3.1. Knocking down EGOT suppresses the in vitro proliferative, migratory, and invasive activity of PC cells
We began by conducting a series of qPCR assays exploring the expression of the lncRNA EGOT in control human pancreatic HPDE cells and in the PANC-1, HPAF-II, AsPC-1, CFPAC-1, and SW1990 PC cell lines. This analysis revealed all five of these PC cell lines to exhibit significant EGOT upregulation relative to HPDE cells (Fig. 1A), with this upregulation being most pronounced in PANC-1 and SW1990 cells, which were thus used for subsequent experiments. To establish the impact of EGOT knockdown on PC cells, we next generated three different EGOT-specific siRNA constructs and transfected these constructs into PANC-1 and SW1990 cells. Subsequent qPCR analyses confirmed the successful knockdown of EGOT in both cell lines (Fig. 1B). We then assessed the proliferation, migration, and invasive activity of these cells using CCK-8, EdU, wound healing and Transwell assays which demonstrated that the knockdown of EGOT significantly impaired the viability, migration, and invasion of both PANC-1 and SW1990 cells (Fig. 1C-H).
3.2. EGOT promotes PC cell proliferative and migratory activity through a miR-214-dependent mechanism in vitro
To explore the association between EGOT and miR-214, we next utilized a program to predict binding sites between these two non-coding RNAs (Fig. 2A). We then conducted luciferase reporter assays to assess interactions between EGOT and miR-214 within cells, revealing that miR-214 mimics were able to suppress luciferase activity driven by wild-type but not mutant EGOT (Fig. 2B), supporting a direct interaction between these RNAs. Relative to control cells, cells in which EGOT had been knocked down exhibited significant increases in miR-214 expression, while EGOT expression levels in cells transfected with miR-214 mimic constructs were conversely reduced (Fig. 2C). A fluorescence in situ hybridization approach was further used to validate the association between EGOT and miR-214 using fluorescence in situ hybridization (Fig. 2D). To evaluate the functional role of miR-214 in our model system, we transfected PC cell lines with a miR-214 inhibitor or corresponding miR-NC control constructs, confirming via qPCR that the former led to significant decreases in miR-214 levels within transfected cells (Fig. 2E). We then transiently co-transfected PC cells with combinations of si-EGOT, miR-214, and/or control constructs and evaluated these cells in CCK-8, EdU, wound healing and Transwell assays. This analysis confirmed that the proliferation, viability, invasion, and migration of PC cells co-transfected with si-EGOT and a miR-214 inhibitor were enhanced as compared to PC cells transfected with si-EGOT alone (Fig. 2F-K). Together, these data suggested that interactions between EGOT and mIR-214 ultimately govern PC cell proliferation, invasion, and migration in vitro.
3.3. PLS3 is a direct miR-214 target in PC cells
We next used predictive software to identify putative miR-214 sites in PLS3 (Fig. 3A). Luciferase reporter assays confirmed that miR-214 mimics were able to suppress wild-type but not mutant PLS3 luciferase reporter activity, confirming the specificity of this interaction (Fig. 3B-C). PC cells were then transfected with miR-214 mimic constructs and PL3 expression was assessed via qPCR and Western blotting, revealing that miR-214 suppressed PLS3 expression at the mRNA and protein levels in these cells relative to control treatment (Fig. 3D-E).
3.4. EGOT promotes the upregulation of PLS3 in PC cells by sponging miR-214
We next evaluated the ability of EGOT to modulate PLS3 expression within PC cells in a manner mediated by miR-214. To that end, we transiently transfected PC cells with si-EGOT, miR-214 inhibitors, and/or control constructs and then examined PLS3 expression via qPCR and Western blotting. This analysis revealed that PLS3 expression was reduced following EGOT knockdown while si-EGOT and miR-214 inhibitor co-transfection was sufficient to inhibit this si-EGOT-mediated reduction in PLS3 expression at the mRNA and protein levels (Fig. 3F-G). These findings indicated that EGOT and miR-214 interact with one another to ultimately shape PLS3 expression within PC cells.
3.5. Knockdown of EGOT suppresses in vivo PC tumor growth
Given the above data emphasizing the oncogenic activity of EGOT in PC cells in vitro, we next sought to extend these results in vivo by implanting mice with either control tumor cells or with tumor cells transfected with si-EGOT. Relative to the control group, si-EGOT tumors grew more slowly (Fig. 4A-D). When we assessed gene expression in tumors from these animals at the end of the study, we confirmed that EGOT knockdown was associated with miR-214 upregulation and PLS3 downregulation, consistent with in vitro results (Fig. 4E-F). These results thus confirmed that the knockdown of EGOT was sufficient to suppress PC tumor growth in vivo.