Med Oncol (2014) 31:164 DOI 10.1007/s12032-014-0164-8
ORIGINAL PAPER
Circulating miR-222 in plasma and its potential diagnostic and prognostic value in gastric cancer Zhengchuan Fu • Fang Qian • Xuhuan Yang Hailiang Jiang • Yu Chen • Sihai Liu
•
Received: 21 July 2014 / Accepted: 7 August 2014 / Published online: 17 August 2014 Ó Springer Science+Business Media New York 2014
Abstract Previous studies have revealed the significance of circulating microRNAs as biomarkers for cancers. The aim of this study was to detect the levels of circulating microRNA-222 (miR-222) in plasma of gastric cancer (GC) patients and evaluate its diagnostic and prognostic value. Levels of circulating miR-222 were detected by using qRTPCR in plasma of 114 GC patients, 36 chronic atrophic gastritis (CAG) patients and 56 healthy controls. The result showed that the expression of circulating miR-222 in plasma was significantly upregulated in GC compared with CAG and healthy controls (all at P \ 0.001). And its high level was significantly correlated with clinical stages (P \ 0.001) and lymph nodes metastasis (P = 0.009). The receiver operating characteristics (ROC) curve analyses revealed that miR-222 had considerable diagnostic accuracy, yielded an AUC (the areas under the ROC curve) of 0.850 with 66.1 % sensitivity and 88.3 % specificity in discriminating GC from healthy controls. Moreover, Kaplan–Meier analysis demonstrated a correlation between increased circulating miR222 level and reduced disease-free survival (P = 0.016) and overall survival (P = 0.012). In multivariate analysis stratified for known prognostic variables, circulating miR-222 was identified as an independent prognostic marker. In conclusion, our findings suggested that circulating miR-222 in plasma might be a potential and useful noninvasive biomarker for the early detection and prognosis of GC. Keywords Gastric cancer Circulating miR-222 Detection Prognosis
Z. Fu (&) F. Qian X. Yang H. Jiang Y. Chen S. Liu Department of Oncology, Zaozhuang Mining Group Central Hospital, Qilianshan Road, Zaozhuang 277000, Shandong, China e-mail: [email protected]
Introduction Gastric cancer (GC) is the forth common malignancy and the second leading cause of cancer-related death in both sexes worldwide [1]. The prognosis of GC varies remarkably by the stage with the 5-year survival rates range from[90 % for stage I to\5 % for stage IV [2]. Thus, early detection of GC is critical to decrease the mortality rate and improve the prognosis for patients with GC. Although gastroscopic screening is currently the most reliable screening tool, the invasive nature and cost incurred have hampered its wide application. On the other hand, the current serum tumor markers such as carcinoembryonic antibody (CEA) and carbohydrate antibody 19-9 (CA19-9) were not sensitive and specific enough for GC screening [3, 4]. Thus, there is an urgent need for discovering novel noninvasive biomarkers to improve early detection or prognostic prediction of GC. MicroRNAs (miRNAs) are 18–24 nucleotide long evolutionarily conserved RNA molecules that regulate gene expression post-transcriptionally by binding to the 3-untranslated regions of target messenger RNAs [5]. miRNAs play important roles in the multistep carcinogenesis process through the dysregulation of oncogenes and tumor suppressor genes [6]. Several recent studies demonstrated that miRNAs are stably detectable in plasma or serum [7, 8]. Mitchell et al. [8] reported that tumorassociated circulating miRNAs are stably detectable in the plasma of human prostate cancer xenograft mouse models and prostate cancer patients, suggesting that their detection could differentiate cancer-bearing individuals from healthy controls. These findings also raised the possibility that assaying miRNAs in plasma or serum could serve as a novel approach for the noninvasive blood-based detection of human cancers. Actually, since the above study, several investigators have reported the significance of some types
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of circulating miRNAs as biochemical markers for human cancers [9–12]. In this study, we focused on miR-222. miR-222, known as an ‘oncomiR,’ has been observed to induce cell growth and cell cycle progression via direct targeting of p27 and p57 [13–15]. Overexpression of miRNA-222 is reported in many types of cancers such as thyroid papillary carcinomas [16], hepatocellular carcinoma [17], melanoma [18] and pancreatic cancer [19]. Also in GC, it was previously reported that the expression was significantly increased in cancer tissues and cell lines and that miRNA-222 targets tumor suppressor genes, such as PTEN and RECK [14, 20, 21]. In other studies, it was also confirmed that plasma circulating miRNA-222 was a useful biomarker for some types of human cancers [22–25]. Thus, we postulated that plasma circulating miRNA-222 expression could be a novel biochemical marker for GC. In the present study, we evaluated the usefulness of plasma circulating miRNA-222 as a biochemical marker for GC by comparing the expression in patients with GC and healthy controls. In addition, we also examined the prognostic significance of plasma circulating miRNA-222 for GC patients.
Materials and methods Patients and samples A total of 114 cases of newly diagnosed GC and 36 cases of chronic atrophic gastritis (CAG) were enrolled in this study between May 2005 and December 2008 in Zaozhuang Mining Group Central Hospital (Zaozhuang, China). Plasma specimens were collected before any therapeutic procedures, including surgery, chemotherapy and radiotherapy. All patients with GC were staged according to the UICC/TNM staging system, including 17 in stage I, 25 in stage II, 34 in stage III and 38 in stage IV. In total, 56 sex- and age-matched healthy subjects were collected as the healthy controls and each subject had no prior diagnosis of any other malignancy. Written informed consent was obtained from all participants, and research protocols were approved by the Ethical Committee of Zaozhuang Mining Group Central Hospital. All patients with GC, which have been reviewed at the intervals of 3 months during the initial 2–3 years and every 6 months thereafter, were evaluated at clinic. The follow-up was completed in all 114 patients until December 2013, and the median follow-up period was 24 months (range 4–60 months).
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was transferred into 1.5-ml Eppendorf tubes, followed by second centrifugation at 12,000g for 10 min at 4 °C to completely remove cellular components. The supernatant plasma was frozen as separate aliquots at -80 °C until use. Total RNA was extracted from 400 ml of plasma using a mirVana PARIS kit (Ambion, Austin, TX, USA) and eluted into 100 ll of pre-heated (95 °C) elution solution according to the manufacturer’s instructions. Quantification of microRNA by quantitative real-time PCR Amounts of miRNAs in plasma samples were quantified by quantitative real-time PCR (qRT-PCR) using the human TaqMan MicroRNA Assay Kit (Applied Biosystems, USA). The reverse transcription reaction was carried out using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) in 15 ll containing 5 ll of RNA extract, 1.5 ll of 109 reverse transcription buffer, 0.15 ll of 100 mM dNTPs, 1 ll of MultiScribe reverse transcriptase, 0.19 ll of RNase inhibitor, 1 ll of gene-specific primer and 4.16 ll of nuclease-free water. For synthesis of cDNA, the reaction mixtures were incubated at 16 °C for 30 min, at 42 °C for 30 min and at 85 °C for 15 min and then held at 4 °C. About 1.33 ll of cDNA solution was amplified by using 10 ll of TaqMan 29 Universal PCR Master Mix with No AmpErase UNG (Applied Biosystems), 1 ll of gene-specific primer and 7.67 ll of nucleasefree water in a final volume of 20 ll. Quantitative PCR was performed in triplicate on a 7,300 real-time PCR system (Applied Biosystems), and the reaction mixtures were incubated at 95 °C for 10 min, followed by 45 cycles of 95 °C for 15 s and 60 °C for 1 min. The cycle threshold (Ct) values were calculated with SDS 2.4 software (Applied Biosystems). The expression levels of the target miRNAs in the plasma were normalized relative to the expression of RNU6B and were calculated using the 2-DDC method [26]. Carcinoembryonic antibody and carbohydrate antibody 19-9 The serum levels of carcinoembryonic antibody (CEA) and carbohydrate antibody 19-9 (CA19-9) are commonly used in the diagnostics of gastrointestinal malignancies. CEA and CA19-9 were measured by chemiluminescent enzyme immunoassays (FUJIREBIO Inc., Tokyo, Japan) according to the manufacture’s instructions.
Samples processing and RNA extraction
Statistical analysis
Up to 8 ml of whole blood from each participant was collected in EDTA tubes. The blood samples were centrifuged at 1,200g for 10 min at 4 °C, and the supernatant
The Kruskal–Wallis test or the Mann–Whitney U test was used to compare miRNAs levels between groups. Receiver operating characteristic (ROC) curve analysis was
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performed to determine the diagnostic performance of miR-222 expression levels in distinguishing patients with GC from the healthy controls. The optimal cutoff thresholds for diagnosis were obtained by Youden index [27]. The Kaplan–Meier method was used to estimate survival rates, and the log-rank tests were used to assess survival differences between groups. Multivariate analysis of the prognostic factors was performed with Cox proportional hazards model. All statistical analyses were carried out by using SPSS 13.0 software (IBM, USA). A P value \0.05 was considered statistically significant.
RNU6B (Fig. 1) among healthy controls (n = 56), CAG patients (n = 36) and GC patients (n = 114), suggesting that RNU6B constitutively expressed in plasma regardless of different disease conditions. Levels of circulating miR-222 were detected by qRT-PCR in plasma of 114 GC patients, 36 CAG patients and 56 healthy controls. Our data indicated that there was a significant difference among GC, CAG and healthy controls (P \ 0.001). As shown in Fig. 2, the levels of circulating miR-222 in GC were higher than in CAG and healthy controls, respectively (P \ 0.001). In addition, the levels of circulating miR-222 were also significantly higher in CAG than in healthy controls (P \ 0.001).
Results
Circulating miR-222 correlates with clinicopathological features of GC
The expression of circulating miR-222 in plasma For accurate quantization of circulating miRNA levels with qRT-PCR, normalization is an important step. As described previously, RNU6B was used as an internal normalization control [12, 28, 29]. Our results demonstrated that no significant difference was observed in terms of Ct values of
The relationship between circulating miR-222 expression and clinicopathological features in GC is summarized in Table 1. The results demonstrated that the level of circulating miR-222 was significantly correlated with clinical
Table 1 Patient characteristics and clinicopathological correlation of circulating miR-222 expression levels Characteristics
No. of patients
miR-222 expression Low
High
P value
Gender Male
54
29
25
Female
60
29
31
Age [50
46
22
24
B50
68
36
32
[5 cm
52
23
29
B5 cm
62
34
28
0.861
0.343
Tumor size
Fig. 1 Ct values of RNU6B among healthy controls (n = 56), CAG patients (n = 36) and GC patients (n = 114). NS not significant
0.425
Differentiation Well and moderate
69
33
36
Poor
45
22
23
T1–T2
49
27
22
T3–T4
65
31
34
0.712
Invasion depth 0.063
Regional lymph nodes metastasis Yes
79
18
61
No
35
26
9
Invaded adjacent organs Yes 78
37
41
No
36
19
17
I–II
42
29
13
III–IV
72
23
49
0.009*
0.431
Clinical stage Fig. 2 Plasma circulating miR-222 expression in 114 GC patients, 36 CAG patients and 56 healthy controls. Expression levels of miR-222 are normalized to RNU6B. The line represents the median value
\0.001*
* P \ 0.05
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Fig. 3 ROC curve analysis for the detection of GC using miR-222, CEA, CA19-9, respectively (a). miR-222 alone or combined with CEA and CA19-9 (b)
stage (P \ 0.001) and lymph nodes metastasis (P = 0.009). However, there was no correlation of miR222 expression with other clinical features, such as age, gender, tumor size, cell differentiation and invasion depth (all at P [ 0.05, respectively). Advanced clinical stage or positive lymph nodes metastasis correlated with higher levels of circulating miR-222. Diagnostic performance of circulating miRNA-222 for GC In order to evaluate the diagnostic capabilities of circulating miRNA-222, CEA or CA19-9 for GC, ROC curve analysis was carried out. At the optimal cutoff value of 2.23 (relative expression in comparison to RNU6B) for circulating miR-222, the sensitivity was 66.1 % and the specificity was 88.3 %, with an AUC of 0.850 in differentiating GC patients from healthy controls. Two conventional tumor markers, CEA and CA19-9, were also measured in all subjects. At a cutoff value of 8.95 for CEA expression level, the optimal sensitivity and specificity were 61.9 and 60.3 %, respectively. At a cutoff value of 45.73 for CA19-9 expression level, the optimal sensitivity and specificity were 72.5 and 61.2 %, respectively. The AUC for the plasma level of circulating miR-222 was significantly larger than that for CEA (0.638) or CA19-9 (0.722) (Fig. 3a), or combination (0.749) (Fig. 3b), indicating that circulating miR-222 was superior to CEA or CA19-9 for the detection of GC. We also tried to combine circulating miR-222, CEA and CA19-9 to improve the diagnostic capability for GC. ROC curves analysis showed that the AUC for this combination was 0.918, which
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significantly larger than that for circulating miR-222 alone (P = 0.024). Correlation between circulating miR-222 and prognosis in GC patients The cumulative 5-year disease-free survival (DFS) and overall survival (OS) in GC patients were 41.321 and 29.468 %, respectively. The optimal cutoff value of circulating miR-222 (2.23) was used to categorize patients with GC into high or low level group. According to the Kaplan–Meier analysis, patients with high levels of circulating miR-222 have a dramatically lower DFS or OS rate than that in the low group [higher (25.267%, 95 % CI 20.932–29.254) vs. lower (44.784 %, 95 % CI 38.708–46.793) for DFS, P = 0.016 and higher (22.428 %, 95 % CI 19.109–26.635) vs. lower (34.17 %, 95 % CI 31.736–40.513) for OS, P = 0.012, respectively] (Fig. 4a, b). Furthermore, univariate Cox proportional hazard regression model analysis revealed that DFS was significantly correlated with clinical stage (P = 0.004) and circulating miR-222 level (P \ 0.001) (Table 2). OS was significantly correlated with lymph node metastasis (P = 0.008), adjacent organs invasion (P = 0.012), clinical stage (P = 0.019) and circulating miR-222 level (P \ 0.001) (Table 2). Parameters significantly related to survival in the univariate analysis were put into the multivariate analysis to identify the independent factors for prognoses. It turned out that circulating miR-222 level still maintained its significance as an independent prognostic factor for DFS (P = 0.023) and OS (P = 0.017) in GC patients.
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Fig. 4 Kaplan–Meier curves for disease-free survival a and overall survival b according to the plasma circulating miR-222 levels. The optimal cutoff value of miR-222 (2.23) was used to categorize the GC patients into high or low level group
Discussion In the present study, we found that the levels of circulating miR-222 were significantly elevated in the plasma of patients with GC. Compared with conventional serum tumor markers, such as CEA or CA19-9, circulating miR222 demonstrated as a more appropriate marker for the detection of GC. Moreover, circulating miR-222 level was demonstrated as an independent factor for poor prognosis in patients with GC. Numerous genetic and epigenetic alterations contribute to oncogenesis and cancer progression. Among the alterations, miRNAs have been proven to regulate gene expressions by targeting messenger RNAs for translational repression or cleavage [5], and numerous studies have shown that aberrant miRNA expressions correlate with development and progression in various types of cancers, including GC [30]. Therefore, miRNAs have recently been recognized as crucial contributory factors in carcinogenesis and can provide new therapeutic strategies as biomarkers and therapeutic targets for cancers. Furthermore, miRNAs were demonstrated to be present in a remarkably stable form in plasma/serum and the expression level of plasma/ serum miRNAs is reproducible and consistent among individuals [7, 8]. In fact, accumulating reports have demonstrated the usefulness of circulating miRNAs as novel noninvasive biomarkers for cancers, such as GC [29], pancreatic cancer [9], breast cancer [12] and papillary thyroid carcinoma [25]. miR-222, known as an ‘oncomiR,’ has been observed to promote cancer cell proliferation by suppressing expression
of the cyclin-dependent kinase (CDK) inhibitors CDKN1B/ p27 and CDKN1C/p57, which are important regulators of cell cycle progression [13–15]. The high expression of miR-222 has been demonstrated in many types of cancers, such as thyroid papillary carcinomas [16], hepatocellular carcinoma [17], melanoma [18] and pancreatic cancer [19]. In pancreatic cancer tissues, patients with overexpressed miR-222 had a poorer clinical prognosis. Also, miR-222 was confirmed to inhibit ER-a translation by direct interaction with the 30 -UTR of ER-a and showed higher levels of expression in ER-a-negative clinical tumors [31]. For GC, Li et al. [20] found that miR-222 was upregulated in H. pylori-infected gastric mucosa and GC tissues, and miR222 could promote GC cell proliferation by targeting RECK. Kim et al. [32] showed that enforced expression of miR-222 enhances the growth of GC xenograft in nude mice, supporting the oncogenic role of miR-222 in the gastro carcinogenesis. In this study, our results demonstrated that the levels of circulating miR-222 in patients with GC were significantly increased, compared with CAG and healthy controls. In addition, higher level of circulating miR-222 had also been found in the plasma of CAG patients compared with healthy controls, indicating the dysregulated expression of miR-222 might be involved in the early events of GC malignancy. Some soluble tumor-associated markers in plasma/ serum have been used in the diagnosis of GC, in which the conventional tumor markers, CEA and CA19-9, were the most widely used ones [3, 4]. However, their low sensitivity and specificity in blood test limited the further application in screening of high-risk individuals and early
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164 Page 6 of 8 Table 2 Univariate and multivariate analysis of prognostic parameters in patients with GC by Cox regression analysis
Med Oncol (2014) 31:164
Variables
Univariate analysis HR (95 % CI)
Multivariate analysis P value
HR (95 % CI)
P value
Disease-free survival Sex Male versus female
0.92 (0.61–1.36)
0.601
0.76 (0.47–1.22)
0.368
1.05 (0.71–1.69)
0.352
0.97 (0.58–1.42)
0.516
T1 ?T2 versus T3 ? T4 Regional lymph nodes metastasis
0.76 (0.43–1.31)
0.426
Yes versus no
2.74 (1.25–4.37)
0.093
1.68 (1.14–2.75)
0.083
2.67 (1.75–4.61)
0.004*
1.73 (1.38–3.65)
0.015*
4.54 (2.55–7.89)
\0.001*
3.38 (1.87–5.23)
0.023*
0.89 (0.58–1.36)
0.581
0.78 (0.50–1.26)
0.341
[5 versus B5 cm
1.27 (0.82–1.88)
0.363
Differentiation Well ? moderate versus poor
0.85 (0.53–1.26)
0.407
0.82 (0.53–1.27)
0.328
2.37 (1.15–4.64)
0.008*
1.69 (0.73–3.81)
0.053
1.79 (1.12–2.89)
0.012*
1.93 (1.14–3.23)
0.009*
3.12 (1.93–5.11)
0.019*
1.86 (1.09–3.21)
0.036*
4.49 (2.68–7.49)
\0.001*
3.41 (1.84–6.16)
0.017*
Age [50 versus B50 years Tumor size [5 versus B5 cm Differentiation Well ? moderate versus poor Invasion depth
Invaded adjacent organs Yes versus no Clinical stage I ? II versus III ? IV MiR-222 expression High versus low Overall survival Sex Male versus female Age [50 versus B50 years Tumor size
Invasion depth T1 ?T2 versus T3 ? T4 Regional lymph nodes metastasis Yes versus no Invaded adjacent organs Yes versus no Clinical stage I ? II versus III ? IV MiR-222 expression * P \ 0.05
High versus low
detection of GC. In our current study, we describe that circulating miR-222 can be used to effectively discriminate between patients with GC and healthy controls. The sensitivity (66.1 %) and specificity (88.3 %) of our test and the AUC value (0.850) that we obtained can be considered good for a blood diagnostic test. Furthermore, the AUC for circulating miR-222 was significantly higher than that for CEA or CA19-9, alone or combined. This gave us a hint that circulating miR-222 could be used as a less expensive
123
and more reliable alternative to combined CEA and CA199 in the diagnosis GC. We also tried to combine miR-222, CEA and CA19-9 for the detection of GC to improve detection capability. The AUC for this combination was significantly larger than that for miR-222 alone, indicating the combination of miR-222, CEA and CA19-9 might improve the diagnostic performance in GC. We also examined the correlation between miRNA-222 expression level in plasma and tumor progression and
Med Oncol (2014) 31:164
prognosis. The results showed that the increased levels of circulating miR-222 were associated with various clinicopathological factors of poor prognosis, such as lymph nodes metastasis and clinical stage. Moreover, patients with high circulating miR-222 levels had poor DFS and OS compared with those with low levels. Cox proportional hazards regression model analyses revealed that the expression level of circulating miR-222 was an independent factor influencing DFS and OS. Our results are consistent with previous studies that have demonstrated the potential of tissue miR-222 as a prognostic and predictive marker for response to radiotherapy in GC [20, 32]. In this respect, circulating miR-222 expression might possess the same potential in prognosis of GC as found with the classical prognostic factors such as clinical stage or lymph node metastasis. Furthermore, technically speaking, plasma is more convenient and noninvasive, which appears to be a desirable source of biomarkers for detecting diseases. In conclusion, our findings demonstrated that circulating miR-222 might potentially serve as a novel and noninvasive biomarker for the early detection and prognostic prediction in GC. Further investigations are necessary to validate results of our study and to evaluate the potential of using circulating miR-222 as a noninvasive marker for treatment monitoring for patients with GC. Conflict of interest
None.
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ORIGINAL PAPER
Circulating miR-222 in plasma and its potential diagnostic and prognostic value in gastric cancer Zhengchuan Fu • Fang Qian • Xuhuan Yang Hailiang Jiang • Yu Chen • Sihai Liu
•
Received: 21 July 2014 / Accepted: 7 August 2014 / Published online: 17 August 2014 Ó Springer Science+Business Media New York 2014
Abstract Previous studies have revealed the significance of circulating microRNAs as biomarkers for cancers. The aim of this study was to detect the levels of circulating microRNA-222 (miR-222) in plasma of gastric cancer (GC) patients and evaluate its diagnostic and prognostic value. Levels of circulating miR-222 were detected by using qRTPCR in plasma of 114 GC patients, 36 chronic atrophic gastritis (CAG) patients and 56 healthy controls. The result showed that the expression of circulating miR-222 in plasma was significantly upregulated in GC compared with CAG and healthy controls (all at P \ 0.001). And its high level was significantly correlated with clinical stages (P \ 0.001) and lymph nodes metastasis (P = 0.009). The receiver operating characteristics (ROC) curve analyses revealed that miR-222 had considerable diagnostic accuracy, yielded an AUC (the areas under the ROC curve) of 0.850 with 66.1 % sensitivity and 88.3 % specificity in discriminating GC from healthy controls. Moreover, Kaplan–Meier analysis demonstrated a correlation between increased circulating miR222 level and reduced disease-free survival (P = 0.016) and overall survival (P = 0.012). In multivariate analysis stratified for known prognostic variables, circulating miR-222 was identified as an independent prognostic marker. In conclusion, our findings suggested that circulating miR-222 in plasma might be a potential and useful noninvasive biomarker for the early detection and prognosis of GC. Keywords Gastric cancer Circulating miR-222 Detection Prognosis
Z. Fu (&) F. Qian X. Yang H. Jiang Y. Chen S. Liu Department of Oncology, Zaozhuang Mining Group Central Hospital, Qilianshan Road, Zaozhuang 277000, Shandong, China e-mail: [email protected]
Introduction Gastric cancer (GC) is the forth common malignancy and the second leading cause of cancer-related death in both sexes worldwide [1]. The prognosis of GC varies remarkably by the stage with the 5-year survival rates range from[90 % for stage I to\5 % for stage IV [2]. Thus, early detection of GC is critical to decrease the mortality rate and improve the prognosis for patients with GC. Although gastroscopic screening is currently the most reliable screening tool, the invasive nature and cost incurred have hampered its wide application. On the other hand, the current serum tumor markers such as carcinoembryonic antibody (CEA) and carbohydrate antibody 19-9 (CA19-9) were not sensitive and specific enough for GC screening [3, 4]. Thus, there is an urgent need for discovering novel noninvasive biomarkers to improve early detection or prognostic prediction of GC. MicroRNAs (miRNAs) are 18–24 nucleotide long evolutionarily conserved RNA molecules that regulate gene expression post-transcriptionally by binding to the 3-untranslated regions of target messenger RNAs [5]. miRNAs play important roles in the multistep carcinogenesis process through the dysregulation of oncogenes and tumor suppressor genes [6]. Several recent studies demonstrated that miRNAs are stably detectable in plasma or serum [7, 8]. Mitchell et al. [8] reported that tumorassociated circulating miRNAs are stably detectable in the plasma of human prostate cancer xenograft mouse models and prostate cancer patients, suggesting that their detection could differentiate cancer-bearing individuals from healthy controls. These findings also raised the possibility that assaying miRNAs in plasma or serum could serve as a novel approach for the noninvasive blood-based detection of human cancers. Actually, since the above study, several investigators have reported the significance of some types
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of circulating miRNAs as biochemical markers for human cancers [9–12]. In this study, we focused on miR-222. miR-222, known as an ‘oncomiR,’ has been observed to induce cell growth and cell cycle progression via direct targeting of p27 and p57 [13–15]. Overexpression of miRNA-222 is reported in many types of cancers such as thyroid papillary carcinomas [16], hepatocellular carcinoma [17], melanoma [18] and pancreatic cancer [19]. Also in GC, it was previously reported that the expression was significantly increased in cancer tissues and cell lines and that miRNA-222 targets tumor suppressor genes, such as PTEN and RECK [14, 20, 21]. In other studies, it was also confirmed that plasma circulating miRNA-222 was a useful biomarker for some types of human cancers [22–25]. Thus, we postulated that plasma circulating miRNA-222 expression could be a novel biochemical marker for GC. In the present study, we evaluated the usefulness of plasma circulating miRNA-222 as a biochemical marker for GC by comparing the expression in patients with GC and healthy controls. In addition, we also examined the prognostic significance of plasma circulating miRNA-222 for GC patients.
Materials and methods Patients and samples A total of 114 cases of newly diagnosed GC and 36 cases of chronic atrophic gastritis (CAG) were enrolled in this study between May 2005 and December 2008 in Zaozhuang Mining Group Central Hospital (Zaozhuang, China). Plasma specimens were collected before any therapeutic procedures, including surgery, chemotherapy and radiotherapy. All patients with GC were staged according to the UICC/TNM staging system, including 17 in stage I, 25 in stage II, 34 in stage III and 38 in stage IV. In total, 56 sex- and age-matched healthy subjects were collected as the healthy controls and each subject had no prior diagnosis of any other malignancy. Written informed consent was obtained from all participants, and research protocols were approved by the Ethical Committee of Zaozhuang Mining Group Central Hospital. All patients with GC, which have been reviewed at the intervals of 3 months during the initial 2–3 years and every 6 months thereafter, were evaluated at clinic. The follow-up was completed in all 114 patients until December 2013, and the median follow-up period was 24 months (range 4–60 months).
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was transferred into 1.5-ml Eppendorf tubes, followed by second centrifugation at 12,000g for 10 min at 4 °C to completely remove cellular components. The supernatant plasma was frozen as separate aliquots at -80 °C until use. Total RNA was extracted from 400 ml of plasma using a mirVana PARIS kit (Ambion, Austin, TX, USA) and eluted into 100 ll of pre-heated (95 °C) elution solution according to the manufacturer’s instructions. Quantification of microRNA by quantitative real-time PCR Amounts of miRNAs in plasma samples were quantified by quantitative real-time PCR (qRT-PCR) using the human TaqMan MicroRNA Assay Kit (Applied Biosystems, USA). The reverse transcription reaction was carried out using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) in 15 ll containing 5 ll of RNA extract, 1.5 ll of 109 reverse transcription buffer, 0.15 ll of 100 mM dNTPs, 1 ll of MultiScribe reverse transcriptase, 0.19 ll of RNase inhibitor, 1 ll of gene-specific primer and 4.16 ll of nuclease-free water. For synthesis of cDNA, the reaction mixtures were incubated at 16 °C for 30 min, at 42 °C for 30 min and at 85 °C for 15 min and then held at 4 °C. About 1.33 ll of cDNA solution was amplified by using 10 ll of TaqMan 29 Universal PCR Master Mix with No AmpErase UNG (Applied Biosystems), 1 ll of gene-specific primer and 7.67 ll of nucleasefree water in a final volume of 20 ll. Quantitative PCR was performed in triplicate on a 7,300 real-time PCR system (Applied Biosystems), and the reaction mixtures were incubated at 95 °C for 10 min, followed by 45 cycles of 95 °C for 15 s and 60 °C for 1 min. The cycle threshold (Ct) values were calculated with SDS 2.4 software (Applied Biosystems). The expression levels of the target miRNAs in the plasma were normalized relative to the expression of RNU6B and were calculated using the 2-DDC method [26]. Carcinoembryonic antibody and carbohydrate antibody 19-9 The serum levels of carcinoembryonic antibody (CEA) and carbohydrate antibody 19-9 (CA19-9) are commonly used in the diagnostics of gastrointestinal malignancies. CEA and CA19-9 were measured by chemiluminescent enzyme immunoassays (FUJIREBIO Inc., Tokyo, Japan) according to the manufacture’s instructions.
Samples processing and RNA extraction
Statistical analysis
Up to 8 ml of whole blood from each participant was collected in EDTA tubes. The blood samples were centrifuged at 1,200g for 10 min at 4 °C, and the supernatant
The Kruskal–Wallis test or the Mann–Whitney U test was used to compare miRNAs levels between groups. Receiver operating characteristic (ROC) curve analysis was
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performed to determine the diagnostic performance of miR-222 expression levels in distinguishing patients with GC from the healthy controls. The optimal cutoff thresholds for diagnosis were obtained by Youden index [27]. The Kaplan–Meier method was used to estimate survival rates, and the log-rank tests were used to assess survival differences between groups. Multivariate analysis of the prognostic factors was performed with Cox proportional hazards model. All statistical analyses were carried out by using SPSS 13.0 software (IBM, USA). A P value \0.05 was considered statistically significant.
RNU6B (Fig. 1) among healthy controls (n = 56), CAG patients (n = 36) and GC patients (n = 114), suggesting that RNU6B constitutively expressed in plasma regardless of different disease conditions. Levels of circulating miR-222 were detected by qRT-PCR in plasma of 114 GC patients, 36 CAG patients and 56 healthy controls. Our data indicated that there was a significant difference among GC, CAG and healthy controls (P \ 0.001). As shown in Fig. 2, the levels of circulating miR-222 in GC were higher than in CAG and healthy controls, respectively (P \ 0.001). In addition, the levels of circulating miR-222 were also significantly higher in CAG than in healthy controls (P \ 0.001).
Results
Circulating miR-222 correlates with clinicopathological features of GC
The expression of circulating miR-222 in plasma For accurate quantization of circulating miRNA levels with qRT-PCR, normalization is an important step. As described previously, RNU6B was used as an internal normalization control [12, 28, 29]. Our results demonstrated that no significant difference was observed in terms of Ct values of
The relationship between circulating miR-222 expression and clinicopathological features in GC is summarized in Table 1. The results demonstrated that the level of circulating miR-222 was significantly correlated with clinical
Table 1 Patient characteristics and clinicopathological correlation of circulating miR-222 expression levels Characteristics
No. of patients
miR-222 expression Low
High
P value
Gender Male
54
29
25
Female
60
29
31
Age [50
46
22
24
B50
68
36
32
[5 cm
52
23
29
B5 cm
62
34
28
0.861
0.343
Tumor size
Fig. 1 Ct values of RNU6B among healthy controls (n = 56), CAG patients (n = 36) and GC patients (n = 114). NS not significant
0.425
Differentiation Well and moderate
69
33
36
Poor
45
22
23
T1–T2
49
27
22
T3–T4
65
31
34
0.712
Invasion depth 0.063
Regional lymph nodes metastasis Yes
79
18
61
No
35
26
9
Invaded adjacent organs Yes 78
37
41
No
36
19
17
I–II
42
29
13
III–IV
72
23
49
0.009*
0.431
Clinical stage Fig. 2 Plasma circulating miR-222 expression in 114 GC patients, 36 CAG patients and 56 healthy controls. Expression levels of miR-222 are normalized to RNU6B. The line represents the median value
\0.001*
* P \ 0.05
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Fig. 3 ROC curve analysis for the detection of GC using miR-222, CEA, CA19-9, respectively (a). miR-222 alone or combined with CEA and CA19-9 (b)
stage (P \ 0.001) and lymph nodes metastasis (P = 0.009). However, there was no correlation of miR222 expression with other clinical features, such as age, gender, tumor size, cell differentiation and invasion depth (all at P [ 0.05, respectively). Advanced clinical stage or positive lymph nodes metastasis correlated with higher levels of circulating miR-222. Diagnostic performance of circulating miRNA-222 for GC In order to evaluate the diagnostic capabilities of circulating miRNA-222, CEA or CA19-9 for GC, ROC curve analysis was carried out. At the optimal cutoff value of 2.23 (relative expression in comparison to RNU6B) for circulating miR-222, the sensitivity was 66.1 % and the specificity was 88.3 %, with an AUC of 0.850 in differentiating GC patients from healthy controls. Two conventional tumor markers, CEA and CA19-9, were also measured in all subjects. At a cutoff value of 8.95 for CEA expression level, the optimal sensitivity and specificity were 61.9 and 60.3 %, respectively. At a cutoff value of 45.73 for CA19-9 expression level, the optimal sensitivity and specificity were 72.5 and 61.2 %, respectively. The AUC for the plasma level of circulating miR-222 was significantly larger than that for CEA (0.638) or CA19-9 (0.722) (Fig. 3a), or combination (0.749) (Fig. 3b), indicating that circulating miR-222 was superior to CEA or CA19-9 for the detection of GC. We also tried to combine circulating miR-222, CEA and CA19-9 to improve the diagnostic capability for GC. ROC curves analysis showed that the AUC for this combination was 0.918, which
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significantly larger than that for circulating miR-222 alone (P = 0.024). Correlation between circulating miR-222 and prognosis in GC patients The cumulative 5-year disease-free survival (DFS) and overall survival (OS) in GC patients were 41.321 and 29.468 %, respectively. The optimal cutoff value of circulating miR-222 (2.23) was used to categorize patients with GC into high or low level group. According to the Kaplan–Meier analysis, patients with high levels of circulating miR-222 have a dramatically lower DFS or OS rate than that in the low group [higher (25.267%, 95 % CI 20.932–29.254) vs. lower (44.784 %, 95 % CI 38.708–46.793) for DFS, P = 0.016 and higher (22.428 %, 95 % CI 19.109–26.635) vs. lower (34.17 %, 95 % CI 31.736–40.513) for OS, P = 0.012, respectively] (Fig. 4a, b). Furthermore, univariate Cox proportional hazard regression model analysis revealed that DFS was significantly correlated with clinical stage (P = 0.004) and circulating miR-222 level (P \ 0.001) (Table 2). OS was significantly correlated with lymph node metastasis (P = 0.008), adjacent organs invasion (P = 0.012), clinical stage (P = 0.019) and circulating miR-222 level (P \ 0.001) (Table 2). Parameters significantly related to survival in the univariate analysis were put into the multivariate analysis to identify the independent factors for prognoses. It turned out that circulating miR-222 level still maintained its significance as an independent prognostic factor for DFS (P = 0.023) and OS (P = 0.017) in GC patients.
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Fig. 4 Kaplan–Meier curves for disease-free survival a and overall survival b according to the plasma circulating miR-222 levels. The optimal cutoff value of miR-222 (2.23) was used to categorize the GC patients into high or low level group
Discussion In the present study, we found that the levels of circulating miR-222 were significantly elevated in the plasma of patients with GC. Compared with conventional serum tumor markers, such as CEA or CA19-9, circulating miR222 demonstrated as a more appropriate marker for the detection of GC. Moreover, circulating miR-222 level was demonstrated as an independent factor for poor prognosis in patients with GC. Numerous genetic and epigenetic alterations contribute to oncogenesis and cancer progression. Among the alterations, miRNAs have been proven to regulate gene expressions by targeting messenger RNAs for translational repression or cleavage [5], and numerous studies have shown that aberrant miRNA expressions correlate with development and progression in various types of cancers, including GC [30]. Therefore, miRNAs have recently been recognized as crucial contributory factors in carcinogenesis and can provide new therapeutic strategies as biomarkers and therapeutic targets for cancers. Furthermore, miRNAs were demonstrated to be present in a remarkably stable form in plasma/serum and the expression level of plasma/ serum miRNAs is reproducible and consistent among individuals [7, 8]. In fact, accumulating reports have demonstrated the usefulness of circulating miRNAs as novel noninvasive biomarkers for cancers, such as GC [29], pancreatic cancer [9], breast cancer [12] and papillary thyroid carcinoma [25]. miR-222, known as an ‘oncomiR,’ has been observed to promote cancer cell proliferation by suppressing expression
of the cyclin-dependent kinase (CDK) inhibitors CDKN1B/ p27 and CDKN1C/p57, which are important regulators of cell cycle progression [13–15]. The high expression of miR-222 has been demonstrated in many types of cancers, such as thyroid papillary carcinomas [16], hepatocellular carcinoma [17], melanoma [18] and pancreatic cancer [19]. In pancreatic cancer tissues, patients with overexpressed miR-222 had a poorer clinical prognosis. Also, miR-222 was confirmed to inhibit ER-a translation by direct interaction with the 30 -UTR of ER-a and showed higher levels of expression in ER-a-negative clinical tumors [31]. For GC, Li et al. [20] found that miR-222 was upregulated in H. pylori-infected gastric mucosa and GC tissues, and miR222 could promote GC cell proliferation by targeting RECK. Kim et al. [32] showed that enforced expression of miR-222 enhances the growth of GC xenograft in nude mice, supporting the oncogenic role of miR-222 in the gastro carcinogenesis. In this study, our results demonstrated that the levels of circulating miR-222 in patients with GC were significantly increased, compared with CAG and healthy controls. In addition, higher level of circulating miR-222 had also been found in the plasma of CAG patients compared with healthy controls, indicating the dysregulated expression of miR-222 might be involved in the early events of GC malignancy. Some soluble tumor-associated markers in plasma/ serum have been used in the diagnosis of GC, in which the conventional tumor markers, CEA and CA19-9, were the most widely used ones [3, 4]. However, their low sensitivity and specificity in blood test limited the further application in screening of high-risk individuals and early
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164 Page 6 of 8 Table 2 Univariate and multivariate analysis of prognostic parameters in patients with GC by Cox regression analysis
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Variables
Univariate analysis HR (95 % CI)
Multivariate analysis P value
HR (95 % CI)
P value
Disease-free survival Sex Male versus female
0.92 (0.61–1.36)
0.601
0.76 (0.47–1.22)
0.368
1.05 (0.71–1.69)
0.352
0.97 (0.58–1.42)
0.516
T1 ?T2 versus T3 ? T4 Regional lymph nodes metastasis
0.76 (0.43–1.31)
0.426
Yes versus no
2.74 (1.25–4.37)
0.093
1.68 (1.14–2.75)
0.083
2.67 (1.75–4.61)
0.004*
1.73 (1.38–3.65)
0.015*
4.54 (2.55–7.89)
\0.001*
3.38 (1.87–5.23)
0.023*
0.89 (0.58–1.36)
0.581
0.78 (0.50–1.26)
0.341
[5 versus B5 cm
1.27 (0.82–1.88)
0.363
Differentiation Well ? moderate versus poor
0.85 (0.53–1.26)
0.407
0.82 (0.53–1.27)
0.328
2.37 (1.15–4.64)
0.008*
1.69 (0.73–3.81)
0.053
1.79 (1.12–2.89)
0.012*
1.93 (1.14–3.23)
0.009*
3.12 (1.93–5.11)
0.019*
1.86 (1.09–3.21)
0.036*
4.49 (2.68–7.49)
\0.001*
3.41 (1.84–6.16)
0.017*
Age [50 versus B50 years Tumor size [5 versus B5 cm Differentiation Well ? moderate versus poor Invasion depth
Invaded adjacent organs Yes versus no Clinical stage I ? II versus III ? IV MiR-222 expression High versus low Overall survival Sex Male versus female Age [50 versus B50 years Tumor size
Invasion depth T1 ?T2 versus T3 ? T4 Regional lymph nodes metastasis Yes versus no Invaded adjacent organs Yes versus no Clinical stage I ? II versus III ? IV MiR-222 expression * P \ 0.05
High versus low
detection of GC. In our current study, we describe that circulating miR-222 can be used to effectively discriminate between patients with GC and healthy controls. The sensitivity (66.1 %) and specificity (88.3 %) of our test and the AUC value (0.850) that we obtained can be considered good for a blood diagnostic test. Furthermore, the AUC for circulating miR-222 was significantly higher than that for CEA or CA19-9, alone or combined. This gave us a hint that circulating miR-222 could be used as a less expensive
123
and more reliable alternative to combined CEA and CA199 in the diagnosis GC. We also tried to combine miR-222, CEA and CA19-9 for the detection of GC to improve detection capability. The AUC for this combination was significantly larger than that for miR-222 alone, indicating the combination of miR-222, CEA and CA19-9 might improve the diagnostic performance in GC. We also examined the correlation between miRNA-222 expression level in plasma and tumor progression and
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prognosis. The results showed that the increased levels of circulating miR-222 were associated with various clinicopathological factors of poor prognosis, such as lymph nodes metastasis and clinical stage. Moreover, patients with high circulating miR-222 levels had poor DFS and OS compared with those with low levels. Cox proportional hazards regression model analyses revealed that the expression level of circulating miR-222 was an independent factor influencing DFS and OS. Our results are consistent with previous studies that have demonstrated the potential of tissue miR-222 as a prognostic and predictive marker for response to radiotherapy in GC [20, 32]. In this respect, circulating miR-222 expression might possess the same potential in prognosis of GC as found with the classical prognostic factors such as clinical stage or lymph node metastasis. Furthermore, technically speaking, plasma is more convenient and noninvasive, which appears to be a desirable source of biomarkers for detecting diseases. In conclusion, our findings demonstrated that circulating miR-222 might potentially serve as a novel and noninvasive biomarker for the early detection and prognostic prediction in GC. Further investigations are necessary to validate results of our study and to evaluate the potential of using circulating miR-222 as a noninvasive marker for treatment monitoring for patients with GC. Conflict of interest
None.
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