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Abstract

BACKGROUND:

miR-126 functions as a tumor suppressor in gastric cancer (GC), however, the clinical significance of serum miR-126 in GC remains unclear.

OBJECTIVE:

To investigate the associations of serum miR-126 level with the clinicopathological characteristics and prognosis of GC patients.

METHODS:

Quantitative real-time polymerase chain reaction was performed to examine the expression levels of miR-126 in 338 GC patients’ tissues and sera, and 50 healthy controls’ sera. The associations of serum miR-126 with clinicopathological characteristics and clinical outcome were evaluated.

RESULTS:

Compared with the matched adjacent non-tumor tissues and normal sera, miR-126 expression was significantly down-regulated in both tumor tissues and sera of GC patients. Importantly, there was a positive correlation between tissue and serum levels of miR-126 in GC patients. A reduced serum miR-126 level statistically correlated with aggressive clinicopathological characteristics, such as larger tumor size, deeper local invasion, more lymph node metastasis, advanced TNM stage, and poorer prognosis. Notably, multivariate analysis identified reduced serum miR-126 level as an independent predictor for the unfavorable prognosis of GC.

CONCLUSIONS:

These results indicate for the first time that serum miR-126 may serve as a novel prognostic biomarker in GC.

Keywords

Gastric cancer microRNA miR-126 serum prognosis

1. Introduction

The incidence of gastric cancer (GC) has dramatically decreased over the past few decades in many regions of the world; however, it still remains one of the most common cancers with almost one million new cases diagnosed per year worldwide [1]. In China, GC is the second most common cancer and the second leading cause of cancer-associated death [2]. In theory, only GC patients at early stage may be cured by R0 resection alone with satisfactory clinical outcomes [3]. However, most GC patients develop into advanced stage at time of diagnosis with dismal clinical outcomes [4]. Thus, the prediction of the prognosis and accurate patient stratification are crucial to optimize personalized treatment.

The prognosis of cancer patients depends on the biological behaviors of the cancer cells. At present, the prediction of prognosis of cancer patients are mainly based on the Tumor, Node Status, Metastasis (TNM) classification system [5]. However, the TNM classification system has been proven to be inadequate to predict the biological behaviors of cancer cells, especially those with high degree of heterogeneity like GC. Thus, modifications of current TNM classification system by the addition of other non-invasive, more sensitive, and specific biomarkers better reflecting the biological behaviors of GC cells are likely to improve the prognostic prediction of GC patients, and help design more reasonable treatment strategy for this devastating illness [6].

MicroRNAs (miRNAs) are a novel class of small (18–24 nucleotides long), evolutionarily conserved non-coding RNA molecules that regulate many physiological and pathological processes, such as proliferation, apoptosis, development and differentiation by regulating target genes post-transcriptionally [7]. Accumulating evidences have revealed that many miRNAs function as either oncogenes or tumor suppressors [8, 9]. In addition, miRNAs have been detected in blood circulation and are more stable than proteins and mRNAs [10]. Therefore, identification of prognosis-related, circulating miRNAs has attracted more and more attentions in the field of cancer research since this provides a quick, non-invasive, and relatively inexpensive method for prognosis prediction. For example, expression levels of miR-15a in esophageal squamous cell carcinoma (ESCC) tumor tissues and patients’ sera were significantly decreased in ESCC patients, and low miR-15a expression was found to be significantly associated with shorter overall survival and disease-free survival of ESCC patients. Thus, serum miR-15a level could be used as a potential prognostic marker of ESCC [11].

Our earlier study demonstrated for the first time that miR-126 was down-regulated in GC, and ectopic expression of miR-126 could inhibit the proliferation and metastatic potential of GC cells both in vitro and in vivo, partly through down-regulating the expression of Crk protein post-transcriptionally [12]. In line with our findings, Li et al. reported subsequently that overexpression of miR-126 inhibited GC cells invasion in part by down-regulating Crk protein expression [13]. Hence, miR-126 may function as a tumor suppressor in GC with Crk as its target gene. Notably, combined miR-126-low/Crk protein-high expression in tumor tissues was found to be an independent unfavorable prognostic factor of GC in our recent study [14]. However, the clinical significance of serum miR-126 in GC patients is yet to be elucidated. To this end, we performed current study to further investigate the associations of serum miR-126 expression level with the clinicopathological characteristics and prognosis of GC patients.

2. Materials and methods

2.1 Patients and clinical samples

In total, 338 patients with primary GC who underwent radical gastrectomy with D2 lymphadenectomy at the Department of General Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (Shanghai, China) from 2010 to 2011 were recruited for this study. None of these GC patients underwent chemotherapy or radiotherapy before sample collection in order to eliminate potential treatment-induced alterations in gene expression profiles. Primary tumor tissues and matched non-tumor tissues were collected immediately after surgery, incubated in RNAlater™ Stabilization Solution and stored at $-$ 80 ${}^{\circ}$ C until total RNA extraction. To obtain serum samples, 10 mL of peripheral blood was drawn into separate gel tubes before surgery and then subjected within 30 min to centrifugation at 1,500 g for 10 min at 4 ${}^{\circ}$ C. The supernatants were transferred to 1.5-mL Eppendorf tubes and stored at $-$ 80 ${}^{\circ}$ C until total RNA extraction. In addition, serum from 50 healthy volunteers, who were not on medication and had no signs or clinical symptoms of cancer, joint, liver, metabolic or endocrine diseases, were also collected.

All clinicopathological factors including age, sex, tumor size, tumor site, histologic type, the depth of invasion, and status of lymph node metastasis were defined according to the Japanese Classification of Gastric Carcinoma (JCGC). Tumors were staged according to the seventh edition of the Union for International Cancer Control tumor, node, metastasis (TNM) classification system.

Follow-up of all patients was carried out for 5 years (or until death) according to our standard protocol (every 3 months in the first two years, every 6 months in the third year, and every 12 months afterwards or until death). No subject was lost to follow-up. The routine check-up during follow-up included physical examination, laboratory tests, chest X-ray, computed tomography (CT) and endoscopy. Disease-specific survival was used for evaluating the association of serum miR-126 level with the prognosis in the GC patients since it allowed controlling for unrelated causes of death.

The study protocol was approved by the Ethic Committee of Ruijin Hospital and signed informed consent was obtained from all study participants. All participants signed an informed consent form for the use of their samples before recruitment. All specimens were handled and made anonymous according to ethical and legal standards.

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

To extract total RNA in serum, 600 $\mu$ L of the serum sample from each participant was subjected to RNA extraction using mirVana™ Paris™ Kit (Ambion) according to the manufacturer’s protocol. RNA extraction from tissue samples were performed as described previously [12]. The quality and quantity of the RNA samples were assessed by standard electrophoresis and spectrophotometric methods. The RNA samples were subjected to a reverse transcription reaction using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems), according to the manufacturer’s instructions. Subsequently, real-time qPCR was carried out using TaqMan 2 $\times$ Universal PCR Master Mix with no AmpErase UNG (Applied Biosystems) on an ABI 7900 Real-Time PCR system (Applied Biosystems). real-time qPCR amplification conditions were set to an initial cycle of 95 ${}^{\circ}$ C for 10 min followed by 40 cycles of 95 ${}^{\circ}$ C for 15 s and 60 ${}^{\circ}$ C for 1 min. Each sample was run in triplicate for analysis. The expression level of mature miR-126 was normalized using U6 small nuclear RNA (Applied Biosystems) by the 2 ${}^{-\Delta\rm CT}$ method. The relative expression ratio of miR-126 in each paired tumor to non-tumor tissue was calculated by the 2 ${}^{-\Delta\Delta\rm CT}$ method. The miR-126 expression level was defined as down-regulation in tumor tissues when the relative expression ratio $<$ 0.5 [12].

2.3 Statistical analysis

Data were expressed as means $\pm$ standard deviation (SD). Student’s $t$ -test was used to analyze the differential expression levels of serum miR-126 between GC patients and healthy controls, and differential expression levels of tissue miR-126 between primary tumor tissues and matched non-tumor tissues. The chi-square test was used to evaluate the correlations between serum miR-126 expression level and the clinicopathological factors. The survival rates were calculated using the Kaplan-Meier method, and comparison was made by the log-rank test. The Cox proportional hazards model (using the forward stepwise procedure for variable selection) was used to determine the independent prognostic factors in a multivariate setting. A two-tailed value of $p<$ 0.05 was considered statistically significant. All statistical analyses were performed using statistical analysis program package (IBM SPSS Statistics 20).

Table 1
Positive correlation between tissue and serum levels of miR-126 in GC

	Tissue miR-126 level		$p$ value
	Low ( $n=$ 176)	High ( $n=$ 162)
Serum miR-126 level
Low ( $n=$ 183)	130	53	0.000*
High ( $n=$ 155)	46	109

* $p<$ 0.01.

Figure 1.

Expression levels of miR-126 were down-regulated both in tumor tissues and sera of GC patients. (A) qRT-PCR for miR-126 expression was carried out using 338 GC patients’ sera (black bar) and 50 healthy controls’ sera (grey bar). The mean and standard deviation of miR-126 expression levels are shown. (B) qRT-PCR for miR-126 expression was carried out using 338 surgical specimens of GC tissues (black bar) and matched adjacent non-tumor tissues (grey bar). The mean and standard deviation of miR-126 expression levels are shown.

3. Results

3.1 Expression levels of miR-126 were down-regulated in both tumor tissues and sera of GC patients

The results of real-time qRT-PCR analysis showed that serum expression levels of miR-126 was significantly lower in GC patients than those in healthy controls (mean $\pm$ SD, GC vs. healthy control: 50.6 $\pm$ 20.8 vs. 116.5 $\pm$ 21.3, $p<$ 0.001, Fig. 1a). In addition, expression levels of miR-126 was significantly lower in tumor tissues than those in matched non-tumor tissues (mean $\pm$ SD, tumor tissues vs. matched non-tumor tissues: 73.1 $\pm$ 7.9 vs. 159.9 $\pm$ 22.3, $p<$ 0.001, Fig. 1b). Notably, there was a positive correlation between serum and tissue levels of miR-126 expression in GC patients ( $p<$ 0.01, Table 1).

3.2 Down-regulation of serum miR-126 was associated with adverse clinicopathological parameters of GC patients

We next examined the association of the serum expression level of miR-126 with the clinicopathological factors of GC. In this study, the mean serum expression level of miR-126 in GC patients was used as a cut-off value to divide all 388 patients into 2 groups: high-expression group (with an expression level above the mean expression level) and a low-expression group (with an expression level below the mean expression level). As shown in Table 2, the serum expression level of miR-126 was significantly associated with tumor size, local invasion, lymph node metastasis, and TNM stage (All $p<$ 0.01). However, the serum expression level of miR-126 was not correlated with age ( $p=$ 0.051), sex ( $p=$ 0.599), tumor site ( $p=$ 0.438), histologic type ( $p=$ 0.726). Thus, our data suggested that the serum level of miR-126 may be used as a biomarker for evaluating the malignant progression of GC.

Table 2
Clinicopathological associations of serum miR-126 expression in GC

		Serum miR-126 level
Factors	No.	Low ( $n$ , %)	High ( $n$ , %)	$p$ value
	of cases
Age
$\leqslant$ 60	179	88 (49.2%)	91 (50.8%)	0.051
$>$ 60	159	95 (59.7%)	64 (40.3%)
Sex
Male	213	113 (53.1%)	100 (46.9%)	0.599
Female	125	70 (56.0%)	55 (44.0%)
Tumor size
$\leqslant$ 5 cm	123	33 (26.8%)	90 (73.2%)	0.000*
$>$ 5 cm	215	150 (69.8%)	65 (30.2%)
Tumor site
Lower	174	99 (56.9%)	75 (43.1%)	0.438
Middle	131	69 (52.7%)	62 (47.3%)
Upper	33	15 (45.5%)	18 (54.5%)
Histologic type
Intestinal	191	105 (55.0%)	86 (45.0%)	0.726
Diffuse	147	78 (53.1%)	69 (46.9%)
Local invasion
T1 $+$ T2	117	25 (21.4%)	92 (78.6%)	0.000*
T3 $+$ T4	221	158 (71.5%)	63 (28.5%)
Lymph node
metastasis
No	100	12 (12.0%)	88 (88.0%)	0.000*
Yes	238	171 (71.8%)	67 (28.2%)
TNM stage
I $+$ II	136	18 (13.2%)	118 (86.8%)	0.000*
III $+$ IV	202	165 (81.7%)	37 (18.3%)

* $p<$ 0.01.

Table 3

Univariate analyses of prognostic factors in GC

Factors	No. of	5-year	$p$ value
	cases	survival (%)
All	338	57.4	N/A
Age			0.104
$\leqslant$ 60	179	61.5
$>$ 60	159	52.8
Sex			0.116
Male	213	60.6
Female	125	52.0
Tumor size			0.000*
$\leqslant$ 5 cm	123	85.4
$>$ 5 cm	215	41.4
Tumor site			0.523
Lower	174	54.6
Middle	131	61.8
Upper	33	54.5
Histologic type			0.955
Intestinal	191	57.1
Diffuse	147	57.8	0.000*
Local invasion
T1 $+$ T2	117	84.6
T3 $+$ T4	221	43.0
Lymph node metastasis			0.000*
No	100	89.0
Yes	238	44.1
TNM stage			0.000*
I $+$ II	136	88.2
III $+$ IV	202	36.6
Serum miR-126 level			0.000*
Low	183	35.5
High	155	83.2

* $p<$ 0.01.

Table 4

Multivariate analyses of prognostic factors in GC

Factors	HR	95% CI	$p$ value
Age	–	–	0.857
Sex	–	–	0.066
Tumor size	2.706	1.612–4.540	0.000*
Tumor site	–	–	0.891
Histologic type	–	–	0.514
TNM stage	3.363	1.824–6.201	0.000*
Serum miR-126 level	0.440	0.269–0.721	0.001*

* $p<$ 0.01, HR: Hazard Ratio, CI: Confidence interval.

Figure 2.

Kaplan–Meier curves of disease-specific survival, stratified by serum miR-126 expression level.

3.3 Down-regulation of serum miR-126 was associated with poor prognosis of GC patients

We further investigated the factors that could predicate the prognosis of GC patients by using the univariate and multivariate analyses. An analysis of the disease-specific survival was performed using theKaplan-Meier approach, with the statistical analysis performed using the log-rank test. The results showed that there was a significant difference in the survival between the miR-126 high-expression group and low-expression group (Fig. 2). The 5-year disease-specific survival rate in the patients with low serum miR-126 level was 35.5%, whereas that in the patients with high serum miR-126 level was 83.2% ( $p<$ 0.01, Table 3). The 5-year disease-specific survival rate with regard to other clinicopathological factors was also compared using the log-rank test. As shown in Table 3, tumor size ( $p<$ 0.01), local invasion depth ( $p<$ 0.01), lymph node metastasis ( $p<$ 0.01), and TNM stage ( $p<$ 0.01) were also found to significantly impact survival, however, age ( $p=$ 0.104), sex ( $p=$ 0.116), tumor site ( $p=$ 0.523), or histologic type ( $p=$ 0.955) did not significantly impact survival. We then employed Cox proportional hazards regression model to identify independent prognostic factors. Since local invasion and lymph node metastasis information was also included in the TNM stage data, therefore, local invasion and lymph node metastasis were not entered into the multivariate analysis. As demonstrated in Table 4, the serum miR-126 expression level (Hazard Ratio, HR: 0.440; 95% Confidence interval, 95% CI: 0.269–0.721; $p<$ 0.01), tumor size (HR: 2.706; 95% CI: 1.612–4.540; $p<$ 0.01), and TNM stage (HR: 3.363; 95% CI: 1.824–6.201; $p<$ 0.01) were found to be independent prognostic factors of GC patients.

4. Discussion

GC remains one of the most common cancers worldwide. Despite remarkable advances in comprehensive treatment approaches, the prognosis of GC patients remains poor, with a 5-year overall survival ranging between 15 and 35% [15]. Currently, clinical indicators, especially TNM staging system, are the main prognostic factors for GC. However, the clinical outcomes, including treatment response, tumor recurrence/meta- stasis, and survival times, of GC patients even with the same TMN stage vary considerably [16]. Therefore, the above clinical parameters are inadequate to predict the biological behaviors of GC cells. In clinical practice, there is currently a lack of reliable, sensitive and specific predictive biomarkers related to the tumor biology for detecting GC progression. Thus, it is urgent to identify novel cancer biomarkers to reflect the biological behaviors of GC cells and predict prognosis of GC patients more accurately, and henceforth design more rational personalized treatment strategy and surveillance plans for them.

miRNAs are a novel class of small non-coding RNAs that regulate gene expression at the post-tran- scriptional level, either by translational repression or by mRNA degradation. Notably, absolute sequence complementarity between the miRNAs and their target messenger RNAs (mRNAs) is not necessary, this flexibility implies that each miRNA could bind and regulate numerous mRNAs. It is estimated that miRNAs regulate about 30% of all protein coding genes in mammals, and govern various important biological functions such as cell proliferation and differentiation [17]. Meanwhile, growing evidences demonstrate that miRNAs exert pivotal roles in the pathogenesis of various human cancers, including GC, either as oncogenes or tumor suppressors [12]. Hence, exploring the cancer-associated miRNAs may broaden our understanding of the underlying mechanisms of tumorigenesis, and shed new light on prognosis prediction.

Circulating tumor-derived miRNAs were first described in peripheral blood by Mitchell et al., who found that tumor-derived miRNAs in serum or plasma can serve as novel biomarkers for cancer detection [18].Mitchell et al. also found that circulating miRNAs were resistant to plasma RNase activity, and were highly stable after prolonged incubation at room temperature and/or after multiple freezing–thawing processes. Since then, the detection of circulating tumor-derived miRNAs has been the focus of clinical studies, and accumulating reports have confirmed the potential of circulating tumor-derived miRNAs as novel tumor biomarkers for prognosis prediction in various cancers, such as leukemia, colorectal, cervical, lung, liver and prostate cancers [19, 20, 21, 22, 23, 24]. From a clinical perspective, the advantages of using circulating miRNAs as tumor biomarkers for prognosis prediction are obvious. First, compared with tissue biopsy specimens, the obtaining of blood specimens is more convenient and less invasive. Second, circulating miRNAs are superior to proteins due to their relatively smaller molecular weight, endogenous power of targeting more broad-range regulators and higher stability in clinical samples. Third, the methods of isolation and detection of circulating miRNAs is relatively cheap and simple.

Located within the seventh intron of epidermal growth factor-like protein 7 gene (EGFL7) on human chromosome 9 [25], miR-126 is one of the most intensively studied cancer-associated miRNAs. First identified as a miRNA regulating human megakaryocytopoiesis [26], miR-126 was found to be commonly down-regulated in several types of cancers and function as tumor suppressor by inhibiting tumor cell proliferation, migration and invasion, through different target genes. For example, miR-126 expression was found to be significantly decreased in papillary thyroid carcinoma tissues and cell lines, and restoration of miR-126 in papillary thyroid carcinoma cells inhibited cell proliferation, colony formations, migration and invasion in vitro, as well as tumor growth in vivo through targeting low-density lipoprotein receptor-related protein 6 (LRP6) [27]. In addition, accumulating reports indicate that aberrantly expressed miR-126 in tumor tissues is also a potentially useful biomarker for cancer screening, diagnosis, and prognosis prediction. For example, Kitano et al. found that miR-126 was down-regulated in thyroid carcinomas compared to benign tumors, the use of miR-126 may help to distinguish thyroid carcinoma from benign thyroid lesions in fine needle aspiration biopsy [28]. Han et al. reported that low expression of miR-126 in tumor tissues was associated with poor prognosis in glioblastoma [29]. Notably, it was found that circulating miR-126 could also serve as a novel biomarker for metastasis detection. For example, Lin et al reported that serum miR-126 level was significantly down-regulated in Stage IV non-small-cell lung cancer patients compared to Stage I/II patients and healthy controls [30]. Yin et al. identified miR-126 as one of the metastasis-associated miRNAs in colorectal cancer, serum miR-126 level was found to be significantly down-regulated in synchronous liver-metastatic colorectal cancer and other organ-metastatic colorectal cancer patients compared to localized colorectal cancer patients [31].

A series of observations have also been reported that miR-126 played an important role in the development of GC as a tumor suppressor. For example, bioinformatics analysis and luciferase reporter assay revealed that miR-126 directly targeted the 3’-untranslated region (3’-UTR) of VEGF-A mRNA in GC. In vivo xenograft mice model experiments clarified the down-regulation of VEGF-A and microvessel density (MVD) as well as inhibition of tumor growth by up-regulation of miR-126 [32].

We have previously demonstrated for the first time that miR-126 may function as a tumor suppressor in human GC with Crk as its direct target gene [12]. Compared with matched adjacent non-tumor tissues, miR-126 was significantly down-regulated while Crk protein was significantly up-regulated in tumor tissues, combined miR-126-low/Crk protein-high expression in tumor tissues was found to be an independent unfavorable prognostic factor of GC in our recent study [14].

Despite growing evidence highlighting its pivotal roles in the progression of GC, no study has systematically explored the prognostic value of circulating miR-126 in GC. The current study thus focused on the clinical significance of preoperative serum level of miR-126 as a prognosis predictor in GC patients. We observed the reduced expression of miR-126 in both GC tumor tissues and patients’ sera comparing to that in adjacent non-tumor tissues and healthy control’s sera, respectively. We also found that serum expression level of miR-126 was positively correlated with that in tumor tissue. We further examined the correlation between serum miR-126 level and the progression and prognosis of GC. Our results demonstrated that the decreased level of serum miR-126 was associated with various clinicopathological factors related with poor prognosis, such as tumor size, local invasion, lymph nodes metastasis and TNM stage. Moreover, GC patients with lower serum miR-126 level had worse prognosis compared with those with higher level. Cox proportional hazards regression model analysis revealed that low expression level of serum miR-126 was an independent factor predicting poor outcome of GC patients. In this respect, serum miR-126 might possess the same potential in prognosis prediction of GC as found with the classical prognostic factors such as lymph node metastasis or TNM stage. Furthermore, technically speaking, serum is more convenient and noninvasive, which appears to be a desirable source of biomarkers for prognosis prediction.

In conclusion, our data offer the convincing evidence for the first time that the serum miR-126 expression was markedly and consistently decreased in GC patients, and low serum miR-126 level was associated with poor prognosis. Serum miR-126 thus has potential clinical utility as a promising biomarker for molecularly monitoring the progression and predicting the clinical outcome of patients with GC. This discovery may help to better identify GC patients with poor prognostic potentials and facilitate personalized treatment of GC patients.

Footnotes

Acknowledgments

We appreciate all subjects that participated in this study. This project was supported, in part, by the grant from Shanghai Municipal Education Commission (12zz102), Yi Gong Jiao Cha Foundation of Shanghai Jiao Tong University (YG2016MS65) and Shanghai Pujiang Program (17PJ1406000).

Conflict of interest

The authors declare no conflict of interest.

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Down-regulated serum miR-126 is associated with aggressive progression and poor prognosis of gastric cancer

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Materials and methods

2.1 Patients and clinical samples

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

2.3 Statistical analysis

Table 1 Positive correlation between tissue and serum levels of miR-126 in GC

3.1 Expression levels of miR-126 were down-regulated in both tumor tissues and sera of GC patients

3.2 Down-regulation of serum miR-126 was associated with adverse clinicopathological parameters of GC patients

Table 2 Clinicopathological associations of serum miR-126 expression in GC

4. Discussion

Footnotes

Acknowledgments

Conflict of interest

References

Table 1
Positive correlation between tissue and serum levels of miR-126 in GC

Table 2
Clinicopathological associations of serum miR-126 expression in GC