functioning as a tumor suppressor was widely reported in various types of cancer. For instance, Wei et al. showed that enhanced miR-373 expression inhibited the migration and invasion capability of glioma cells via inversely regulating CD44 and TGFBR2 [15]. In T cell lymphoma, miR-373 was dramatically down-regulated in cancer tissue compared to adjacent normal tissue. Moreover, inhibition of miR-373 promoted cell growth by up-regulating CCND1 [16].

Interestingly, miR-373 was demonstrated to act as an oncogene in many other tumors. In hepatocellular carcinoma, Wu and colleagues showed that increased miR-373 expression was found in cancer tissues, and miR-373 overexpression stimulated cancer cell growth activity in vitro. Moreover, the protein phosphatase 6 catalytic subunit was identified as the downstream target [17]. In gastric cancer, miR-373 expression was elevated in both cancer cell lines and tissues. Furthermore, down-regulation of miR-373 inhibited cancer cell proliferation by regulating tumor necrosis factor, $\alpha$ -induced protein 1 [18]. Similarly, Wang et al. revealed that miR-373 was up-regulated in both cervical cancer tissues and cell lines. Decreased miR-373 expression suppressed cancer cell growth, colony formation in vitro and inhibited tumorigenicity in vivo through targeting YOD1 [19]. In oral squamous cell carcinoma, miR-373 overexpression was observed in cancer tissues compared to the normal mucosa. Moreover, upregulation of miR-373 expression was associated with unfavorable clinical parameters and poor survival [20]. In esophageal squamous cell carcinoma, Liu et al. found that miR-373 was over-expressed both in cancer tissues and the plasma of patients. Reduced miR-373 level suppressed cancer cell proliferation, migration, invasion and induced cell apoptosis [21]. These reports reflect that miR-373 has diverse functions in the tumorigenesis of different cancers.

In summary, our findings clearly demonstrated that serum miR-373 was significantly decreased in PC patients. Furthermore, low serum miR-373 expression was closely associated with poorer clinical outcome of PC patients. Therefore, the results indicated that serum miR-373 might serve as a promising diagnostic and prognostic marker in PC.

Footnotes

Acknowledgments

This study was supported by the Grants from the National Natural Science Foundations of China (Grant No. 81560474), Ningxia Natural Science foundation (NZ15281) and the Medical Guide Project of the Shanghai Municipal Commission for Science and Technology (15401932400).

Conflict of interest

We deny any conflict of interest.

References

Michaud

D.S.

, Epidemiology of pancreatic cancer, Minerva Chir 59 (2004), 99–111.

Chen

Zheng

Baade

P.D.

Zhang

Zeng

Bray

Jemal

X.Q.

and He

, Cancer statistics in China, 2015, CA Cancer J Clin 66 (2016), 115–132.

Sultana

Tudur Smith

Cunningham

Starling

Neoptolemos

J.P.

and Ghaneh

, Meta-analyses of chemotherapy for locally advanced and metastatic pancreatic cancer: results of secondary end points analyses, Br J Cancer 99 (2008), 6–13.

Jemal

Siegel

Ward

Hao

and Thun

M.J.

, Cancer statistics, 2009, CA Cancer J Clin 59 (2009), 225–249.

Heinemann

Boeck

Hinke

Labianca

and Louvet

, Meta-analysis of randomized trials: evaluation of benefit from gemcitabine-based combination chemotherapy applied in advanced pancreatic cancer, BMC Cancer 8 (2008), 82.

Lai

E.C.

, Micro RNAs are complementary to 3’ UTR sequence motifs that mediate negative post-transcriptional regulation, Nat Genet 30 (2002), 363–364.

Hanahan

and Weinberg

R.A.

, Hallmarks of cancer: the next generation, Cell 144 (2011), 646–674.

Zhou

Gou

Xiong

Wang

Yan

and Liu

, MiR-744 increases tumorigenicity of pancreatic cancer by activating Wnt/β-catenin pathway, Oncotarget 6 (2015), 37557–37569.

Liu

Gong

and Wang

, MicroRNA-127 is aberrantly downregulated and acted as a functional tumor suppressor in human pancreatic cancer, Tumour Biol 37 (2016), 14249–14257.

10.

Hussein

N.A.

Kholy

Z.A.

Anwar

M.M.

Ahmad

M.A.

and Ahmad

S.M.

, Plasma miR-22-3p, miR-642b-3p and miR-885-5p as diagnostic biomarkers for pancreatic cancer, J Cancer Res Clin Oncol 143 (2017), 83–93.

11.

Deng

Yuan

Zhang

Yao

Wang

Liu

and Ba

, Identification of circulating miR-25 as a potential biomarker for pancreatic cancer diagnosis, Cell Physiol Biochem 39 (2016), 1716–1722.

12.

Liu

Chen

Yao

Shen

Wang

Zhuang

Ning

Zhang

Yuan

Zen

and Zhang

C.Y.

, Serum microRNA expression profile as a biomarker in the diagnosis and prognosis of pancreatic cancer, Clin Chem 58 (2012), 610–618.

13.

Place

R.F.

L.C.

Pookot

Noonan

E.J.

and Dahiya

, MicroRNA-373 induces expression of genes with complementary promoter sequences, Proc Natl Acad Sci USA 105 (2008), 1608–1613.

14.

Nakata

Ohuchida

Mizumoto

Aishima

Oda

Nagai

and Tanaka

, Micro RNA-373 is down-regulated in pancreatic cancer and inhibits cancer cell invasion, Ann Surg Oncol 21(Suppl 4) (2014), S564–574.

15.

Wei

Wang

Huang

Luan

and Wang

, miR-373 inhibits glioma cell U251 migration and invasion by down-regulating CD44 and TGFBR2, Cell Mol Neurobiol 36 (2016), 1389–1397.

16.

Tian

Y.Y.

Jia

C.M.

Wang

Jiang

and Liu

A.C.

, Restoration of microRNA-373 suppresses growth of human T-cell lymphoma cells by repressing CCND1, Eur Rev Med Pharmacol Sci 20 (2016), 4435–4444.

17.

Liu

Fan

Liu

Zhong

and Tang

, MicroRNA-373, a new regulator of protein phosphatase 6, functions as an oncogene in hepatocellular carcinoma, FEBS J 278 (2011), 2044–2054.

18.

Zhang

Tan

Liu

Yang

Ding

Zhou

Xiang

Zhou

and Zhang

, MicroRNA-373 is upregulated and targets TNFAIP1 in human gastric cancer, contributing to tumorigenesis, Oncol Lett 6 (2013), 1427–1434.

19.

Wang

L.Q.

Zhang

Yan

Liu

K.J.

and Zhang

, MicroRNA-373 functions as an oncogene and targets YOD1 gene in cervical cancer, Biochem Biophys Res Commun 459 (2015), 515–520.

20.

H.F.

Chang

K.W.

Cheng

H.W.

and Liu

C.J.

, Upregulation of miR-372 and -373 associates with lymph node metastasis and poor prognosis of oral carcinomas, Laryngoscope 125 (2015), E365–370.

21.

Liu

Chen

Zhang

Wei

Zhang

Wang

Meng

Zhu

and Li

, MicroRNA-373 promotes migration and invasion in human esophageal squamous cell carcinoma by inhibiting TIMP3 expression, Am J Cancer Res 6 (2015), 1–14.

Low serum miR-373 predicts poor prognosis in patients with pancreatic cancer

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

2. Materials and methods

2.1 Study population and sample collection

2.2 RNA isolation and RT-qPCR

2.3 Statistical analysis

3. Results

3.1 Expression level and ROC curve of serum miR-373 in PC patients

3.3 Potential utility of serum miR-373 as a prognostic factor for patients with PC

Footnotes

Acknowledgments

Conflict of interest

References