The abnormal expression of SNHG6 and miR-944 in NPC
Here, the abnormal expression of SNHG6 and miR-944 was detected examined in NPC. SNHG6 expression was increased in NPC tissues compared with normal tissues (Fig. 1A). Additionally, SNHG6 was upregulated in stage III/IV and distant metastasis tissues (Fig. 1B, 1C). It indicates that SNHG6 expression may be related to TNM stage and distant metastasis in NPC patients. Meanwhile, upregulation of SNHG6 was found in C666-1 NPC cells contrast to normal nasopharyngeal epithelial cell NP69 (Fig. 1D). Next, miR-944 was found to be downregulated in NPC tissues compared to normal tissues (Fig. 1E). Furthermore, low miR-944 expression was measured in stage III/IV and distant metastasis tissues (Fig. 1F, 1G), suggesting that miR-944 expression may be related to TNM stage and distant metastasis in NPC patients. Decreased expression of miR-944 was also detected in C666-1 cells compared to NP69 cells (Fig. 1H). Based on these results, we suspect that SNHG6 and miR-944 may be involved in the pathogenesis of NPC.
SNHG6 acts as a molecular sponge of miR-944.
Next, SNHG6 was found to have a binding site with SNHG6 in starBase database (http://starbase.sysu.edu.cn/, Fig. 2A). Dual luciferase reporter showed that miR-944 mimics only reduced the luciferase activity of wt-SNHG6, but had little effect on mut-SNHG6 in C666-1 cells (Fig. 2B). Additionally, SNHG6 was negatively correlated with miR-944 expression in NPC tissues (Fig. 2C). To further confirm their relationship, si-SNHG6, SNHG6 vector, miR-944 mimics or miR-944 inhibitor was transfected into C666-1 cells, respectively. We found that miR-944 expression was inhibited by SNHG6 vector and enhanced by si-SNHG6 in C666-1 cells (Fig. 2D). Meanwhile, miR-944 mimics reduced SNHG6 expression, while miR-944 inhibitor increased SNHG6 expression in C666-1 cells (Fig. 2E). These findings reveal that the tendency of mutual restrain existed between SNHG6 and miR-944 expression in NPC.
SNHG6/miR-944 axis regulates NPC progression.
Then, the regulatory mechanism of SNHG6/miR-944 was investigated in NPC cells. First, miR-944 inhibitor was transfected into C666-1 cells containing si-SNHG6. We found that SNHG6 expression was decreased by si-SNHG6. But miR-944 inhibitor restored this decreased expression of SNHG6 (Fig. 3A). In addition, knockdown of SNHG6 was found to restrain C666-1 cell proliferation. But miR-944 inhibitor abolished the inhibitory effect of si-SNHG6 on cell proliferation (Fig. 3B). Meanwhile, cell migration and invasion were also suppressed by knockdown of SNHG6. And the inhibition of cell migration and invasion was recovered by miR-944 inhibitor (Fig. 3C, 3D). To explore the role of miR-944 in NPC cells, miR-944 mimics and SNHG6 vector were transfected into C666-1 cells. MiR-944 expression was increased by its mimics. Upregulation of SNHG6 reduced the increased expression of miR-944 (Fig. 3E). Functionally, overexpression of miR-944 restrained cell proliferation, migration and invasion in C666-1 cells. Furthermore, SNHG6 vector weakened the inhibitory effect of miR-944 in Y79 cells (Fig. 3F, 3G, 3H). The results imply that SNHG6/miR-944 axis plays an important role in NPC progression.
MiR-944 directly targets RGS17.
In addition, RGS17 was predicted to be a potential target for miR-944 in TargetScan database (http://www.targetscan.org, Fig. 4A). Luciferase reporter assay showed that miR-944 mimics decreased the luciferase activity of wt-RGS17, indicating that miR-944 can bind with the 3’-UTR of RGS17 (Fig. 4B). Meanwhile, RGS17 expression was declined by miR-944 mimics and enhanced by miR-944 inhibitor in C666-1 cells (Fig. 4C). However, upregulation of SNHG6 increased RGS17 expression, whereas SNHG6 downregulation inhibited RGS17 expression in C666-1 cells (Fig. 4D). Besides that, upregulation of RGS17 was found in NPC tissues compared with normal tissues (Fig. 4E). Furthermore, a negative correlation between miR-944 and RGS17 expression was identified in NPC tissues (Fig. 4F). At the same time, SNHG6 was found to be positively correlated with RGS17 expression in NPC tissues (Fig. 4G). The results reveal that RGS17 may be involved in the regulation of SNHG6/miR-944 axis.
SNHG6/miR-944 is involved in NPC tumorigenesis by regulating RGS17.
To investigate the role of RGS17 in NPC as well as its interaction with SNHG6/miR-944, SNHG6 vector or miR-944 inhibitor was transfected into C666-1 cells containing si-RGS17. We found that the decreased expression of RGS17 induced by si-RGS17 was restored by SNHG6 vector or miR-944 inhibitor (Fig. 5A). Additionally, CCK-8 assay suggested that knockdown of RGS17 inhibited C666-1 cell proliferation. This inhibition of cell proliferation was recovered by SNHG6 upregulation or miR-944 downregulation (Fig. 5B). Meanwhile, cell migration and invasion were also suppressed by RGS17 downregulation. SNHG6 vector or miR-944 inhibitor also abolished the inhibitory effect of si-RGS17 on C666-1 cell migration and invasion (Fig. 5C, 5D). Collectively, SNHG6/miR-944 axis regulates NPC progression by regulating RGS17 expression.