Neurol Sci DOI 10.1007/s10072-016-2727-2

ORIGINAL ARTICLE

The effects of CD147 on the cell proliferation, apoptosis, invasion, and angiogenesis in glioma Haoyuan Yin1 • Ying Shao2 • Xuan Chen1

Received: 20 July 2016 / Accepted: 30 September 2016 Ó Springer-Verlag Italia 2016

Abstract To analyze the effects of extracellular matrix metalloproteinase inducer (CD147) on glioma proliferation, apoptosis, invasion, and angiogenesis. Tissue samples were obtained from 101 glioma cases while normal brain tissues were obtained from 30 brain injury cases. Immunohistochemical assay was performed to detect the expressions of CD147, CD34, and VEGF in tissue samples. QRT-PCR was performed to detect the relative expression of CD147 mRNA in human glioma cell lines. CD147 siRNA was transfected into glioma cell line U251. Cell proliferation, apoptosis, invasion, and angiogenesis were tested by MTT, flow cytometry, Transwell assay, and vasculogenic mimicry assay, respectively. Expressions of relative proteins were analyzed with western blot. CD147 was positively expressed with the percentage of 0, 37.5, 44.8, 67.9, and 85.7 % in normal tissues and glioma tissues with WHO grades I–IV, respectively, and the scores of MVDand VEGF were associated with the expression of CD147. CD147 was significantly upregulated in the human glioma cell lines (P \ 0.05). Downregulated the expression of CD147 suppressed cell proliferation, blocked cell cycle, induced apoptosis, inhibited cell invasion and angiogenesis in glioma cells in vitro. The expression of CD147 was significantly associated with WHO tumor grade and angiogenesis; silencing of CD147 contributed to inhibition of glioma proliferation, invasion, and angiogenesis. Our

& Xuan Chen [email protected] 1

Department of Neurovascular Surgery, The First Hospital of Jilin University, No71 Xinmin Road, Changchun, Jilin 130021, China

2

Department of Plastic Surgery, The First Hospital of Jilin University, Changchun, Jilin 130021, China

study provided firm evidence that CD 147 is a potential glioma target for anti-angiogenic therapies. Keywords Glioma  CD147  Proliferation  Apoptosis  Invasion  Angiogenesis

Introduction Glioma is a typical class of primary brain tumor, which is able to affect the surrounding brain tissues and astrocytic glioma accounts for the largest proportion of glioma cases [1, 2]. The incidence rate for all histologic types of glioma ranges from 4.67 to 5.73 per 100,000 people [3]. Glioma cases are further divided into four categories based on histological classification: two low-grade astrocytomas (WHO grade I–II), anaplastic astrocytomas (WHO grade III), and glioblastoma (GBM, WHO grade IV) [4]. Although there have been noticeable advancements in current multimodal treatments including radical surgery, chemotherapy, and radiotherapy in the past several decades, the prognosis of glioblastoma remains tremendously poor with a median survival approximately 12–15 months and a 5-year survival rate \3 % [5, 6]. Several factors may trigger resistance to glioma therapies, including invasiveness of glioma cells through white matter tracts, immortalized cellular growth, and abnormal angiogenesis [7]. Although a number of molecular markers have been discovered, their prediction power is very ambiguous [8]. Hence, we proposed this study for discovering new diagnostic markers that are able to provide reliable information with respect to prognosis prediction. Furthermore, the investigation of prognostic markers may also explain potential molecular mechanisms involved in the progression of glioma [9, 10]. Recently, various

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studies have reported that high expressions of various matrix metalloproteinases (MMPs) were related to advanced tumor stage, invasion, metastasis, and shortened survival of human glioma due to degrading effects of their extracellular matrix (ECM) on both primary tumor site and secondary tumor colonization site [11]. The investigation of MMP-inducing mechanism in tumor cells resulted in the identification of ECM metalloprotease inductor (EMMPRIN/basigin), also known as CD147, which is a significant inductor of the MMPs production [12]. CD147, a transmembrane glycoprotein and a member of the immunoglobulin (IgG) superfamily, is comprised of a transmembrane region which is essential to the counter receptor binding activity and another short cytoplasmic region which interacts with Cav-1 [13]. It has been confirmed that CD147 has a higher expression in a variety of malignancies including glioma when compared with surrounding normal tissues [14]. The invasive effect of CD147 on tumors has also been reported by immunohistochemistry in various categories of carcinoma cells and surrounding tissues [15]. Cancer cells interact with surrounding normal cells to produce MMPs through CD147 on their surface, and then they affect both lymphatic tissues and blood vessels, eventually permeating into adjacent organs through the process of ECM [16]. Owing to the important role of CD147 in the progression of tumor, a variety of research has indicated that CD147 is related to the prognosis of various human malignancies, including salivary duct carcinoma, prostate cancer, bladder cancer, breast cancer, colorectal cancer, and pulmonary adenocarcinoma [17–19]. However, the effects of CD147 on human glioma have not been identified. Herein, we analyzed the association between the expression of CD147 and different grades of glioma patients. Also, we studied the potential mechanisms and roles of CD147 in the angiogenesis of malignant glioma through experiment in vitro.

documents. This study was approved by the First Hospital of Jilin University ethics committee. Cell culture and transfection The human glioma cell lines including U251, U87, SHG44, U-118, and normal human astrocyte cell line HEB (American Type Culture Collection, ATCC) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Invitrogen) with 10 % FBS (Invitrogen) at 37 °C in humidified atmosphere with 5 % CO2. Cells were cultured in 6-well plates for 24 h and transfected with CD147siRNA or negative control siRNA using LipofectamineTM 2000 (Invitrogen) according to the manufacturer’s instructions for another 6 h. Then the fresh culture medium was added for further culture and harvested in all experiments. Immunohistochemical analysis

Materials and methods

Streptavidin-peroxidase (SP) immunohistochemical staining method was adopted to determine the expressions of CD147, CD34, and VEGF in the 101 brain glioma tissue samples and 30 brain injury tissue samples. In accordance with the kit (Maxin, China), rabbit anti-human CD147, CD34, and VEGF antibodies (dilution 1:1000, BD, USA) were used as primary antibodies. PBS buffer instead of the primary antibody was utilized as a negative control. Horseradish peroxidase-conjugated (HRP-conjugated) goat anti-rabbit IgG antibody (dilution 1:800, Zhongshan Biology Company, Beijing) was used as the secondary antibody. Then samples were analyzed by 3, 30 -diaminobenzidine (DAB) substrate kit (Maxin, China) according to the manufacturer’s instructions. All specimens were observed with five horizons and the average value was utilized in the study. The results were analyzed according to the label index. Label index(%) = count (positive cell)/count (total cell) 9 100 %. MVD was assessed by calculating the microvascular structures consisting of cells with positive expressions of CD34.

Clinical specimens

Total RNA extraction and qRT-PCR

101 glioma tissues from glioma patients who underwent tumor removal surgery in the First Hospital of Jilin University during 2014 and 2016 were involved in this study (59 males and 42 females, mean age 33 years old). The number of cases which were classified as WHO grade I, II, III, and IV were 16, 29, 28, and 28, respectively. The control group (normal brain tissues) was comprised of 30 individuals who underwent brain surgery as a result of brain injury (18 males and 12 females, mean age 30 years old). All of the patients signed consent forms and related

The total RNA of cells collected in logarithmic phase was extracted using Trizol (Invitrogen, USA). For CD147 mRNA detection, cDNA was synthesized using miRNA reverse transcription kit (Promega, USA). QRT-PCR was performed using SYBR-Green PCR kit (TransGen Biotech, China) following the manufacturer’s instructions. b-actin was used to normalize the expression of CD147 mRNA. The relative expression level of CD147 mRNA was calculated by relative quantification (2-DDCt) method. Primers used for qRT-PCR were shown in Table 1.

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Neurol Sci Table 1 Primers used for qRT-PCR Gene

Primers

CD147 (forward)

50 -GAGAGCAGGTTCTTCGTGAGTTC-30

CD147 (reverse)

50 -GCCTTTGTCATTCTGGTGCTG-30

b-actin (forward)

50 -CCACGAAACTACCTTCAACTCCA-30

b-actin (reverse)

50 -GTGATCTCCTTCTGCATCCTGTC-30

with matrigel for cell attachment. The above steps were followed by incubation and tube formation observation for 1 h. Once the formation of lumen was achieved in the control group, the number of microtubules was counted for five times to obtain the average value. Finally, a microscope was used to visualize the formation of tube in each group and the corresponding photographs were recorded. Western blot

Cell proliferation assay by MTT Cells were seeded and incubated for 24, 48, 72, and 96 h. After the medium was replaced, 20 ll MTT (0.5 mg/ml) was added into each micropore and incubated for another 4 h. The supernatant was discarded and 150 ll dimethylsulfoxide (DMSO) was added into each cell micropore, then placed into a shock shaker at the speed of 20 rpm for 10 min. The corresponding absorbance value was detected by the microplate reader at the wavelength of 490 nm. Experiments were carried out for three times. Cell cycle and apoptosis analysis in vitro For cell cycle assay, after U251 cells were transfected for 96 h, trypsin digestion was performed and the corresponding cell density was adjusted to a concentration level of 5 9 106/ml. The cells were combined with 70 % cold ethanol overnight at 4 °C and then washed with PBS three times and followed by incubation with PBS containing 0.5 mg/ml RNase A and 10 mg/ml propidium iodide for 30 min at 37 °C in the dark. The cell cycle was measured with FACS cytometry (BD Biosciences, USA). Each experiment was performed in triplicate. For cell apoptosis assay, 5 9 106 cells in each group were collected and then stained with Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences), the apoptosis rates of cells were acquired using FACS cytometry. Each experiment was performed in triplicate. Transwell assay Transwell chambers were prepared in accordance with the instructions and cell density was adjusted for preparing the cell suspension solution. In addition, Transwell chambers were incubated for 12 h after inoculation and the medium was removed and washed gently by PBS for three times. The corresponding data during these procedures were recorded. Vasculogenic mimicry (VM) experiment On completion of transfection for 72 h, cells were trypsinized to single cell suspension. After adjusting the cell density for each group, cells were inoculated in 24-well plates pre-coated

Cells were collected and lysated to obtain the total protein after the culture medium was discarded. Then the protein concentration was determined with a BCA kit (Thermo Scientific, America). Proteins were separated on SDS-PAGE by Bio-rad system, and then transferred to the PVDF membranes, and blocked with 5 % skim milk (Millipore, USA). Target proteins were detected by incubation overnight at 4 °C with primary antibodies (abcam, USA, Catalogue Number: ab666) and GAPDH (abcam, USA, Catalogue Number: ab8245) as a normalizer. Then the membranes were washed and incubated with secondary antibodies for another 2 h. Once the hybridization membrane was processed, it was placed on a transparent plastic plate and the fluorescent chemical luminescent substrate was uniformly added onto the surface of the membrane. The reaction process lasted for 5 min and the filter paper provided by the kit was used to absorb excessive substrate solution appeared on the surface of the membrane. The membrane was inserted into the cartridge and the integral optical density value of the protein bands that are related to the invasion, migration, and angiogenesis process was determined. Statistical analysis The results were depicted as mean value ± standard deviation; the t test or non-parametric rank sum test (Mann–Whitney test) was used to assess difference in continuous variables between two groups whereas the analysis of variance (ANOVA) or non-parametric Kruskal– Wallis test was performed to calculate difference in continuous variables among multiple groups. Counting data were assessed using the Chi-square (v2) test. GraphPad Prism 6.0 software (GraphPad Software, Inc. La Jolla, USA) was employed for data analysis and image production. P \ 0.05 was considered statistically significant.

Results CD147 expression was correlated to tumor grades and angiogenesis in glioma patients We used immunohistochemistry assay to detect the levels of CD147 and angiogenesis (CD34 and VEGF) in the

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normal brain tissues and glioma tissues. As shown in Fig. 1a, the expression of CD147 in glioma tissues was significantly higher than those in normal tissues, and an increased expression of CD147 on average appeared to be associated with the progression of glioma grades. In addition, there were lower levels of VEGF and MVD in earlystage tumors (WHO grade I–II) than those in more malignant stage tumors (WHO grade III–IV) (Fig. 1b, c). According to the status of CD147 expression, the glioma tissues were divided into four groups (-, n = 39; ?, n = 21; ??, n = 18; ???, n = 23), and the scores of MVD (CD34 positive rate) and VEGF were associated with

CD147 expression, which are shown in Fig. 1d, e. Obviously, there were significant differences in MVD and VEGF scores among the different groups, which suggested CD147 expression was significantly correlated with MVD and VEGF. Therefore, we hypothesized that there is a positive correlation between CD147, glioma tumor grades, and angiogenesis.

Fig. 1 CD147 expression and scores of MVD and VEGF were measured by Immunohistochemistry. a Immunohistochemistry assay utilized to determine the expression of CD147 in normal brain tissues and glioma tissues with different WHO grades (I, II, III, IV) (940). b MVD comparison between glioma tissues with different WHO grades. c VEGF comparison between glioma tissues with different

WHO grades. ***P \ 0.0005 versus WHO grades I glioma tissues. d MVD comparison between glioma tissues with different CD147 expression intensity. e VEGF comparison between glioma tissues with different CD147 expression intensity. **P \ 0.001, ***P \ 0.0005 versus CD147 expression intensity

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CD147 was upregulated in human glioma cells We used qRT-PCR to detect the expression of CD147 in human glioma cell lines. As shown in Fig. 2, CD147 was

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SiRNA interference resulted in an upregulation of the protein expression of P53, Bax, and Mdm2 and a downregulation of Bcl-2 (Fig. 3d). These results revealed that CD147 expression profile was closely related to the proliferation of glioma, and CD147 silencing could inhibit the proliferation of glioma and induce the apoptosis of glioma cells. Effect of CD147 silencing on glioma cell invasion and angiogenesis Fig. 2 qRT-PCR was performed to detect the relative expression of CD147 mRNA in human glioma cell lines including U251, U87, SHG-44, U-118. *P \ 0.05 versus HEB

significantly upregulated in human glioma cells (P \ 0.05) compared with HEB. U251 was selected for further experiments in terms of its highest expression. CD147 silencing suppressed cell proliferation, blocked cell-cycle, and induced apoptosis in glioma cells in vitro After CD147-siRNA transfection for 24 h, the proliferation of U251 cells was significantly inhibited (P \ 0.01, Fig. 3a), while the cell apoptosis was significantly enhanced (P \ 0.05, Fig. 3b). In addition, the cell cycle analysis showed that CD147 silencing caused cell mitosis inhibition, the cell cycle was arrested in G0/G1 phase, and cells in S phase was significantly decreased (Fig. 3c).

Fig. 3 Effects of CD147 silencing on the proliferation, apoptosis and cell cycle of glioma cell line. a MTT method employed to test cell viability after glioma cell lines transfected by CD147-siRNA; b flow cytometry utilized to detect the apoptosis after glioma cell lines were

Results from Transwell assay showed that after silencing CD147 by siRNA transfection, the number of glioma cells penetrating across the membrane was significantly lower than those in the control and negative control group which was transfected with empty vector (P \ 0.05, Fig. 4a). The results in this section suggested that the invasion ability of the glioma cells were significantly suppressed by the silencing of CD147. The results of in vitro VM experiment showed that the microtubule density of siRNA group was significantly lower than that of the control and negative control group in glioma cell lines (P \ 0.05, Fig. 4b). The results in this section confirmed that the vasculogenic mimicry ability of glioma cell lines was inhibited after CD147 silencing. To study the mechanisms of glioma invasion and angiogenesis inhibition caused by silencing CD147, western blot assay was performed to detect the expressions of proteins that are closely related with invasion and vasculogenic mimicry ability. Finally, western blot results showed that the expressions of MMP-2, MMP-

transfected by CD147-siRNA; c cell cycle changes after CD47siRNA transfection; d Western blot utilized to check the expression of proteins related to apoptosis

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Fig. 4 Effects of CD147 silencing on glioma cell invasion and angiogenesis. a Transwell assay results of glioma cells in different groups after CD147-siRNA transfection. b Vasculogenic mimicry assay results of glioma cells in different groups after CD147-siRNA transfection. c Detection of expression levels of proteins associated

with cell invasion and vasculogenic mimicry after glioma cell lines transfected by CD147-siRNA. d Detection of cell invasion and vasculogenic mimicry in three groups. ***P \ 0.001, ****P \ 0.0001 versus siRNA–NC group

9, VEGFR-1, and VEGFR-2 in siRNA group cell lines were significantly inhibited compared with those in the control group (Fig. 4c).

One of the specific characteristics of glioma is the development of well-perfused blood vessels in the brain of very high density, which distinguishes glioma from many other categories of cancers. Angiogenesis is the formation of new blood vessels accompanied with pre-existing vasculature, which is crucial for occurrence and development of tumors [22]. The specific process occurs as follows: tumor cells produce excessive angiogenic factors, leading to endothelial cell (EC) activation and resulting in angiogenic phenotype. Vascular extracellular matrix is altered and basement membrane is degraded, stimulating the proliferation and migration in ECs. Eventually, vascular ECs form a capillary loop and tubular lumen [23]. In brief, angiogenic factor secretion of tumor cells and activation of ECs play important roles in tumor angiogenesis. Glioblastoma is a vascular tumor and it is particularly important to suppress the secretion of angiogenesis-related factors in glioma cells, and subsequently restrain vascular EC proliferation and migration when glioma treatments are involved. Glioblastoma is usually characterized by a focal

Discussion Glioma is the most prevalent astrocytic brain tumor and is characterized by extremely destructive invasion and aggressive malignancy with both high incidence and mortality [20, 21]. Although there have been strong improvements and innovations in chemoradiotherapy, immunotherapy, surgical techniques, and targeted therapy, the 5-year overall survival rate for glioma patients remains poor, especially in malignant glioma such as glioblastoma (GBM). Therefore, there is an urgent need for greater understanding of the molecular factors involved in the therapy and prognosis of glioblastoma, which will contribute to appropriate therapeutics and accurate diagnostic and prognostic predictive abilities.

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lesion with central necrosis surrounded by an angiogenic tumor rim, and this category of tumor affects the surrounding extracellular matrix by both white matter tracts and blood vessels as substrates. Pre-existing evidence has suggested that angiogenesis is crucial for the growth of malignant glioma [24, 25]. The first goal of our present study was to confirm the relationship between CD147 and glioma. Accumulating studies indicated that the expression level of CD147 protein and mRNA in glioma-affected brain was higher than that in normal cases [14, 26]. These studies demonstrated that CD147 might exert an oncogenic effect on human glioma. In addition, the protein expression of CD147 was significantly correlated with WHO grade of glioma since intensive CD147 staining was more likely to be detected in the advanced grade of glioma [27]. Previous study proved the same trend in the mRNA expression level of CD147 indicating that CD147 mRNA expression was also related to the progression of tumor [28]. These researches showed that the mRNA and protein expression level of CD147 was highest in grade IV of glioma followed by grade III and low grade of glioma and this trend may be explained by the increase in the invasion and metastasis ability of glioma at advanced grades. Consistent with above research, our study found that the expression of CD147 was positively correlated with the WHO grading of glioma and the angiogenesis, suggesting that CD147 may act as a marker for evaluating the grading and prognosis of glioma. CD147 is expressed at the surface of human tumor cells, and is secreted by these cells, interacting with stromal fibroblasts to activate them to produce MMP-1, -2, -3, -9, 14, and -15 [29]. The expression of CD147 mRNA and protein is believed to be elevated in some tumors, such as breast cancer, hepatoma, colorectal cancer, ovarian cancer, and esophageal squamous cell carcinoma, and can promote tumor invasion via facilitating MMP synthesis in surrounding stromal cells [30, 31]. Associations of CD147 expression with prognosis have also been proved in seminomas, pulmonary adenocarcinoma, prostate cancer, salivary duct carcinoma, colorectal cancer, bladder cancer, and breast cancer [19, 32]. Concerning glioma, one previous study has found that CD147 may stimulate tumor cell invasion [28]. CD147 has been shown to be involved in the invasiveness and angiogenesis of glioma, and modulate glycolysis in glioma [33–35]. Consistent with findings of Tian et al., we found that silencing of CD147 by the transfection of CD147 SiRNA in glioma cell line U251 can downregulate the levels of Bcl-2, and upregulate the levels of P53, Bax, Mdm2 in glioma cells, further inducing the apoptosis of glioma cells. In addition, we found that silencing of CD147 through the transfection of CD147 SiRNA in glioma cell line U251 can downregulate the levels of MMP-2, MMP-9,

VEGFR-1, and VEGFR-2 in glioma cells, which inhibits the degradation of extracellular matrix components and thereby suppressing the invasion and angiogenesis in tumor tissues. Therefore, we propose that CD147 might be a novel therapeutic target for glioma. However, this study was concerned with only 101 cases and human glioma cell line U251 cell was used to detect the influence of CD147 and its downstream molecules on tumor proliferation, apoptosis, invasiveness, and angiogenesis. So there were still some limitations in the present study, which need to be improved upon in future research. The molecular mechanism of CD147 and its downstream molecules such as MMPs and VEGFs on angiogenesis of glioma will need to be studied further. In conclusion, the expression level of CD147 was positively correlated with both the progression of glioma and angiogenesis. Moreover, the silencing of CD147 can inhibit cell proliferation, angiogenesis, and vascular mimicry, but induce apoptosis in glioma. Therefore, CD147 is s potential diagnostic and therapeutic target for glioma patients. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest.

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