LncRNA SNHG16 is elevated in PC tissues and cell lines
In this study, the expression level of SNHG16 in human PC tissues was firstly evaluated by qRT-PCR. Compared with the pair-matched adjacent normal samples, SNHG16 expression was significantly increased in PC tissues (Fig. 1A). Moreover, the expression level of SNHG16 was up-regulated in four PC cell lines BxPC3, Panc-1, MIA Paca-2 and SW1990, compared with HPY-Y5 (Fig. 1B). In addition, Kaplan-Meier analysis indicated that PC patients with increased SNHG16 shown a shorter over-all survival than those with decreased SNHG16 (Fig. 1C). The above results indicate that elevated SNHG16 might be a critical role in the progression of PC.
SNHG16 affects PC cells proliferation and apoptosis
In order to explore the roles of SNHG16 in PC process, we established BxPC3 and Panc-1 cells that stably silenced SNHG16, which were named sh-SNHG16 (Fig. 2A). Next, CCK8 and EdU assays were employed to determinate the proliferation of PC cells, and it was revealed that knockdown of SNHG16 inhibited BxPC3 and Panc-1 cells proliferation (Fig. 2B and 2C). Consistently, the protein levels of cell proliferation markers, PCNA and Ki-67, were decreased as SNHG16-silencing (Fig. 2D). Then, flow cytometry cell apoptosis analysis demonstrated that SNHG16 knockdown increased the proportion of apoptotic PC cells (Fig. 2E), and western blot analysis revealed that SNHG16 knockdown promoted the expression of apoptosis-related proteins Bax, cleaved-caspase-3 and cleaved-caspase-9, while inhibited the expression of Bcl-2 (Fig. 2F). Above these, SNHG16 knockdown suppressed PC cells proliferation and promoted apoptosis.
Knockdown of SNHG16 suppresses PC cells migration and invasion
Subsequently, we examined the roles of SNHG16 in migration and invasion. Wound healing assay indicated that the motility of PC cells that stably silenced SNHG16 was significantly decreased (Fig. 3A). Transwell assay was implemented to evaluate the abilities of PC cells migration and invasion, and it is observed the reduced migration and invasion abilities of PC cells that stably silenced SNHG16 (Fig. 3B). The results of western blot demonstrated that reduced SNHG16 inhibited the expression of ICAM-1, VCAM-1 and MMP-9 (Fig. 3C). In general, SNHG16 knockdown suppressed PC cells migration and invasion.
SNHG16 acts as a sponge to regulate miR-302b-3p expression in PC cells
It is well-known that the commonly molecular mechanism of lncRNAs is acted as molecular sponges of miRNAs. Previous researches confirmed that SNHG16 regulated miRNA expression. Therefore, we hypothesized that SNHG16 might exert functions via interacting with miRNAs in PC. The online software ENCORI was used for predicting the miRNAs which might be regulated by SNHG16, and qRT-PCR was performed to determine the expression levels of these predicted miRNAs in PC cells. It was found that only miR-302b-3p was downregulated in PC cells (Fig. 4A), and the biding site of miR-302b-3p on SNHG16 were displayed in Fig. 4B. The expression of miR-302b-3p was increased in SNHG16-knockdown PC cells (Fig. 4C). Dual luciferase reporter assays and RIP were performed to verify whether miR-302b-3p binds to SNHG16 directly. The luciferase activity in PC cells co-transfected with SNHG16-WT and miR-302b-3p mimics were weaker than these in cells co-transfected with SNHG16-WT and NC mimics, or co-transfected with SNHG16-Mut and miR-302b-3p (Fig. 4D). The results of RIP revealed that SNHG16 and miR-302b-3p were highly enriched in anti-Ago2 beads compared with IgG beads (Fig. 4E). In addition, miR-302b-3p was reduced both in PC tissues and cell lines (Fig. 4F and 4G). Taken together, above results proved that SNHG16 acts as a molecular sponge to regulate miR-302b-3p expression in PC cells.
Overexpression of miR-302b-3p inhibits PC cells proliferation, migration and invasion, and promotes apoptosis
The effects of miR-302b-3p on biological phenotypes of PC cells were further explored in PC cells transfected with miR-302b-3p mimics or negative controls. The transfection efficiency of miR-302b-3p was detected by qRT-PCR (Fig. 5A). The results of CCK8 and EdU assays indicated that overexpressed miR-302b-3p inhibited the proliferation of PC cells (Fig. 5B and 5C). Apoptosis experiment revealed that miR-302b-3p overexpression induces PC cells apoptosis (Fig. 5D). And miR-302b-3p overexpression attenuated the capacities of PC cells migration and invasion (Fig. 5E).
MiR-302b-3p targets SLC2A4 and inhibits its expression directly
Three online tools were used to screen seven targets of miR-302b-3p, including SLC2A4, RSBN1, ELK4, NFIA, CELF1, WEE1 and NTN4. Among them, SLC2A4 (Solute carrier family 2 member 4) was the most significantly elevated gene in PC cells (Fig. 6A). The putative binding site between miR-302b-3p and SLC2A4 was indicated in Fig. 6B. Dual luciferase reporter assay verified that the target binding of miR-302b-3p to SLC2A4 mRNA 3’UTR (Fig. 6C). Moreover, transfection of miR-302b-3p mimics inhibited the mRNA level of SLC2A4, as well as protein level (Fig. 6D and 6E). In addition, elevated SLC2A4 was observed in PC tissues and cell lines (Fig. 6F and 6G). These data proved that miR-302b-3p targets SLC2A4 and inhibits its expression directly.
SNHG16 promotes PC progression partly through miR-302b-3p/SLC2A4 axis
To confirm the correlation of SNHG16/miR-302b-3p/SLC2A4 axis in PC progression, PC cells that stably silenced SNHG16 were transfected with miR-302b-3p inhibitor, miR-302b-3p inhibitor plus sh-SLC2A4, or NC inhibitor, and biological phenotypes of PC cells were analyzed. Firstly, after transfected with sh-SNH616 in Bx-PC3 and Panc-1 cells, miR-302b-3p inhibitors significantly inhibited miR-302b-3p expression, and sh-SLC2A4 decreased the expression of SLC2A4 (Fig. 7A). CCK-8 and EdU assays revealed that SLC2A4 knockdown reversed the promotion of PC cells proliferation increased by miR-302b-3p inhibitor (Fig. 7B and 7C). The inhibition of apoptosis by miR-302b-3p inhibitor was also abolished in PC cells co-transfected with miR-302b-3p inhibitor and sh-SLC2A4 (Fig. 7D). Meanwhile, the increased migration and invasion abilities of PC cells with miR-302b-3p knockdown were suppressed by sh-SLC2A4 (Fig. 7E). These results confirmed that SNHG16 participated in the progression of PC via targeting miR-302b-3p/SLC2A4 axis.