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 significantly 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 significantly 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 showed that circ_0048856 expression (HR=2.001, 95%CI: 1.258-3.184, P=0.003 and HR=1.669, 95% CI: 1.018-2.736, P=0.042) and lymphatic node metastasis (HR=2.118 95% CI: 1.340-3.348, P=0.001 and HR=1.766, 95% CI: 1.086-2.874, P=0.022) were useful prognostic indicators for overall survival in cases with NSCLC.
Table 2.
Cox regression analyses for circ_0048856 in NSCLC patients
Factors
|
Univariate analysis
|
Multivariate analysis
|
HR(95% CI)
|
P values
|
HR(95% CI)
|
P values
|
circ_0048856 expression
|
2.001 (1.258–3.184)
|
0.003
|
1.669 (1.018–2.736)
|
0.042
|
Age
|
-
|
0.332
|
-
|
-
|
Gender
|
-
|
0.999
|
-
|
-
|
Smoking history
|
-
|
0.935
|
-
|
-
|
Tumor size
|
-
|
0.666
|
-
|
-
|
TNM stage
|
-
|
0.072
|
-
|
-
|
Lymph node metastasis
|
2.118 (1.340–3.348)
|
0.001
|
1.766 (1.086–2.874)
|
0.022
|
Differentiation
|
-
|
0.554
|
-
|
-
|
Note: - indicated no data. |
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 significantly 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 significantly 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.