|Year : 2015 | Volume
| Issue : 3 | Page : 630-634
Plasma miR-185 as a predictive biomarker for prognosis of malignant glioma
Hailin Tang1, Qing Liu2, Xiaoping Liu1, Feng Ye1, Xinhua Xie1, Xiaoming Xie1, Minghua Wu2
1 Department of Breast Oncology, Sun Yat-Sen University Cancer Center; Sun Yat-Sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou, Guangdong, China
2 The Center for Skull Base Surgery and Neurooncology, Hunan Province, China
|Date of Web Publication||9-Oct-2015|
The Center for Skull Base Surgery and Neurooncology, Hunan Province, 87 Xiangya Road, Changsha, 410078
Source of Support: None, Conflict of Interest: None
Introduction: Specific microRNA (miRNA) expression signatures have been identified in a variety of human cancers. More recently, increasing evidence shows that miRNAs exist in human blood serum and plasma.
Materials and Methods: Levels of miR-185 in plasma were measured by quantitative reverse-transcriptase polymerase chain reaction in 66 glioma patients, 11 pituitary adenoma patients, 32 meningioma patients, and 14 acoustic neuroma patients.
Results: The plasma levels of miR-185 were significantly altered in glioma patients compared to normal controls. However, its levels were not observably changed in patients with other brain tumors such as meningioma, acoustic neuroma, or pituitary adenoma. Furthermore, the plasma levels of miR-185 in glioblastoma multiforme patients with operation and chemo-radiation almost revived to normal levels. Finally, we also demonstrated that low plasma miR-185 levels are correlated with poor survival in glioma patients.
Conclusion: These findings suggest that plasma miR-185 has become potential biomarkers for glioma and may be useful in clinical management for glioma patients.
Keywords: Biomarker, glioma, miR-185, plasma
|How to cite this article:|
Tang H, Liu Q, Liu X, Ye F, Xie X, Xie X, Wu M. Plasma miR-185 as a predictive biomarker for prognosis of malignant glioma. J Can Res Ther 2015;11:630-4
Hailin Tang and Qing Liu contributed equally to this work
| > Introduction|| |
Gliomas are the most common type of primary brain tumors in adults and persist as serious clinical and scientific problems. Survival depends heavily on the histological grade of the tumor, but patients afflicted with the most malignant glioma, glioblastoma multiforme, survive on average about 15 months.  Based on their histology and morphological features, glioma is divided into four clinical grades: Grade I comprises slowly growing benign glioma, Grade II represents the least malignant cases and Grades III and IV (glioblastoma multiforme) are highly malignant. , Therefore, malignant glioma is in urgent need of some noninvasive, clinically applicable detection and prognosis biomarkers. 
Recently, it has been identified a class of endogenous, small, nonprotein coding single-stranded RNA molecules, termed microRNA (miRNA), which played a crucial role in the posttranscriptional regulation of gene expression. An increasing evidence suggested that circulating miRNAs of serum or plasma (extracellular miRNAs) could be used as potential biomarkers for detection, identification, and classification of cancers and other diseases due to their stability and detectability in plasma.  Recent publications have shown that glioblastoma cells release microvesicles containing a select subset of cellular proteins and RNAs including miRNAs, and miRNAs' characteristic of gliomas could be detected in serum microvesicles of glioblastoma patients. ,, miRNAs in plasma or serum could be developed as a novel class of blood-based biomarker to diagnose and monitor glioma. , miR-185, one of intensively studied miRNAs in cancer biology, was reported to be down-regulated in glioma tissues and cells. miR-185 can directly target DNA methyltransferases 1, thereby leading to a reduction in global DNA methylation and regulating the expression of the promoter-hypermethylated genes in glioma cells.  Overexpression of miR-185 inhibited the proliferation and invasion of glioma cell lines by targeting CDC42 and RhoA. Furthermore, miR-185 might be associated with the overall survival (OS) of glioma patients. 
Based on our previous work, we asked whether miR-185, one of the most investigated miRNAs in cancer biology, existed in the plasma of glioma patients comparing with healthy controls. We assess the potential of miR-185 as a noninvasive biomarker for prognosis of malignant gliomas.
| > Materials and methods|| |
Plasma samples for miRNAs detection were collected from patients with pathologically confirmed malignant glioma (Grades I-IV) (n = 66), pituitary adenoma (n = 11), and meningioma (n = 32), acoustic neuroma (n = 14). In addition, plasma samples of glioblastoma multiforme patients (n = 19) were obtained in preoperation, 2 weeks after surgery and a month after X-ray radiotherapy and temozolomide chemotherapy, respectively. The detailed characteristics of these patients are shown in [Table 1]. Plasma samples from 20 control individuals were recruited from a large pool of individuals seeking a routine health check-up. Participants who showed no evidence of disease were selected as noncancer controls and were matched to the patients by age, sex and ethnicity. The blood samples were obtained and centrifuged for 10 min at 1500 g within 2 h after collection, and the supernatant was removed to RNase-free tubes and further centrifuged for 10 min at 12,000 g and 4°C to remove cells and debris. Plasma was stored at −80°C until further processing.
|Table 1: Analysis of the correlation between expression of miR-185 in primary glioma and its clinicopathological parameters|
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RNA isolation and quantitative reverse-transcriptase polymerase chain reaction
Those procedures were carried out as previously described.  For all experiments, 500 μL of human plasma was used, and total RNA was extracted and eluted in 60 μL of RNase-free water using a mirVana PARIS kit (number 1556; Ambion) following the manufacturer's protocol for liquid samples.
Using spiked-in C. Elegans miRNAs as internal reference controls, plasma samples were used for the validation of differentially expressed miRNAs in plasma. Given the early stage of plasma miRNA study, no established endogenous small miRNAs were acted as the controls for normalization of technical variations in sample processing or potential variation in sample quality. Normalization by matching the amount of loaded RNA in the RT reaction is not a feasible approach because the RNA content in plasma varies considerably with physical or pathological states. During the RNA extraction for quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) analysis, cel-miR-39 and cel-miR-238 were spiked at a fixed concentration as internal control. We, therefore, chose to use a fixed volume of RNA elute (5 μL) from a given volume of starting plasma (500 μL), and normalized by the added exogenous cel-miR-39 and cel-miR-238. MiScript PCR System kit series (miScript Reverse Transcription Kit, miScript SYBR Green PCR Kit, Qiagen) were used to quantify specific miRNAs. The fold change of differentially expressed miRNA level was calculated with 2 -ΔΔCT .
The statistical analysis was performed by SPSS version 16.0. The Mann-Whitney test was used to assess the differences between the healthy group, glioma and other brain tumor patients. P < 0.05 was considered statistically significant. The Bonferroni correction was applied for multiple comparisons for between normal control group, glioma II, III and IV grades, or different brain tumors, P values are significant at the 0.008 level adjusted for multiple comparisons with Bonferroni correction. The relative levels of miRNA were quantified using the 2 -ΔΔCT method.
| > Results|| |
The expression of plasma miR-185 in glioma and the relationship with clinical parameters
To evaluate the expression of plasma miR-185 in glioma patients, qRT-PCR was used to detect 66 gliomas, 20 control individuals with a matched distribution of age and sex served as controls. In comparison with the control individuals, the results showed that plasma level of miR-185 was down-regulated in 62.1% (41/66) of the glioma patients: Thirty-eight samples in ≤45 years group, 18 samples in >45 years group; 32 males and 34 females; 23 Grade I + II, 43 Grade III + IV. The reduction of miR-185 expression was not correlated with tumor grade, sex or age [Table 1]. But the expression level was much lower in Grade III + IV than in Grades I + II (P < 0.001).
As can be seen, a markedly reduced OS was observed in the glioma patients who had lost miR-185 expression compared with the patients who exhibited high expression levels [Figure 1]a. These results suggest that reduced miR-185 expression is a frequent event in human glioma tissues and may be involved in glioma carcinogenesis. Furthermore, miR-185 may function as a potential marker for the initiation of a glioma.
|Figure 1: The expression level of plasma miR-185 was specifically correlated with glioma. (a) Plasma level of miR-185 in normal cohorts, meningioma cohorts, pituitary adenoma cohorts, acoustic neurinoma and glioma cohorts. miR-185 was normalized to cel-miR-39 and cel-miR-238 and analyzed using 2-ΔΔCT method. Statistically significant differences were determined using the Mann-Whitney U-test. Data were represented as mean ± standard deviation (SD) from three independent experiments. **P < 0.01. (b) miR-185 expression level in plasma of healthy controls (n = 20), glioma I + II (n = 23) and glioma IV + III (n = 43) patients. miR-185 was normalized to cel-miR-39 and cel-miR-238 and analyzed using 2-ΔΔCT method. Statistically significant differences were determined using the Mann-Whitney U-test. Data were represented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01. (c) The miR-185 expression in preoperation (n = 19), postoperation (n = 19), and chemo-radiation (n = 19) plasma samples. miR-185 is normalized to cel-miR-39 and cel-miR-238 and analyzed using 2-ΔΔCT method. Statistically significant differences were determined using the Mann-Whitney U-test. Data were represented as mean ± SD from three independent experiments **P < 0.01|
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The expression level of plasma miR-185 was specifically correlated with glioma
To evaluate whether levels of plasma miR-185 is specifically correlated with glioma. First, we selected control individuals (n = 20) and histopathological grade of glioma: Grade I + II (n = 23), Grade IV + III (n = 43) and then detected the level of miR-185 using qRT-PCR. Our data showed that miR-185 was significantly lower in glioma samples than in normal control samples, and much lower level of miR-185 in high grades glioma (Grades IV + III) than in low grades glioma (Grades I + II) [[Figure 1]b, P < 0.001]. Furthermore, we detected plasma level of miR-185 in control individuals (n = 20), glioma cohorts (n = 66), pituitary adenoma patients (n = 11) and meningioma patients (n = 32), acoustic neurinoma patients (n = 14). It was found that the plasma level of miR-185 was significantly lower in glioma samples than in normal control samples [[Figure 1]a, P < 0.001]. In addition, there was no significant difference between control individuals and meningioma patients, pituitary adenoma patients or acoustic neurinoma patients [P > 0.05, [Figure 1]a]. The data suggest that miR-185 is specifically associated with glioma.
In order to further explore the expression of miR-185 in glioma patients, we chose 20 GBM (Grade IV) patients and collected their plasma before/after operation, and after chemo-radiation. We found that the level of miR-185 was markedly up-regulated after operation and chemo-radiation [[Figure 1]c, P < 0.001].
Low plasma miR-185 level is correlated with poor survival in glioma patients
To analyze the significance of miR-185 in terms of clinical prognosis, a Kaplan-Meier survival analysis was performed using patient OS and disease free survival (DFS). It was demonstrated that patients with low plasma miR-185 had shorter mean months of OS and DFS than patients with high plasma miR-185 [P = 0.000 for OS, P = 0.000 for DFS, [Figure 2]]. Our results indicate that expression level of miR-185 is significantly associated with patient OS and DFS.
|Figure 2: Plasma miR-185 level was correlated with poor survival in glioma patients. overall survival (a) and disease free survival (b) curves for all studied patients with high or low miR-185 expression|
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To find the impact of each variable on OS and DFS, univariate Cox regression [Table 2] was used. In glioma patients, low plasma miR-185 significantly correlated with shorter OS (hazard ratio [HR] =0.179, 95% confidence interval [CI]: 0.071-−0.450, P = 0.000) and DFS (HR = 0.160, 95%CI: 0.064-0.399, P = 0.000). Meanwhile, glioma histopathology (P = 0.029, P = 0.037) was correlated with shorter OS and DFS in patients.
|Table 2: Univariate cox regression analysis of OS and DFS (cox proportional hazards regression model)|
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| > Discussion|| |
There is a burgeoning interest in circulating biomarkers, given their potential in the translational area and because they might constitute representative readouts of both primary tumor and metastatic deposits, and provide ways to expedite the discovery and validation of clinically useful predictive biomarkers.  Increasing number of papers reported that circulating miRNAs can serve as noninvasive biomarkers for cancer detection. Recently, Wang et al. reported that cell-free miR-21, miR-128, and miR-342-3p of plasma are specificity and sensitivity to diagnosis of glioblastoma multiforme, suggesting that these miRNAs may be used as noninvasive biomarkers in glioblastoma multiforme.  In this manuscript, we showed that miR-185 was down-regulated in plasma samples of human glioma tumors comparing with healthy controls, In order to further identify the relationship between plasma level of miR-185 and classification and treatment effect of glioma, we next performed statistical analysis of miR-185 expression data. There was a significant difference in plasma level of miR-185 between the earlier stages (Grades I + II) and the later subgroups (Grades III and IV). In addition, the miR-185 can specifically discriminate glioma from other brain tumors such as meningioma, pituitary adenoma, and acoustic neurinoma.
Based on genome-wide miRNA expression-profiling studies using high-throughput technologies, it is widely accepted that miRNAs are commonly dysregulated in various human cancers, acting either as oncogenes or tumor suppressor genes. miR-185, located on chromosome 22q11.21, is one of the characterized tumor suppressor miRNAs. miR-185 has been reported to be down-regulated in a variety of cancers, including colorectal cancer. , hepatocellular carcinoma,  epithelial ovarian cancer,  breast cancer,  prostate cancer  and gastric cancer.  As our previous report, plasma miR-185 level was correlated with malignant glioma cancer stages. We also demonstrated by univariate analysis that low plasma miR-185 was significantly correlated with shorter OS and DFS. Meanwhile, glioma histopathology was correlated with shorter OS and DFS in patients. It has been confirmed that altered expression levels of circulating miR-425*, miR-302b, let-7c,  and miR-122  might have great potential to serve as novel, noninvasive biomarkers for early detection of breast cancer and may possibly allow optimized chemotherapy treatments and preventive anti-metastasis interventions in future clinical applications.  miR-221 and miR-210, circulating expression levels of which were associated with trastuzumab sensitivity, tumor presence, and lymph node metastases, are of great interest in the search for a relevant noninvasive biomarker to predict and, perhaps, monitor response to breast cancer patients therapies.  But we found that plasma miR-185 was increased slightly after operation than before the operation, no significant difference existed. miR-185 is observably decreased after further treatment with chemo-radiation. Thus, these data suggest a possible association between miR-185 and treatment effect.
In summary, our data showed that the cell-free miR-185 has a strong correlation with classification and clinical course and aid in therapeutic decisions for glioma patients through detecting miR-185 level in plasma. We believe that miR-185 may represent a valuable plasma marker for malignant glioma, with the potential to be translated into clinical applications including prognosis, monitoring response to therapy. Further validation in a larger cohort of samples and a prospective study are required to determine conclusively whether miR-185 serves as a predictive biomarker for prognosis of malignant glioma.
| > Acknowledgments|| |
This work was supported by the National Natural Science Foundation of China (31100935, 81171932, 81101643 81272514), the Key Program of National Natural Science Foundation of China (31030061) and the China Postdoctoral Science Foundation (2012M520075).
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[Figure 1], [Figure 2]
[Table 1], [Table 2]