Role of long intergenic non-protein coding RNA 00152 in pancreatic cancer glycolysis via the manipulation of the microRNA-185-5p/Krüppel-like factor 7 axis

Abstract

The current study set out to investigate the role of long intergenic non-protein coding RNA (LINC) 00152 in pancreatic cancer (PC) cell glycolysis with the microRNA (miR)-185-5p/Krüppel-like factor 7 (KLF7) axis. Firstly, PC tissues and cells as well as the control ones were collected from 53 PC patients, and assessed for LINC00152 expression patterns. Besides, PC cells with the most differentially expressed LINC00152 were selected for further experiments. When LINC00152 was silenced or overexpressed, PC cell glucose consumption, lactic acid production, adenosine triphosphate and levels of glycolysis-associated enzymes were detected. In addition, the binding relation between LINC00152 and miR-185-5p as well as the target relation between miR-185-5p and KLF7 was clarified and validated. Additionally, xenograft transplantation was performed to confirm the in vitro experiments. It was found that LINC00152 was over-expressed in PC, and it predicted a poor prognosis. Besides, LINC00152 knockdown inhibited PC cell glycolysis. Moreover, LINC00152 could specifically targeted miR-185-5p. Meanwhile, LINC00152 exhaustion blocked PC cell glycolysis through the up-regulation of miR-185-5p. Lastly, LINC00152 inhibition targeted miR-185-5p to quench KLF7, therefore suppressing PC cell tumorigenesis and glycolysis. Collectively, our findings indicated that silencing LINC00152 restricted PC cell glycolysis via promoting miR-185-5p and reducing KLF7.

Keywords: Competing endogenous RNA; Glycolysis; KLF7; Long intergenic non-protein coding RNA 00152; Pancreatic cancer; Subcellular localization; microRNA-185-5p.

Figure 1

LINC00152 is over-expressed in PC, and it predicts poor prognosis. A, LINC00152 over-expression in PC predicted by GEPIA website. B, LINC00152 expression measured by qRT-PCR, the paired t-test was employed to test statistical significance; n=53. The n specifies the number of tissue case. C, The relation between LINC00152 expression and PC patients' prognosis analyzed by Kaplan-Meier and log-rank test; n=53. The n specifies the number of tissue case. D, LINC00152 expression in human normal pancreatic ductal cells and PC cells assessed through qRT-PCR; n=3. The n specifies the replicate. The results were exhibited as mean ± standard deviation. One-way ANOVA was used to analyze data. Tukey's multiple comparisons test was applied for post hoc test. * p<0.05.

Figure 2

LINC00152 knockdown inhibits PC cell glycolysis. A, LINC00152 expression in PANC-1 cells with si-LINC00152 detected qRT-PCR. B, C and D, Glucose consumption (B), lactic acid production (C) and ATP level (D) in PANC-1 cells with si-LINC00152. E, Levels of glycolysis-related enzymes in PANC-1 cells with si-LINC00152 assessed by western blot analysis. F, LINC00152 expression in Capan-1 cells with oe-LINC00152 detected qRT-PCR. G, H and I, Glucose consumption (G), lactic acid production (H) and ATP level (I) in Capan-1 cells with oe-LINC00152. J, Levels of glycolysis-correlated enzymes in PANC-1 cells with si-LINC00152 assessed by western blot analysis. In panels A-J, n=3. The n specifies the replicate. The results were exhibited as mean ± standard deviation. One-way ANOVA was used to analyze data in panel A. Two-way ANOVA was used to analyze data in panels E and J. Tukey's multiple comparisons test was applied for post hoc test. Independent t-test was used to analyze data in B, C, D, F and I * p<0.05.

Figure 3

LINC00152 sponges miR-185-5p, whose overexpression reduces PC cell glycolysis. A, LINC00152 principal localization at cytoplasm predicted by website (http://lncatlas.crg.eu/). B, LINC00152 subcellular localization in PANC-1 and Capan-1 certified by FISH, 400×, scale bar: 25 µm. C, the binding relation between LINC00152 and miR-185-5p predicted by RNA22 website. D, The target relation of LINC00152 and miR-185-5p confirmed by dual-luciferase reporter gene assay. E, The target relation of LINC00152 and miR-185-5p detected by RIP. F, miR-185-5p expression in human normal pancreatic ductal cells and PC cells determined by qRT-PCR. G, miR-185-5p expression in PANC-1 transfected with mimic NC or miR-185-5p mimic examined through qRT-PCR. H, I and J, Glucose consumption (H), lactic acid production (I) and ATP level (J) in PC cells with mimic NC and miR-185-5p mimic. K, Levels of glycolysis-related enzymes detected by western blot analysis. In panels B/D-K, n=3. The n specifies the replicate. The results were exhibited as mean ± standard deviation. Two-way ANOVA was used to analyze the data in panels D, E, F and K. Tukey's multiple comparisons test was applied for the post hoc test. Independent t-test was used to analyze data in G, H, I and J. * p<0.05.

Figure 4

Silencing of LINC00152 suppresses PC cell glycolysis via encouraging miR-185-5p expression. A, miR-185-5p expression verified by qRT-PCR. B, C and D, Glucose consumption (B), lactic acid production (C) and ATP level (D) in PC cells. E, Levels of glycolysis-related enzymes determined by western blot analysis. In panels A-E, n=3. The n specifies the replicate. The results were exhibited as mean ± standard deviation. One-way ANOVA was used to analyze the data in panels A, B, C and D. Two-way ANOVA was used to analyze the data in panel E. Tukey's multiple comparisons test was applied for post hoc test. * p<0.05.

Figure 5

Silencing of LINC00152 downregulates KLF7 expression via the upregulation of miR-185-5p. A, miR-185-5p binding to KLF7 at 3'UTR predicted by TargetScan website. B, miR-185-5p binding to KLF7 at 3'UTR verified via dual-luciferase reporter gene assay. C, miR-185-5p binding to KLF7 detected via RIP. D, KLF7 expression in PANC-1 cells transfected with mimic NC and miR-185-5p mimic assessed by qRT-PCR. E, KLF7 expression measured by western blot analysis. F, Highly expressed KLF7 in PC predicted by GEPIA website. G, H, I and J, KLF7 expression in PC cells examined via qRT-PCR (G and I) and western blot analysis (H and J). In panels B-E and G-J, n=3. The n specifies the replicate. The results were exhibited as mean ± standard deviation. The independent t-test was used to analyze the data in panels D and E. One-way ANOVA was used to analyze the data in panels G, H, I and J. Two-way ANOVA was used to analyze the data in panels B and C. Tukey's multiple comparisons test was applied for the post hoc test. * p<0.05.

Figure 6

Silencing of LINC00152 reduces PC cell glycolysis by inhibiting KLF7 expression. A, KLF7 expression measured by western blot analysis. B, C and D, glucose consumption (B), lactic acid production (C) and ATP level (D) in PC cells. E, Levels of glycolysis-related enzymes determined by western blot analysis. In panels A-E, n=3. The n specifies the replicate. The results were exhibited as mean ± standard deviation. One-way ANOVA was used to analyze the data in panels A, B, C and D. Two-way ANOVA was used to analyze the data in panel E. Tukey's multiple comparisons test was applied for the post hoc test. * p<0.05.

Figure 7

LINC00152 knockout discourages PC cell tumorigenesis and glycolysis by quenching KLF7 expression. The lentivirus interference vector (LV-sh-LINC00152) and lentivirus overexpression vector KLF7 (LV-oe-KLF7) as well as their controls were infected with PANC-1 cells. Then the stably-expressed cells were screened for subcutaneous tumor formation in nude mice. A, Tumor growth; n=12, the n specifies the mouse number. B, Representative images of tumor. C, Tumor weight; n=12, the n specifies the mouse number. D, Positive rate of Ki67 in tumor tissue detected by Immunohistochemistry. E, Expression of LINC00152, miR-185-5p and KLF7 measured by qRT-PCR. F, Glycolysis-related enzyme levels and KLF7 assessed via western blot analysis; the image on the left is a representative image of Western blot in tumor tissue. In panels D-E, n=6, the n specifies the mouse number; the results were exhibited as mean ± standard deviation. One-way ANOVA was used to analyze the data in panel C. Two-way ANOVA was used to analyze the data in panels A, D and E. Tukey's multiple comparisons test was applied for the post hoc test. * p<0.05.

Figure 8

Mechanism diagram. LINC00152 promotes the expression of KLF7 through the competitive binding to miR-185-5p in pancreatic cancer cells, thereby promoting the glycolysis process of pancreatic cancer cells and promoting the occurrence of pancreatic tumor.