top of page

Contact Atlanta Group

Public·50 members
Emil Sitnikov
Emil Sitnikov

GREEN ROADS 1294



Visit your Burnsville ALDI for low prices on groceries and home goods. From fresh produce and meats to organic foods, beverages and other award-winning items, ALDI makes the flavorful affordable. Plus, with new limited-time ALDI Finds added to shelves each week, there's always something new to discover. Shop online with curbside pickup or delivery at participating locations, or swing by your neighborhood market at 1294 County Road 42 W. to start saving on quality products.




GREEN ROADS 1294



The Tollway is committed to building green and minimizing the environmental impact of construction by reducing, recycling and reusing materials. In addition to reducing the cost of this work, reuse of these materials reduces the need for virgin asphalt materials and reduces energy consumption, greenhouse gases and the volume of material that would otherwise be sent to landfills.


Guinea pigs are very susceptible to vitamin C deficiency, sometimes called "scurvy." They must have a special guinea pig diet (they cannot eat rabbit food) which must be supplemented with fresh leafy green vegetables, such as kale.


Graphical circular map of the chromosome for (A) MY1483/09 and (B) Phil1136/12. From the outside to the centre: scaffolds (green), genomic islands (red), putative genes in forward (blue), putative genes in reverse (purple), tRNA (dark blue), rRNA (dark red), GC plot (green) and GC skew (orange and black).


Long respected as one of the finest facilities on the Peninsula, this 5,400 yard, par-69 course is convenient to both Williamsburg and Southside communities. Add 52 sand bunkers and you have a challenge for duffers and professionals. The clubhouse offers a pro shop, snack bar restaurant, meeting room and access to a spacious practice putting green.


CircEYA3 was explored based on Gene Expression Omnibus (GEO) dataset analysis. qRT-PCR was applied to determine the expression of circRNAs, miRNAs and mRNAs in PDAC cells and tissues. The biological roles of circEYA3 in vitro and in vivo were determined by performing a series of functional experiments. Further, dual luciferase reporter, fluorescence in situ hybridization (FISH), RNA pull-down assays, and RNA immunoprecipitation (RIP) assays were used to confirm the interaction of circEYA3 with miR-1294.


CircEYA3 was elevated in PDAC tissues and cells, and a higher level of circEYA3 was significantly associated with a poorer prognosis in patients with PDAC. Functionally, circEYA3 increased energy production via ATP synthesis to promote PDAC progression in vitro and in vivo. Mechanistically, circEYA3 functions as an endogenous miR-1294 sponge to elevate c-Myc expression, thus exerting its oncogenic functions.


In our present study, we focused on the upregulated circRNAs in PDAC based on GEO dataset analysis for the first time and characterized a novel oncogenic circRNA, circEYA3 (hsa_circ_0007895). CircEYA3 was derived from the EYA3 gene, exhibited an apparently elevated level in PDAC and was closely correlated with the prognosis of PDAC patients. Mechanistically, circEYA3 functioned as a sponge for has-miR-1294 (miR-1294) to elevate the level of c-Myc. c-Myc is highly important in PDAC [16], and circEYA3 was found to exert its oncogenic functions by elevating ATP production in PDAC cells. Collectively, our findings indicated that circEYA3 may be a novel prognostic biomarker and promising therapeutic target for PDAC.


Existing studies have demonstrated that many circRNAs exert their biological effects by sponging miRNAs and subsequently regulating miRNA expression in PDAC [13, 14, 25]. Given that circEYA3 exhibited high stability and was localized mainly in the cytoplasm, as described above, we hypothesized that circEYA3 facilitated malignant biological behaviours of PDAC cells by sponging miRNAs. Therefore, to validate this hypothesis, RIP assays using an anti-Ago2 antibody and IgG were performed in PANC-1 cells and MiaPaCa-2 cells. We found that endogenous circEYA3 was pulled down by anti-Ago2, indicating that circEYA3 bound to miRNAs via the Ago2 protein (Fig. 3A). Subsequently, three databases (starBase, miRanda, and circBank) were used for bioinformatic analysis to predict the potential target miRNAs of circEYA3. Then, 16 candidate miRNAs were identified from the intersection of the miRNAs identified in these three databases, as shown in Fig. 3B. RNA pull-down experiments were subsequently performed using a biotin-labelled probe specific for circEYA3 and a random oligo negative control probe in PANC-1 and MiaPaCa-2 cells transfected with the pLCDH-circEYA3 overexpression plasmid and the corresponding pLCDH-circ control plasmid. A greater amount of circEYA3 was pulled down in both cell lines after transfection with the pLCDH-circEYA3 overexpression plasmid (Fig. 3C and D). The expression levels of the 16 candidate miRNAs in the immunoprecipitates were determined using a biotin-labelled probe specific for circEYA3. The qRT-PCR results indicated that miR-1294 was the only miRNA that was statistically abundant in both PANC-1 and MiaPaCa-2 cells, suggesting that miR-1294 specifically interacted with circEYA3 (Fig. 3E). Next, luciferase reporter plasmids containing WT circEYA3 and circEYA3 with a mutated miR-1294 binding site were constructed (Fig. 3F). The luciferase activity of WT circEYA3 was markedly suppressed in PANC-1 cells transfected with the miR-1294 mimic but elevated in MiaPaCa-2 cells transfected with the miR-1294 inhibitor. Conversely, the luciferase activity of the circEYA3 mutant was not significantly changed among the miR-1294 mimic, miR-1294 inhibitor and corresponding negative control groups, indicating the possibility of a direct interaction between miR-1294 and circEYA3 (Fig. 3G). To further validate this interaction, a RIP assay using an anti-AGO2 antibody and negative control IgG was performed in PANC-1 cells transiently overexpressing miR-1294 to pull down circEYA3 and miR-1294, and qRT-PCR was then performed to analyse the levels of circEYA3 and miR-1294 levels in the precipitates. Both circEYA3 and miR-1294 were pulled down in significantly higher quantities by the anti-Ago2 antibody compared with IgG (Fig. 3H). Furthermore, circEYA3 and miR-1294 were dramatically more abundant in cells transfected with the miR-1294 mimic compared with cells transfected with the mimic negative control (mimic NC) (Fig. 3H). Next, the expression level of miR-1294 was determined by qRT-PCR in 104 pairs of PDAC tumor and paracarcinoma tissues, and miR-1294 expression was found to be obviously downregulated in PDAC tissues (Fig. 4A). Pearson correlation analysis indicated that miR-1294 expression was negatively associated with circEYA3 expression, as determined by qRT-PCR, in these 104 pairs of fresh frozen PDAC tissues (Fig. 3I). We also found that downregulation of circEYA3 increased miR-1294 expression, while upregulation of circEYA3 decreased miR-1294 expression (Fig. S3A). Furthermore, the FISH results confirmed the co-localization of circEYA3 and miR-1294 mainly in the cytoplasm of PANC-1 cells (Fig. 3L). Taken together, these findings indicated that circEYA3 can directly bind with miR-1294 in PDAC cells.


Reports have confirmed that miR-1294 acts as a tumor suppressor in different cancers [27,28,29]. A study by Yi et al. also indicated that miR-1294 expression was lower in PDAC tissue samples than in the paired adjacent normal tissues, as determined by qRT-PCR [30], consistent with our analysis of a 104-case cohort of fresh frozen PDAC tissues (Fig. 4A). However, no obvious relationships were observed between miR-1294 expression and PDAC clinicopathological features based on our qRT-PCR results (Additional file 1: Table S5). To determine the specific biological roles of miR-1294 in PDAC, we further explored its functions in PDAC cells. qRT-PCR analysis showed that miR-1294 expression was apparently lower in PDAC cells than in HPDE cells. Among the tested cell lines, PANC-1 exhibited the lowest level and MiaPaCa-2 cells exhibited the highest level of miR-1294 (Fig. 4B). Thus, we selected PANC-1 and MiaPaCa-2 cells for further research. PANC-1 and MiaPaCa-2 cells were transfected with the miR-1294 mimic or mimic NC or with the miR-1294 inhibitor or inhibitor NC, respectively. Then, the transfection efficiency was confirmed by qRT-PCR (Fig. S3B and S3C). The CCK-8 and colony formation assays showed that upregulated expression of miR-1294 inhibited PANC-1 cell proliferation, whereas downregulated expression of miR-1294 led to the opposite effects (Fig. 4C and D). Consistent with these findings, the EdU incorporation assay showed that the percentage of EdU-positive cells was significantly decreased by upregulation of miR-1294 but increased by downregulation of miR-1294 (Fig. 4E). Moreover, the apoptosis assay showed that upregulation of miR-1294 promoted the apoptosis of PANC-1 cells, whereas downregulation of miR-1294 exerted the opposite effect on MiaPaCa-2 cells (Fig. 4F). The Transwell assay showed that miR-1294 mimic transfection notably decreased the migratory and invasive abilities of PANC-1 cells, while miR-1294 inhibitor transfection resulted in the opposite trends in MiaPaCa-2 cells (Fig. 4G). These results demonstrated that miR-1294 may function as a tumor suppressor in PDAC in vitro.


Our previous study confirmed that in pancreatic cancer, c-Myc can increase energy metabolism/ATP production, which is essential for many cellular processes [22]. Therefore, we investigated whether circEYA3 performs its biological functions by affecting the cellular production of ATP. Specifically, downregulation of circEYA3 significantly decreased the production of ATP in PANC-1 cells, while upregulation of circEYA3 exerted the opposite effect in MiaPaCa-2 cells (Fig. 6L). As expected, downregulation of miR-1294 also markedly increased but upregulation of miR-1294 decreased the production of ATP (Fig. 6M). Furthermore, the miR-1294 inhibitor and mimic considerably reversed the alterations in ATP production caused by downregulation and upregulation of circEYA3, respectively (Fig. 6N). Specifically, these findings collectively demonstrated that circEYA3 may increase the production of ATP to perform its oncogenic biological functions via the miR-1294/c-Myc axis.


About

Welcome to the group! You can connect with other members, ge...

Members

bottom of page