Different expression of miR-29b and VEGFA in glioma

Abstract

Glioma is one of the most common carcinomas in terms of both incidence and mortality worldwide. This is a case–control study with 240 cases and age- and gender-matched controls in the rate of 1:1. The results of this present word indicated that the expressions of both miR-29b and VEGFA in blood were significantly different compared with the control group, and thus may help to differentiate glioma cases from the controls. In addition, the diagnostic role of miR-29b and VEGFA was important for the clinical application. In conclusion, circulating miR-29b and VEGFA could be used as diagnosis biomarkers.

Keywords:

• case–control
• diagnosis
• glioma
• microRNA

Introduction

Glioma is the most common aggressive adult primary tumor of the central nervous system (Barbarin et al. 2014, Liu et al. 2015b). Treatment of glioma includes surgery, radiotherapy, and adjuvant temozolomide chemotherapy (Towner et al. 2015). The mortality of glioma occupies the top position among the malignant tumors worldwide (Chen et al. 2015, Leng et al. 2015, Malone and Bruce 2014). During the early stages (I and II) when the tumor is still small, the patients with glioma were usually asymptomatic (Wolking et al. 2013). Grades III and IV gliomas, such as glioblastoma (GBM), are aggressive and lethal malignant neoplasms. Unfortunately, many glioma cases detected in the clinical were in the late stage and the prognosis was very likely to be poor (Awad et al. 2014). Although tumor markers greatly improve diagnosis, the invasive, unpleasant, and inconvenient nature of current diagnostic procedures limits their application (McGovern et al. 2014). Hence, there is a great need for identification of novel non-invasive biomarkers for early tumor detection.

MicroRNAs (miRNAs) are short RNA molecules with 18–25 nucleotides in length, which are implicated in post-transcriptional regulation of gene expression (Liu et al. 2015a). Currently, it is clear that miRNAs are also present in circulation (e.g., in plasma or serum), and these circulating miRNAs are found to be either encapsulated in membranous microvesicles or associated with RNA-binding proteins or lipoprotein complexes, rendering circulating miRNAs relatively resistant to enzymatic degradation (Keirstead et al. 2015, Ovchinnikova et al. 2015). An increasing number of studies demonstrate that miRNA expression profiles may be specific to certain types of cancer and tumor-derived miRNAs may be stably detected in the plasma or serum (Ma et al. 2013, Xiao et al. 2014). These findings highlight the potential of circulating miRNAs as biomarkers for the diagnosis of cancer.

MiR-29b, a member of the miR-29 family, was found to be highly associated with the development and progression of kinds of cancers. A study by Xu et al. showed that miR29b and miR-29c levels were higher in T24 cells than normal urothelial cells. Knockdown of miR-29b-1* or miR-29c suppressed T24 cell growth. Bioinformatic analysis showed that miR-29b-1* and miR-29c co-regulated a subset of putative target genes, about 10% of which have been experimentally validated (Xu et al. 2013). Using global miR expression profiling, it was also observed differently miR-29b expressed in colon cancer cells. Since miR-29b plays a role in regulating the migration of cancer cells, miR-29b expression to target matrix metalloproteinase-2 thereby suppressing the migration of colon cancer cells (Poudyal et al. 2013). It was found that miR-29b induced apoptosis and inhibited invasion and angiogenesis of trophoblast cells. Further studies confirmed that miR-29b regulated the expression of MCL1, MMP2, VEGFA and ITGB1 genes by directly binding to their 3′-UTRs (Li et al. 2013). VEGFA, which was reported to be involved in the development of glioma, was predicated to be one of the target genes of miR-29b. The relation between miR-29b and VEGFA is presented in Figure 1.

Figure 1. The relation between miR-29b and VEGFA.

In this study, miR-29b was chosen to ascertain whether its serum expression can distinguish glioma from cancer-free controls. We conducted circulating miR-29b expression by a stem-loop qRT-PCR assay. Besides, the serum VEGFA was also detect in this study. The relationship between both circulating miR-29b and VEGFA and glioma was investigated by a case–control study.

Materials and methods

Ethics statement

The study was conducted according to the principles approved by the Medical Ethics Committee of Xiangya Hospital, Central South University, Changsha, Hunan, P.R. China. The written informed consents were obtained from all the patients prior to participation. Besides, the study was conducted in accordance with the Declaration of Helsinki.

Patient samples

In this case–control study, a total of 240 glioma cases and age- and gender-matched healthy controls were included. All the glioma cases were from Department of Neurosurgery, Xiangya Hospital, Central South University, Changsha, Hunan, P.R. China, while the healthy cases were from healthy medical examination center. All the cases were obtained between January 2013 and May 2014. Among the 240 glioma cases, blood samples of pre-operation and post-operation were obtained from 124 cases. All the blood samples were collected after the informed consent was obtained from the patients or the legal guardian.

Sample processing

The peripheral venous blood samples were collected into EDTA-containing tubes and processed within 2 h. Blood samples were processed as described previously. Whole blood was first centrifuged at 1600 g for 10 min, and then the supernatant was transferred into a fresh tube and centrifuged again at 13,000 g for 10 min. The clear plasma was aliquoted and stored at −80 °C until use. All procedures were carried out on ice.

RNA extraction

Total RNA extraction was performed using a mirVana PARIS kit (Life Technologies, Carlsbad, CA), following the manufacturer’s instructions (Scheffer et al. 2014). The qualification and quantification of RNA were verified by measuring ultra-violet (UV) absorbance (A260 and A280) on the NanoDrop Spectrophotometer 2000 (Thermo Scientific, Wilmington, DE). The A260/A280 ratio should be close to 2.0 (ratios between 1.8 and 2.1 are acceptable).

RT-qPCR analysis processing

Quantification of miR-29b was conducted by RT-qPCR analysis. The RT-PCR processing was conducted with TaqMan miRNA-RT Kit (Applied Biosystems, Carlsbad, CA), according to the manufacturer’s instructions. In this study, U6 small RNA (RNU6) was adopted as an endogenous control for normalizing the levels of target miR-29b. RNU6 is a widely used endogenous reference RNA in miRNA quantification. The 20 μl PCR reaction system included 1 × TaqMan Universal PCR Master Mix (Applied Biosystems), and the corresponding TaqMan probes for the target genes. The reactions were incubated in a 96-well plates and the cycling began with template denaturation and hot start Taq activation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The Ct value was defined as the fractional cycle number at which the fluorescence passes the fixed threshold. All assays were conducted in triplicate. Relative expression was calculated using the comparative Ct method. First, the Ct values for all samples were determined. Then, the ^ΔΔCt methods were used for the determination of miRNA content. The ^ΔΔCt values were calculated: ^ΔΔCt = ^ΔCt_(cases) − ^ΔCt _(controls). The investigators were blinded to the results of clinical and pathological diagnosis.

VEGFA assay

Clinical venous blood samples of all participants were collected using the blood collection system. Venous blood was centrifuged for 10 min at 1700 × g and then the serum was isolated. The serum samples were stored frozen at −80°C until use. Serum samples were thawed and then used for the advanced analyses. The concentrations of VEGFA in all the serum samples were determined using a VEGFA ELISA kit (63103, Sigma-Aldrich Co., St. Louis, MO). All the procedures were performed according to the manufacturer’s instructions. Mean of the duplicate values was used for analysis. All samples were assayed in duplicate.

Statistical analysis

All the data in this study were presented by mean ± standard deviation (SD). For continuous variables, the t-test was used to explore the difference between two groups. However, when the comparisons of over three groups were conducted, the one-way ANOVA test was used. Clinical characteristics by categorical variables were compared by the chi-squared test. Receiver operating characteristic (ROC) curve analysis was used for the diagnosis of the related biomarkers. All P values were shown with the results of two-sided tests and P < 0.05 was considered statistically significant. All statistical analyses and graph editing were conducted using SPSS 13.0 (SPSS, Chicago, IL) and GraphPad5 software (La Jolla, CA). 

Results

The circulating miR-29b and VEGFA in glioma cases and controls

In this study, we found that the circulating miR-29b level in glioma patients is significantly lower compared with the control group. In advanced, compared with the samples from the controls, the miR-29b level is 0.85 ± 0.20 in stages I–II and 0.74 ± 0.15 in stages III–IV. However, advanced study showed that VEGFA expression was higher in the glioma (321.0 ± 49.4 pg/mL in stages I–II and 552.0 ± 51.6 pg/mL in stages III–IV) compared with the control group (220.7 ± 34.4 pg/mL). The detailed data is shown in Figure 2.

Figure 2. Expression of circulating miR-29b and VEGFA in glioma cases and healthy controls. (A) Expression of circulating miR-29b in stages I and II, stages III and IV glioma cases and healthy controls. (B) Expression of circulating VEGFA in stages I and II, stages III and IV glioma cases and healthy controls.

Circulating miR-29b and VEGFA and the clinicopathologic characters of glioma patients

In addition, we also detect the levels of miR-29b and VEGFA and the clinicopathologic characters of glioma patients. When the miR-29b was considered, no significant differences were detected between the different gender (P = 0.535) and age group (P = 0.455). However, the miR-29b levels were different among varied WHO stage groups. The glioma cases in stages I and II were reported to have a higher rate of high miR-29b compared with the never (P < 0.001). When the tumor size was considered, the tumor size ≥5 cm have a lower rate of high miR-29b level compared with the group with <5 group (P < 0.001). However, no significant differences were detected between KPS groups (P = 0.081), extent of resection (P = 0.480) and adjuvant treatment group (P = 0.946).

Besides, we also detected VEGFA level and clinicopathologic characters of glioma patients. No significant differences were detected between the different gender (P = 0.523) and age group (P = 0.194) when VEGFA level was considered. It was reported that higher levels of VEGFA in the patients with higher WHO stage (stages III–IV vs. stages I–II, P < 0.001) and less KPS score (≥80 group vs. <80 group, P = 0.004). In addition, we also find that the tumor size (P = 0.195), extent of resection (P = 0.392) and adjuvant treatment group (P = 0.550) were not associated with the higher levels of VEGFA (Table I).

Table I. Correlation of serum miR-29b and VEGFA expression with clinicopathological features of glioma.

Clinicopathological features	N	miR-29b		P	VEGFA		P
		High	Low		High	Low
Gender	240	123	117	0.535			0.528
Female	131	67	64		63	68
Male	109	56	53		52	57
Age (years)		112	128		130	110
<50	103	49	54	0.455	52	51	0.194
≥50	137	63	74		78	59
WHO grade		115	125		119	121
WHO I–II	156	90	66	<0.001	65	91	<0.001
WHO III–IV	84	25	59		54	30
KPS		124	116		116	124
≥80	126	71	55	0.081	50	76	0.004
<80	114	53	61		66	48
Tumor size
≥5 cm	79	30	49	<0.001	50	29	0.195
<5 cm	161	112	49		91	70
Extent of resection		125	115		130	110
Total resection	189	99	90	0.480	101	88	0.392
Partial resection	51	26	25		29	22
Adjuvant treatment		124	116		123	117
Radiotherapy	108	64	44	0.946	57	51	0.550
Chemotherapy	33	20	13		19	14
Radiotherapy combined with Chemotherapy	99	59	40		47	52

The P value in bold was statistically significant.

The association of circulating miR-29b and VEGFA

As well, we also detected the association between circulating miR-29b and VEGFA in the patients with glioma. As shown in Figure 3, we find a significant inverse correlation between circulating miR-29b and VEGFA (r² = 0.864, P < 0.001).

Figure 3. The association between circulating miR-29b and VEGFA in glioma cases.

The change of the circulating miR-29b and VEGFA after the surgery

A total of 108 glioma cases were obtained to detect the change of the circulating miR-29b and VEGFA after surgery. Compared with the pre-operative expression, the miR-29b demonstrated a significant augmentation (P = 0.001). Besides, the level of VEGFA showed significant suppression after the surgery (P = 0.012). All the detailed data are shown in Figure 4.

Figure 4. ROC curves of circulating miR-29b and VEGFA for the diagnosis of glioma.

The diagnostic value of miR-29b and VEGFA for glioma

As indicated above, we found that miR-29b was down-regulated, while VEGFA was up-regulated in glioma cases. Thus, we conducted the ROC analyses for miR-29b and VEGFA in the diagnosis of glioma. Figure 5(A) indicates the AUC of the ROC curves, in order to evaluate the diagnostic effect of miR-29b and VEGFA levels of glioma patients. In order to discriminate the glioma cases from healthy controls, the AUC for circulating miR-29b was 0.913 (P < 0.001; 95% CI, 0.862–0.968). Besides, the AUC for circulating VEGFA was 0.752 (P < 0.001; 95% CI, 0.592–0.862, Figure 5B).The result indicated that miR-29b and VEGFA could be used as a potential diagnostic biomarker for glioma patients.

Figure 5. The circulating miR-29b and VEGFA level of pre- and post-operation of the glioma patients.

Clinicopathological features and circulating biomarkers as risk factors of glioma

We also conducted advanced analyses to further demonstrate that clinicopathological features and circulating biomarkers as risk factors of glioma. As shown in Table II, we found that no association between age or gender with the incidence of glioma. It was also elevated that alcohol drinking was not significantly associated with risk of glioma. However, it was reported that tobacco smoking significantly increased glioma incidence rate in this study (P = 0.029). In addition, we also conduct advanced researches to study the circulating risk factors of glioma cases. We find that miR-29b expression was associated lower glioma risk (RR = 0492; 95% CI, 0.238–0.882). However, the VEGFA expression was found to be associated higher incidence of glioma (RR = 2.826; 95% CI, 1.93–4.545).

Table II. Clinicopathological features and serum biomarkers as risk factors of glioma.

Parameter	Risk ratio	95% Confidence interval	P
Gender	1.539	0.664–2.274	0.629
Age	2.236	0.831–3.925	0.547
Tobacco smoking	2.861	1.323–4.894	0.026
Alcohol drinking	2.425	0.863–4.381	0.476
Radiation contact	0.653	0.342–2.763	0.341
Glioma history	0.912	0.452–2.183	0.362
miR-29b expression	0.492	0.238–0.882	0.001
VEGFA expression	2.826	1.93–4.545	<0.001

The P value in bold was statistically significant.

Discussion

Altered expression of miR-29b and VEGFA has been found in every malignancy examined and reported to play important roles in the development of kinds of cancers. There is increasing evidence that both miR-29b and VEGFA have potential as both diagnostic biomarkers for kinds of cancers. Here, we report that elevated it is the expression of miR-29b and VEGFA and their diagnostic effects for the glioma patients.

The results of this present word indicated that the expression of both miR-29b and VEGFA in blood was significantly different compared with the control group, and thus may help to differentiate glioma cases from the controls. Advanced study showed that worse tumor WHO stage and tumor size was associated with lower level of miR-29b; while the higher VEGFA expression was associated with poorer tumor stage and worse KPS score. In addition, the diagnostic role of miR-29b and VEGFA was important for the clinical application. Compared with the pre-operative expression, the expression of miR-29b and VEGFA demonstrated significant modification. In the study, we focused on the expression level of the two kinds of biomarkers and the detailed mechanisms should be studied by advanced well-designed experiments.

In recent years, the effects of miR-29b in the development of different cancers have been reported (Grant et al. 2014, Okamoto et al. 2013). A recent study was conducted to determine the clinical significance of miR-29b expression in primary breast cancer patients (Wang et al. 2014b). The results showed that miR-29b and certain target genes of miR-29b, such as DNA methyltransferase 3A (DNMT3A), ten–eleven translocation 1 (TET1) and thymine DNA glycosylase (TDG), was performed in 94 primary breast cancer samples. Low expression of miR-29b in primary tumors was significantly associated with poorer disease-free survival and overall survival. Multivariate analysis indicated that miR-29b expression was an independent prognostic factor for overall survival (Shinden et al. 2015). Another study involving a total of 54 patients diagnosed with CRC and 42 healthy controls showed the different miR-29b expression (Wang et al. 2014a). For the expression analysis, total RNA was extracted from FFPE tissue samples and converted to cDNA. All expression analyses were assessed by using Fluidigm Microfluidic Dynamic Array chips for 96 samples and the reactions were held in Fluidigm BioMark™ HD System Real-Time PCR. As a result of the study, miR-29b-3p was one of the 18 deregulated miRNAs in CRC (Kara et al. 2015).

There were also several previous indicated the potential association between miR-29b and glioma (Aldaz et al. 2013, Ouyang et al. 2015). GBM is the most frequent and malignant brain tumor, characterized by an elevated capacity for cellular proliferation and invasion. MiRNAs are responsible for the development, progression and therapeutic resistance of human GBM. A study by Cortez et al. reported that miR-29b and miR-125a are down-regulated in GBM and also in CD133-positive cells. Taken together, these results suggest that miR-29b and miR-125a represent potential therapeutic targets in GBM (Cortez et al. 2010). As GBM was one of the subtypes of glioma, it was possible that miR-29b might produce certain effects in the glioma.

In this study, we also found that both miR-29b and VEGFA were differently expressed between the glioma cases and controls. Advanced study showed that the expression of miR-29b and VEGFA was negatively associated in the glioma cases and healthy controls. Thus, it could be inferred that miR-29b might be used as an effective biomarker for the diagnosis of glioma cases. As VEGFA was one of the potential target genes of miR-29b, the effect of miR-29b might be associated with the modification of VEGFA. For instance, overexpression of miR-29b suppressed MG63 cell proliferation, migration and invasion. Meanwhile, miR-29b could induce apoptosis of MG63. Besides, miR-29b directly targets VEGF and over-expression of miR-29b led to down-regulation of VEGF protein level, In conclusions, miR-29b may play an important role in osteosarcoma progression, which might negatively regulate the expression of VEGF and suppresses proliferation and induces apoptosis of MG63 cell line (Zhang et al. 2014). Accordingly, miR-29b is enriched in luminal breast cancers and loss of miR-29b, even in GATA3-expressing cells, increases metastasis and promotes a mesenchymal phenotype. Mechanistically, miR-29b inhibits metastasis by targeting a network of pro-metastatic regulators involved in angiogenesis, collagen remodeling and proteolysis, including VEGFA, ANGPTL4, PDGF, LOX and MMP9 (Chou et al. 2013). In this study, the association between miR-29b and VEGFA showed that potential effects in the development of glioma. Also, the detailed effects might be studied in advanced studies.

The change of the circulating miR-29b and VEGFA after the surgery showed that the level of miR-29b increased compared with the pre-operative condition. By now, the source of miR-29b in blood is not totally clear. The significant association of between glioma stage and tumor size with miR-29b expression in the glioma cases showed that glioma tumor tissue might modify the expression of miR-29b. Besides, the VEGFA levels decreased after the surgery and these results indicate that serum VEGFA is released from tumor cells, rather than from healthy cells affected by cancer.

Conclusion

In conclusion, stable and detectable circulating miR-29b and VEGFA of patients with glioma have been reported in this study. MiR-29b could be a useful biomarker for the incidence of glioma. If validated in a large-scale case–control study, this test may be useful in future as a non-invasive screening test for glioma. The association between miR-29b and VEGFA suggested the potential mechanism.

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.