The online version of this article (doi:10.1186/s13045-017-0422-2) contains supplementary material, which is available to authorized users.
Circular RNAs are a subgroup of non-coding RNAs and generated by a mammalian genome. Herein, the expression and function of circular RNA circ-TTBK2 were investigated in human glioma cells.
Fluorescence in situ hybridization and quantitative real-time PCR were conducted to profile the cell distribution and expression of circ-TTBK2 and microRNA-217 (miR-217) in glioma tissues and cells. Immunohistochemical and western blot were used to determine the expression of HNF1β and Derlin-1 in glioma tissues and cells. Stable knockdown of circ-TTBK2 or overexpression of miR-217 glioma cell lines (U87 and U251) were established to explore the function of circ-TTBK2 and miR-217 in glioma cells. Further, luciferase reports and RNA immunoprecipitation were used to investigate the correlation between circ-TTBK2 and miR-217. Cell Counting Kit-8, transwell assays, and flow cytometry were used to investigate circ-TTBK2 and miR-217 function including cell proliferation, migration and invasion, and apoptosis, respectively. ChIP assays were used to ascertain the correlations between HNF1β and Derlin-1.
We found that circ-TTBK2 was upregulated in glioma tissues and cell lines, while linear TTBK2 was not dysregulated in glioma tissues and cells. Enhanced expression of circ-TTBK2 promoted cell proliferation, migration, and invasion, while inhibited apoptosis. MiR-217 was downregulated in glioma tissues and cell lines. We also found that circ-TTBK2, but not linear TTBK2, acted as miR-217 sponge in a sequence-specific manner. In addition, upregulated circ-TTBK2 decreased miR-217 expression and there was a reciprocal negative feedback between them in an Argonaute2-dependent manner. Moreover, reintroduction of miR-217 significantly reversed circ-TTBK2-mediated promotion of glioma progression. HNF1β was a direct target of miR-217, and played oncogenic role in glioma cells. Remarkably, circ-TTBK2 knockdown combined with miR-217 overexpression led to tumor regression in vivo.
These results demonstrated a novel role circ-TTBK2 in the glioma progression.
Additional file 1: Figure S1. Circ-TTBK2 was resistant to RNase R treatment, and the transfection efficiency of each target. a and b Expression level of TTBK2 mRNA in glioma tissues and cells (data are presented as the mean + SD (n = 5, each group)). c Expression level of circ-TTBK2 in glioma cells with RNase R treatment (data are presented as the mean + SD (n = 5, each group), ** P < 0.01 vs. control in normal human astrocytes group; ## P < 0.01 vs. RNase R in Normal human astrocytes group). d Expression level of TTBK2 in glioma cells treated with RNase R (data are presented as the mean + SD (n = 5, each group), ** P < 0.01 vs. control group respectively). e qRT-PCR was used to detect the transfection efficiency of circ-TTBK2 (data are presented as the mean + SD (n = 5, each group). ** P < 0.01 vs. circ-TTBK2 (+)-NC group; ## P < 0.01 vs. circ-TTBK2 (−)-NC group). f qRT-PCR was used to detect the transfection efficiency of sh-TTBK2 (data are presented as the mean + SD (n = 5, each group). ** P < 0.01 vs. sh-NC group). g qRT-PCR was conducted to investigate the transfection efficiency of miR-217 (data are presented as the mean + SD (n = 5, each group). ** P < 0.01 vs. pre-NC group; ## P < 0.01 vs. anti-NC group). h Western blot was used to investigate the transfection efficiency of HNF1β (data are presented as the mean + SD (n = 5, each group). ** P < 0.01 vs. HNF1β (+)-NC group; ## P < 0.01 vs. HNF1β (−)-NC group). i Western blot was used to investigate the transfection efficiency of Derlin-1 (data are presented as the mean + SD (n = 5, each group). ** P < 0.01 vs. Derlin-1 (+)-NC group; ## P < 0.01 vs. Derlin-1 (−)-NC group). (TIF 781 kb)13045_2017_422_MOESM1_ESM.tif
Additional file 2: Figure S2. Correlation between miR-217 and TTBK2. a qRT-PCR was conducted to detect expression levels of TTBK2 in glioma cell treated with circ-TTBK2 (+) and circ-TTBK2 (−) (n = 5, each group). b Expression level of circ-TTBK2 in cells treated with sh-TTBK2 (n = 5, each group). c Expression level of TTBK2 in cells treated with pre-miR-217 and anti-miR-217 (n = 5, each group). d The predicted two miR-217 binding sites in TTBK2 (TTBK2-Wt and TTBK2-Wt1) and the designed mutant sequences (TTBK2-Mut and TTBK2-Mut1) were indicated. e and f Luciferase reporter assays of HEK 293T cells co-transfected with TTBK2-Wt (or TTBK2-Wt1) or TTBK2-Mut (or TTBK2-Mut1), and miR-217 or the miR-217-NC (n = 5, each group). (TIF 408 kb)13045_2017_422_MOESM2_ESM.tif
Additional file 3: Additional materials and methods. (DOCX 32 kb)13045_2017_422_MOESM3_ESM.docx
Additional file 4: Primers used for chromatin immunoprecipitation assay. (XLSX 9 kb)13045_2017_422_MOESM4_ESM.xlsx
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- TTBK2 circular RNA promotes glioma malignancy by regulating miR-217/HNF1β/Derlin-1 pathway
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