Aberrant Expression of SCARA5 in Lung Adenocarcinoma and its Clinical Signicance

Background Lung adenocarcinoma, the main subtype of non-small cell lung cancer, leading one of the most aggressive and fatal causes of malignant deaths around the world. Scavenger receptor class A member 5 (SCARA5), a newly discovered tumor suppressor gene, belonged to the SR family. The present study’s object was to explore the clinical impacts of SCARA5 in lung adenocarcinoma treatment. Methods SCARA5 expressions in 120 paired lung adenocarcinoma patients’ tissues and cell lines were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC). CCK-8, EdU, ow cytometry, and western blot assays further demonstrated the signicance of SCARA5 on A549 cell growth. Then, the relationships between the expression level of SCARA5 and pathological factors were analyzed. Finally, the receiver operating characteristic (ROC) curve and overall survival analysis was carried out to verify the diagnostic and prognostic signicance of SCARA5 in lung adenocarcinoma.


Abstract
Background Lung adenocarcinoma, the main subtype of non-small cell lung cancer, leading one of the most aggressive and fatal causes of malignant deaths around the world. Scavenger receptor class A member 5 (SCARA5), a newly discovered tumor suppressor gene, belonged to the SR family. The present study's object was to explore the clinical impacts of SCARA5 in lung adenocarcinoma treatment.
Methods SCARA5 expressions in 120 paired lung adenocarcinoma patients' tissues and cell lines were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC). CCK-8, EdU, ow cytometry, and western blot assays further demonstrated the signi cance of SCARA5 on A549 cell growth. Then, the relationships between the expression level of SCARA5 and pathological factors were analyzed. Finally, the receiver operating characteristic (ROC) curve and overall survival analysis was carried out to verify the diagnostic and prognostic signi cance of SCARA5 in lung adenocarcinoma.

Results
SCARA5 was prominently decreased in lung adenocarcinoma cells and tissues compared with human bronchial epithelial cells and para-carcinoma non-tumor tissues. Meanwhile, SCARA5 expression was strongly correlated with smoking (P = 0.0011), TNM stage (P < 0.0001) and lymph node metastasis (P = 0.0005). Furthermore, SCARA5 may be a crucial biomarker for lung adenocarcinoma diagnosis with an area under the curve (AUC) of 0.9102 while SCARA5 could signi cantly reduce overall survival (OS; P = 0.0006). In vitro experiments, we found that SCARA5 overexpressing could signi cantly hinder A549 cell proliferation but facilitate apoptosis through the AKT signaling pathway.

Conclusions
SCARA5 might be an important diagnostic and prognostic biomarker for patients with lung adenocarcinoma.

Background
Lung adenocarcinoma was more common in Asian women patients with a smoking history (1,2). The incidence of lung adenocarcinoma was on a rise year by year, accounting for over 40% of all lung cancer, with the overall survival rate remaining low (3). Lung adenocarcinoma patients usually had no obvious particular symptoms at an early stage, generally consisted of fever, hemoptysis, chest pain, shortness of breath, and other respiratory symptoms or asymptomatic (4). Hence, it was easy to be ignored. In recent years, lung adenocarcinoma became common among women, non-smokers, and young adults (5). Most lung cancers were frequently diagnosed at an advanced stage due to the absent symptoms leading to poor prognosis (6,7). Therefore, it is urging to investigate or reveal diagnostic and prognostic molecular biomarkers for lung adenocarcinoma, Scavenger receptors (SR) represented a group of heterogeneous molecules located on the surface of phagocytes (8), which could identify oxidized or acetylated modi ed low-density lipoprotein (LDL). SR was rst discovered in 1979 by studying the binding sites on macrophages that ingested and degraded acetylated LDL (9). Later, it was classi ed as class A scavenger receptor (SR-A) (10). Gradually, several SR-As were subsequently discovered, and these SRs formed an SR family. Scavenger receptor class A member 5 (SCARA5) was a member of the SR family (11), which was ubiquitous in the presence of testis, bladder, trachea, adrenal gland, skin, lung, ovary, thymus, and small intestine (12,13). Studies reported that the essential functions of SCARA5 included the role of organism defense (14). A variety of ndings also veri ed that SCARA5 exerted a pivotal role in tumor growth and angiogenesis inhibition (inhibition of tumorigenesis, colony formation, and cell in ltration) and iron transport (15,16). More importantly, the inhibitory effect of SCARA on the tumor was strong, whose tumor inhibitory effect was 69.4%, the tumor proliferation index was reduced by 23.3%, and the apoptosis index was 47.3% angiogenesis was signi cantly reduced (17). Previous studies reported that SCARA5 was dysregulated in various cancers, including colorectal cancer (18), breast cancer (19), oral squamous cell carcinoma (20), hepatocellular carcinoma (21), and lung cancer (17,22). However, the clinical impacts of SCARA5 in lung adenocarcinoma has not been elucidated yet.
The purpose of this study was to reveal the eccentric expression and roles of SCARA5 lung adenocarcinoma.

Methods
Patients 120 paired tumor tissues and para-carcinoma non-tumor tissues were obtained from patients with lung adenocarcinoma who had undergone surgical resection without receiving any preoperative adjuvant therapy in the Second A liated Hospital, Zhejiang University School of Medicine from May 2013 to April 2015. Written informed consent was obtained from each patient, and the Ethics Committee approved the present study of the Second A liated Hospital, Zhejiang University School of Medicine. The pathological parameters of all patients were presented in Table 1.

Quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR)
The RT-qPCR analysis was used to analyze the abnormal expressions of SCARA5 in tissues and cells, while glyceraldehyde 3-phosphate dehydrogenase (GAPDH) functioned as the internal control. Brie y, total RNA was isolated and extracted from tissues or cells using TRIzol reagent (Thermo Fisher Scienti c, Waltham, MA, USA) under the manufacturer's instructions. Then, RNA was reverse transcribed into cDNA with the ExScript RT-PCR kit (Takara, Dalian, China). The expression of SCARA5 was detected by SYBR protocol. All the ampli cation and detections were performed in the CFX96 Real-Time PCR system (Bio-Rad, Hercules, CA, USA), and the thermo-cycling pro les were described as follows: 95℃ for 10 seconds, 40 cycles of 95℃ for 5 seconds, and 62℃ for 30 seconds. Statistical analysis was calculated using the 2 -△△CT method.

Immunohistochemistry (IHC)
Tissues gathered from 120 patients with lung adenocarcinoma were xed and embedded in para n. Immunohistochemistry was carried out using a rabbit anti-SCARA5 antibody (1: Cell viability measured by CCK-8 assay A549 cells were plated in a 96-well plate with a density of 1×10 4 cells/well. After the cells reached 70% con uency, cells were transfected with pcDNA3.1 or pcDNA3.1-SCARA5. Then, at 0 hour, 12 hours, 24 hours, and 48 hours, 10 μL CCK-8 reagent was added to each plate and incubated for another 30 minutes at room temperature. Finally, the cell viability was analyzed by measuring the absorbance at 450 nm using a Microplate Reader.

Cell proliferation rated evaluated by EdU staining
The transfected cells were inoculated in a 96-well plate with a density of 4×10 3 cells/well. Then, 100 μL 50 μM EdU reagent was added to each well and cultured for 2 hours. After washed by PBS twice, 50 μL PBS containing 4% paraformaldehyde solution was added to each well and cultured for 30 minutes at room temperature. Subsequently, 100 μL Apollo® 567 Imaging Kit (Riobio) and Hoechst 33342 were added and cultured at 37℃ for 30 minutes. Afterward, 100 μL PBS containing 0.5% Triton X-100 was added and cultured for another 5 minutes at room temperature. Finally, all images were captured with uorescence microscopy.

Cell apoptosis detected by ow cytometry analysis
After transfection, cells were cultured in a 6-well plate at 37℃ in a humidi ed atmosphere. At 48 hours post-incubation, cells were stained with Annexin V/PI using an apoptosis kit. Finally, the cell apoptosis rate was analyzed using ow cytometry following the instructions.

Statistical analysis
All analyses were performed using SPSS 17.0 (Chicago, IL, USA) to analyze the data. The expression level of SCARA5 was presented as a mean ± standard deviation via the student's t-test. The 2 test was used to determine the correlation of SCARA5 expression and pathological factors in lung adenocarcinoma. The ROC curve de ned by the log-rank test and the overall survival by Kaplan-Meier analysis was applied to determine the diagnostic and prognostic value of SCARA5 in lung adenocarcinoma, respectively. A P value < 0.05 was considered to be statistically signi cant.

Results
The expression of SCARA5 was downregulated in lung adenocarcinoma tumor tissues compared with para-carcinoma tissues As shown in Fig. 1A, the mRNA expression level of SCARA5 was prominently decreased in lung adenocarcinoma tumor tissues compared with para-carcinoma non-tumor tissues (P<0.001). Consistent with the mRNA result, the protein measured by IHC in Fig. 1B presented a similar trend.

Relationship of SCARA5 expression with pathological factors in lung adenocarcinoma
As shown in Table 1, high expression of SCARA5 presented a strong correlation with smoking (P = 0.0011), TNM stage (P < 0.0001) and lymph node metastasis (P = 0.0005). Whereas, there were no signi cant differences in gender, age, and tumor size between the high and low expression of SCARA5 groups.

SCARA5 was a diagnostic and prognostic biomarker in lung adenocarcinoma
As shown in Fig. 2A, the AUC value was 0.9102 (95% CI = 0.8681~0.9523), suggesting that SCARA5 exerted its remarkable function concerning lung adenocarcinoma diagnosis. Meanwhile, Fig. 2B further demonstrated that the overall survival determined by Kaplan-Meier analysis of lung adenocarcinoma patients with low SCARA5 expression was lower than those with high expression.
SCARA5 was signi cantly downregulated in lung adenocarcinoma cell compared with normal bronchial epithelial cell Through RT-qPCR analysis, we detected the eccentric levels of SCARA5 in lung adenocarcinoma cells. As shown in Fig. 3A, the SCARA5 level was remarkably decreased in A549 lung adenocarcinoma cell compared with normal bronchial epithelial BEAS-2B cell (P<0.01). Furthermore, the transfection e ciency con rmed that SCARA5 levels were prominently increased after transfection with pcDNA3.1-SCARA5, suggesting the transfection was successful ( Fig. 3B; P<0.01).
SCARA5 overexpressing inhibited A549 cell proliferation but promote cell apoptosis via AKT signaling pathway After transfection, we further detected the effects of SCARA5 on A549 cell proliferation and apoptosis. Fig. 4 and Fig. 5 showed that SCARA5 overexpressing markedly suppressed A549 cell proliferation compared with the pcDNA3.1 group (P<0.01); however, ow cytometry results in Fig. 6 veri ed that upregulation of SCARA5 could promote A549 cell apoptosis (P<0.01). As shown in Fig. 7, compared with the pcDNA3.1 group, SCARA5 overexpressing could reduce p-AKT and Bax levels but increase Bcl-2.

Discussion
Recently, studies concerning cancer biomarkers have been massively reported for its clinical role in the early diagnosis and prognosis of various cancers (23)(24)(25), including lung adenocarcinoma (26,27). As known, the majority of lung adenocarcinoma cancer-related death frequently occurred at an early stage.
Contributions and development of speci c biomarkers for lung adenocarcinoma for early diagnosis and prognosis might improve overall survival and clinical situations.
In the present study, the mRNA and protein expression of SCARA5 was downregulated in lung adenocarcinoma tumor tissues compared with para-carcinoma non-tumor tissues, consistent with a previous study (17). Meanwhile, SCARA5 expression was strongly correlated with smoking, TNM stage, and lymph node metastasis. Our ndings agreed with the earlier studies on the roles of SCARA5 in hepatocellular carcinoma and breast cancer (19,28). Furthermore, we investigated the diagnostic and prognostic value of SCARA5 in lung adenocarcinoma. As demonstrated, the ROC curve presented an AUC value of and the Kaplan-Meier analysis revealed that patients with low SCARA5 expression's overall survival were lower than those with higher expression. Thus, the downregulation of SCARA5 in patients may be associated with poor prognosis, and SCARA5 may exert its function as a crucial biomarker concerning lung adenocarcinoma early diagnosis and prognosis.
Consistent with our ndings, the clinical signi cance of SCARA5 was veri ed in various cancers before. For instance, Liu et al. (18) proposed that SCARA5 was downregulated in colorectal cancer tissues, and low SCARA5 expression was correlated with unfavorable prognosis in colorectal cancer. Yan et al. (17) con rmed that SCARA5 was frequently decreased in various cancer, functioning as potential antimetastatic agents for cancer treatment. In our study, we found that SCARA5 was downregulated in A549 lung adenocarcinoma compared with BEAS-2B normal bronchial epithelial cells; meanwhile, SCARA5 overexpressing could inhibit lung adenocarcinoma cell proliferation but promote apoptosis through the AKT signaling pathway.
The present study identi ed the clinical value of SCARA5 in predicting lung adenocarcinoma diagnosis and prognosis, suggesting that it may be an essential biomarker for therapeutics of lung adenocarcinoma.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.