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. 2017 Dec 5:10:5783-5794.
doi: 10.2147/OTT.S150678. eCollection 2017.

CircRNA_100782 regulates pancreatic carcinoma proliferation through the IL6-STAT3 pathway

Genwen Chen #  1 , Yanting Shi #  2 , Yu Zhang  1 , Jianyong Sun  1
Affiliations

CircRNA_100782 regulates pancreatic carcinoma proliferation through the IL6-STAT3 pathway

Genwen Chen et al. Onco Targets Ther. .

Abstract

Circular RNAs (circRNAs) are a novel class of noncoding RNAs that play an important role in cancer. However, the mechanisms by which circRNAs regulate gene expression in pancreatic ductal adenocarcinoma (PDAC) remain unclear. This study seeks to elucidate the role that circRNAs play in the proliferation of PDAC cells. On the basis of previous studies of circRNA expression profiles in PDAC, we found that the circRNA_100782 was markedly upregulated in PDAC tissue. Functional experiments revealed that circRNA_100782 down-regulation inhibited BxPC3 cell proliferation and colony formation. Loss-of-function studies showed that knockdown of circRNA_100782 inhibited cell proliferation by downregulating the microRNA-124 (miR-124) target genes interleukin-6 receptor (IL6R) and signal transducer and activator of transcription 3 (STAT3). Overexpression of miR-124 also inhibited BxPC3 cell proliferation by reducing the expression of IL6R and STAT3, which was consistent with the result of silencing circRNA_100782. In addition, luciferase assay revealed that miR-124 was a direct target of circRNA_100782. Silencing STAT3 inhibited BxPC3 cell proliferation and colony formation. Cell viability was reduced in BxPC3 cells treated with si-circRNA_100782 and miR-124 mimic, and this effect could be attenuated by activating STAT3. In vivo study validated that circRNA_100782 knockdown suppressed BxPC3 xenografts in nude mice. Taken together, these results suggest that circRNA_100782 regulates BxPC3 cell proliferation by acting as miR-124 sponge through the IL6-STAT3 pathway.

Keywords: IL6-STAT3 pathway; circular RNA; microRNA; pancreatic ductal adenocarcinoma; proliferation; sponge.

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Conflict of interest statement

Disclosure The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Identification and expression of circRNA_100782 in BxPC3 and HPDE cells. Notes: (A) The divergent primer used for detecting circRNA_100782. (B) The expression of circRNA_100782 in BxPC3 and HPDE cells. (C) Agarose gel electrophoresis verified the qRT-PCR product of circRNA_100782. (D) Sanger sequence showing the back-splice junction of circRNA_100782. Error bars represent mean ± SD from at least 3 independent experiments. *p<0.05. Abbreviations: F, forward primer; HPDE, human pancreatic duct epithelial cells; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; R, reverse primer.
Figure 2
Figure 2
Function of circRNA_100782 in BxPC3 cells. Notes: (A) Knockdown efficiency of si-circRNA_100782. (B) Effect of circRNA_100782 on cell proliferation by CCK-8 assay. (C) Effect of circRNA_100782 on BxPC3 cell colony formation. Error bars represent mean ± SD from at least 3 independent experiments. *p<0.05. Abbreviations: CCK-8, Cell Counting Kit-8; Ctrl, control.
Figure 3
Figure 3
CircRNA_100782 functions by binding to miR-124. Notes: (A) The schematic graph illustrates the putative complementary site within circRNA_100782 and miR-124. (B) Relative expression of miR-124 in BxPC3 and HPDE cells. (C) Expression of miR-124 after transfection with miR-124 mimic for 48 h in HEK293T cells. (D) Relative luciferase activity cotransfected pmirGLO-circRNA_100782 or pmirGLO-vector with miR-124 mimic in HEK293T cells. (E) mRNAs expression of STAT3 and IL6R 48 h after transfection with si-Ctrl and si-circRNA_100782. (F) Expression of IL6R, JAK2, p-JAK2, STAT3 and p-STAT3 48 h after transfection with si-Ctrl and si-circRNA_100782 by Western blot analysis. (G) MiR-124 inhibitor rescued si-circRNA_100782 induced reduction of p-JAK2 and p-STAT3 by Western blot analysis. Error bars represent mean ± SD from at least 3 independent experiments. *p<0.05. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPDE, human pancreatic duct epithelial cells; IL6R, interleukin 6 receptor; JAK2, janus kinase 2; miR-124, microRNA-124; p-JAK2, phosphorylation of JAK2; STAT3, signal transducer and activator of transcription 3.
Figure 4
Figure 4
IL6-JAK2-STAT3 pathway is responsible for BxPC3 cell proliferation. Notes: (A) Relative expression level of STAT3 in BxPC3 and HPDE cells. (B) Western blot analysis of p-JAK2 and p-STAT3 by IL6 treatment for 20 min. (C) Effect of activated STAT3 by IL6 on BxPC3 cell proliferation by CCK-8 assay. (D) Knockdown efficiency of si-STAT3 by Western blot compared with si-Ctrl. (E) Effect of si-STAT3 on BxPC3 cell proliferation by CCK-8 assay and (F) colony formation. (G) IL6 rescued si-circRNA_100782 induced reduction of p-JAK2 and p-STAT3 and (H) BxPC3 cell proliferation by CCK-8 assay. Error bars represent mean ± SD from at least 3 independent experiments. *p<0.05. #p<0.05 vs si-circRNA_100782. Abbreviations: CCK-8, Cell Counting Kit-8; Ctrl, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPDE, human pancreatic duct epithelial cells; IL6R, interleukin 6 receptor; JAK2, janus kinase 2; p-JAK2, phosphorylation of JAK2; STAT3, signal transducer and activator of transcription 3.
Figure 4
Figure 4
IL6-JAK2-STAT3 pathway is responsible for BxPC3 cell proliferation. Notes: (A) Relative expression level of STAT3 in BxPC3 and HPDE cells. (B) Western blot analysis of p-JAK2 and p-STAT3 by IL6 treatment for 20 min. (C) Effect of activated STAT3 by IL6 on BxPC3 cell proliferation by CCK-8 assay. (D) Knockdown efficiency of si-STAT3 by Western blot compared with si-Ctrl. (E) Effect of si-STAT3 on BxPC3 cell proliferation by CCK-8 assay and (F) colony formation. (G) IL6 rescued si-circRNA_100782 induced reduction of p-JAK2 and p-STAT3 and (H) BxPC3 cell proliferation by CCK-8 assay. Error bars represent mean ± SD from at least 3 independent experiments. *p<0.05. #p<0.05 vs si-circRNA_100782. Abbreviations: CCK-8, Cell Counting Kit-8; Ctrl, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPDE, human pancreatic duct epithelial cells; IL6R, interleukin 6 receptor; JAK2, janus kinase 2; p-JAK2, phosphorylation of JAK2; STAT3, signal transducer and activator of transcription 3.
Figure 5
Figure 5
miR-124 mediates BxPC3 cell proliferation by IL6-JAK2-STAT3 pathway. Notes: (A) Effect of miR-124 on BxPC3 cell proliferation by CCK-8 assay and (B) colony formation. (C) Expression of IL6R and STAT3 mRNA levels 48 h after transfection with miR-124 mimic. (D) Western blot analysis of IL6R, JAK2, p-JAK2, STAT3 and p-STAT3 caused by miR-124. (E) IL6 rescued miR-124-induced reduction of p-JAK2 and p-STAT3 by Western blot analysis and (F) BxPC3 cell proliferation by CCK-8 assay. Error bars represent mean ± SD from at least 3 independent experiments. *p<0.05. #p<0.05 vs miR-124 mimic. Abbreviations: CCK-8, Cell Counting Kit-8; Ctrl, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL6R, interleukin 6 receptor; JAK2, janus kinase 2; miR-124, microRNA-124; p-JAK2, phosphorylation of JAK2; STAT3, signal transducer and activator of transcription 3.
Figure 5
Figure 5
miR-124 mediates BxPC3 cell proliferation by IL6-JAK2-STAT3 pathway. Notes: (A) Effect of miR-124 on BxPC3 cell proliferation by CCK-8 assay and (B) colony formation. (C) Expression of IL6R and STAT3 mRNA levels 48 h after transfection with miR-124 mimic. (D) Western blot analysis of IL6R, JAK2, p-JAK2, STAT3 and p-STAT3 caused by miR-124. (E) IL6 rescued miR-124-induced reduction of p-JAK2 and p-STAT3 by Western blot analysis and (F) BxPC3 cell proliferation by CCK-8 assay. Error bars represent mean ± SD from at least 3 independent experiments. *p<0.05. #p<0.05 vs miR-124 mimic. Abbreviations: CCK-8, Cell Counting Kit-8; Ctrl, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL6R, interleukin 6 receptor; JAK2, janus kinase 2; miR-124, microRNA-124; p-JAK2, phosphorylation of JAK2; STAT3, signal transducer and activator of transcription 3.
Figure 6
Figure 6
CircRNA_100782 regulates tumor growth in nude mice. Notes: (A) CircRNA_100782 depletion markedly repressed tumor growth in nude mice. (B) Sh-circRNA_100782 significantly reduced tumor volume compared with sh-Ctrl. (C) Sh-circRNA_100782 markedly reduced tumor weight. (D) Western blot analysis of IL6R, STAT3 and p-STAT3 in xenograft tumors. Error bars represent mean ± SD. *p<0.05. Abbreviations: Ctrl, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL6R, interleukin 6 receptor; STAT3, signal transducer and activator of transcription 3.
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References

    1. Memczak S, Jens M, Elefsinioti A, et al. Circular RNAs are a large class of animal RNAs with regulatory potency. Nature. 2013;495(7441):333–338. - PubMed
    1. Hsu MT, Coca-Prados M. Electron-microscopic evidence for the circular form of rna in the cytoplasm of eukaryotic cells. Nature. 1979;280(5720):339–340. - PubMed
    1. Rybak-Wolf A, Stottmeister C, Glazar P, et al. Circular RNAs in the mammalian brain are highly abundant, conserved, and dynamically expressed. Molecular Cell. 2015;58(5):870–885. - PubMed
    1. Wang K, Long B, Liu F, et al. A circular RNA protects the heart from pathological hypertrophy and heart failure by targeting miR-223. Eur Heart J. 2016;37(33):2602–2611. - PubMed
    1. Hansen TB, Kjems J, Damgaard CK. Circular RNA and miR-7 in cancer. Cancer Res. 2013;73(18):5609–5612. - PubMed