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Abstract

BACKGROUND:

Few biomarkers are available for the prediction of prognosis and recurrence in lymph node (LN)-negative gastric cancer (GC) currently. miR-126 functions as a tumor suppressor in GC, however, its clinical significance in LN-negative GC remains unknown.

AIM:

To investigate the associations of tissue miR-126 level with the clinicopathological characteristics and clinical outcome of LN-negative GC patients.

METHODS:

Quantitative real-time polymerase chain reaction was performed to examine the tissue miR-126 level in 315 LN-negative GC patients who underwent curative gastrectomy with D2 lymphadenectomy. The associations of tissue miR-126 level with clinicopathological characteristics and clinical outcome were evaluated.

RESULTS:

Compared with matched adjacent non-tumor tissues, miR-126 expression was significantly down-regulated in tumor tissues. A reduced tissue miR-126 level statistically correlated with aggressive clinicopathological characteristics, including larger tumor size, deeper local invasion, and poorer prognosis. Notably, multivariate analysis identified advanced T stage and low miR-126 level as independent predictors of the unfavorable prognosis and recurrence of LN-negative GC.

CONCLUSIONS:

These results indicate for the first time that advanced T stage and low miR-126 level are predictors of unfavorable prognosis and recurrence in LN-negative GC patients. These parameters should be taken into account to stratify patients for adjuvant therapy and close follow-up.

Keywords

Gastric cancer lymph node negative prognosis recurrence miR-126

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, especially in Japan, South Korea and China [1, 2]. Despite the tremendous advances in the understanding of the mechanism of pathogenesis, early detection and comprehensive cancer treatment approaches of GC, the overall 5-year survival rate for GC patients still remains unsatisfactory [3].

Currently, the prediction of prognosis of GC patients is mainly determined by the traditional Tumor, Node Status, Metastasis (TNM) classification system. And the lymph node (LN) metastasis is widely recognized as one of the most important determinants of prognosis in GC patients following curative gastrectomy [4, 5]. In theory, GC patients with localized tumor without LN and distant metastasis may be cured by R ${}_{0}$ resection alone. Several reports have shown that GC patients without LN metastasis demonstrate substantially better outcome than those with LN metastasis [6, 7]. However, we can’t ignore the fact that a subgroup of LN-negative GC patients still experience recurrence, metastasis and cancer-related death [8, 9]. Obviously, there are several limitations in the traditional TNM staging system in terms of prognosis and recurrence prediction. Therefore, it is of prime importance to identify novel biomarkers for the prediction of prognosis and recurrence of LN-negative GC patients, and henceforth design more appropriate adjuvant therapy and surveillance plans for them.

microRNAs (miRNAs) are a novel class of small non-coding RNAs that regulate gene expression posttranscriptionally, either by translational repression or by mRNA degradation [10]. It is estimated that miRNAs may regulate about 30% genes in mammals, and govern important cellular processes, including cell differentiation, proliferation, and apoptosis [11]. 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, 13].

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 with Crk as its direct target gene [14]. Of note, we also found miR-126 down-regulation and Crk protein up-regulation may be synergistically associated with tumor progression in GC and may predict unfavorable prognosis of GC [15]. However, the prognostic value of miR-126 in LN-negative GC patients is yet to be elucidated. The aim of this study is to identify the role of clinicopathological factors in predicting the clinical outcome of LN-negative GC patients undergoing curative resection, with particular focus on the effect of miR-126 on LN-negative GC patients’ prognosis and recurrence.

2. Materials and methods

2.1 Patients and tissue samples

A total of 315 LN-negative GC patients who underwent curative gastrectomy plus D2 lymphadenectomy with more than 15 LNs dissected from 2006 to 2011 at the Department of General Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (Shanghai, China) were enrolled in this study. Inclusion criteria for this study were listed as follows: (1) Primary gastric adenocarcinoma confirmed pathologically. (2) No evidence of LN, peritoneal and distant metastasis. (3) No other organs malignancy. (4) Pathologically negative resection margins. (5) No history of neoadjuvant chemotherapy or radiotherapy in order to eliminate potential treatment-induced alterations in gene expression profiles. (6) Patient who wasn’t lost during follow-up. 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.

All the operations were performed by well-trained surgeons. LNs were meticulously dissected from the en bloc specimens by the surgeons. Dissected LNs were fixed in 10% formalin, embedded in paraffin, stained with hematorylin-eosin, then assessed independently by two gastroenterology pathologists blinded to the clinical data according to the Japanese General Rules for Gastric Cancer Study in Surgery and Pathology [16].

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 associations of miR-126 levels and clinicopathological factors with the prognosis in the LN-negative GC patients since it allowed controlling for unrelated causes of death.

Ethical approval was granted by the Ethics Committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (Shanghai, China). Signed informed consent was obtained from all patients for the acquisition and use of specimens and clinical data.

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

RNA isolation and qRT-PCR were performed as described previously [15]. Briefly, total RNA was isolated from tissue samples using mirVana™ miRNA Isolation Kit (Ambion) according to the manufacturer’s instructions. The quality and quantity of the RNA samples were assessed by standard electrophoresis and spectrophotometric methods. The expression level of mature miR-126 was measured by qRT-PCR according to the Taqman MicroRNA Assays protocol (Applied Biosystems) and normalized using U6 small nuclear RNA (RNU6B; Applied Biosystems) by the 2 ${}^{-\Delta\text{CT}}$ method. The relative expression ratio of miR-126 in each paired tumor to non-tumor tissue was calculated by the 2 ${}^{-\Delta\Delta\text{CT}}$ method. The miR-126 expression level was defined as down-regulation in tumor tissue when the relative expression ratio $<$ 0.5 [15].

2.3 Clinical data collection

The medical records of the 315 patients were retrospectively analyzed to determine the associations of tissue miR-126 level with the clinicopathological characteristics (including age at operation, sex, tumor size, tumor site, T stage, and histologic type) and clinical outcome of LN-negative GC patients. The exact T stage was determined according to the TNM staging system of the 7 ${}^{\text{th}}$ UICC/AJCC TNM Classification. Histologic types were classified as differentiated (papillary adenocarcinoma, or moderately or well-differentiated adenocarcinoma) or undifferentiated (poorly differentiated or undifferentiated adenocarcinoma, signet-ring cell carcinoma, or mucinous adenocarcinoma) based on the Japanese Classification of Gastric Carcinoma (JCGC) [16].

2.4 Statistical analysis

The chi-square test was used to evaluate the association between categorical variables. The cumulative probability of disease-specific survival was estimated by means of the Kaplan-Meier method. Univariate analyses of prognostic factors associated with disease-specific survival were performed using log-rank tests. Factors with significant effects on disease-specific survival in the univariate analyses were then subjected to multivariate analysis using a Cox proportional hazards model, using the forward stepwise procedure for variable selection, to identify independent prognostic factors. Univariate analyses of risk factors potentially associated with recurrence were performed using chi-square test. Factors found to be significant in univariate analysis were included in subsequent multivariate logistic regression analysis to identify independent risk factors associated with recurrence. $p$ value less than 0.05 was considered statistically significant. All statistical analyses were performed with SPSS statistical analysis program package (IBM SPSS Statistics 20).

3. Results

3.1 Clinicopathological characteristics

Clinicopathological characteristics of 315 gastric cancer patients in this study were summarized in Table 1. Of the 315 patients, 218 (69.2%) were male and 97 (30.8%) were female. One hundred and thirty-four (42.5%) were 60 years of age or younger at the time of surgery and 181 (57.5%) were older than 60 years. With respects to tumor site, the tumor was found in the lower third of the stomach in 156 (49.5%) patients, in the middle third of the stomach in 110 (34.9%) patients, in the higher third of the stomach in 49 (15.6%) patients. For T staging, 65 (20.6%) patients were T1 stage, 85 (27.0%) patients were T2 stage, 97 (30.8%) patients were T3 stage, and 68 (21.6%) patients were T4 stage. In terms of histologic type, 116 (36.8%) patients presented with differentiated tumor while 199 (63.2%) patients presented with undifferentiated tumor.

3.2 miR-126 expression was down-regulated in tumor tissues of LN-negative GC

The results of real-time qRT-PCR analysis showed that the range and median of expression level of miR-126 in tumor tissues were 95.2 and 115.3, respectively. The range and median of expression level of miR-126 in matched adjacent non-tumor tissues were 162.7 and 193.5, respectively. The expression level of miR-126 was significantly lower in tumor tissues than that in matched adjacent non-tumor tissues (mean $\pm$ SD, tumor tissues vs. matched non-tumor tissues: 112.6 $\pm$ 21.7 vs. 190.5 $\pm$ 37.8, $p<$ 0.05, Fig. 1).

Table 1
Clinicopathological associations of miR-126 expression in LN-negative GC

		miR-126 level
Factors	No. of	Low ( $n$ , %)	High ( $n$ , %)	$p$ value
	cases
Age
$\leqslant$ 60	134	30 (22.4%)	104 (77.6%)	0.401
$>$ 60	181	48 (26.5%)	133 (73.5%)
Sex
Male	218	50 (22.9%)	168 (77.1%)	0.260
Female	97	28 (28.9%)	69 (71.1%)
Tumor size
$\leqslant$ 5 cm	220	30 (13.6%)	190 (86.4%)	0.000*
$>$ 5 cm	95	48 (50.5%)	47 (49.5%)
Tumor site
Lower	156	30 (19.2%)	126 (80.8%)	0.009*
Middle	110	28 (25.5%)	82 (74.5%)
Upper	49	20 (40.8%)	29 (59.2%)
Histologic type
Differentiated	116	30 (25.9%)	86 (74.1%)	0.730
Undifferentiated	199	48 (24.1%)	151 (75.9%)
T stage
T1	65	2 (3.1%)	63 (96.9%)	0.000*
T2	85	9 (10.6%)	76 (89.4%)
T3	97	32 (33.0%)	65 (67.0%)
T4	68	35 (51.5%)	33 (48.5%)

* $p<$ 0.05 was considered statistically significant.

Figure 1.

Down-regulation of miR-126 expression in GC tissues compared with matched adjacent non-tumor tissues. qRT-PCR for miR-126 expression was carried out using 315 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.

3.3 Down-regulation of miR-126 was associated with adverse clinical parameters of LN-negative GC

As shown in Table 1, we found that the miR-126 low-expression group favored larger tumor size, and deeper local invasion (both $p<$ 0.05), however, tissue miR-126 level wasn’t significantly correlated with sex, age, and histologic type (all $p>$ 0.05). These results suggested that miR-126 might act as a tumor suppressor in the progression of LN-negative GC.

3.4 Univariate survival analysis

The 5-year disease-specific survival rate of the entire cohort was 78.7%. The clinicopathological factors tested in the univariate analysis were shown in Table 2. Factors having significant influence on the survival included tumor size, T stage and tissue miR-126 level (all $p<$ 0.05). However, sex, age, tumor site, and histologic type had no significant influence on the survival.

Table 2
Univariate analyses of prognostic factors in LN-negative GC

Factors	No. of cases	5-year survival (%)	$p$ value
All	315	78.7	N/A
Age			0.321
$\leqslant$ 60	134	81.3
$>$ 60	181	76.8
Sex			0.517
Male	218	79.8
Female	97	76.3
Tumor size			0.000*
$\leqslant$ 5 cm	220	84.5
$>$ 5 cm	95	65.3
Tumor site			0.231
Lower	156	78.8
Middle	110	82.7
Upper	49	69.4
Histologic type			0.950
Differentiated	116	78.4
Undifferentiated	199	78.9
T stage			0.000*
T1	65	95.4
T2	85	89.4
T3	97	75.3
T4	68	54.4
miR-126 level			0.000*
Low	78	46.2
High	237	89.5

* $p<$ 0.05 was considered statistically significant.

Table 3

Multivariate analyses of prognostic factors in LN-negative GC

Factors	HR	95% CI	$p$ value
Tumor size	–	–	0.590
T stage	1.822	1.346–2.466	0.000*
miR-126 level	0.218	0.129–0.369	0.000*

* $p<$ 0.05 was considered statistically significant.

3.5 Multivariate survival analysis

All the three factors considered significant in univariate analysis (tumor size, T stage, and tissue miR-126 level) were included in the Cox proportional hazards model to determine the independent prognostic factors. As a result, there were two independent, statistically significant prognostic factors: T stage and tissue miR-126 level. The hazard ratio and their 95% confidence interval were listed in Table 3.

3.6 Down-regulation of miR-126 was associated with poor prognosis of LN-negative GC patients

As shown in Fig. 2, there was a significant difference in the survival between the miR-126 high-expression group and low-expression group. The 5-year disease-specific survival rate in the patients with low miR-126 level was 46.2%, whereas that in the patients with high miR-126 level was 89.5% ( $p<$ 0.05).

Table 4
Clinicopathological features associated with recurrence in LN-negative GC

Factors	No. of	Recurrence	Recurrence	$p$ value
	cases	( $-$ ), $n$ (%)	( $+$ ), $n$ (%)
Age
$\leqslant$ 60	134	103 (76.9%)	31 (23.1%)	0.185
$>$ 60	181	127 (70.2%)	54 (29.8%)
Sex
Male	218	162 (74.3%)	56 (25.7%)	0.437
Female	97	68 (70.1%)	29 (29.9%)
Tumor size
$\leqslant$ 5 cm	220	176 (80.0%)	44 (20.0%)	0.000*
$>$ 5 cm	95	54 (56.8%)	41 (43.2%)
Tumor site
Lower	156	117 (75.0%)	39 (25.0%)	0.058
Middle	110	84 (76.4%)	26 (23.6%)
Upper	49	29 (59.2%)	20 (40.8%)
Histologic type
Differentiated	116	85 (73.3%)	31 (26.7%)	0.937
Undifferentiated	199	145 (72.9%)	54 (27.1%)
T stage
T1	65	57 (87.7%)	8 (12.3%)	0.000*
T2	85	75 (88.2%)	10 (11.8%)
T3	97	64 (66.0%)	33 (34.0%)
T4	68	34 (50.0%)	34 (50.0%)
miR-126 level
Low	78	29 (37.2%)	49 (62.8%)	0.000*
High	237	201 (84.8%)	36 (15.2%)

* $p<$ 0.05 was considered statistically significant.

Figure 2.

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

3.7 Relationship between clinicopathologic characteristics and recurrence

Table 5 showed the relationship between clinicopathological characteristics and recurrence. Recurrence was significantly correlated with tumor size, T stage, and tissue miR-126 level (all $p<$ 0.05, Table 4). A multivariate logistic regression analysis showed that T stage and tissue miR-126 level were independent predictors of recurrence (both $p<$ 0.05, Table 5).

Table 5
Independent predictors of recurrence in LN-negative GC

Factor	OR	95% CI	$p$ value
T stage	1.611	1.182–2.196	0.003*
miR-126 level	0.155	0.084–0.288	0.000*

* $p<$ 0.05 was considered statistically significant.

4. Discussion

Despite the recent advances in diagnosis, surgical techniques, perioperative management, and concept of chemotherapy, GC remains one of the most common malignant tumors and the third leading cause of cancer death worldwide [17]. The LN metastasis is generally regarded as one of the most powerful prognostic factors for GC, and GC patients without LN metastasis usually had a better survival than those with LN metastasis. It has been reported that the 5-year disease-specific survival rate of LN-negative GC varied from 73 to 86.1% [18, 19]. The 5-year disease-specific survival in our group of patients was 78.7%, which was similar to previous studies.

However, recurrence and metastasis are still noted in a subset of LN-negative GC patients after curative resection, these patients may benefit from more aggressive adjuvant therapy, which has been shown to improve survival of GC patients when compared with gastrectomy alone [20, 21]. Therefore, the identification of novel biomarkers associated with poor prognosis and recurrence in LN-negative GC patients will help to select patients after curative resection for adjuvant therapy and surveillance, and ultimately improve the prognosis. Nevertheless, biomarkers useful for progression monitoring and prognosis prediction of LN-negative GC patients are still lacking today.

The discovery that miRNAs expression is frequently dysregulated in a variety of cancers, and miRNAs have multiple target genes that are involved in cell growth and signaling pathways may broaden our understanding of the underlying mechanisms of carcinogenesis, and shed new light on prognosis prediction. As far as GC is concerned, various studies have shown that miRNAs play critical roles in the development of GC, and are promising indicators of prognosis. For example, upregulation of miR-125b [22], or downregulation of miR-338-3p is associated with poor prognosis of GC [23]. However, to date, few studies have specifically addressed the significance of miRNAs in the prognosis and recurrence of LN-negative GC.

Located on chromosome 9 within the seventh intron of the epidermal growth factor-like domain 7 (EGFL7), miR-126 is one of the most intensively studied cancer-related miRNAs in cancer patients [24]. First identified as a miRNA regulating human megakaryocytopoiesis [25], miR-126 was found to be commonly down-regulated in various cancers, and was thought to be a tumor suppressor through its ability to modulate angiogenesis, tumor growth, and metastasis. For example, miR-126 was found to be highly expressed in low-grade glioma tissues but lowly expressed in high-grade ones, and miR-126 inhibited the migration and invasion of glioma cells by suppressing GATA4 protein expression [26]. Compared to normal B cells, miR-126 was downregulated in chronic lymphocytic leukemia (CLL) cells, and overexpression of miR-126 in leukemia cell lines significantly downregulated p85 $\beta$ expression and decreased activation of prosurvival mitogen-activated protein kinase (MAPK) signaling [27]. miR-126 negatively regulated the PIK3R2 expression and further inhibited the PI3K/Akt signaling pathway, thereby reducing proliferation, migration, and invasion of bladder carcinoma cells and promoting cell apoptosis [28]. Furthermore, accumulating evidences demonstrates that aberrantly expressed miR-126 is a potentially useful biomarker for cancer screening, diagnosis, and prognosis prediction. For example, combination of circulating miR-126 and AFP was a promising noninvasive diagnostic biomarker for detection of hepatitis B virus infected hepatocellular carcinoma [29]. Low miR-126 expression was associated with poorer survival in patients with colorectal cancer [30].

miR-126 also play vital roles in the progression of GC. For example, down-regulation of miR-126 was found to inversely correlate with an increased microvessel density (MVD) and vascular endothelial growth factor A (VEGF-A) expression in GC tissues. miR-126 could suppress tumor growth and tumor angiogenesis of GC through VEGF-A signaling [31]. Our previous studies demonstrated for the first time that miR-126 may function as a tumor suppressor in human GC through down-regulation of Crk [14]. In addition, combined miR-126-low/Crk protein-high expression in tumor tissues was found to be an independent unfavorable prognostic factor of GC [15]. But there have been no reports describing the prognostic relevance of miR-126 in LN-negative GC. Hence, in current study, we studied the clinical implication of miR-126 expression in LN-negative GC using a relatively large number of samples.

In most cases, it is the pathologist who retrieves and examines LNs. However, according to our own experience, the retrieved LNs number per specimen varies between surgeons and pathologists, surgeons retrieved more LNs than pathologists did. It has been found that increased number of retrieved LNs can reduce stage deviation and guide decision making for chemotherapy. Jiang and colleagues reported that compared with retrieval performed by pathologists, postoperative LNs retrieval performed by surgeons was associated with significant increase in the total number of LNs in stage I patients, the numbers of positive and total LNs in stage II and III patients. As a result, survival rate of stage II and III patients were significantly higher in the LNs retrieval performed by surgeons group than that in the LNs retrieval performed by pathologist group due to more accurate staging, which helped to facilitate a decision regarding the subsequent chemotherapy treatment [32]. In order to improve quality control in retrieving LNs postoperatively, and determine N stage more accurately, LNs were routinely retrieved by surgeons and then submitted to the pathologists for further examination in our department. In addition, all the patients enrolled in current study successfully underwent radical resection plus D2 lymphadenectomy with pathologically negative resection margins to ensure complete resection. Furthermore, none of patients had undergone preoperative adjuvant therapy before surgery. As a result, statistical bias was decreased and the results of current study were more objective.

Among various clinicopathological factors that have been reported to influence the survival of LN-negative GC, depth of tumor invasion is consistently reported as an adverse prognostic factor [33]. Depth of invasion, especially the presence of serosa involvement was found to be the independent negative predictor of survival [9]. Study by Lee et al. [34] demonstrated that patients without serosal invasion (T1-2) and with serosal invasion (T3-4) had a large difference in survival rates; the depth of invasion was an independent predictor of poor outcome in LN-negative GC patients. As the depth of tumor invasion increases, the tumor would be more likely to invade the blood vessels, thus increasing the likelihood of metastasis. Once tumor further perforates serosa, peritoneal seeding may occur. In addition, tumor associated-stroma secretes several factors such as stromal cell-derived factor 1 and cytokines to promote tumor invasion and stimulate cancer cell metastasis [35]. All of these factors mentioned above increased the possibility of metastasis and recurrence, leading to poorer prognosis. Our data also demonstrated that the depth of invasion was an independent prognostic factor of LN-negative GC. Of note, the findings of our study clearly showed that the down-regulation of miR-126 was significantly associated with advanced T stage. Moreover, the survival analysis demonstrated that the disease-specific survival rate of LN-negative GC patients with low miR-126 expression level was significantly lower than that of the patients with high miR-126 expression level. To eliminate the impact of mixed factors correlated with prognosis on statistical analysis, the Cox proportional hazards model was performed to establish the independent prognostic factors. The results of multivariate analysis confirmed that low miR-126 expression level and advanced T stage were independent unfavorable prognostic factors of LN-negative GC.

Unlike other solid tumors such as breast and lung cancer, tumor size is not currently included in the tumor-node-metastasis (TNM) staging system of GC, and the prognostic significance of tumor size has yet to be determined in LN-negative GC. In our current study, tumor size failed to demonstrate a significant association with survival in the multivariate analysis. Other investigators such as Lee et al. [9] also found that tumor size didn’t independently influence survival of LN-negative GC patients.

In our previous studies, we found that miR-126 expression was closely related to LN metastasis, and reduced miR-126 level was an independent predictor for the unfavorable prognosis of GC [15, 36, 37]. In current study, we found reduced miR-126 level was also an independent predictor for the unfavorable prognosis of LN-negative GC. The explanation for this phenomenon might be that down-regulation of miR-126 may be associated with LN micrometastasis in histologically LN-negative GC patients because of its strong association with LN metastasis. Due to advance in technology, such as immunohistochemistry (IHC) staining and reverse transcription-polymerase chain reaction (RT-PCR) assay, the detection of LN micrometastasis of GC has increased significantly [38, 39]. It was reported that LNs micrometastasis was detected by immunohistochemical examinations among 10%–43% patients with LN-negative GC diagnosed by routine histologic examination [40, 41]. LN micrometastasis was found to be associated with poor outcome in patients with histologically node-negative GC [42, 43]. Therefore, we speculate that miR-126 down-regulation may represent the presence of LN micrometastasis in the histologically LN-negative GC patients, which lead to the poor outcome. Therefore, in the future, we plan to analyze the association between miR-126 expression and LN micrometastasis in LN-negative GC patients to determine whether such patients with low miR-126 level tend to have more micrometastatic LNs than those with high miR-126 level.

Despite the favourable prognosis of LN-negative GC, some patients still developed recurrence. The recurrence rates were reported to vary from 9.4% to 29.4% in LN-negative GC patients [44]. The recurrence rate in our study group is 27.0%, which is consistent with other publications in this field. In addition, we found advanced T stage and low miR-126 level were the independent risk factors for recurrence.

In conclusion, although the LN-negative GC patients generally have an excellent prognosis, some patients may still have recurrence and die. To the best of our knowledge, the current study demonstrated for the first time that miR-126 expression was markedly and consistently decreased in primary tumor, and low miR-126 expression could predict the poor outcome and recurrence in patients with LN-negative GC. miR-126 thus has potential clinical utility as a promising biomarker for molecularly monitoring the progression and predicting the clinical outcome of patients with LN-negative GC. This discovery may help to better identify LN-negative GC patients with poor prognostic potentials and facilitate personalized treatment of these 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).

References

Torre

L.A.

Bray

Siegel

R.L.

Ferlay

Lortet-Tieulent

and Jemal

, Global cancer statistics, 2012, CA Cancer J Clin 65 (2015), 87–108.

Chen

Zheng

Zhang

Zeng

Zuo

Xia

et al., Cancer incidence and mortality in China in 2013: An analysis based on urbanization level, Chin J Cancer Res 29 (2017), 1–10.

Kusano

Shiraishi

Shiroshita

Etoh

Inomata

and Kitano

, Poor prognosis of advanced gastric cancer with metastatic suprapancreatic lymph nodes, Ann Surg Oncol 20 (2013), 2290–2295.

Lee

J.H.

Kang

J.W.

Nam

B.H.

Cho

G.S.

Hyung

W.J.

Kim

M.C.

et al., Correlation between lymph node count and survival and a reappraisal of lymph node ratio as a predictor of survival in gastric cancer: A multi-institutional cohort study, Eur J Surg Oncol 43 (2017), 432–439.

Wang

Zheng

C.H.

Fang

Xie

J.W.

et al., Influence of total lymph node count on staging and survival after gastrectomy for gastric cancer: An analysis from a two-institution database in China, Ann Surg Oncol 24 (2017), 486–493.

Kim

D.Y.

Seo

K.W.

Joo

J.K.

Park

Y.K.

Ryu

S.Y.

Kim

H.R.

et al., Prognostic factors in patients with node-negative gastric carcinoma: A comparison with node-positive gastric carcinoma, World J Gastroenterol 12 (2006), 1182–1186.

Deng

Liang

Sun

Zhang

Zhan

and Wang

, Prognosis of gastric cancer patients with node-negative metastasis following curative resection: Outcomes of the survival and recurrence, Can J Gastroenterol 22 (2008), 835–839.

Dittmar

Schüle

Koch

Rauchfuss

Scheuerlein

and Settmacher

, Predictive factors for survival and recurrence rate in patients with node-negative gastric cancer – a European single-centre experience, Langenbecks Arch Surg 400 (2015), 27–35.

Lee

I.S.

Yook

J.H.

Kim

T.H.

Kim

H.S.

Kim

K.C.

S.T.

et al., Prognostic factors and recurrence pattern in node-negative advanced gastric cancer, Eur J Surg Oncol 39 (2013), 136–140.

10.

Jonas

and Izaurralde

, Towards a molecular understanding of microRNA-mediated gene silencing, Nat Rev Genet 16 (2015), 421–433.

11.

Dong

Lei

Ding

Wen

Zhang

, MicroRNA: Function, detection, and bioanalysis, Chem Rev 113 (2013), 6207–6233.

12.

Huang

Sun

and Li

, miR-135b-5p promotes gastric cancer progression by targeting CMTM3, Int J Oncol 52 (2018), 589–598.

13.

Gao

Wang

Jing

Zhan

and Wang

, microRNA-203 suppresses invasion of gastric cancer cells by targeting ERK1/2/Slug/E-cadherin signaling, Cancer Biomark 19 (2017), 11–20.

14.

Feng

Chen

et al., miR-126 functions as a tumour suppressor in human gastric cancer, Cancer Lett 298 (2010), 50–63.

15.

Feng

Sah

B.K.

Beeharry

M.K.

Yuan

Jin

et al., Dysregulation of miR-126/Crk protein axis predicts poor prognosis in gastric cancer patients, Cancer Biomark 21 (2018), 335–343.

16.

Japanese Gastric Cancer Association, Japanese classification of gastric carcinoma: 3rd English edition, Gastric Cancer 14 (2011), 101–112.

17.

Ferlay

Soerjomataram

Dikshit

Eser

Mathers

Rebelo

et al., Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012, Int J Cancer 136 (2015), E359–386.

18.

Kooby

D.A.

Suriawinata

Klimstra

D.S.

Brennan

M.F.

and Karpeh

M.S.

, Biologic predictors of survival in node-negative gastric cancer, Ann Surg 237 (2003): 828–835; discussion 835–837.

19.

Baiocchi

G.L.

Tiberio

G.A.

Minicozzi

A.M.

Morgagni

Marrelli

Bruno

et al., A multicentric Western analysis of prognostic factors in advanced, node-negative gastric cancer patients, Ann Surg 252 (2010), 70–73.

20.

Sasako

Sakuramoto

Katai

Kinoshita

Furukawa

Yamaguchi

et al., Five-year outcomes of a randomized phase III trial comparing adjuvant chemotherapy with S-1 versus surgery alone in stage II or III gastric cancer, J Clin Oncol 29 (2011), 4387–4393.

21.

GASTRIC (Global Advanced/Adjuvant Stomach Tumor Research International Collaboration) Group Paoletti

Oba

Burzykowski

Michiels

Ohashi

et al., Benefit of adjuvant chemotherapy for resectable gastric cancer: A meta-analysis, JAMA 303 (2010), 1729–1737.

22.

J.G.

Wang

J.J.

Jiang

Lan

J.P.

X.J.

Wang

H.J.

et al., MiR-125b promotes cell migration and invasion by targeting PPP1CA-Rb signal pathways in gastric cancer, resulting in a poor prognosis, Gastric Cancer 18 (2015), 729–739.

23.

Liu

Suo

Wang

Sun

Wang

et al., Downregulation of tissue miR-338-3p predicts unfavorable prognosis of gastric cancer, Cancer Biomark 21 (2017), 117–122.

24.

Wang

Aurora

A.B.

Johnson

B.A.

McAnally

Hill

J.A.

et al., The endothelial-specific microRNA miR-126 governs vascular integrity and angiogenesis, Dev Cell 15 (2008), 261–271.

25.

Garzon

Pichiorri

Palumbo

Iuliano

Cimmino

Aqeilan

et al., MicroRNA fingerprints during human megakaryocytopoiesis, Proc Natl Acad Sci USA 103 (2006), 5078–5083.

26.

Dai

and Liang

, miR-126 affects the invasion and migration of glioma cells through GATA4, Artif Cells Nanomed Biotechnol 45 (2017), 1–7.

27.

Guinn

Lehman

Fabian

Maddocks

Andritsos

L.A.

et al., The regulation of tumor-suppressive microRNA, miR-126, in chronic lymphocytic leukemia, Cancer Med 6 (2017), 778–787.

28.

Xiao

Lin

H.Y.

Zhu

Y.Y.

Zhu

Y.P.

and Chen

L.W.

, MiR-126 regulates proliferation and invasion in the bladder cancer BLS cell line by targeting the PIK3R2-mediated PI3K/Akt signaling pathway, Onco Targets Ther 9 (2016), 5181–5193.

29.

Ghosh

Datta

Dasgupta

Das

Ray

et al., Hepatic miR-126 is a potential plasma biomarker for detection of hepatitis B virus infected hepatocellular carcinoma, Int J Cancer 138 (2016), 2732–2744.

30.

Ebrahimi

Gopalan

Wahab

C.T.

Smith

R.A.

and Lam

A.K.

, Deregulation of miR-126 expression in colorectal cancer pathogenesis and its clinical significance, Exp Cell Res 339 (2015), 333–341.

31.

Chen

Wang

Lei

et al., Reduced miR-126 expression facilitates angiogenesis of gastric cancer through its regulation on VEGF-A, Oncotarget 5 (2014), 11873–11885.

32.

Jiang

Yao

Zhang

Zhao

Zhai

et al., Comparison of lymph node number and prognosis in gastric cancer patients with perigastric lymph nodes retrieved by surgeons and pathologists, Chin J Cancer Res 28 (2016), 511–518.

33.

Zhou

Cui

J.G.

Huang

Zhang

Zhao

Z.C.

et al., Prognostic factors for survival in node-negative gastric cancer patients who underwent curative resection, Scand J Surg 106 (2017), 235–240.

34.

Lee

C.C.

C.W.

S.S.

Chen

J.H.

A.F.

Hsieh

M.C.

et al., Survival predictors in patients with node-negative gastric carcinoma, J Gastroenterol Hepatol 22 (2007), 1014–1018.

35.

Luga

and Wrana

J.L.

, Tumor-stroma interaction: Revealing fibroblast-secreted exosomes as potent regulators of Wnt-planar cell polarity signaling in cancer metastasis, Cancer Res 73 (2013), 6843–6847.

36.

Feng

Beeharry

M.K.

Sah

B.K.

Yuan

Yan

et al., Down-regulated serum miR-126 is associated with aggressive progression and poor prognosis of gastric cancer, Cancer Biomark 22 (2018), 119–126.

37.

Feng

Sah

B.K.

Beeharry

M.K.

Zhang

Yan

et al., Serum miR-126 level combined with multi- detector computed tomography in the preoperative prediction of lymph node metastasis of gastric cancer, Cancer Biomark (2018), DOI: 10.3233/CBM-181324.

38.

Kim

J.J.

Song

K.Y.

Hur

J.I.

Park

S.M.

and Park

C.H.

, Lymph node micrometastasis in node negative early gastric cancer, Eur J Surg Oncol 35 (2009), 409–414.

39.

Shimizu

Takeuchi

Sakakura

Saikawa

Nakahara

Mukai

et al., Molecular detection of sentinel node micrometastases in patients with clinical N0 gastric carcinoma with real-time multiplex reverse transcription-polymerase chain reaction assay, Ann Surg Oncol 19 (2012), 469–477.

40.

Kim

J.H.

Park

J.M.

Jung

C.W.

Park

S.S.

Kim

S.J.

Mok

Y.J.

et al., The significances of lymph node micrometastasis and its correlation with E-cadherin expression in pT1-T3N0 gastric adenocarcinoma, J Surg Oncol 97 (2008), 125–130.

41.

Kikuchi

Tsuchiya

Ando

Yoshida

and Takenosita

, Immunohistochemical detection of lymph node microinvolvement in node-negative gastric cancer, Gastric Cancer 2 (1999), 173–178.

42.

Yasuda

Adachi

Shiraishi

Inomata

Takeuchi

and Kitano

, Prognostic effect of lymph node micrometastasis in patients with histologically node-negative gastric cancer, Ann Surg Oncol 9 (2002), 771–774.

43.

Lee

C.M.

Cho

J.M.

Jang

Y.J.

Park

S.S.

Park

S.H.

Kim

S.J.

et al., Should lymph node micrometastasis be considered in node staging for gastric cancer?: The significance of lymph node micrometastasis in gastric cancer, Ann Surg Oncol 22 (2015), 765–771.

44.

Huang

C.M.

Lin

J.X.

Zheng

C.H.

Xie

J.W.

Lin

B.J.

et al., Prognostic impact of dissected lymph node count on patients with node-negative gastric cancer, World J Gastroenterol 31 (2009), 3926–3930.

miR-126: An indicator of poor prognosis and recurrence in histologically lymph node-negative gastric cancer

Abstract

BACKGROUND:

AIM:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Materials and methods

2.1 Patients and tissue samples

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

2.3 Clinical data collection

2.4 Statistical analysis

3. Results

3.1 Clinicopathological characteristics

3.2 miR-126 expression was down-regulated in tumor tissues of LN-negative GC

Table 1 Clinicopathological associations of miR-126 expression in LN-negative GC

3.4 Univariate survival analysis

Table 2 Univariate analyses of prognostic factors in LN-negative GC

3.6 Down-regulation of miR-126 was associated with poor prognosis of LN-negative GC patients

Table 4 Clinicopathological features associated with recurrence in LN-negative GC

Table 5 Independent predictors of recurrence in LN-negative GC

Footnotes

Acknowledgments

References

Table 1
Clinicopathological associations of miR-126 expression in LN-negative GC

Table 2
Univariate analyses of prognostic factors in LN-negative GC

Table 4
Clinicopathological features associated with recurrence in LN-negative GC

Table 5
Independent predictors of recurrence in LN-negative GC