SNRPB regulates cisplatin-induced cell proliferation inhibition of NSCLC cells
We first examine the SNRPB protein expression in a panel of 5 NSCLC cell lines. We find a higher expression level of SNRPB in H1299 and H23 cells and a lower expression level of SNRPB in H460 cells (Fig. 1A). We thus knock out of SNRPB in H1299 cells or overexpress it in H460 cells (Fig. 1B and 1C). Next, we test whether manipulate SNRPB expression can affect cisplatin-induced inhibition of cell growth in NSCLC cells using the CCK-8 assay. Consistent with our previous finding , overexpression of SNRPB promotes while knockout of SNRPB inhibits cell growth in non-treated H1299 and A549 cells, respectively (Fig. 1D, 1E). Interestingly, after the treatment with cisplatin for 24 or 48 hours, the inhibitory rate increased in H1299 cells with SNRPB knockout (Fig. 1D). Conversely, the inhibitory rate decreased in H460 cells with SNRPB overexpression (Fig. 1E). The cisplatin IC50 values were also determined in these two cell lines with or without SNRPB manipulation. As is shown in Fig. 1F, the IC50 increased from 16.56 µM to 27.39 µM when SNRPB knocked out in H1299 cells. However, the IC50 declined from 4.28 µM to 2.46 µM when SNRPB overexpressed in H460 cells (Fig. 1G). Taken together, these results indicate that SNRPB is a mediator of NSCLC cells in response to cisplatin.
SNRPB regulates cisplatin response in NSCLC cells by affecting cell cycle progression
To explore the potential mechanisms responsible for the function of SNRPB, cell cycle analyses were conducted by flow cytometry in H1299 and H460 cells. As is shown in Fig. 2A and 2B, cisplatin treatment increased the G1/G2 but decreased the S phase population of SNRPB knockout H1299 cells compared to control cells. However, overexpression of SNRPB decreased the percentage of G1/G2 but increased the percentage of S phase in H460 cells after treatment with cisplatin (Fig. 2C and 2D).
A previous study demonstrated that knockdown of SNRPB affects cancer genes expression, many of which are cell cycle-related genes . We thus selected several dysregulated cell cycle-associated genes upon SNRPB knockdown for further confirmation and then examined whether their expression will change in SNRPB knocked out or overexpressed H1299 and H460 cells after treatment with cisplatin. Cisplatin treatment induced a much higher expression of CCNB1 and CDK4 in a dose-dependent manner in SNRPB knockout H1299 cells compared to control cells (Fig. 2E). However, overexpression of SNRPB reduced cisplatin-induced CCNB1 and CDK4 expression in H460 cells (Fig. 2F). These results suggest that SNRPB may modulate cell cycle progression of NSCLC cells by regulating cell cycle-related gene expression.
Snrpb Modulates Cisplatin-induced Apoptosis In Nsclc Cells
We next measured cisplatin-induced apoptosis by flow cytometry in H1299 and H460 cells with altered SNRPB expression. The results showed that knockout of SNRPB markedly decreased cisplatin-induced apoptosis in H1299 cells (Fig. 3A and 3B). While in H460 cells, overexpression of SNRPB significantly increased cisplatin-induced apoptosis compared to control cells (Fig. 3C and 3D). We also assessed the activity of the molecular marker of apoptosis PARP by western blotting assay. As shown in Fig. 3E, cisplatin treatment induced higher expression of cleaved PARP in SNRPB knockout H1299 cells compared to the wild type cells. However, ectopic expression of SNRPB in H460 cells decreased cisplatin-induced cleaved PARP expression (Fig. 3F). We obtained similar results when examined the expression of the DNA damage marker γ-H2AX (Fig. 3E and 3F), suggesting that SNRPB negatively regulates cisplatin-induced apoptosis in NSCLC cell.
SNRPB regulates ERK signaling pathway activity in NSCLC cells after treatment with cisplatin
Previous studies have reported that activated ERK signaling pathway is responsible for the resistance of NSCLC cells to cisplatin [16, 17]. To investigate whether SNRPB regulates cisplatin resistance in NSCLC via ERK signaling pathway, we detected the pERK level in H1299 and A549 cells treated with multiple concentrations of cisplatin. As shown in Fig. 4A and 4B, cisplatin treatment activates ERK in a dose-dependent manner in control H1299 cells. However, knockout of SNRPB significantly decreased the cisplatin-induced pERK level. In control of H460 cells, cisplatin treatment did not affect ERK pathway activity. While in SNRPB overexpressing H460 cells, high concentration (0.5, 1 µg/ml) cisplatin could induce pERK expression (Fig. 4C, 4D). These results suggest that SNRPB may regulate the toxicity effect of cisplatin via ERK signaling pathway in the NSCLC cell.
SNRPB-mediated xenograft NSCLC tumors were more effectively inhibited by cisplatin
Due to SNRPB-knocked out H1299 cells could hardly form tumors in nude mice , we then tested whether SNRPB influence anti-tumor effects of cisplatin in H460-mediated xenograft tumor model. In this regard, control and SNRPB overexpressing H460 cells were subcutaneous injected into 6-week old nude mice. Tumor volume was measured 12 days after cancer cells injected. Consistent with our previous findings, overexpression of SNRPB significantly promote tumor growth in nude mice (H460-Vector Ns Vs H460-SNRPB Ns, Fig. 5A) . Cisplatin treatment markedly reduced tumor growth in the SNRPB-overexpressing group. While in the vector expressing group, cisplatin treatment only had a modest impact on tumor growth (Fig. 5A). The tumor inhibition rate of cisplatin is higher in the SNRPB-overexpressing group than in the control group (H460-Vector + Cisplatin Vs H460-SNRPB + Cisplatin, Fig. 5B). Also, the tumor volume and weight are bigger and heavier in the SNRPB-overexpressing group than in the vector-overexpressing group. However, cisplatin only has a significant impact on tumor growth when SNRPB overexpressed in tumor cells (Fig. 5C, 5D). These results suggest that SNRPB enhances cisplatin-induced tumor inhibition in vivo.