SFRP1 Decline in Non-Small Cell Lung Cancer (NSCLC) and its Importance in Prognoses

Background: In the present study, we sought to detect the expression of secreted zzled-related protein-1 (SFRP1) and investigate its role in the progression and prognosis of patients with non-small cell lung cancer (NSCLC). Methods: The expression of SFRP1 at both mRNA and protein level were examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Immunohistochemistry analysis, respectively. The relationship between SFRP1 expression and clinical factors of patients with NSCLC was analyzed by chi-square test. Transwell assay was conducted to determine the inuences of SFRP1 on migratory and invasive of NSCLC cells. Kaplan-Meier analysis was used to describe the overall survival of NSCLC patients. Cox regression analysis was conducted to estimate the prognostic value of SFRP1 in NSCLC. Results: The expression of SFRP1 was down-regulated in NSCLC tissues compared to that in adjacent normal controls both at mRNA and protein level (P<0.05). And the low SFRP1 expression was related to the distant metastasis, vascular invasion and TNM stage. The overexpression of SFRP1 in vitro signicantly inhibited the migration and invasion of NSCLC cells. The overall survival of patients with high SFRP1 expression was was proved to be longer than those with low expression (log rank test, P<0.001). In addition, univariate and multivariate analyses suggested that SFRP1 was an independent prognostic molecule marker for NSCLC patients. Conclusion: Taken together, our ndings indicated that the decreased of SFRP1 could inuence the cell migration and invasion as well as be regarded as an independent prognostic marker in NSCLC.


Background
Lung cancer is the most frequent cancer that causes the majority of cancer-related deaths all over the world [1]. It is categorized into several subtypes and non-small cell lung cancer (NSCLC) is the most common type, which accounts for about 80-85% of all lung cancers [2,3]. NSCLC is a kind of molecularly heterogeneous malignancy with high proliferation rate, even patients with the same tumor node metastasis (TNM) stage appear varied clinical prognosis and outcomes [4,5]. Standard therapies for NSCLC are mainly surgery, radiation therapy, and chemo therapy combined or alone [6,7]. Recently, though great and e cient advancements and improvements have been made in treatment modalities, the prognosis of NSCLC patients remains poor [8,9]. The ve-year survival rate of NSCLC patients is limited to 15%, even the 5-year post-resection survival rate is only 20-30% [10]. Therefore, it is urgently need to discovery biomarkers that could be used as prognostic factors for NSCLC patients.
SFRP1 is a member of SFRPs family which are regarded as inhibitors of the Wnt signaling pathway that participate in multiple biological progressions such as embryogenesis and tumorigenes and is important for the maintenance of gut homeostasis and embryonic development [11][12][13]. It is located at chromosome 8p12-p11.1 and can encode a secreted glycoprotein with the molecular mass of 35 kDa [14][15][16]. SFRP1 is a type of newly discovered candidate anti-oncogene and its aberrant expression has been found in several cancers, such as glioma and prostate cancer and [17,18]. However, it had mostly focused on the methylation of SFRP1 in NSCLC according to previous studies [19,20], the prognostic value of SFRP1 was never reported.
In this study, we detected the expression of SFRP1 in NSCLC patients and analyzed its relationship with clinical facotrs of patients with NSCLC. Besides, we estimated its in uence on cell migration and invasion of NSCLC cells. What's more, the prognostic value of SFRP1 for NSCLC patients was evaluated.

Methods And Materials
Patients and specimens A total of 125 patients with pathologically diagnosed NSCLC who suffered from surgical resection Cangzhou Central Hospital were recruited in this study, including 59 males and 66 females. None of them had received any radio-or chemo-therapy before surgery. Besides, 51 out of 125 patients were randomly selected to resect the adjacent normal tissues which were taken as internal controls. This study was approved by the Ethic Committee of the hospital and all participants signed the written informed consents in advance.
Tumor tissues and adjacent normal tissues were collected from patients with NSCLC and frozen in liquid nitrogen immediately. Then the tissues samples were severally stored at -80℃for RNA extraction.
Detailed information of patients including gender, smoking status, histology, vascular invasion, distant metastasis, and TNM stage were summarized in a database. A 5-years' follow-up was conducted with all patients and those who were died from unexpected events or other diseases were excluded from our study.

Cell lines and cell culture
The NSCLC cell line A549 was obtained from America Type Culture Collection and maintained in DMEM (Dulbecco's modi ed Eagle's medium), which was supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37℃ under a 5% CO 2 atmosphere.

Plasmid construction and cell transfection
The SFRP1 expressing plasmid pEF6/V5-SFRP1 and empty pEF6/V5 (negative control) were obtained from Yoshitaka Sekido (Nagoya University, Nagoya, Japan). Cells were transfected by these two types of vectors with FuGene HD Transfection Reagent (Roche, Mannheim, Germany) based on the instructions and maintained in normal medium.
RAN extraction and qRT-PCR analysis RNA was rst isolated from NSCLC tissues and adjacent normal tissues using Trizol reagent (Invitrogen) and then used to synthesize the rst strand of cDNA through reverse transcription using a PrimeScript RT reagent Kit (Takara, Dalian, China) following the manufacture's guides. RT-PCR reaction was performed with PRISM 7900 Sequence Detection System (Applied Biosystems). GAPDH served as the endogenous control. The relative mRNA expression of SFRP1 was calculated by -2 ∆∆CT method. Each sample was in triplicate.

Immunohistochemistry (IHC)
IHC assay was performed to detect the expression of SFRP1 at protein level. In brief, 4-µm sections were prepared and then depara nized and rehydrated. Following, the sections were incubated with primary antibody and secondary antibody in order. Finally, all sections were stained with DAB and later counterstained by hematoxylin. Tissues were grouped according the percentages of stained cells. Those tissues with the percentage more than 30% belonged to the positive group and others were of the negative group.

Transwell assay
Transwell assay was used to investigate the role of SFRP1 in migration and invasion of NSCLC cells. The transfected cells was rst seeded into the upper chamber of the Transwell plates (BD Biosciences, San, Jose, CA, USA). Medium with 10% FBS was added into the lower chamber as a chemoattractant. After migration for 24h and invasion for for 48h, cells that migrated or invaded into the surface of the lower chamber were xed by 90% alcohol, stained with 0.1% crystal violet and then counted in seven randomly selected elds. For invasion assay, the membrane should be per-coated by Matrigel (BD Biosciences, NJ, USA).

Statistical analysis
Statistical analyses were performed by SPSS 18.0 and Sigmaplot software. The differences between SFRP1 expression and clinical factors were analyzed by students' t test. Chi-square test was adopted to estimate the relationship between SFRP1 expression and clinical factors of NSCLC patients. The overall survival of NSCLC patients were compared with the Kaplan-Meier analysis. The prognostic signi cance of SFRP1 was evaluated using the cox regression analysis. P<0.05 was regarded to be statistically signi cant.

Results
The expression of SFRP1 was decreased in NSCLC tissues at both mRNA and protein levels The relative expression of SFRP1 at mRNA and protein level were detected by qRT-PCR and IHC analyses, respectively. The results showed that the mRNA expression of SFRP1 was signi cantly lower in NSCLC tissues than that in adjacent normal tissues (1.48 ± 0.38 vs 3.24 ± 0.51) (Fig. 1, P < 0.05). We further determined the expression of SFRP1 at protein level in NSCLC tissues and normal controls using IHC method. The positive staining of SFRP1 protein mainly presented in the cytoplasm of cells. As shown in Table 1, the positive rate of SFRP1 was 27.2% (91/125) in NSCLC tissues and 72.5% (37/51) in adjacent normal tissues, indicating that SFRP1 expression at protein level was also down-regulated in NSCLC tissues (P < 0.05). The association of SFRP1 expression and clinical features of NSCLC patients was listed and summarized in Table 2. It was found that the down-regulation of SFRP1 was signi cantly associated with distant metastasis (P = 0.043), vascular invasion (P = 0.021) and TNM stage (P = 0.013). However, no correlation was observed between SFRP1 expression and gender, smoking status and histology (P > 0.05). The effects of SFRP1 on migration and invasion of NSCLC cells in vitro A549 cells were transfected with pEF6/V5-SFRP1 and pEF6/V5-NC. After transfection, the SFRP1 expression was elevated by in cells with pEF6/V5-SFRP1. Then transwell assay was conducted to analyze the cell migration and invasion after being gtransfected. The outcomes demonstrated that the overexpression of SFRP1 in viro signi cantly inhibited the migration and invasion of NSCLC cells compared to that in cells transfected with pEF6/V5-NC ( Fig. 2A and Fig. 2B, P < 0.05).

The prognostic value of SFRP1 in patients with NSCLC
To further examine the correlation between SFRP1 expression and overall survival of NSCLC patients, a 5years' follow-up was conducted. Based on the data of follow-up, Kaplan-Meier analysis was carried out. It displayed that patients with low SFRP1 expression had a signi cantly shorter overall survival than those with high expression (Fig. 3, log rank test, P < 0.001). Besides, univariate and multivariate analyses adjusted for clinical factors using cox regression analysis was performed to assess the prognostic value of SFRP1 in NSCLC. As shown in Table 3, vascular invasion (P = 0.002, HR = 2.170, 95%CI = 1.344-3.504), TNM stage (P = 0.008, HR = 1.892, 95%CI = 1.184-3.023) and SFRP1 expression (P = 0.000, HR = 4.860, 95%CI = 2.407-9.813) were identi ed as important prognostic markers in NSCLC (Table 3).

Discussion
NSCLC is the most lethal human cancer and almost half of patients with newly diagnosed NSCLC have metastatic [21]. Despite great progress have obtained in the diagnosis and treatments, the prognosis of NSCLC remains poor as it often at advanced stages when diagnosed [22,23]. It is therefore of great importance to nd some effective and accuracy bio-markers for the diagnosis and prognosis of NSCLC so that provide new therapy target for this cancer.
Recently, attention is shifted onto candidate molecules for radical treatment of various cancers. So far, a variety of biomarkers have been discovered to function as indicators in NSCLC. According to Zhan et al., high expression of Orai1 was proved to be a promoter for cell proliferation and predicted a poor prognosis in NSCLC [24]. Fan et al., reported that ELMO3 was a candidate prognostic and diagnostic predictor for NSCLC [25]. Luo et al., revealed that FSCN1 played crucial roles on the development of NSCLC and might act as an e cient biomarker for patients with this disease [26].
SFRP1 was expressed in various tissues, such as the brain, kidney and heart, and its transcript was present both in the adult and during embryogenesis [27,28]. It was also reported to abnormal expression in various of diseases. For instance, Rogler et al., found the expression of SFRP1 was inhibited by hypermethylation and it could predict the outcome of patients with bladder cancer [29]. Zheng et al., claimed the expression of SFRP1 was decreased and it could be a prognostic marker combing with the expression of β-catenin in prostate cancer [30]. The methylation of SFRP1 was proved to be an useful diagnostic indicator in cholangiocarcinoma [31]. An et al., detected the expression of SFRP1 and its down-regulation was related to the progression of acute myeloid leukemia [32]. In the present study, we measured the SFRP1 expression and found it was decreased in NSCLC tissues compared to that in adjacent normal controls. The result suggested SFRP1 was a tumor suppressor in NSCLC. All our ndings were in accordance with the previous reports.
Then we analyzed the relationship between SFRP1 expression and clinical characteristics of NSCLC patients. The low SFRP1 expression appeared to be signi cantly associated with vascular invasion, distant metastasis and TNM stage, suggesting that SFRP1 expression might be involved in the development and progression of NSCLC. Based on this, we further investigated the role of SFRP1 on cells migration and invasion by transwell assay. The up-regulation of SFRP1 was considered to signi cantly inhibited migration and invasion of NSCLC cells.
Subsequently, we estimated the prognostic value with the data from follow-up. Kaplan-Meier analysis displayed that patients with high SFRP1 expression had a longer overall survival than those with low expression. What's more, cox regression analysis demonstrated that SFRP1 was a novel biomarker for the prognosis of NSCLC patients. However, although the prognostic role in NSCLC has been validated, the precise mechanism of SFRP1 on development and progresses of NSCLC is still unclear and a sophisticated issue. It is known to all that Wnt signaling pathway not only affects the expression of genes but also in uences cell migration [33,34]. As a newly discovered antagonist of Wnt signaling pathway in recent years, SFRP1 has important inhibitory effects on the transmission of Wnt pathway [35]. Therefore, we inferred that SFRP1 might function on NSCLC through the Wnt signaling pathway, which still need more and further studies.

Conclusions
All in all, the expression of SFRP1 is decreased and it participates in the development and progression of NSCLC via regulating the cell migration and invasion. Moreover, the low expression of SFRP1 is con rmed to be an independent prognostic bio-marker in NSCLC. However, its mechanism is unclear and the sample scale is small in this study. Some further more studies are need in future. The subjects had been informed the objective. Certainly, written consents were signed by every subject in this study.

Consent for publication
We obtaining permission from participants to publish their data.

Availability of data and materials
Data sharing is not applicable to this article as no datasets were generated or analysed during the current study.

Competing interests
The authors declare that they have no competing interests.

Funding
Not applicable. The relative expression of SFRP1 at mRNA level in NSCLC tissues and adjacent normal controls was determined by qRT-PCR. SFRP1 mRNA expression was down-regulated in NSCLC tissues compared to that in adjacent normal controls (P<0.05).