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Abstract

BACKGROUND AND OBJECTIVE:

MicroRNAs (miRNAs) are under investigation as novel diagnostic and prognostic biomarkers in several malignancies, including gastric cancer (GC). miR-25 was overexpressed in GC tissues, and higher miR-25 expression was statistically correlated with aggressive clinicopathological characteristics. In our study, we investigate the associations of serum miR-25 level with the clinicopathological characteristics, diagnosis and prognosis of GC patients.

METHODS:

Serum samples from 184 GC patients, 56 gastritis patients and 78 healthy controls were subjected to real-time quantitative polymerase chain reaction (RT-qPCR), and the relationship between micR-25 level and cliniopathological characteristics including diagnosis and prognosis was explored.

RESULTS:

Compared with the gastritis and healthy patients, serum miR-25 level was significantly up-regulated in patients with GC. Using a cut-off of 0.042, the level of miR-25 was significantly increased in serum samples from cancer patients; Using this test cancer patients were identified with 67.3–69.4% sensitivity and 80.4%–81.0% specificity. High serum miR-25 level was significantly associated with depth of invasion, lymph node metastasis and stage of disease. In univariate and multivariate analyses, miR-25 was an independent prognostic factor for overall survival (OS). Moreover, high serum miR-25 level was correlated with poor prognosis in patients subgroups stratified by tumor size, depth of invasion and lymph node metastasis. Serum miR-25 level was increased in both prominent serosal invasion group and lymph node metastasis group. Furthermore, stratified analysis showed that the TNM stage I–VI patients with high serum miR-25 level had poor prognosis than those with low serum miR-25 level.

CONCLUSIONS:

Serum levels of miR-25 could improve gastric cancer screening, and as the better diagnostic and prognostic marker of gastric cancer.

Keywords

Gastric cancer miR-25 prognosos diagnosis

1. Introduction

Gastric carcinoma (GC) is the second leading cause of cancer death throughout the world [1]. Global statistics showed that in 2008 alone, nearly 989,000 people were diagnosed with GC, and approximately 464,000 people died from this disease [2]. Currently, GC prognosis is primarily determined based on the clinical data and pathological stages of patients at the time of diagnosis and treatment [3]. However, successful management of GC patients is still hampered by the lack of highly sensitive and specific biomarkers capable of predicting prognosis and likelihood of metastasis.

Evidence of microRNA (miRNA) dysregulation in malignant tumors has emerged in recent years. miRNAs are small non-coding RNA molecules that modulate the expression of multiple target genes and play important roles in various physiological and pathological processes, such as development, differentiation, cell proliferation, apoptosis, organogenesis, and homeostasis [4, 5]. After the discovery of miRNA in 1993 [6], its importance in malignant disease was suggested in 2004 when miRNA genes were found to be specifically deleted in leukemia [7, 8]. Subsequent reports have demonstrated that miRNAs are dysregulated in many malignant tumors, and these miRNAs can initiate carcinogenesis or drive progression [8, 9].

miR-25 was reported to be associated with tumor carcinogenesis, including breast cancer [10], cholangiocarcinoma [11], ovarian cancer [12], esophageal squamous cell intraepithelial neoplasia [13], pancreatic cancer [14] and hepatocellular carcinoma [15]. A number of studies found unequivocally that miR-25 is overexpressed in human GC cell lines, tissue and serum/plasma samples as well. In addition, overexpression of the miR-25 resulted in a decreased response of gastric cancer cells to TGF- $\beta$ interfering with the synthesis of cell cycle inhibitor p21 and the pro-apoptotic Bim [16]. Interestingly, another study using human gastric cell lines also reported that the amplification of MCM7 and its intron miR-25 might be the major molecular switchers in the development of gastric cancer [17]. Li et al. showed overexpression of miR-25 in plasma and tissue samples of GC patients which promoted gastric cancer migration, invasion and proliferation by directly targeting the tumor suppressor TOB1 and correlated with poor survival [18]. In contrast, a single nucleotide polymorphism rs41274221 in the mature miR-25 gene was associated with reduced tumor growth and metastasis in GC patients [19]. This evidence on the potential of miR-25 is transforming the field of clinical biomarkers. However, comprehensive profiling studies of circulating miR-25 in GC are limited and a consistent diagnostic signature for circulating miR-25 is not available.

In this study, we evaluated whether the serum miR-25 could serve as a non-invasive diagnostic and prognostic marker for GC. We also evaluated the sensitivity of serum miR-25 in detection of GC patients.

2. Materials and methods

2.1 Patients and samples

A total of 184 patients with gastric cancer (GC) from 3 hospital [the affiliated hospital of Qingdao university ( $n=$ 78), the people’s hospital of Ji-ning ( $n=$ 50) and the central hospital of Lin-yi ( $n=$ 64)] in China were included in this study. All the patients were underwent radical resection for gastric cancer between Apri-2-2006 and Jan-10-2010. All patients underwent endoscopic diagnosis and biopsy. The mean age of the patients at initial surgery was 62 years (range, 33–81 years); 112 men and 72 women were included in this study. The mean duration of follow-up was 37 months (range, 1–75 months). Survival time was calculated from the date of surgery to the date of death or last follow-up. All patients had follow-up after surgery at 6 to 12 month intervals; Overall survival was defined as was defined as the period between the time of surgery and death or was censored at the last follow-up. Clinical information was collected from the medical records of each patient and shown in Table 1. The study was approved by the local Institution Review Boards (IRB) at each institution in accordance with the Declaration of Helsinki and its later revision. All patients provided written, informed consent authorizing the collection and use of their samples for research purposes unless the IRB permitted a waiver. Patients with pre-operative chemotherapy were not included in the study. Normal or gastritis biopsies (Nor/G) from 78 or 56 outpatients (age-matched) at the people’s hospital of Ji-Ning were used as the cancer-free controls. The 2003 UICC-TNM (tumor-node-metastasis, TNM) system was used for the classification of GCs [20].

2.2 RNA extraction

Peripheral blood (7 ml) was obtained from each patient at the time of diagnosis or before surgery, as well as from the healthy volunteers. The blood was transferred into sodium heparin tubes (BD Vacutainer, Franklin Lakes, NJ, USA) and immediately subjected to the three-spin protocol (1500 rpm for 30 min, 3000 rpm for 5 min, and 4500 rpm for 5 min) to prevent contamination of the cellular nucleic acids. Plasma was stored at $-$ 80 ${}^{\circ}$ C until further processing. Four hundred fifty microliters plasma of each sample was placed in a new 2 ml RNase free EP tube after thawing, thoroughly mixed with same volume of 2 $\times$ denaturing solution, and incubated on ice for 5 min before adding 900 $\mu$ l phenol-chloroform solution. The mixture was vortexed for 20 s, and centrifuged at 12,000 g, room temperature for 5 min. Six hundred microliters supernatant were placed in a new 2 ml RNase free EP tube and added with 750 $\mu$ l 100% ethanol and mixed thoroughly. Six hundred eighty microliters of the mixture were placed in a filter column, followed by centrifugation at 12,000 g for 30 s and the filtrate was discarded. This step was repeated until all mixture of the sample passed through the filter column. Seven microliters wash buffer 1 was added to the filter column and spun down at 12,000 g for 20 s and the filtrate was discarded. Five hundred microliters wash buffer 2/3 was added to the filter column and spun down at 12,000 g for 20 s and the filtrate was discarded. The above step was repeated once. The filter column was centrifuged at 12,000 g for 1 min and added with 50 $\mu$ l preheated elution solution (95 ${}^{\circ}$ C water bath) before centrifugation at 12,000 g, room temperature for 1 min to obtain total RNA. A volume of 1.5 $\mu$ l extracted RNA sample was used to measure the purity of RNA using a NanoDrop 1000 spectrophotometer. The ratio of OD260/OD280 should be ranged 1.8–2.1. Extracted total RNA was stored in a $-$ 80 ${}^{\circ}$ C freezer for later experiments.

2.3 Reverse transcription and qRT-PCR

Reverse transcription and qRT-PCR were performed according to the manufacture’s manual (Biomics, Jiangsu, Nantong, China). U6 siRNA was used as internal control in this experiment. The 25 $\mu$ l qRT-PCR reaction system contained 3 $\mu$ l total RNA extract, miR-25- and U6-specific primers, forward and reverse primers, and 2 $\times$ master mix. RNase-free DEPC water was used in the qRT-PCR reaction system. The prepared reaction system was placed in a PCR reaction strip, containing eight PCR tubes to start the qRT-PCR reaction in Mx300P qRT-PCR detection system. PCR conditions included reverse transcription at 42 ${}^{\circ}$ C for 30 min; 95 ${}^{\circ}$ C denaturation for 10 min; 40 cycles of 95 ${}^{\circ}$ C denaturation for 20 s, 60 ${}^{\circ}$ C annealing for 30 s, and 72 ${}^{\circ}$ C elongation for 30 s. Fluorescent signals were collected at 72 ${}^{\circ}$ C. The cycle threshold (Ct) value was defined as the number of cycles needed for the fluorescent signal intensity to cross the defaulted threshold in a quantitative PCR tube. Data were analyzed using $\Delta$ Ct $=$ Ct ${}^{\text{miR-25-}}$ Ct ${}^{\text{U6}}$ method. The 2- $\Delta$ Ct represented the relative miR-25 expression in patients’ plasma. Each experimental sample was assessed in triplicate in three reaction tubes, and each experiment was repeated three times.

2.4 Statistical analysis

All statistical analyses were performed using the SPSS statistical software package (version 22; SPSS Inc.). All data were expressed as mean $\pm$ SD. The unpaired t test or Mann-Whitney test was used to analyze the differences between subjects and controls or the correlation between the mean of miR-25 levels and patient clinicopathological features. Receiver-operating characteristic (ROC) curves and area under the ROC curve (AUC) values were used to assess the feasibility of using plasma miR-25 levels as a diagnostic tool for detecting GC. The Youden index was used to determine the cut-off value for the plasma miR-25 levels. The resulting value of the cut-off point for each evaluation was applied to the determination of the sensitivity, specificity and odds ratio. Consequently, 95% confidence intervals were calculated. For the survival rate analysis, Kaplan-Meier survival curves were constructed for groups based on univariate predictors, and differences between the groups were analyzed with the log-rank test or the Wilcoxon test. Univariate and multivariate survival analyses were performed using the likelihood ratio test of the stratified Cox proportional hazards model. All $P$ values were 2-sided, and those less than 0.05 were considered statistically significant.

3. Results

3.1 Patient characteristics

We firstly detected serum miR-25 levels in 184 gastric cancer patients, as compared with the levels in 78 healthy controls and 56 gastritis patients. The age (year) was 63.6 years (range, 33–81 years) in patients with GC, 62.8 (32–80) in 78 healthy controls ( $p=$ 0.192) and 62.7 (34–79) in 76 patients with gastritis ( $p=$ 0.106). The number of male and female was 112 (60.9 %) and 72 (39.1 %) in patients with GC, 44 (56.4 %) and 34 (43.6 %) in healthy controls, and 42 (53.8 %) and 36 (46.2 %) in patients with gastritis. There was no significant difference between the gender in the groups of patients with GC and healthy controls ( $p=$ 0.376), GC and gastritis ( $p=$ 0.118).The clinicopathological parameters of the patients are listed in Table 1.

Figure 1.

Kaplan-Meier survival analysis showing the relationship between miR-25 and overall survival in all patients (A) patients at TNM I–II stage (B) and patients at TNM III–IV stage (C).

Table 1

Clinicopathologic correlation of miR-25 levels in gastric cancer

Groups	Number	Mean (SD)	$p$ -value
Gender			0.536
Male	112	0.063 $\pm$ 0.041
Female	72	0.062 $\pm$ 0.039
Age (Year)			0.327
$<$ 60	80	0.064 $\pm$ 0.040
$\geqslant$ 60	104	0.061 $\pm$ 0.039
Pathology			0.162
Well or moderately-	64	0.061 $\pm$ 0.038
differentiated
Poorly differentiated	99	0.070 $\pm$ 0.037
Mucinous adenocarcinoma	21	0.063 $\pm$ 0.040
Tumor size (cm)			0.095
$<$ 4	71	0.060 $\pm$ 0.038
$\geqslant$ 4	113	0.066 $\pm$ 0.037
Vessel invasion			0.165
Yes	142	0.066 $\pm$ 0.034
No	42	0.063 $\pm$ 0.041
T stage			0.026
T1–T2	56	0.052 $\pm$ 0.031
T3–T4	128	0.083 $\pm$ 0.046
Lymph node metastasis			0.003
Yes	97	0.050 $\pm$ 0.034
No	87	0.087 $\pm$ 0.040
Distal metastasis			0.253
Yes	15	0.062 $\pm$ 0.035
No	169	0.063 $\pm$ 0.041
TNM stage			0.002
I/II	81	0.058 $\pm$ 0.035
III/IV	103	0.076 $\pm$ 0.030
Lauren’s classification			0.129
Intestinal type	110	0.062 $\pm$ 0.037
Diffuse type	52	0.059 $\pm$ 0.036
Mixture	22	0.060 $\pm$ 0.036
Tumor site			0.423
Cardia	92	0.061 $\pm$ 0.039
Corpus	37	0.063 $\pm$ 0.040
Antrum	43	0.063 $\pm$ 0.038
Two or more sites	12	0.064 $\pm$ 0.034

3.2 Correlation of serum miR-25 levels with clinicopathological features

We next evaluated serum expression levels of miR-25 by qRT-PCR in a validation cohort of 184 GC patients, 56 gastritis patients and 78 healthy controls. The miR-25 levels in healthy controls was 0.012 $\pm$ 0.008 (median 0.009, range 0.001–0.018), and 0.014 $\pm$ 0.010 (median 0.028, range 0.001–0.019) in patients with gastritis, and 0.063 $\pm$ 0.040 (median 0.042, range 0.010–0.483) in GC. Serum concentrations of miR-25 in GC patients were significantly higher than that in healthy controls ( $P=$ 0.006) and gastritis ( $P=$ 0.006). Our results suggested that serum concentrations of miR-25 was increased in patients with gastric cancer. To verify the functions of miR-25 in gastric cancer, we correlated miR-25 with other widely recognized clinicopathologic features (Table 1). High serum concentrations of miR-25 was positively related to T stage ( $P=$ 0.026) lymph node metastasis ( $P=$ 0.002) and stage of disease ( $P=$ 0.003) in gastric cancer (all $P<$ 0.001) (Table 1).

3.3 High serum concentrations of miR-25 correlated with poor prognosis in gastric cancer

We next explored the relationship between serum miR-25 and overall survival using Kaplan-Meier analysis. We used the median serum miR-25 value 0.042 as the cutoff value in GC patients. Increased serum miR-25 levels were found in 65% (120/184) of patients with GC and 35% (64/106) was found less 0.042. The results demonstrated that high serum miR-25 showed poor survival in gastric cancer patients (Fig. 1a, $P=$ 0.0028). To evaluate further the efficiency of serum miR-25 in stratifying patients with different TNM stages, we divided the patients into early (I–II) and advanced (III–IV) groups, respectively. In both the TNM I $+$ II and TNM III $+$ IV subgroups, serum miR-25 showed statistically significant value in predicting the outcomes of gastric cancer patients (Fig. 1b and c). These data suggested that serum miR-25 is correlated with overall survival for patients with gastric cancer.

Table 2
Univariate and multivariate analysis of clinicopathological factors for overall survival

	Univariate				Multivariate
	HR	95% CI	$P$		HR	95% CI	$P$
Gender (female vs. male)	1.38	0.46–2.83	0.	36
Age (years) ( $\geqslant$ 60 vs $<$ 60)	1.83	0.91–4.02	0.	184
Differentiation (poorly vs well/moderately/mucinous	1.68	0.94–3.72	0.	098
adenocarcinoma)
Vessel invasion (yes vs no)	2.47	2.546–4.74	0.	0023	1.26	0.94–2.38	0.086
T stage (T3–T4 vs T1–T2)	2.95	2.17–4.95	0.	001	1.42	1.24–10.37	0.036
Lymph node metastasis (yes vs no)	5.42	2.38–15.36	$<$ 0.	001	3.23	1.27–9.96	0.028
Distant metastasis (yes vs no)	17.46	1.37–102.5	0.	039	1.26	0.453–4.88	0.284
TNM stage (III–IV vs I–II)	4.38	1.85–7.89	$<$ 0.	001	2.13	0.87–5.69	0.28
Tumor size (cm) ( $<$ 4 vs $>$ 4)	3.82	1.84–7.92	0.	004	2.46	1.62–5.38	0.012
Lauren’s classification (diffuse $+$ mixture vs intestinal)	1.67	1.17–2.628	0.	274
Tumor site (antrum vs others)	2.52	1.27–4.90	0.	0038	2.06	1.2–4.46	0.042
miR-25 level ( $<$ 0.042 vs $>$ 0.042)	2.54	1.32–4.85	0.	0063	1.84	1.16–3.78	0.037

We also conducted univariate Cox analysis to identify the prognostic significance of clinicopathological factors for overall survival. Tumor location, tumor size, Vessel invasion, T stage, lymph node metastasis, distant metastasis, TNM stage, and serum miR-25 were found to be risk factors for survival in patients with gastric cancer (Table 2). Further adjustment of covariate factors using multivariate Cox analysis identified Tumor location, tumor size, T stage, lymph node metastasis and serum miR-25 as independent risk factors for gastric cancer (Table 2). These data indicate that high serum miR-25 is an independent factor that predicts poor prognosis in patients with gastric cancer.

Figure 2.

ROC curves for miR-25 to distinguish GC cases from healthy and gastritis controls.

3.4 Detection value of the serum miR-25 for gastric cancer patients vs. healthy controls

Next, we performed ROC curve analysis to evaluate the diagnostic value of serum miR-25. First, we identified a cut-off value that distinguished 184 GC patients from 78 healthy and 56 gastritis sex- and age-matched controls. The ROC curve data showed that serum miR-25 could distinguish the GC patients from the healthy and gastritis controls. The serum level of miR-25 had a sensitivity of 69.4% and a specificity of 84.6% to differentiate the GC patients from the healthy controls, with an AUC of 0.768 at the optimal cut-off point ( $P<$ 0.0001, 95% confidence interval [CI]: 0.662–0.817) (Fig. 2). In addition, miR-25 had a sensitivity of 67.3% and a specificity of 80.4% to differentiate the GC patients from the gastritis controls, with an AUC of 0.732 at the optimal cut-off point ( $P<$ 0.0001, 95% confidence interval [CI]: 0.653–0.794) (Fig. 2).

4. Discussion

During the past two decades, lack of highly sensitive and noninvasive diagnostic biomarkers led to only modestly improved prognosis for gastric cancer. miRNAs can be released from cancer cells to body fluids via secreting exosomes particles, which could protect them from RNase degradation in circulation. Several circulating miRNAs have been detected in sera, plasma, urine, tears, amniotic fluid and gastric juice [21, 22]. The different expression patterns of circulating miRNA in body fluids might originate from different cell types under certain physiological status [23]. Therefore, miRNA might be a useful noninvasive biomarker for diagnosis and recurrent gastric cancer. Mitchell et al. [22] demonstrated that expression levels of circulating miRNAs in serum are consistent with gastric tumour tissues, and they could serve as a biomarker for cancer detection. In gastric cancer, several circulating miRNAs have been studied as potential diagnostic biomarkers by evaluating their amount in serum, plasma and gastric juice. Accumulating recent studies have shown that miRNAs is a promising biomarker that could be used to determine the prognosis of gastric cancer patients and predict the survival rate and recurrence of patients with gastric cancer. Ueda et al. [24] reported that low expression of let-7 g and miR-433 and high expression of miR-214 were associated with poor overall survival independent of clinical covariates, including lymph node metastasis and stage. Li et al. [25] analysed seven miRNAs expression profiles (miR-10b, miR-21, miR-223, miR-338, miR-30a-5p and miR-126) by real-time PCR in 100 gastric cancer patients and showed that seven-miRNA signatures could predict relapse-free and overall survival of patients with gastric cancer.

It has recently found that under pathophysiological conditions, miR-25 is a well described oncogenic miRNA. It plays a crucial role in the development and spread of many tumor types including brain tumors, lung, breast, ovarian, prostate, thyroid, esophageal, gastric, colorectal, hepatocellular cancers, etc. [26]. Clinical studies have also found that miR-25 is elevated in tissue and/or plasma/serum samples of GC patients (stage I–III) [27]. In our study, we found that serum concentrations of miR-25 was increased in patients with gastric cancer, and high serum concentrations of miR-25 was positively related to T stage, lymph node metastasis, and stage of disease in gastric cancer. However, there was no statistical significance between the serum expression of this marker and other clinicopathological elements. In addition, the prognosis analysis showed that the patients with high serum concentrations of miR-25 had poorer survival than that with low serum concentrations of miR-25. According to the results of multivariate analysis, serum concentrations of miR-25 was an independent indicator for poor OS and RFS in gastric cancer patients. Furthermore, the prognostic significance of serum concentrations of miR-25 in different risk of subgroups based on tumor size, depth of invasion and lymph node metastasis was assessed, which appeared that serum concentrations of miR-25 could be a negative prognostic biomarker for different risks of gastric cancer patients. Our finding concluded that serum concentrations of miR-25 could serve as a feasible prognostic biomarker of gastric cancer. The ROC curve analysis also confirmed that serum miR-25 levels could help differentiate patients with PC from healthy controls and gastritis with high sensitivity and specificity. Therefore, serum miR-25 levels could be a diagnostic biomarker of GC. However the sensitivity and specificity of the serum miR-25 to differentiate patients with PC from healthy controls and gastritis is still relatively low. If serum miR-25 combines clinical serum markers commonly used in gastric cancer, such as sCEA, CA19-9, CA72-4, CA125 and alpha-fetoprotein etc., it may improve the diagnosis rate of gastric cancer, which need further investigation. Although serum miR-25 alone has relatively low sensitivity and specificity, it has great advantage in judging tumor stage, lymph node metastasis and prognosis, it is very meaningful for guiding the treatment of advanced gastric cancer.

In conclusion, serum miR-25 levels were upregulated in GC patients and showed favorable sensitivity and specificity, correlating with poor prognosis. Serum levels of miR-25 could improve gastric cancer screening, and as the better diagnostic and prognostic marker of gastric cancer. The clinical development of this methodology as a noninvasive diagnostic or monitoring strategy may be promising for clinicians and patients with malignant diseases.

Footnotes

Acknowledgments

This study was supported by grants from Shandong Natural Scientific Research Funds (no: 2016ZRB230 36).

Conflict of interest

None.

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Clinical significance of serum miR-25 as a diagnostic and prognostic biomarker in human gastric cancer

Abstract

BACKGROUND AND OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Materials and methods

2.1 Patients and samples

2.2 RNA extraction

2.3 Reverse transcription and qRT-PCR

2.4 Statistical analysis

3. Results

3.1 Patient characteristics

3.3 High serum concentrations of miR-25 correlated with poor prognosis in gastric cancer

Table 2 Univariate and multivariate analysis of clinicopathological factors for overall survival

4. Discussion

Footnotes

Acknowledgments

Conflict of interest

References

Table 2
Univariate and multivariate analysis of clinicopathological factors for overall survival