Positive Expression of Retinol-Binding Protein 4 Is Related to the Malignant Clinical Features Leading to Poor Prognosis of Glioblastoma

Backgrounds Retinol-binding protein 4 (RBP4) is a monomeric-binding protein belonging to the lipocalin protein family, which has been reported to be dysregulated in several malignancies such as breast cancer and lung cancer. However, the expression and function of RBP4 in glioblastoma (GBM) are completely unknown. Materials and Methods TCGA datasets were used for analyzing the mRNA level of RBP4 in GBM and its clinical relevance. A retrospective GBM cohort (n = 73) was enrolled from our hospital to test the protein expression profile of RBP4 in GBM tissues as well as its correlation with patients' prognoses. Two human GBM cell lines, LN229 and U251, were collected to conduct overexpression and knockdown experiments targeting RBP4. The tumor-related effects of RBP4 in GBM were finally evaluated by proliferation and invasion assays. Results Both the higher mRNA level and protein level of RBP4 in GBM tissues were significantly correlated with poorer patients' overall survival. Multivariate analysis identified RBP4 as a novel independent prognostic predictor in GBM patients. Overexpression of RBP4 resulted in enhanced GBM proliferation capacity, which was consistent with clinical findings on the positive correlation between RBP4 level and tumor size. Meanwhile, overexpressing RBP4 promoted GBM cell migration and invasion, while silencing RBP4 led to the opposite results. Conclusions RBP4 overexpression in tumor tissues is correlated with poorer prognosis of GBM patients, which functions by promoting GBM proliferation and invasion, thus, may serve as an invaluable predictive biomarker and therapeutic target.


Introduction
Te most common malignancy of the central nervous system in adults is glioma, which originates in the brain or glial tissues. Pathologically, gliomas can be further classifed as astrocytomas, ependymomas, oligodendrogliomas, and glioblastomas (GBM), and so on. Among them, GBM is the most aggressive type of glioma [1]. GBM treatment depends on size, location, patient status, and other clinical and pathological factors. Major therapies include surgery, radiotherapy, and chemotherapy [2]. Due to the aggressive phenotype of GBM, the median survival time of patients is only 15 months despite advances in therapeutic development [3]. Failing to precisely predict patients' survival is another contributor for unsatisfed outcomes. Tus, there is still a great need to further understand the pathology of GBM and to employ more efective methods for prognostic prediction as well as disease treatment.
Retinol-binding protein 4 (RBP4) is a monomericbinding protein of 21 kilo Dalton, which belongs to the lipocalin family and was initially identifed as the specifc carrier for retinol in plasma [4]. Dysregulated RBP4 results in numerous diseases such as retinal dystrophy, iris Coloboma, comedogenic acne syndrome, and microphthalmia [5,6]. Moreover, an aberrant level of plasma RBP4 has been discovered in several malignancies. For example, a signifcant elevation in the serum level of RBP4 was found in pancreatic cancer patients, which was signifcantly decreased after operation than before operation [7]. Of note, the serum level of RBP4 showed no statistically diferent between patients with pancreatic adenocarcinoma or chronic pancreatitis, indicating it may not be a suitable biomarker for pancreatic cancer [8].
However, the fact that RBP4 is altered in several tumor types is contradictory. On the other hand, the serum concentration of RBP4 in ovarian cancer patients was signifcantly lower than that in healthy volunteers, according to mass spectrometry data [9]. On the other hand, Cheng et al. reported a signifcantly higher serum RBP4 level in ovarian cancer patients than those in healthy individuals [10]. Similar contradictory fndings were reported in renal cell carcinoma (RCC). Choi et al. reported that RBP4 was higher in RCC cases than that in healthy controls [11], while the lower serum RBP4 level was correlated with unfavorable overall survival (OS) and disease-free survival (DFS) according to Sobotka's data [12].
Moreover, it appears that the expression pattern of RBP4 in solid tumor tissues difers from its serum concentrations. For example, patients with breast cancer may showed higher serum RBP4 level [13], although another study found no statistically signifcant association between serum RBP4 and breast cancer development [14]. Intriguingly, bioinformatic analyses demonstrated a lower expression of RBP4-mRNA in breast cancer tissues, and lower RBP4 indicated poorer overall survival of breast cancer patients [15]. Similarly, hepatocellular carcinoma (HCC) patients with higher serum RBP4 levels exhibited poorer OS and DFS compared to those with lower serum RBP4 levels [16]. In contrast to the serum level, RBP4 expression was low in HCC tissues compared with normal tissues, and low RBP4 expression levels were associated with advanced tumor stages and poorer overall survival [17].
Terefore, RBP4 may play multiple roles in diferent types of cancer. Here, we aimed to initially investigate the expression and potential function of RBP4 in GBM from both the TCGA dataset and our retrospective cohort. A univariate and multivariate analysis was conducted to evaluate the clinical signifcance and prognostic predictive role of RBP4 in GBM, which revealed its independent prognostic efect. Additionally, we assessed the tumor-related function of RBP4 in GBM cell lines by testing their proliferation, migration, and invasion after RBP4 interference.

Online Data
Mining. Te RBP4-mRNA in GBM tissues was extracted from the TCGA dataset and presented as FPKM (fragments per kilobase of transcript per Million) according to the RNA-seq data. Te Kaplan-Meier method was used to evaluate the prognostic efect of RBP4-mRNA. MethSurv web tool (version MethSurv©2017; https://biit.cs. ut.ee/methsurv/) was used to perform survival analysis based on RBP4 methylation using TCGA data [18].

Patients' Information and Ethics.
We retrospectively enrolled a cohort of GBM cases (n � 73) from our hospital, including 31 females and 42 males. All cases were pathologically diagnosed as single lesion GBM without other malignancy history. All cases underwent surgical resection (39 cases underwent local resection, 16 cases with radical resection, and 18 cases with lobectomy). None of the cases possessed distant metastasis before surgery. None of the cases underwent neoadjuvant chemotherapy or radiotherapy, but 12 cases underwent postoperative adjuvant chemotherapy while the other 61 cases rejected chemotherapy or unknown. Te median age was 63 years old at the time of diagnoses. Te median tumor size was 4.3 cm according to pathological records. As for the tumor location, 13 tumors located in the parietal lobe, 25 tumors in temporal lobe, 32 cases in the frontal lobe, and 3 unclear based on the surgical record. Tis study was approved by the Ethic Committee of Guangdong 999 Brain Hospital. Written informed consent was provided by each patient or immediate family member at the start of the study, and all procedures in this study were performed in accordance with the Declaration of Helsinki.

Immunohistochemistry (IHC) Staining.
A total of 73 GBM tissue samples were obtained, and tissue sections were treated for antigen retrieval using EDTA bufer according to a standard procedure [19], and then the tissues were incubated with a primary anti-RBP4 antibody (1 : 300, sc-48384, Santa Cruz Biotechnology) at 4°C overnight. Next, the sections were incubated with a secondary antibody and then visualized using diaminobenzidine (DAB) and hematoxylin counterstaining. Te immunohistochemical results were evaluated by two pathologists and defned as negative staining unless there were more than 50% positively stained cells with moderate to dark staining intensity. Te two pathologists who analyzed IHC data were blinded to the patient's information in this study.

Cell Lines and Transfection.
Human originating GBM cell lines LN229 and U251 were obtained from ATCC (American Type Culture Collection) and cultured in DMEM medium (Invitrogen) supplemented with 10% FBS at 37°C in a humidifed atmosphere with 5% CO2. Knockdown and overexpression were achieved by using Lipo3000 transfection reagent (Termo Fisher Scientifc) according to the manufacturer's instructions. Te siRNA targeting human RBP4 was synthesized by GenePharma (Shanghai, China) as described by others [20]. Te cDNA of RBP4 was cloned into a pcDNA3.0 vector by GenePharma. For the control group, cells were treated with transfection reagent and underwent the same experimental procedure.

Western Blot (WB).
Total protein in transfected cells was extracted with RIPA bufer (Beyotime, Shanghai, China), and its concentration was tested via a BCA protein assay kit (Termo Fisher Scientifc). Protein samples were then separated by SDS-PAGE and transferred to PVDF membrane (Millipore, MA, USA). Afterwards, the membrane was blocked with 5% nonfat milk and incubated with primary antibodies (RBP4, 1 : 1000, sc-48384;beta-actin, 1 : 2000, sc-8432; both from Santa Cruz Biotechnology) at 4°C overnight 2 Genetics Research followed by secondary antibody incubation for 1 h in room temperature. Te immunoreactivity was visualized by ECL reagents [21].

MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) Assay.
Te MTT assay was used to measure the proliferation of transfected cells. Briefy, 200 μL of transfected cells were incubated in a 96-well plate (2,000 cells/ well) for 1-5 days. On each day, 20 μL of MTT solution (5 mg/ mL) was added to each well and incubated for 4 h. Te optical density was measured at 570 nm using a microplate reader. Each experiment was conducted for three independent times.

Colony Formation.
Te colony formation assay was used to evaluate the clone-forming capacity of transfected cells. Briefy, 500 cells were inoculated into a 6-well plate and cultured for 14 days, during which period the medium was changed every fve days. Afterwards, the colonies were immobilized with methanol and dyed with 0.2% crystal violet. Finally, the clone number was counted. Each experiment was conducted for three independent times.

Transwell Assay.
Transfected cells were resuspended in serum-free DMEM medium (100 μL, containing 5,000 cells) and seeded into the upper chamber of the 24-well plate Transwell chamber (Corning, Cambridge, MA, USA), while the lower was added with 200 μl DMEM containing 10% FBS. Cells were cultured in 5% CO 2 at 37°C for 48 h, and then, the chamber was taken out, cells on the upper surfaces of the chamber were wiped of with cotton swabs, and cells from the bottom surfaces were fxed and stained with crystal violet for 15 min. Migrated cells were counted under an inverted microscope. Te cell invasion assay was also conducted as described above except that the membrane was precoated with diluted Matrigel (BD Biosciences, San Jose, CA, USA). In addition, the cell seeding number for invasion experiments was 20,000 cells/well. Each experiment was conducted for three independent times.

Statistics.
Statistical analyses were carried out using SPSS 16.0 software. Te cancer-specifc survival was defned as the length of time from the date of diagnosis to the date of death from GBM. Te overall survival was defned as the length of time from the date of diagnosis to the date of death from any causes. We also added the descriptions in our revised main text. Kaplan-Meier method was used to generate survival curves and compared by the log-rank test. Multivariable Cox regression analysis was used to test the independent prognostic efects of variables. Two tails of the student's t-test was used to compare the diferences between groups in cellular experiments. P < 0.05 was chose to be statistically signifcant.

High RBP4-mRNA Level in GBM Tissues Indicates Poor
Prognosis. We frst evaluated the potential clinical relevance of RBP4 by retrieving its mRNA level from TCGA datasets.
Accordingly, GBM patients with low RBP4-mRNA showed poorer overall survival (Figure 1(a), P � 0.03). Meanwhile, higher methylation of RBP4-mRNA was also correlated with poorer overall survival (Figure 1(b), P � 0.029). Considering lower mRNA levels were generally correlated with higher methylation, the mRNA data and methylation data were consistent.

Protein Expression of RBP4 in GBM Tissues.
To further explore the role of RBP4 in GBM, we next tested its protein expression pattern in GBM tissues from a retrospective cohort (n � 73). As revealed by IHC results, GBM tissues exhibited distinct RBP4 protein expression levels. According to the immunoreactivity, 31 cases were classifed as negative RBP4 expression (Figure 1(c)), while the other 42 cases were defned as positive RBP4 protein expression (Figure 1(d)). Interestingly, gliomas with larger tumor sizes were more prevalent to exhibit positive RBP4 expression compared with the smaller ones (P < 0.001, Table 1), indicating that RBP4 may participate in GBM progression.

Prognostic Factors of GBM Cohort.
Kaplan-Meier survival curves were next plotted to assess prognostic factors of GBM ( Figure 2). Te median age was 63 years old (ranging from 34-79 years old) at the time of diagnosis. Te median follow up time was 3 months, ranging from 0-97 months.

RBP4 Can Promote Proliferation, Migration, and Invasion of GBM Cells.
Here, we also conducted cellular experiments to validate the tumor-related role of RBP4 in GBM. Te transfection efciencies of overexpression and knockdown were tested by Western blot (Figure 3(a)). Comparing with control cells, RBP4-overexpression cells showed 3.8 folds and 4.1 folds increase on the RBP4 protein levels in LN229 cells and U251 cells, respectively. In contrast, siRNA transfection led to an 83% and 69% decrease on the RBP4 expression levels compared with the control groups in LN229 and U251 cells, respectively. After then, the cell viability was analyzed by the MTT assay and colony formation assay (Figures 3(b) and 3(c)). Accordingly, overexpressing RBP4 can signifcantly enhance both LN229 and U251 cell proliferation. Oppositely, silencing RBP4 resulted in impaired cell viability. Furthermore, we assessed the effects of RBP4 on GBM migration and invasion via Transwell strategies (Figures 3(d) and 3(e)). As a result, sRBP4overexpression enhanced cell migration and invasion, while RBP4-knockdown inhibited the migration and invasion processes of both GBM cell lines.

Discussion
Since RBP4 was originally identifed as a circulation protein in plasma and showed a predictive role of in several malignancies including lung cancer [22] and colon cancer [23], we were interested to investigate its role in GBM. However, our preliminary ELISA data showed no statistically significant diference regarding the serum level of RBP4 in GBM patients and healthy volunteers (data not shown). Interestingly, a higher RBP4 level in stool was also identifed as a biomarker for noninvasive detection of colorectal adenomas with a high risk of progression [24]. Since literature research demonstrated that RBP4 can also be localized in solid organs such as livers, and its abnormal expression in tissues was also correlated with malignancies. For example, immunohistochemistry results demonstrated that RBP4protein expression levels in ovarian cancer tissues were higher than those in normal ovarian tissue and exerted prognostic predictive roles [10,25]. Consistently, our data demonstrated that higher RBP4-mRNA and protein expression in GBM tissues was correlated with poorer survival after surgical treatment.
Besides the expression levels, online data mining revealed that the methylation of RBP4 in tumor tissues was positively correlated the overall survival of GBM patients (P � 0.025). Similarly, methylation of RBP4 in esophageal squamous cell carcinoma seems more frequent than that in adjacent esophagus tissues, however, the diference was not statistically signifcant (P � 0.08) [26]. Nevertheless, dysregulated methylation may help explain the abnormal expression of RBP4 in malignancies, which deserve more investigation.
A recent study reported that downregulating RBP4 in colon cancer cells decreased the fraction of cancer stem cells [27], therefore we also tested the cellular efects of RBP4 in GBM cells. According to the MTT assay and colony formation assay, knockdown of RBP4 resulted in impaired proliferation capacity of GBM cells, while RBP4-overexpression showed the opposite efects. Our data was consistent with the reported functions of RBP4 in prostate cancer cells [20] and ovarian cancer cells [25]. Besides proliferation, RBP4 also participates in the metastasis of malignancies. For example, genomic studies revealed that liver metastases from the colon adenocarcinomas showed signifcantly higher RBP4 transcript than that in paired primary colorectal carcinomas [28]. According to the data by Uehara et al. tissue expression level of RBP4 was identifed to be increased in a prostate cancer bone metastasis mice model [20]. By comparing diferent cell lines, Wang et al. also demonstrated that RBP4 protein was overexpressed in HCC cell lines compared with normal liver cell line and correlated with metastatic potential [16]. Additionally, RBP4 was reported to enhance the metastatic potential of breast cancer through both direct efect on cancer cells and through impairing endothelial blood vessels within the tumor [29]. According to the migration and invasion tests, our data also revealed a similar conclusion on the potential metastasis-promoting efect of        Indeed, serum level of RBP4 was upregulated after LA-12, a platinum-based anticancer agent, treatment in rat models [30]. Similarly, Phase I clinical trials of LA-12 indicated that circulating RBP4 levels correlated well with platinum levels in the human plasma of 12 randomly selected patients with solid tumors [30]. Since retinol metabolism is closely associated with many malignancies, targeting RBP4 may represent a novel direction for drug development. Our study has several limitations. First, RBP4 was closely correlated with metabolism [27] and has been recognized as a link between adiposity and cancer [31], however, here we didn't fnd any signifcant correlation between the body mass index with the RBP4 levels. Whether RBP4 participate in high-fatdiet-induced malignancies remains further investigation. Second, our study lacks sufcient clinical cohorts to validate the conclusions generated by the retrospective cohort with limited case numbers. Tird, we did not fully dig into RBP4's mechanism for afecting GBM progression through systematic molecular biology experiments.

Conclusions
In conclusion, our results suggested that RBP4-overexpression in tumor tissues is correlated with poorer prognosis of GBM patients. Terefore, RBP4 may serve as an invaluable predictive biomarker and therapeutic target for malignancies.

Data Availability
Te data are made available from the corresponding author upon reasonable request.  Figure 3: RBP4 promotes GBM proliferation, migration, and invasion. (a) LN229 and U87 cells were transfected with pcDNA3.0-RBP4 plasmids (overexpress), transfection reagent (control), or RBP4-siRNA, respectively. Te transfection efciencies were tested by western blot. (b) Proliferation of transfected LN229 and U87 cells were tested via MTT method. Te proliferation curves indicated that overexpressing RBP4 promotes cell proliferation while silencing RBP4 inhibits cell proliferation. (c) Colony formation assays were also conducted to evaluate cell proliferation capacity. Comparing with control cells, clone-forming capacities of RBP4-overexpressed cells were signifcantly enhanced while impaired in RBP4-knockdown cells. (d) Migration capacities of transfected cells were tested by Transwell assay, which demonstrated that RBP4 can positively regulation the migration process. (e) Invasion abilities of GBM cells were assessed by Matrigel-Transwell method. Accordingly, overexpressing RBP4 increased the number of invaded cells while silencing RBP4 decreased the number of invaded cells. Data were presented as mean ± SD. Each experiment was conducted for three independent times. * P < 0.05 compared to control group. Genetics Research 9