Down-regulation of MiR-539 Indicates Poor Prognosis in Patients with Pancreatic Cancer.

It has been demonstrated that miR-539 plays an important role in the development and progression of tumors. The purpose of this study was to analyze the correlation between the expression level of miR-539 and the clinicopathological features and prognosis of patients with pancreatic cancer. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze the expression level of miR-539 in 60 patients with pancreatic cancer. It was found that miR-539 gene expression was down-regulated in pancreatic cancer compared with that in paracancerous tissues. In addition, the expression level of miR-539 was inversely correlated with tumor differentiation (poorly to moderately differentiated vs. well differentiated, P=0.006), lymph node metastasis (positive vs. negative, P=0.006), clinical stage (III-IV vs. I-II, P=0.002), CA199 (≥200 vs. <200, P=0.019) and distant metastasis (positive vs. negative, P=0.035). The survival time of pancreatic cancer patients with low expression of miR-539 was significantly shorter than that of patients with high expression of miR-539. Multivariate analysis suggested that miR-539 expression level was an independent prognostic indicator for patients with pancreatic cancer (P=0.025). Down-regulation of miR-539 may be a potentially unfavorable prognostic factor for patients with pancreatic cancer, and further studies are needed to confirm our conclusion in the future.

Introduction

Pancreatic cancer (PC) is a highly malignant gastrointestinal cancer. In recent years, although a variety of new methods have been applied to the diagnosis and treatment of pancreatic cancer, the treatment of pancreatic cancer is still unsatisfactory and the prognosis is extremely poor. In the United States, pancreatic cancer ranks the fourth among the mortality rate of malignant tumors in males or females in 2014. From 2006 to 2012, the five-year survival rate of pancreatic cancer patients is only 8%, of which 52% of patients have distant metastases and their five-year survival rate is only 3% [1]. According to the statistics in China, the incidence and mortality of pancreatic cancer had an upward trend in males from 2000 to 2011 [2]. Surgical resection is the preferred method for radical treatment of pancreatic cancer. However, most patients are already in the advanced stage at the time of their first visit due to the late onset of clinical symptoms, early local invasion and high metastatic potential, and less than 20% of patients can undergo surgery. For patients who cannot receive surgical therapy, interventions and routine cytotoxic chemotherapy are of limited benefit [3, 4]. Pancreatic cancer has become a major disease to be overcome in the 21st century, and therefore, it is extremely important to find a new treatment method. Previous studies have shown that new biomarkers have great potential in the diagnosis and prognosis of pancreatic cancer, such as miR-185 [5], miR-107 [6], miR-217 [7], DNA topoisomerase II alpha (TOP2A) [8], coiled-coil domain containing 34 (CCDC34) [9], and miR-183 [10], etc. They play a key role in the proliferation, infiltration or metastasis of pancreatic cancer. Therefore, it is of crucial importance to find more novel biomarkers of pancreatic cancer.

MicroRNA is a class of single-stranded non-coding RNA molecules composed of 19-25 nucleotides, which has become one of the hotspots in the current tumor study regarding the mechanism of tumorigenesis and development. It has been shown that there are some abnormally expressed miRNAs in pancreatic cancer, which mostly target the genes and signaling pathways related to the development and progression of pancreatic cancer, thereby regulating the pathogenesis of pancreatic cancer in a wide variety of manners [11]. For example, Radha Krishnan et al. demonstrated that miR-200c inhibits the proliferation and metastasis of pancreatic cancer cells by inhibiting the expression of MUC4 and MUC16 [12]; Pham et al. found that miR-143 inhibits the proliferation and metastasis of pancreatic cancer by inhibiting cyclooxygenase 2 and prostaglandin E2 [13]. Wang et al. found that the silencing of miR-124 expression mediated by methylation promotes the progression and metastasis of pancreatic cancer [14]. It has been demonstrated that the abnormal expression of miRNA is closely related to the clinicopathological features (type, stage and grade) and prognosis of pancreatic cancer, suggesting that miRNA may be used not only for the diagnosis and individualized treatment of pancreatic cancer, but also as a tool for the prediction of the prognosis of the disease. Giovannetti et al. found that miR-211 may serve as a prognostic factor for pancreatic cancer after resection, and the down-regulation of miR-211 expression is an independent factor of poor prognosis [15]. Therefore, it is expected that the identification and characterization of the miRNAs and their targets that play an important role in the progression of pancreatic cancer may provide a new approach for the treatment of pancreatic cancer. Recently, miR-539 has been found to be up-regulated in failing heart tissue and could inhibit O-GlcNAcase expression [16]. MiR-539 suppresses the proliferation, invasion and migration of osteosarcoma, prostate cancer and colorectal cancer [17, 18, 19], however, the effects of miR-539 on the pancreatic cancer have not been explored.

In the present study, we first determined the expression level of miR-539 in pancreatic and paracancerous tissues. The relationship between miR-539 expression and the clinicopathological features and prognosis of patients with pancreatic cancer was then further analyzed with the purpose of elucidating the role of miR-539 in the pathogenesis of pancreatic cancer. Our study will contribute to a better understanding of the pathogenesis of pancreatic cancer.

Materials and Methods

Clinical samples

In this study, 60 pairs of pancreatic and paracancerous tissues were enrolled, which were collected from patients who underwent pancreatic cancer resection at Wenzhou Central Hospital and Nanjing General Hospital from January 2011 to February 2016. All samples were snapfrozen immediately in liquid nitrogen and stored at -80 °C until further molecular analysis. In these 60 cases of pancreatic cancer, there were 37 males and 23 females, and the median age was 63.72 years (42-76 years). None of the patients received preoperative chemotherapy or radiotherapy, and the clinical follow-up time was 6 to 33 months. All the patients signed the subject informed consent form for the use of the clinical samples and this study has been approved by the ethics committees of Wenzhou Central Hospital and Nanjing General Hospital. The histopathological diagnosis of all samples was confirmed independently by two experienced pathologists. The clinicopathological features of the patients are summarized in the Table 1.

Table 1

Correlation between the clinicopathologic characteristics and expression of miR-539 in PC

Parameters	Number of cases	miR-539 expression		Chi2	P value
		Low	High
Age (years)
≥60	41	17	24	0.116	0.734
<60	19	7	12
Gender
Male	37	16	21	0.423	0.515
Female	23	8	15
Differentiated degree
Low and middle	15	11	4	7.500	0.006
High	45	13	32
Lymph node metastasis
+	13	10	3	7.565	0.006
_	47	14	33
Clinical stage
I-II	44	12	32	9.237	0.002
III-IV	16	12	4
CA199
<200	22	4	18	5.529	0.019
≥200	38	20	18
Distant metastasis
+	7	6	1	3.403	0.035
_	53	19	34
Tumor size (cm)
≥2.5	20	16	4	4.441	0.208
<2.5	40	24	16

Informed consent: Informed consent has been obtained from all individuals included in this study

Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the Helsinki Declaration, and has been approved by the ethics committees of Wenzhou Central Hospital and Nanjing General Hospital.

RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from tissues stored at -80°C using RNAiso Plus (Takara, Japan), which was then reverse transcribed using the OneStep PrimeScript miRNA cDNA Synthesis Kit according to the manufacturer’s protocol. For qRT-PCR of miR-539, U6 small nuclear RNA (snRNA) was used as an internal control and the forward primers for miR-539 and U6 were 5’-GGAGAAATTATCCTTGGTGTGT-3’ and 5’-CTCGCTTCGGCAGCACATA-3’, respectively. Uni-miR qPCR primer, which was included in the kit, was used as the reverse primer according to manufacturer’s instructions. The PCR profile was 30 seconds at 95°C, followed by 40 cycles at 95°C for 5 seconds and 60°C for 20 seconds. The 2−ΔΔCt method was used for the relative quantitation of miRNA expression and the U6 snRNA was selected as the endogenous reference for normalization. All qRT-PCR reactions were performed in triplicate.

Statistical analysis

Statistical analysis was performed using SPSS 19.0 software. The paired t-test was used to compare the levels of miRNA

in pancreatic and paracancerous tissues, and the Chi-square test was used to analyze the correlation between clinical features and miR-539 expression levels. The Kaplan-Meier method and log-rank test were used to analyze the correlation between miR-539 expression levels and survival of patients with pancreatic cancer. The Cox survival analysis was then used to analyze the hazard ratio (HR). A P value less than 0.05 was considered statistically significant.

Results

Comparison of the expression level of miR-539 in pancreatic cancer tissues and normal adjacent tissues

QRT-PCR showed that the expression level of miR-539 was significantly lower in pancreatic cancer tissues than that in the adjacent normal tissues in the 60 pairs of the paracancerous normal tissues and pancreatic cancer tissues (0.65 ± 0.02 vs. 1.06 ± 0.03, P<0.001) (Figure. 1).

Figure 1

The expression level of miR-539 in pancreatic cancer and matched normal tissue samples.

Relationship between miR-539 expression and clinicopathological features of pancreatic cancer

The association of miR-539 expression with clinicopathological features in patients with pancreatic cancer is summarized in Table 1. It was found that the expression level of miR-539 was inversely correlated with tumor differentiation (poorly to moderately differentiated vs. well differentiated, P=0.006), lymph node metastasis (positive vs. negative, P=0.006), clinical stage (III-IV vs. I-II, P=0.002), CA199 (≥200 vs. <200, P=0.019) and distant metastasis (positive vs. negative, P=0.035). However, no significant correlation was found the expression of miR-539 and the patient’s age (P=0.734), gender (P=0.515), and tumor size (P=0.208) in 60 cases pancreatic cancer.

Relationship between miR-539 expression and survival of patients with pancreatic cancer

The Kaplan-Meier analysis and log-rank test were employed to analyze the correlation between the patient’s survival and the miR-539 expression level and it was found that there was a significant association between miR-539 expression and the overall survival (OS) in patients with pancreatic cancer (figure 2). The median OS value of pancreatic cancer patients with low expression of miR-539 was 10.89 months, which was significantly lower than that of patients with high expression of miR-539 (20.48 months) (Table 2). In addition, other clinicopathological features, including cell differentiation, clinical stage, lymph node metastasis, CA199 level, distant metastasis, and tumor size were also significantly associated with the patient’s survival. To determine further whether miR-539 was an independent prognostic factor for pancreatic cancer, a multivariate analysis of miR-539 expression was carried out using the same set of parameters and the results showed that miR-539 expression level was an independent prognostic factor for the OS of patients (HR (95% CI) 1.728 (1.225-2.712), P=0.025) (Table 3).

Figure 2

Kaplan–Meier survival analysis of overall survival time in 60 PC patients according to miR-539 expression.

Table 2

Analyses of factors regarding overall survival.

Parameters	Number of cases	Median overall survival	Log-rank test chi2 value	P value
Age (years)
≥60	41	19.24	0.457	0.613
＜60	19	19.84
Gender
Male	37	21.89	1.213	0.092
Female	23	17.09
Differentiated degree
Low and middle	15	10.60	20.751	0.001
High	45	23.20
Lymph node metastasis
+	13	10.15	22.761	0.001
_	47	22.79
Clinical stage
I-II	44	23.45	23.332	0.001
III-IV	16	10.69
CA199
<200	22	22.92	9.233	0.029
≥200	38	15.09
Distant metastasis
+	7	7.00	33.161	0.000
_	53	21.50
Tumor size (cm)
≥2.5	20	12.55	18.965	0.002
<2.5	40	23.8
Expression of miR-539
High expression	33	20.48	21.262	0.001
Low expression	27	10.89

Table 3

Summary of univariate and multivariate analyses of overall survival time.

Parameters	Univariate analysis
	HR (95% CI)	P value
Univariate analysis
Age (≥60 vs. <60)	0.875(0.478-1.587)	0.896
Gender(Male vs. Female)	0.455(0.322-0.982)	0.103
Differentiated degree (Low and middle vs. High)	2.219(1.256-3.528)	0.004*
Lymph node metastasis (yes vs. no)	2.012(1.134-3.236)	0.004*
Clinical stage (III-IV vs I-II.)	0.667(0.433-1.521)	0.001*
CA199 (≥200 vs. <200)	2.113(1.421-3.489)	0.023*
Distant metastasis (yes vs. no)	0.489(0.322-1.132)	0.038*
Tumor size (cm) ( ≥2.5 vs.<2.5 )	0.672(0.467-1.871)	0.036*
Mir-539 (low vs. High expression)	2.159(1.025-3.546)	0.003*
Multivariate analysis
Differentiated degree (Low and middle vs. High)	1.342(0.434-1.934)	0.025*
Lymph node metastasis (yes vs. no)	1.524(0.834-2.216)	0.009*
Clinical stage (III-IV vs I-II.)	0.443(0.133-0.929)	0.021*
miR-539 (low vs. High expression)	1.728(1.225-2.712)	0.025*

Abbreviation: HR=hazard ratio; CI= confidence interval

Discussion

A large number of studies have shown that the development of pancreatic cancer is a process of continuous accumulation of genetic defects, and each abnormal gene is considered a potential target for treatment. After years of hard work, new treatments have emerged endlessly but the therapeutic effect on pancreatic cancer has not yet been improved significantly. Prognostic molecular biomarkers may help individualize treatment of patients with pancreatic cancer. However, the biomarkers currently used are not satisfactory. Therefore, the identification and characterization of new biomarkers is of particular importance.

Recent studies have shown that miR-539 is down-regulated in various cancers, including breast cancer [20], lung cancer [21], colon cancer [22], esophageal cancer [23], liver cancer [24], osteosarcoma [25], nasopharyngeal cancer [26], prostate cancer [27], and thyroid cancer [28]. However, the relationship between the expression of miR-539 and the pathological features of pancreatic cancer patients is still not clear. The results of this study showed that the expression of miR-539 in pancreatic cancer tissues was significantly lower than that in paracancerous normal tissues, and in 60 cases of pancreatic cancer patients, the down-regulation of miR-539 was significantly correlated with tumor differentiation, lymph node metastasis, clinical stage, CA199 and distant metastasis. Similarly, Ye et al. also found that the expression level of miR-539 is inversely correlated with the TNM stage of renal cell carcinoma, and miR-539 may function as a tumor suppressor to inhibit proliferation and induce the apoptosis of renal cell carcinoma cells through the action of its target gene HMGA2 [29]. In breast cancer, miR-539 expression is down-regulated and abnormally expressed miR-539 is associated with lymph node metastasis; miR-539 inhibits breast cancer growth in vivo and in vitro by targeting the epidermal growth factor receptor (EGFR) gene [30]. In addition, the expression of miR-539 is significantly down-regulated in non-small cell lung cancer cells and miR-539 inhibits the growth of non-small cell lung cancer by targeting the WNT8B gene in vivo and in vitro [31]. Similarly, miR-539 inhibits the progression of colon cancer by targeting the regulation of RUNX2 [32]. These findings consistently indicate that miR-539 may play an important role in the pathogenesis of various cancers.

In this study, it was found that the expression level of miR-539 was significantly associated with the overall survival rate of 60 patients with pancreatic cancer. According to the results of multivariate analysis, the down-regulation of miR-539 expression was an important predictor of poor prognosis in patients with pancreatic cancer. The results of our study were consistent with previous findings by Mirghasemi et al. in patients with osteosarcoma. In their study consisting of 35 patients with osteosarcoma, the high expression of miR-539 is a protective factor and the overall survival of patients with low expression of miR-539 is decreased. At the same time, they also found that the expression level of miR-539 is significantly associated with the pathological features of patients with osteosarcoma [25].

Although this study received some interesting results, there were also a few limitations. First the miR-539 targets had not been evaluated in the patients’ RNA samples. In future studies, we would evaluate the levels of miR-539 targets in patients’ RNA samples. Second, the sample size is relatively low, which would be increased in subsequent investigation. Third, this study only evaluated levels of miR-539 in cancer tissues, but not in the blood of patients. In further investigations, we would examine the levels of miR-539 in the blood of patients. Fourth, based on the larger number of samples collected in subsequent investigation, we would construct a web-based clinical database.

In summary, miR-539 is a potentially unfavorable prognostic factor for patients with pancreatic cancer. However, the sample size in our study was relatively small, and it is necessary to carry out further studies with larger sample sizes to confirm that miR-539 is a reliable predictor of clinical prognosis in patients with pancreatic cancer. In addition, more studies are also needed to investigate the possibility that miR-539 could be a new therapeutic target for the prevention of the metastasis and progression of pancreatic cancer.