Circ_0013401 was identified as highly expressed in NB
To investigate the expression changes of the related circRNAs in NB, we firstly examined circ_0013401, circ_ 0045997, circ_0077578 and circ_0080307 expressions in a total of 8 GN and NB tissue samples. As displayed in Fig. 1A, only circ_0013401 was significantly upregulated in NB tissues compared to that in GN tissues (p < 0.01). Next, we adopted FISH assay to identify the expression of circ_0013401 in GN and NB tissues, the results also uncovered that circ_0013401 was highly expressed in NB tissues relative to GN tissues (Fig. 1B). Additionally, Ki67 and PAK2 expressions were monitored using IHC assay, the results discovered that Ki67 and PAK2 expressions were also markedly elevated in NB tissues with respect to GN tissues (Fig. 1C and 1D). Consequently, we verified that circ_0013401, Ki67 and PAK2 were highly expressed in NB.
Circ_0013401, as an oncogene, prominently accelerated NB proliferation
Given that circ_0013401 was down-regulated in NB, we further investigated the possible functional roles mediated by circ_0013401 overexpression and knockdown in NB cells. We first determined circ_0013401 expression in different neuroblastoma cells (SK-N-BE, GNP, SH-SY5Y, IMR-32, LAN-1 and SK-N-SH) using RT-qPCR, and the results proved that circ_0013401 was significantly upregulated in SH-SY5Y and SK-N-BE cells compared to other NB cells. And SH-SY5Y and SK-N-BE cells were applied in subsequent experiments (p < 0.01, Fig. 2A). RT-qPCR analysis was then conducted to identify the transfection effects of circ_0013401-overexpressed plasmid or circ_0013401 shRNAs in SH-SY5Y and SK-N-BE cells. As presented in Fig. 2B, circ_0013401 was observably upregulated in circ_0013401 overexpression group compared to overexpression-NC group, and circ_0013401 was also signally downregulated in circ_0013401 knockdown group versus sh-NC group in SH-SY5Y and SK-N-BE cells (p < 0.01, Fig. 2B). Afterwards, we detected the influences of circ_0013401 overexpression or knockdown on the proliferation of SH-SY5Y and SK-N-BE cells. The CCK-8 results manifested that the viability of SH-SY5Y and SK-N-BE cells was memorably increased in circ_0013401 overexpression group with respect to overexpression-NC group, and dramatically decreased in circ_0013401 knockdown group relative to sh-NC group (p < 0.05, p < 0.01, Fig. 2C). Consequently, the EdU + number of circ_0013401 overexpressing SH-SY5Y and SK-N-BE cells was significantly more than those of overexpression-NC group, and the Edu + number of circ_0013401 silencing cells was dramatically less than those of sh-NC group (p < 0.05, p < 0.01, Fig. 2D). Meanwhile, clone formation assay revealed that circ_0013401 overexpression observably induced NB proliferation, and circ_0013401 knockdown prominently suppressed NB proliferation (p < 0.05, p < 0.01, Fig. 2E). Hence, we testified that circ_0013401 played a significant role in inducing the proliferation of NB cells.
Circ_0013401 markedly facilitated migration and invasion, and prevented apoptosis and autophagy of NB cells
Besides, we further verified the changes of migration, invasion, apoptosis and autophagy in SH-SY5Y and SK-N-BE cells after circ_0013401 overexpression or knockdown. Firstly, the migration and invasion were certified by adopting Transwell assay, and the data disclosed that overexpression of circ_0013401 could result in prominent enhancements, and knockdown of circ_0013401 could lead to remarkable reductions in the migration and invasion capabilities of SH-SY5Y and SK-N-BE cells (p < 0.05, p < 0.01, Fig. 3A and 3B). The data in flow cytometer detection exhibited that circ_0013401 overexpression notably reduced the apoptosis rate, circ_0013401 knockdown prominently raised the apoptosis rate in SH-SY5Y and SK-N-BE cells (p < 0.05, p < 0.01, Fig. 3C). Moreover, the TEM results testified that compared with the control group, the autophagosomes in circ_0013401-overexpressed SH-SY5Y and SK-N-BE cells were significantly reduced, and a large number of autophagosomes were formed in circ_0013401-silenced SH-SY5Y and SK-N-BE cells (Fig. 3D). Thus, we indicated that circ_0013401 could signally induce migration and invasion, and repressed apoptosis and autophagy of SH-SY5Y and SK-N-BE cells.
Circ_0013401 dramatically regulated miR-195/PAK2 axis, autophagy and apoptosis-related proteins in NB cells
Subsequently, we explored the underlying regulatory pathway of circ_0013401 in NB. Through bioinformatics predictions, we discovered that circ_0013401 might be a miRNA response element (MRE) of miR-195, which might bind with circ_0013401; and PAK2 might be the most likely target gene of miR-195. So, miR-195 and PAK2 became our research targets in mechanism. To further confirm the miR-195 expression in NB, the level of miR-195 was analyzed in different neuroblastoma cells using RT-qPCR. As indicated in Fig. 4A, miR-195 was signally downregulated in SH-SY5Y and SK-N-BE cells relative to other NB cells (p < 0.01). In addition, we demonstrated that miR-195 could be observably downregulated by circ_0013401 overexpression, and memorably upregulated by circ_0013401 knockdown in SH-SY5Y and SK-N-BE cells (p < 0.05, p < 0.01, Fig. 4B). While PAK2 could be markedly upregulated by circ_0013401 overexpression, and dramatically downregulated by circ_0013401 knockdown in SH-SY5Y and SK-N-BE cells (p < 0.05, p < 0.01, Fig. 4C). Meanwhile, the IF results further indicated that circ_0013401 overexpression prominently raised PAK2 expression and reduced LC3B expression; meanwhile, circ_0013401 knockdown notably lowered PAK2 expression and elevated LC3B expression in SH-SY5Y and SK-N-BE cells (Fig. 4D). More importantly, western blotting results showed that circ_0013401 overexpression observably upregulated PAK2, p62 and Bcl-2, and downregulated LC3B II/I, Beclin1, Bax and cleaved Caspase-3, while circ_0013401 knockdown could lead to the opposite effects of its overexpression on these proteins in SH-SY5Y and SK-N-BE cells (Fig. 4E). On the whole, we manifested that circ_0013401 dramatically suppressed autophagy and apoptosis, and regulated miR-195/PAK2 axis in NB cells.
Circ_0013401 sponged miR-195, and PAK2 was a target gene of miR-195
To verify the relationship between miR-195 and circ_0013401 or PAK2, luciferase reporter assay was performed. We proved that miR-195 could significantly lower the luciferase activity of WT-circ_0013401, not Mut-circ_0013401 in SH-SY5Y and SK-N-BE cells (p < 0.01, Fig. 5A). Similarly, the luciferase activity of WT-PAK2 was signally repressed by miR-195, while the luciferase activity of Mut-PAK2 was not affected by miR-195 in SH-SY5Y and SK-N-BE cells (p < 0.01, Fig. 5B). Thus, we proved that circ_0013401 can significantly regulate miR-195/PAK2 axis through targeted binding.
MiR-195 inhibitor prominently reversed the inhibitory action of circ_0013401 knockdown on the proliferation of NB cells
Next, the rescue assays were conducted to confirm whether circ_0013401 induced NC proliferation by targeting miR-195. MiR-195 inhibitor were transfected into circ_0013401-silenced SH-SY5Y and SK-N-BE cells, and the RT-qPCR results demonstrated that miR-195 inhibitor markedly downregulated miR-195, which was induced by circ_0013401 shRNA in SH-SY5Y and SK-N-BE cells (p < 0.01, Fig. 6A). Then EdU staining results uncovered that the proliferation of SH-SY5Y and SK-N-BE cells was memorably increased in the co-transfection group of miR-195 inhibitor and circ_0013401 shRNAs compared with that in the transfection group of circ_0013401 shRNA (p < 0.01, Fig. 6B and 6C). Likewise, the results of clone formation assay also revealed that miR-195 inhibitor could prominently facilitate NB cell proliferation mediated by circ_0013401 knockdown (p < 0.01, Fig. 6D). Therefore, we testified that the inhibition of cell proliferation mediated by circ_0013401 shRNA could be significantly reversed by miR-195 inhibitor in NB.
MiR-195 was involved in the inhibition of migration and invasion, and induction of apoptosis mediated by circ_0013401 knockdown in NB cells
Additionally, through Transwell assay, we discovered that the migration and invasion abilities of SH-SY5Y and SK-N-BE cells were markedly higher in the co-transfection group of miR-195 inhibitor and circ_0013401 shRNA than that in the transfection group of circ_0013401 shRNA (p < 0.01, Fig. 7A and 7B). Flow cytometer results testified that miR-195 inhibitor signally reduced the apoptosis of SH-SY5Y and SK-N-BE cells, which have been induced by miR-491-5p mimics (p < 0.01, Fig. 7C). In general, we certified that circ_0013401 knockdown prevented migration and invasion, and accelerated apoptosis of NB cells by miR-195.
MiR-195 inhibitor memorably attenuated the downregulation effects of circ_0013401 knockdown on PAK2, autophagy and apoptosis-related proteins in NB cells
Furthermore, we identified the downstream regulatory molecules of circ_0013401/miR-195 axis in NB cells. SH-SY5Y and SK-N-BE cells were co-transfected circ_0013401 shRNA and miR-195 inhibitor, and RT-qPCR was first conducted to confirm PAK2 expression. And the data indicated that PAK2 expression mediated by circ_0013401 knockdown could be dramatically upregulated by miR-195 inhibitor in SH-SY5Y and SK-N-BE cells (p < 0.01, Fig. 8A). Simultaneously, western blot results proved that after transfection with miR-195 inhibitor, PAK2, p62 and Bcl-2 were observably upregulated, LC3BII/I, Beclin1, Bax and cleaved Caspase-3 signally were downregulated in circ_0013401 shRNA-transfected SH-SY5Y and SK-N-BE cells (Fig. 8B). As a result, we suggested that circ_0013401 knockdown could reduce PAK2 expression and induce autophagy and apoptosis-related proteins by miR-195 in NB cells.
Overexpression of PAK2 signally suppressed apoptosis and autophagy mediated by miR-195 in NB cells
Whereafter, we adopted the rescue assays to verify the impacts of miR-195/PAK2 axis on apoptosis and autophagy in NB cells. miR-195 mimics or/and PAK2-overexpressed plasmids were transfected into SH-SY5Y and SK-N-BE cells, and PAK2 expression was assessed using RT-qPCR. As shown in Fig. 9A, overexpression of PAK2 prominently aggrandize PAK2 expression in miR-195 mimics-transfected SH-SY5Y and SK-N-BE cells (p < 0.01). Meanwhile, the data of flow cytometry revealed that the apoptosis rate could be markedly weakened by PAK2 overexpression in SH-SY5Y and SK-N-BE cells after transfection with miR-195 mimics (p < 0.01, Fig. 9B). In the mechanism, we disclosed that overexpression of PAK2 also could memorably increase p62 and Bcl-2 expressions, signally decrease LC3BII/I, Beclin1, Bax and cleaved Caspase-3 expressions in miR-195 mimics-transfected SH-SY5Y and SK-N-BE cells (Fig. 9C). Overall, we indicated that PAK2 was significantly involved in the effects of miR-195 on apoptosis and autophagy of NB cells.
Verification of circ_0013401/miR-195/PAK2 axis in vivo experiment
Based on the results of in vitro experiments, we further verified the regulatory effects of circ_0013401 on tumor growth and miR-195/PAK2 expressions in NB. We first established SH-SY5Y cells where circ_0013401 were overexpressed or silenced, then the transfected SH-SY5Y cells were injected into BALB/c nude mice. On the 28th day, the mice were killed and the tumor was removed. As displayed in Fig. 10A and 10B, NB tumor growth was markedly promoted by circ_0013401 overexpression, and suppressed by circ_0013401 knockdown (p < 0.01). RT-qPCR results also uncovered that circ_0013401 and PAK2 were significantly upregulated, miR-195 was dramatically downregulated in circ_0013401-overexpressed group relative to the control group; circ_0013401 and PAK2 were notably downregulated, miR-195 was memorably upregulated in circ_0013401-silenced group versus the control group (p < 0.05, p < 0.01, Fig. 10C). IHC results also testified that circ_0013401 overexpression observably elevated PAK2 and Ki67 expressions, circ_0013401 knockdown dramatically lowered PAK2 and Ki67 expressions in NB tumor tissues (Fig. 10D). Moreover, we further verified that overexpression of circ_0013401 markedly upregulated PAK2, p62 and Bcl-2, and downregulated LC3B II/I, Beclin1, Bax and cleaved Caspase-3 in mice tumor tissues; the influences of circ_0013401 knockdown on the expressions of these proteins were contrary to its overexpression in mice tumor tissues (Fig. 10E). Hence, we further certified that circ_0013401 signally downregulated miR-195 and upregulated PAK2, and inhibited apoptosis and autophagy-related proteins in vivo.