Serum exosomal lncRNA SNHG7 is increased in NSCLC and predicts unfavorable prognosis

Background Exosomal long non-coding RNAs (lncRNAs) are proposed as promising non-invasive biomarkers for clinical applications. Aims In this study, we aimed to explore the potential of serum exosomal lncRNA SNHG7 for the diagnosis and prognosis prediction of non-small-cell lung cancer (NSCLC). Methods A total of 128 patients with NSCLC and 80 healthy volunteers were enrolled. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to measure the expression level of serum exosomal lncRNA SNHG7 in all participants. Receiver operating characteristic (ROC) analysis was used to analyze the diagnostic value of serum exosomal lncRNA SNHG7 and CEA for NSCLC. The relationship between serum exosomal lncRNA SNHG7 expression and clinical characteristics of NSCLC was also assessed.


Introduction
In 2018, 774,323 new lung cancer cases and 690,567 new lung cancer deaths are projected to occur in China. Lung cancer accounts for 18% of all new diagnosed cancer and 24% of all cancer-related deaths [1]. Among all lung cancer cases, approximately 85% were non-small cell lung cancer (NSCLC) [2]. Despite the improvement of treatments in the past decades, the prognosis of this malignancy remains dismal.
Unfortunately, most NSCLC cases are diagnosed at advanced clinical stage, the 5-year survival rates for this disease are unfavorable [3,4]. Thus, it is urgently needed to identify novel markers for the accurate diagnosis and prognosis prediction of NSCLC, which are important to improve the long-term survival rates.
Long non-coding RNAs (lncRNAs) is a type of non-coding RNAs of more than 200 nucleotides in length [5]. Many reports have revealed that lncRNAs are widely involved in the regulation of various biological processes, such as cell proliferation, invasion and metastasis [6,7]. Dysregulation of lncRNAs has been found to be associated with the cancer progression, and lncRNAs might play as oncogenes or tumor suppressors in NSCLC. For instance, lncRNA PTAR upregulation signi cantly increased NSCLC cell proliferation, growth and functioned as an oncogene in NSCLC [8], while lncRNA LIFR-AS1 overexpression markedly restrained the carcinogenesis process and served as a tumor suppressor in NSCLC [9].
Exosomes are extracellular vesicles with a diameter about 40-100 nm and contain different molecules including lncRNAs. Recent studies have shown a number of exosomal lncRNAs are aberrantly expressed in NSCLC, such as 19H and DLX6-AS1 [10,11].
Small nucleolar RNA host gene 7 (SNHG7) is located on chromosome 9q34.3 and identi ed as an oncogene in NSCLC [12]. For example, lncRNA SNHG7 was con rmed to be signi cantly increased in NSCLC tissues and cells [13][14][15]. However, the potential clinical signi cance of serum exosomal lncRNA SNHG7 remains unknown. In this study, the levels of serum exosomal lncRNA SNHG7 were rstly detected, and the associations between serum exosomal SNHG7 expression and the prognosis of NSCLC were further analyzed.

Study design and subjects
The current study was approved by the Ethics Committee of Beijing Chao-yang Hospital, Capital Medical University, and each participant provided the signed written informed consent. A total of 128 patients with NSCLC and 80 healthy volunteers as controls were recruited. No patient had received any chemotherapy or radiotherapy before rst time serum sample collection. The patients consisted of 102 males and 26 females with a medium age at 56.3 years old. Tumor stage was determined was according to the seventh edition of TNM staging system of the American Joint Committee on Cancer (AJCC). The demographic characteristics of all patients were summarized Table 1. All cases received regular follow-up. Overall survival (OS) time was calculated from the date of the initial surgery until the date of death or last followup. Peripheral blood samples were collected from all NSCLC patients and controls in EDTA-containing tubes. The samples were centrifuged at 1500 g for 10 min at 4°C, and then stored at -80°C until exosome isolation. In addition, post-operative blood samples were drawn from all NSCLC patients 30 days after surgery.

Isolation of exosome
Total exosomes were isolated using Total Exosome Isolation Reagent for Serum (Life Technologies, Austin, USA). Brie y, serum was thawed on ice and centrifuged at 2,000 g for 30 min to remove cells and debris. Next, ExoQuick Solution was added to serum samples, and the mixture was centrifuged at 1,500 g for 30min after incubation. The supernatant was removed, followed by centrifugation at 1,500 g for 5 min to remove residual liquid. The exosome-containing pellets were resuspended in PBS and stored at −80°C until RNA isolation.
RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)

Results
Serum exosomal lncRNA SNHG7 expression was signi cantly higher NSCLC Serum exosomal lncRNA SNHG7 levels were compared between NSCLC patients and healthy volunteers. Figure 1A showed that serum exosomal lncRNA SNHG7 levels were signi cantly higher in NSCLC patients (P<0.0001). In addition, increased serum exosomal lncRNA SNHG7 levels were found in NSCLC patients in the advanced stage (P<0.0001, Figure 1B), or with positive lymphatic invasion (P=0.0131, Figure 1C) or with positive lymph node metastasis (P=0.0006, Figure 1D).
Diagnostic values of serum exosomal lncRNA SNHG7 and CEA in NSCLC patients ROC curve analysis demonstrated that serum exosomal lncRNA SNHG7 effectively discriminated NSCLC patients from normal controls with an AUC of 0.856. The sensitivity was 77.34% and the speci city was 83.75%, respectively ( Figure 2A). Additionally, CEA had a sensitivity of 76.56% and a speci city of 82.50% for identifying NSCLC subjects from controls, with an AUC of 0.784 ( Figure 2B). Combination of serum exosomal lncRNA SNHG7 expression and CEA revealed an increased AUC value of 0.932, with a sensitivity of 88.28% and a speci city of 86.25% ( Figure 2C). Therefore, the combination of these two makers had better performance to differentiate NSCLC patients from healthy controls.

Association of serum exosomal lncRNA SNHG7 with clinical variables in NSCLC patients
To analyze the association between serum exosomal lncRNA SNHG7 levels and the clinical characteristics, all 128 NSCLC patients were divided into high expression group (n=64) and low expression group (n=64) based on the median serum exosomal lncRNA SNHG7 expression. As illustrated in Serum exosomal lncRNA SNHG7 levels before and after surgery As presented in Figure 3A, serum exosomal lncRNA SNHG7 expression levels in the post-operative blood samples were markedly reduced following surgery (P<0.0001). During the follow-up, 12 patients relapsed (12/64, 18.8%) among patients with low serum exosomal lncRNA SNHG7 expression, while 46 patients relapsed (46/64, 71.9%) among patients with high serum exosomal lncRNA SNHG7 expression. Blood samples were obtained from 58 patients with disease relapse, and serum exosomal lncRNA SNHG7 expression was detected with qRT-PCR. Serum exosomal lncRNA SNHG7 levels in the relapse samples (n=58) were greatly higher than in the pre-operative blood samples (P<0.0001, Figure 3B).

Discussion
Nowadays, clinical scientists still face the challenge of improving the early diagnosis and prognosis prediction of NSCLC. Some tumor markers, such as CEA, have low speci city and sensitivity for the early detection of this disease. Thus, exploring novel biomarkers for NSCLC are urgently required. In this study, we showed that serum exosomal SNHG7 expression was signi cantly higher in NSCLC patients. High serum exosomal SNHG7 expression occurred more frequently in NSCLC patients with advanced TNM stage, positive lymphatic invasion and positive lymph node metastasis. In addition, serum exosomal SNHG7 showed high accuracy in differentiating NSCLC patients from normal controls, and a combination of serum exosomal SNHG7 and CEA could yield improved the diagnosis of NSCLC. Moreover, increased serum exosomal SNHG7 expression was strongly associated with aggressive clinical parameters.
Furthermore, serum exosomal SNHG7 levels were remarkably decreased after surgery, and patients with relapse exhibited signi cantly higher serum exosomal SNHG7 levels. Finally, NSCLC patients with high serum exosomal SNHG7 expression had worse survival, and serum exosomal SNHG7 was considered as an independent prognostic indicator for OS.
Our ndings were consistent with previously reported results. For instance, Pang et al showed SNHG7 was signi cantly overexpressed in NSCLC tissues and cells. Downregulation of SNHG7 greatly inhibited cell proliferation, metastasis and epithelial to mesenchymal transition (EMT) in vitro and carcinogenesis in vivo [13]. Likewise, SNHG7 was involved in the NSCLC progression by sponging miR-181a-5p. In vitro and in vivo analysis showed that SNHG7 inhibition or miR-181a-5p upregulation markedly decreased cell proliferation, stimulated cell apoptosis and attenuated tumor growth, and vice versa [14,15]. The data showed SNHG7 might serve as an oncogene in NSCLC.
Accumulating evidence have also shown the oncogenic activities of SNHG7 in different types of cancer. In hepatocellular carcinoma (HCC), SNHG7 expression levels were remarkably upregulated in HCC tissues in comparison with adjacent normal tissues. SNHG7 overexpression was strongly associated aggressive clinical parameters and worse prognosis of HCC patients, and SNHG7 knockdown signi cantly decreased cancer cell proliferation, migration and invasion by inversely regulating RBM5 and RPL4 expression [16][17][18]. In colorectal cancer (CRC), SNHG7 overexpression was found both in CRC tissues and cell lines. SNHG7 upregulation signi cantly stimulated cell proliferation, metastasis in vitro and promoted tumorigenesis in vivo [19,20]. In addition, SNHG7 expression was remarkably increased in cervical cancer (CC) tissues and cell lines. SNHG7 inhibition signi cantly attenuated cancer cell proliferation, invasion, migration and induced cell apoptosis through miR-485-5p/JUND axis. CC patients with higher lncRNA SNHG7 expression had shorter survival time [21,22]. Moreover, SNHG7 expression was upregulated in breast cancer (BC) tissues and cells. High SNHG7 expression was closely associated with tumor stage, lymph node metastasis and worse overall survival of BC patients. SNHG7 inhibition enhanced the sensitivity of BC cells to trastuzumab, reduced cell proliferative, invasive abilities and promoted cell apoptosis by upregulating miR-186 [23,24]. In gastric cancer (GC), high expression of SNHG7 occurred more frequently in in cancerous tissues and cell lines. Increased SNHG7 expression was highly associated with advanced clinical stage, lymph node metastasis and distant metastasis. In vitro and in vivo evidence demonstrated that SNHG7 suppression markedly inhibited the cancer progression of GC [25,26].

Conclusion
In conclusion, we have demonstrated that serum exosomal SNHG7 expression is dramatically higher in NSCLC, and its upregulation is strongly associated with unfavorable prognosis of NSCLC. Therefore, serum exosomal SNHG7 might serve as a non-invasive and robust biomarker for NSCLC diagnosis and prognosis prediction.    Compared to the patients in the relapse group, patients in the non-relapse group had better survival.