IL-10 Promotes Glioma Cell Growth and Invasion via Upregulation of KPNA2 In vitro

Background Glioma is one of the leading causes of death worldwide incidence, recurrence and IL-10 is a cytokine with dual function in many types of tumors. Although IL-10 is overexpressed and promotes tumor progression in human primary brain tumor, the mechanisms are largely unknown. Methods Glioma cells were treated with different dosages of IL-10. The cell growth was detected by CCK8, and the invasion was measured by Transwell. The relative expression of mRNAs was detected by Quantitative real-time PCR (q-PCR). Results We found that IL-10 treatment significantly enhanced glioma cell growth and invasion. And KPNA2 was significantly upregulated after treatment with IL-10. By performing knockdown experiments, we found that the glioma cell growth and invasion were significantly declined. The results indicated that knockdown of KPNA2 significantly inhibited the growth and invasion of glioma cells. And IL-10 promotes glioma progression via upregulation of KPNA2. This study will be of important significance and provides a potential target for treatment of patients with glioma.

U87 cells were seeded in 6-well plates and cultured overnight. Next day, cells were transfected with three siRNA for KPNA2 at optimal concentration. Forty-eight hours later, cells were used for further experiments.

Statistical analysis
The Student's t-test was used to quantify significant differences. GraphPad Prism 5 software was used for statistical analysis, Data are presented as the mean ± SD, and a value of P < 0.05 were considered statistically significant.

1.
IL-10 promotes the proliferation of glioma cells Cancer progression is a complex process involving cell growth, migration, invasion, colony formation, and metastasis. To explore whether IL-10 affected glioma progression, we first detected the role of IL-10 in glioma cell growth. We cocultured glioma cell line U87 with different dosages of IL-10. After 24 hours, CCK-8 assay was performed. We found that IL-10 promoted the cell growth of U87 in a dosedependent manner ( Figure 1A). As shown in Figure 1A, IL-10 at the concentration of 50 ng/ml enhanced the proliferation of U87 cells much significantly than others ( Figure 1A). Therefore, we used IL-10 at 50 ng/ml for further experiments. We also found that IL-10 significantly promoted the cell growth of U87 cells at day4 compared with negative control cells (NC) ( Figure 1B). Thus, these data demonstrated that IL-10 promotes the proliferation of glioma cells.

IL-10 enhances the invasion of glioma cells
Next, we assessed the role of IL-10 in glioma cell invasion by cell invasion assay. As compared with negative control cells, IL-10 treatment remarkably increases the number of invasion cells (Figure 2A, . These results demonstrated that IL-10 treatment enhanced the invasion of human glioma cells.

IL-10 increases the expression of kpna2 in glioma cells
We further explored the mechanisms by which IL-10 promoted the glioma cell proliferation and invasion. Many studied have shown that KPNA2 played a role in cell growth and invasion of many cancer cells, including human brain tumour [26, 27, 29, 31, 32]. Therefore, we examined the expression of kpna2 in IL-10 treated U87 cells. We found that IL-10 significantly increased kpna2 mRNA levels in U87 cells compared with untreated control cells ( Figure 3).

Knockdown of KPNA2 inhibits the proliferation of glioma cells
To investigate the role of KPNA2 in glioma cell growth, we used siRNA to specifically silence KPNA2 in U87 cells. We first examined the knockdown efficiency of KPNA2. As shown in Figure 4A, the expression of KPNA2 was significantly inhibited in U87 cells. We found that knockdown of KPNA2 significantly inhibited the cell growth of U87 cells at day3 and day4 compared with negative control cells (NC) ( Figure 4B). Taken together, these data demonstrated that KPNA2 affected the glioma cell growth.

Knockdown of KPNA2 suppressed the invasion of glioma cells
We also assessed the effect of KPNA2 on cell invasion of U87 cells. The results showed that knockdown of KPNA2 significantly decreased the cell invasion of U87 cells. Thus, we confirmed that kpna2 contributes the invasion of glioma cells.
Taken together, we found that IL-10 promoted glioma cell progress via upregulation of KPNA2.

Discussion
In this study, we report that IL-10 promotes cell growth and invasion in glioma cells. We found that IL-    The experiment was performed in quintuplicate. Data were shown as mean±SD. * p<0.05.

Figure 5
KPNA2 knockdown inhibited the glioma cell invasion -U87 cells infected with the control (NC) or KPNA2-shRNA lentivirus were seeded in matrigel-coated Transwell chamber overnight. Then, cell invasion assay was performed. Invasion cells were counted from at least five random fields Data were presented as mean±SD. * p<0.05.