Boosting In uences From circ_0048856 On Non- Small Cell Lung Cancer Advancement Via Aiming At miR-1287-5p


 Background: Non small cell lung cancer (NSCLC) is one of the main causes of death in human diseases. In this study, we aimed to explore the prognostic value of serum circ_0048856 in patients with NSCLC and its relationship with miR-1287-5p.Methods: The expression levels of circ_0048856 and miR-1287-5p in serum of NSCLC patients and healthy people were detected by qRT-PCR. The relationships between circ_0048856 expression and clinical data were evaluated via χ2 test. Overall survival analysis was carried out via Kaplan-Meier method with log rank test. Cox regression analysis was taken to evaluate the prognostic ability of circ_0048856 in NSCLC. Luciferase reporter gene was used to detect the targeting relationship between circ_0048856 and miR-1287-5p.Results: The expression of circ_0048856 in serum of NSCLC patients was significantly higher than that of healthy volunteers (P < 0.001). And its high expression was significantly correlated with TNM stage (P = 0.002) and lymph node metastasis (P < 0.001). In addition, Kaplan Meier log rank test analysis showed that the overall survival of patients with high expression of circ_0048856 was significantly shorter than that of patients with low expression of circ_0048856 (log rank test, P = 0.002). Serum circ_0048856 expression is an independent prognostic biomarker in NSCLC patients. Furthermore, we found that miR-1287-5p is a direct downstream miRNA of circ_0048856 through luciferase reporter gene analysis, and there is a negative correlation between circ_0048856 and miR-1287-5p.Conclusion: Serum circ_0048856 is an independent prognostic biomarker in patients with NSCLC. Circ_0048856 can participate in the progression of NSCLC by targeting miR-1287-5p.

Background Lung cancer is a highly heterogeneous cancer [1]. Every year, about 1.8 million people are diagnosed with lung cancer and 1.6 million people die of lung cancer. The 5-year survival rate is very low, which is usually between 4-17% due to the stage and regional differences [2]. Lung cancer mainly includes non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), accounting for 76% and 13% respectively [3].
Smoking is a major risk factor for lung cancer. Of course, the occurrence of lung cancer is also related to environmental and genetic factors [4,5]. At present, surgical treatment is still the best treatment for early lung cancer [6]. In recent years, with the progress of medical technology, the treatment methods of NSCLC are diverse, and the mortality has been reduced [7]. However, NSCLC patients are prone to metastasis.
Metastatic NSCLC is an advanced tumor that is di cult to cure, with low survival rate, almost no cure possibility and poor prognosis [8]. Therefore, it is urgent to nd new prognostic markers and improve the treatment level of NSCLC. C ircular RNAs (circRNAs) are a class of covalently closed RNA loops widely expressed in eukaryotic cells.
They are produced by a process called reverse splicing, and they are evolutionarily conserved [9,10]. Studies have shown that circRNAs are microRNA (miRNA) sponges, protein regulators, transcription regulators, protein templates, etc [11]. CircRNAs have a variety of biological functions and participate in a variety of physiological and pathological processes, including tumorigenesis and development [12][13][14].
The emergence of the circRNAs family provides a new entry point for the research and treatment of tumors. Studies have found that many circRNAs are involved in the progress of NSCLC. For example, Xu et al. pointed out that circ_0001869 promotes the progress of NSCLC through sponge miR-638 and promoting the expression of FOSL2 [15]. Sun and colleagues found circ_0000376 is highly expressed in NSCLC clinical specimens, which is related to adverse pathological parameters, and it can signi cantly promote the proliferation and metastasis of NSCLC cells, and enhance the drug resistance of cells [16].
Among them, circ_ 0048856 is up-regulated in NSCLC samples, which may participate in the occurrence and development of NSCLC as a cancer promoting factor [17]. However, the prognostic value and molecular mechanism of circ_0048856 in NSCLC are still unclear. Therefore, the study of serum circ_0048856 as a prognostic marker of NSCLC has important clinical signi cance.
This study examined the prognostic signi cance of circ_0048856 in serum of patients with NSCLC. Further veri cation found that circ_0048856 and miR-1287-5p have a targeted relationship, and found that there is a negative correlation between them, which provides a new theoretical basis for the treatment of patients.

Subjects and samples
A total of 121 cases pathologically diagnosed with NSCLC and 113 healthy individuals were collected in this study. Before sampling, they did not receive any treatment including radiotherapy or chemotherapy. 5mL fasting blood specimen were obtained from all subjects, then serums were separated from whole blood via centrifugation. The supernate was immediately stored at -80℃ until for use. All patients were followed up by telephone for 5 years. The clinicopathological features of the cases were shown in Table   1. This study was approved by the Ethic committee of Inner Mongolia People's Hospital. The written informed consent forms were provided from each participant in the present study prior to sampling. Cell culture NSCLC cell lines A549 and H1299, as well as human bronchial epithelial cell (HBE) were purchased from American Type Culture Collection (ATCC). The cells were cultured in DMEM medium containing 10% fetal bovine serum in 37 ℃ and 5% CO 2 saturated humidity incubator for subculture once every two days.
When the cells were covered with the bottom of the culture bottle and fused into a single layer, and the density reached 70% ~ 80% fusion, the cells were digested with 0.25% trypsin and 1:3 ratio is used for subculture.
qRT-PCR RNA Isolation Kit (Qiagen, Hilden, Germany) was used to isolate total RNA based on operation manual. The qualitative integrity of puri ed total RNA was tested by capillary electrophoresis using 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). RevertAid TM First Strand cDNA Synthesis Kit (Ferments, Vilnius, Lithuania) was adopted to perform the reverse transcription. The expression of gene in specimens were measured via quantitative real-time PCR (qRT-PCR). The reaction was carried out with SYBR Premix Ex Taq TM (TaKaRa, Dalian, China) kit in ABI 7500 instrument (ABI). The experiment was repeated at least 3 times. Finally, the levels of gene in NSCLC patients or healthy control group were calculated via the 2 −ΔΔCt method.

Cell transfection
Cell lines in exponential growth period were conducted cell transfection. The transfection vectors were si-circ_0048856 and si-NC were purchased from Guangzhou RiboBio Co., Ltd.,(Guangzhou, China). They were transfected into NSCLC cells by LipofectamineTM2000 (Invitrogen, CA, USA) following the instruction of manufacturer, then cultured in 37℃humididied atmosphere with 5% CO 2 for 6h. The medium was replaced and they were transfected for 48h. Ultimately, qRT-PCR was employed to measure transfection e ciency.

Luciferase reporter assay
To verify the targeting relationship between circ_0048856 and miR-1287-5p, we performed a luciferase reporter analysis. Circ_0048856-WT or circ_0048856-MUT (100 ng) were co-transfected with 100 nM miR-1287-5p mimics or miR-1287-5p mimics NC into NSCLC cells using Lipofectamine 2000 (Invitrogen, CA, USA) following the instruction of manufacture. 48 hours after transfection, the cells were harvested and detected by Dual-Luciferase Reporter Assay System Kit (Promega, Madison, WI, USA). The speci c operation was carried out according to the instructions.

Statistical analysis
We used the software of SPSS 18.0 (SPSS. Chicago. IL, USA) was performed to analyze all data, and all graphs were plotted via GraphPad Prism (GraphPad, San Diego, CA, USA). The correlation between circ_0048856 expression and clinicopathological features was assessed via χ 2 test. Difference between groups was compared by Student's t-test. Kaplan-Meier and cox regression analyses were conducted to assess the prognostic ability of circ_0048856 in NSCLC. P<0.05 was seen as obviously signi cant difference.

Results
Expression of circ_0048856 is up-regulated in the serum of NSCLC patients The relative expression of circ_0048856 in serum samples of NSCLC subjects and healthy individuals were measred via qRT-PCR. All data revealed that the expression of circ_0048856 in NSCLC serum samples was signi cantly up-regulated than that in healthy volunteers' serum specimens (P<0.001, Figure  1).

Relationship between circ_0048856 expression and clinical features
The patients diagnosed with NSCLC were divided into low expression group (n=58) and high expression group (n=63), according to their median serum levels of circ_0048856. In addition, we examined the relationship between circ_0048856 levels and the clinical features of NSCLC patients using chi-square test. All data proved that circ_0048856 expression was signi cantly correlated with TNM stage (P=0.002) and lymphatic node metastasis (P<0.001). Nevertheless, circ_0048856 expression was not found to be associated with other clinical features including age, gender, smoking history, tumor size and differentiation (all P>0.05, Table 1).

Prognostic value of circ_0048856 expression in patients with NSCLC
To evaluate the prognostic ability of circ_0048856 for NSCLC cases, we assessed the connection between circ_0048856 levels and overall survival. The result of Kaplan-Meier with log rank test analysis revealed that the overall survival of patients with high circ_0048856 expression was obviously shorter than those with low circ_0048856 expression (log rank test, P = 0.002, Figure 2).
Besides, we used the Cox regression analysis to assess the prognostic ability of circ_0048856 in NSCLC patients. Both univariable and multivariable analysis results listed in Table 2  Circ_0048856 directly targeted miR-1287-5p We predicted that miR-1287-5p might be the direct downstream miRNA of circ_0048856 through online software Starbase v2.0 ( Figure 3A). To further verify the relationship between circ_0048856 and miR-1287-5p was evaluated by luciferase reporter assay. Circ_0048856-3'UTR-WT or circ_0048856-3'-UTR-MUT were co-transfected with 100 nM miR-1287-5p mimics or mimics NC into A549 and H1299 cells using Lipofectamine 2000 following the instruction of manufacture. The data revealed that in the cells transfected with circ_0048856-3'UTR-WT, miR-1287-5p mimics had signi cantly lower luciferase activity; when the binding site was mutated, the above inhibition was lost ( Figure 3B and C). The experiment shows that miR-1287-5p is the direct downstream miRNA of circ_0048856.
Circ_0048856 has a negative correlation with miR-1287 To further explore the relationship between miR-1287-5p and circ_0048856, we detected the expression level of miR-1287-5p in serum of patients with NSCLC, and found that the expression level of miR-1287-5p was signi cantly decreased ( Figure 4A), which was contrary to the expression pattern of circ_0048856 in serum of patients with NSCLC. We also detected the expression of miR-1287-5p and circ_0048856, we found that the expression of miR-1287-5p in A549 and H1299 cells was lower than that in the control group, while the expression of circ_0048856 in A549 and H1299 cells was higher than that in the control group ( Figure 4B). Furthermore, knockdown of circ_0048856 in NSCLC cells resulted in up regulation of miR-1287-5p expression ( Figure 4C). Analysis of the correlation between circ_0048856 and miR-1287-5p mRNA expression in serum of NSCLC showed that there was a negative correlation between them. ( Figure  4D). Our results proved that circ_0048856 is negatively correlated with miR-1287-5p.

Discussion
NSCLC is a kind of malignant tumor threatening the safety of human life, which has a high incidence and mortality. The disease is prone to metastasis and poor prognosis [18]. At present, many prognostic markers have been found, but the clinical application is less [19][20][21]. In this study, we con rmed that the expression of circ_0048856 in serum of NSCLC was increased, and the expression of circ_0048856 can be used as an independent prognostic biomarker for the overall survival rate of NSCLC patients, and it can also play a role by targeting miR-1287-5p.
In recent years, circRNAs have become a research hotspot. The role of circRNAs is to isolate or "sponge" other gene expression regulators, especially miRNAs. CircRNAs play an important role in a variety of biological functions and have broad application prospects [22,23]. The accumulated data con rmed that circRNAs may become a new indicator for prognosis and diagnostic evaluation of lung cancer patients [24].
Previous studies have found that a large number of circRNAs are dysregulated in NSCLC. Although circRNAs are constantly found, their biological functions in NSCLC have just been explored [25][26][27]. We detected the expression level of circ_0048856 in serum of NSCLC by qRT-PCR, and found that the expression level of circ_0048856 was signi cantly up-regulated, which was consistent with previous studies [17]. The high expression of circ_0048856 was signi cantly correlated with TNM stage and lymph node metastasis. Survival analysis showed that the overall survival of patients with high expression of circ_0048856 was signi cantly shorter than that of patients with low expression of circ_0048856. Further analysis showed that circ_0048856 expression and lymph node metastasis were independent prognostic biomarkers of overall survival in NSCLC patients. A study by Wang et al. found that circ_0067934 was upregulated in NSCLC tissues and cells, and its high expression was closely related to TNM stage, lymph node status and distant metastasis, indicating poor prognosis of NSCLC [28]. Geng et al. reported that the expression of circ-MTHFD2 was up-regulated in NSCLC samples, and its high expression indicates poor prognosis of patients, which can be used as a diagnostic, prognostic marker and therapeutic target of NSCLC patients [29]. Liu et al. con rmed that the low expression of circ_0001649 is associated with poor prognosis of NSCLC patients, and can be used as a prognostic marker of NSCLC [30].
In addition, we predicted the direct related miRNAs of circ_0048856 by online prediction software Starbase V2.0, and found that miR-1287-5p may be the direct downstream miRNA of circ_0048856. Furthermore, we con rmed that miR-1287-5p is a direct downstream miRNA of circ_0048856 by luciferase reporter gene analysis. Moreover, the expression of miR-1287-5p was signi cantly down regulated in NSCLC serum and cell lines, and the expression trend was opposite to circ_0048856, showing a negative correlation. We speculate that circ_0048856 is involved in the progression of NSCLC by targeting miR-1287-5p.
Previous studies have shown that circRNAs can play a role in NSCLC by targeting miRNAs. Li et al. identi ed a new circRNA, circ_0002483, which can sponge multiple miRNAs and inhibit the progression of NSCLC through sponge miR-182-5p [31]. Another study found that circ_0000735 had a targeting relationship with miR-1179/1182. Circ_0000735 combined with miR-1179/1182 and participated in the biological process of NSCLC cells [32]. Gao et al. con rmed that circ_0074027 is carcinogenic in NSCLC, and its high expression indicates poor prognosis. Circ_0074027 can promote the process of NSCLC cells as competitive endogenous RNA through miR-185-3p/BRD4/MADD pathway [33]. These studies have con rmed that circRNAs has a signi cant biological function in NSCLC, and can be used as a target for the diagnosis, prognosis and treatment of NSCLC.
There are several limitations in this study that cannot be ignored. Firstly, we recruit fewer patients and volunteers, which may affect the accuracy of the results. Secondly, we did not explore the speci c mechanism of circ_0048856 in NSCLC. The pathogenesis of NSCLC is very complex. In the next step, we will expand the sample size and further explore the molecular mechanism of circ_0048856 in the occurrence and development of NSCLC, so as to bring new hope for the treatment of patients.

Conclusion
To sum up, circ_0048856 may be an independent prognostic biomarker in patients with NSCLC. There is a negative correlation between circ_0048856 and miR-1287-5p. circ_0048856 may participate in the occurrence and development of NSCLC by targeting miR-1287-5p. Our study provides a new target for the treatment and prognosis of the disease, and provides a new theoretical basis for the prevention and treatment of the disease. 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
All data generated or analysed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.   Survival analysis. The overall survival of patients with high circ_0048856 expression was signi cantly shorter than those with low circ_0048856 expression (log rank test, P=0.002).