==== Front Open Med (Wars) Open Med (Wars) med Open Medicine 2391-5463 De Gruyter med-2020-0128 10.1515/med-2020-0128 Research Article GABRD promotes progression and predicts poor prognosis in colorectal cancer Niu Gengming 1 Deng Li 1 Zhang Xiaotian 1 Hu Zhiqing Han Shanliang Xu Ke Hong Runqi Meng He Ke Chongwei dr_kecw@163.com Department of General Surgery, the Fifth People’s Hospital of Shanghai, Fudan University, 801 Heqing Road, Shanghai, 200240, People’s Republic of China Department of Animal Science, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, 200240, People’s Republic of China 1 Contributed equally to this work. 21 11 2020 2020 15 1 1172 1183 29 3 2020 22 7 2020 13 8 2020 © 2020 Gengming Niu et al., published by De Gruyter2020Gengming Niu et al., published by De GruyterThis work is licensed under the Creative Commons Attribution 4.0 International License.Supporting InformationAbstract Little is known about the functional roles of gamma-aminobutyric acid type A receptor subunit delta (GABRD) in colorectal cancer (CRC). The expression of GABRD between CRCs and adjacent normal tissues (NTs), metastasis and primary tumors was compared using public transcriptomic datasets. A tissue microarray and immunohistochemical staining (IHC) were used to determine the clinical and prognostic significance of the GABRD in CRC. We used gain-of-function and loss-of-function experiments to investigate the in vitro roles of GABRD in cultured CRC cells. We characterized the potential mechanism of GABRD’s activities in CRC using a Gene Set Enrichment Analysis (GSEA) with The Cancer Genome Atlas Colon Adenocarcinoma (TCGA-COAD) dataset. We found that the GABRD expression was significantly increased in CRCs compared to that in NTs, but was similar between metastasis and primary tumors. Overexpression of GABRD was significantly associated with later pTNM stages and unfavorable patient survival. Overexpression of GABRD accelerated while knock-down of GABRD inhibited cell growth and migration. Mechanistically, the function of GABRD might be ascribed to its influence on major oncogenic events such as epithelial–mesenchymal transition (EMT), angiogenesis, and hedgehog signaling. Collectively, GABRD could be a novel prognostic predictor for CRC that deserves further investigation. Keywords GABRDcolorectal cancerprognosisgrowthmigration ==== Body 1 Introduction Colorectal cancer (CRC) is one of the most common malignancies in both east and west societies [1,2]. Despite recent progress in early screening and treatment [3], it remains a great challenge due to disease recurrence, drug resistance, and distal metastasis [4]. Identification of prognostic factors for CRCs may help select patients at a higher risk and develop more personalized therapies. Gamma-aminobutyric acid (GABA) is the predominant inhibitory chemical neurotransmitter in the central nervous system. Originally, three groups of GABA receptors, namely GABAA, GABAB, and GABAC, were identified. The term GABAC is no longer in use and now it is recognized that GABAA is more prevalent and functionally related to GABA. GABAA receptors are heteropentamers formed by five types of subunits, with a central chloride ion-selective channel gated by GABA [5,6]. Gamma-aminobutyric acid type A receptor subunit delta (GABRD), which encodes the GABAA receptor δ subunit, has been suggested as a susceptibility gene to childhood-onset mood disorders and generalized epilepsies [7,8]. Several recent studies have revealed the possible functional roles of GABRD in tumors. In a cohort of patients with corticotroph adenomas, Bujko et al. demonstrated that GABA-related genes including GABRD were enriched in tumors with USP8 mutations, which are driver mutations in corticotrophinomas [9]. Using data from The Cancer Genome Atlas (TCGA), Gross et al. conducted a pan-cancer analysis and found that GABRD was overexpressed in nearly 90% of the patients included [10]. In another TCGA-based bioinformatic study, GABRD expression was significantly decreased in IDH wild-type diffuse low-grade gliomas compared with that in IDH mutant tumors, while patients with a high expression of GABRD had better prognosis than those with a low expression of GABRD [11]. Although Fagerberg et al. carried out a human tissue-specific expression analysis and demonstrated that GABRD mRNA was relatively abundant in the colon under physiological conditions [12], little is known about the involvement of GABRD in tumorigenesis and progression of CRCs. In the present study, we analyzed the GABRD expression in CRCs and peritumoral normal tissues (NTs) with transcriptomic datasets from gene expression omnibus (GEO) and the cancer genome atlas (TCGA). We used clinically resected samples to validate these results with quantitative polymerase chain reaction (q-PCR). We also compared the expression levels of GABRD between primary and metastatic CRCs. We investigated the correlation between GABRD expression and patient survival with a tissue microarray by immunohistochemistry (IHC) and validated the result with the combined TCGA-colon adenocarcinoma (COAD) and rectal adenocarcinoma (READ) dataset. We investigated the in vitro roles of GABRD on cell proliferation and migration in cultured CRC cells using gain-of-function and loss-of-function assays. We characterized the possible mechanisms of GABRD’s function in CRC carcinogenesis using a gene set enrichment analysis (GSEA) with the TCGA-COAD dataset. We aimed to evaluate the prognostic value and oncogenic roles of GABRD in CRCs. 2 Patients and methods 2.1 Human CRC samples and cell lines This study was approved by the Shanghai Fifth People’s Hospital Institutional Ethics Committee (Ethical Approval Form Number: 2017-097) and adhered to the principles listed in the Declaration of Helsinki. Informed consent was obtained before the collection of tissues. Sixteen paired tumor and NTs were collected from CRC patients at the Shanghai Fifth People’s Hospital (Shanghai, China) between 2016 and 2018. The samples were snap-frozen in liquid nitrogen and stored at −80°C. The corresponding formalin-fixed and paraffin-embedded tissues were retrieved, and 4-µm tissue sections were prepared by the Department of Pathology at the same hospital. Tissue microarrays were prepared by Shanghai Outdo Biotech (Shanghai, China) and contained CRCs from 100 patients with a median follow-up of 30 months. Detailed information about these samples is summarized in Table S1. Five human CRC cell lines COLO205, COLO320DM, HCT116, HT15, and HT29, as well as a colon epithelial cell line FHC were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in McCoy’s 5A or DMEM supplemented with 10% FBS, 100 µg/mL of penicillin, and 100 mg/mL of streptomycin at 37°C with 5% CO2 in a humidified incubator (Thermo Fisher Scientific, Waltham, MA). 2.2 IHC Sections were stained with a polyclonal antibody against GABRD (1:50 dilution; rabbit anti-human, mouse, and rat; abs141150; Absin Bioscience Inc., Shanghai, China) by IHC based on a standard protocol in the Department of Pathology at our institution [13]. Briefly, formalin-fixed and paraffin-embedded microarray sections were deparaffinized, dehydrated, and immersed in sodium citrate buffer (pH 6.0) for antigen retrieval, blocked with 3% hydrogen peroxide for 10 min at room temperature to inactivate endogenous peroxidase, rinsed with phosphate-buffered saline for 10 min, and pretreated in a microwave oven for 10 min. Then the slides were incubated at 4°C overnight with the primary antibody. After staining with a two-step plus Poly-HRP anti-Rabbit IgG Detection System (Elabscience Biotechnology Co. Ltd, Wuhan, China), the slides were visualized by reacting with the DAB chromogen (Biocare Medical LLC, Pacheco, CA, USA) counterstained with hematoxylin and covered with glycerin gel, and then photographed under a microscope. A modified H score system was used to semi-quantify GABRD expression, as previously described [14]. Briefly, the maximal intensity of staining (0, negative; 1, weak; 2, moderate; and 3, strong) was multiplied by the percentage of positive tumor cells (0–100%) to generate the modified H score (range: 0–300). The GABRD expression was classified into high or low by the median H score. Data interpretations were made independently by two pathologists who had been blinded to each other’s findings and to the original pathology reports. 2.3 Access to public datasets We comprehensively searched transcriptomic datasets of CRCs in the GEO database [15]. Only those that compared gene transcription between NTs and CRCs, or between primary and metastatic CRCs were selected and further screened. Datasets that contained less than 50 tissue samples or provided incomplete information were ruled out. For repetitive datasets, after each data matrix was checked and compared with regard to sample ID and data type, only the one with most samples was kept. Ten datasets were finally included to compare the GABRD expression between NTs and CRCs, including GSE3629 [16], GSE6988 [17], GSE21510 [18], GSE28000 [19], GSE31279 [20], GSE37182 [21], GSE41258 [22], GSE44861 [23], GSE87221 [24], and GSE106582 [25]. In addition, the Colon Adenocarcinoma and Rectum Adenocarcinoma Projects of The Cancer Genome Atlas (TCGA-COAD and READ) were retrieved from UCSC Xena (https://xenabrowser.net/heatmap/) and combined into one CRC dataset.1 The results here are in whole or part based upon data generated by the TCGA Research Network: http://cancergenome.nih.gov/ These 11 datasets include 707 NTs and 1358 CRCs. Besides, 15 datasets containing primary and metastatic CRCs, including GSE6988 [17], GSE18105 [26], GSE21510 [18], GSE27854 [27], GSE28722 [28], GSE29623 [29], GSE38832 [30], GSE40967 [31], GSE41258 [22], GSE41568 [32], GSE62322 [33], GSE64258 [34], GSE71222 [34], GSE81582 [35], and GSE81986 [36], were retrieved from GEO. These 15 datasets include 1607 primary and 581 metastatic CRCs. 2.4 Ectopic expression or silencing of GABRD, and transfection Lentiviral plasmids expressing GABRD (using GV144 vector), short hairpin RNA (shRNA) oligos of GABRD (using GV248 vector), or respective controls were constructed by Shanghai Genechem Co., Ltd (Shanghai, China). The target sequences were CAGACACCATTGACATTTA (shGABRD-1), CTCATTTCAACGCCGACTA (shGABRD-2), TGACGATGACCACGCTCAT (shGABRD-3), GTTACTCATCGGAGGACAT (shGABRD-4), and TTCTCCGAACGTGTCACGT (scramble control). Transient transfection of cells was performed using Lipofectamine 3000 from Invitrogen (L3000015, San Diego, CA, USA) according to the manufacturer’s instruction. To select stable transfectants, puromycin with a concentration of 0.4 µg/mL was used (abs42025969, Absin Bioscience Inc., Shanghai, China). The medium containing puromycin was changed every two days to remove dead cells. Once establishment of stable transfectants was confirmed, puromycin treatment was reduced to 0.2 µg/mL. 2.5 RNA extraction and the quantitative polymerase chain reaction (q-PCR) Tissue and cellular RNA extraction and q-PCR were performed, as previously described [37]. The sequences for q-PCR primers were: GABRD forward primer, 5′-GCATCCGAATCACCTCCACTG-3′; and GABRD reverse primer, 5′-GATGAGTAACCGTAGCTCTCCA-3′. The specificity of primers was validated by electrophoresis and sequencing. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal reference. Experiments were performed three times in duplicate. 2.6 Western blotting Total cellular protein extraction and western blotting were performed, as previously described [37]. The following antibodies were used: a rabbit anti-GABRD polyclonal antibody (1:1,000 dilution; abs141150; Absin Bioscience Inc., Shanghai, China) and a rabbit anti-GAPDH polyclonal antibody (1:2,000 dilution; Beyotime Biotechnology, Shanghai, China) as the loading control. 2.7 Cell proliferation assay Stably transfected HCT15 and HCT116 cells (5 × 103 cells/well) were seeded in 96-well plates and cultivated overnight. Then proliferation assays were performed, as previously described [37]. 2.8 Scratch wound healing assay Stably transfected HCT15 and HCT116 cells (4 × 105 cells/well) were seeded in 12-well plates and cultivated until 100% confluence. Then monolayer scratch wound healing assays were performed, as previously described [37]. 2.9 Statistical analysis Paired or unpaired Student’s t tests were used for continuous variables. The Fisher exact test and chi-square tests were utilized for categorical comparisons. Survival analyses were evaluated by the Kaplan–Meier method. Univariate and multivariate survival analyses were conducted with the Cox proportional hazards regression model. All tests and reported p values were two-sided, and p < 0.05 was defined as statistically significant. Statistical analyses were performed with the Microsoft Excel 2010 (Microsoft, Redmond, WA, USA), GraphPad Prism7 (GraphPad, San Diego, CA, USA), and SPSS software version 22 for Windows (SPSS Inc., Chicago, Ill, USA). 3 Results 3.1 Expression of GABRD was increased in CRCs As GABRD had not been investigated in CRCs previously, we first explored the expression pattern of GABRD in CRCs compared to that in NTs using 11 public transcriptomic datasets. As shown in Figure 1, GABRD was significantly upregulated in seven datasets (Figure 1a–g) and decreased in two datasets (Figure 1h–i), and was similar in two datasets (Figure 1j–k), in CRCs compared to that in NTs. We further compared the mRNA expression of GABRD between CRCs and NTs using clinically resected samples. The result confirmed that GABRD was increased in CRCs than in NTs (Figure 1l). We then wanted to make sure whether this was also the case in metastatic CRCs. In a panel of 15 transcriptomic datasets, no significant difference was observed with regard to GABRD expression in 14 of them in metastatic CRCs compared to that in primary CRCs (Figure S1a–n). While in one dataset that included different disease stages, GABRD expression was significantly increased in polyps and primary tumors compared to that in mucosae, and slightly but significantly decreased in metastatic CRCs compared to primary tumors, but was not significantly different between primary tumors and polyps (Figure 1o). Taken together, these results demonstrated that GABRD was upregulated in CRCs and might be involved in early tumorigenesis. Figure 1 GABRD transcription was increased in CRCs. Expression of GABRD was compared between CRCs and adjacent NTs using transcriptomic data from one combined TCGA-COAD and READ dataset and 10 datasets in GEO. The results demonstrated that the GABRD expression was significantly increased in seven datasets (a–g), decreased in two datasets (h–i), and similar in two datasets (j–k) in CRCs compared to that in NTs. Sixteen paired CRCs and NTs from clinically resected samples were used to validate results from the transcriptomic datasets using q-PCR (l). Abbreviations: CRC, colorectal cancer; GABRD, gamma-aminobutyric acid type A receptor subunit delta; GEO, gene expression omnibus; NS, not significant; NT, adjacent normal tissue; q-PCR, quantitative polymerase chain reaction; TCGA-COAD and READ, the cancer genome atlas colon adenocarcinoma and rectal adenocarcinoma. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. 3.2 Overexpression of GABRD predicted unfavorable patient prognoses in CRC patients We next determined the relationship between the overexpression of GABRD and patient outcome. We first performed IHC on 16 paired CRCs and NTs from clinically resected samples. As shown in Figure 2a, GABRD staining was weak or negative in NTs and localized to the cytoplasm and membrane (Figure 2a and b). By contrast, GABRD expression was stronger and localized to the nuclei, cytoplasm, and membrane in CRCs (Figure 2c and d), consistent with those observed from the Human Protein Atlas database (http://www.proteinatlas.org/ENSG00000187730-GABRD/pathology). An evaluation of the sections using H score revealed significant difference between CRCs and NTs with regard to GABRD staining (p < 0.000, Figure 2b). We then evaluated the correlation between GABRD expression and clinicopathological variables. As shown in Table 1, overexpression of GABRD was significantly correlated with later pTNM stages. We next determined the prognostic role of GABRD in CRC patients using a TMA. As shown in Figure 2c, a high expression of GABRD was associated with unfavorable overall survival (OS) in these patients (estimated mean OS 44.9 [95% CI, 36.2–53.7] months vs 63.7 [95% CI, 54.5–73.0] months, log-rank p = 0.001). In the multivariate analysis using a Cox proportional hazards model, GABRD overexpression was significantly and independently associated with shorter OS, after adjustment by age, tumor size, and tumor stage (Table 2). Besides, GABRD overexpression was significantly associated with shorter OS and recurrence-free survival (RFS) in the combined TCGA-COAD and READ cohort, which supported the observation in the TMA cohort (Figure 2d and e). Collectively, these results demonstrated that the overexpression of GABRD was predictive of unfavorable prognoses in CRC patients. Figure 2 Overexpression of GABRD was associated with poor patient prognosis. The protein expression and localization of GABRD were characterized by IHC in 16 paired NTs and CRCs. GABRD expression was weak or negative in NTs and mainly localized to the cytoplasm and membrane (a, a and b). By contrast, GABRD expression was stronger and localized to the nuclei, cytoplasm, and membrane in CRCs (a, c and d; b). The prognostic value of GABRD in CRCs was explored with a TMA and validated using the combined TCGA-COAD and READ dataset. Kaplan–Meier plots demonstrated that the overexpression of GABRD was associated with unfavorable patient OS in the TMA cohort (c) and TCGA cohort (d), and unfavorable patient RFS in the TCGA cohort (e). P values were obtained by using the log-rank test. Censored data are indicated by the + symbol. Patients were stratified into GABRD low and high expression according to the median H score or GABRD mRNA expression (