lncRNA TTN‑AS1 upregulates RUNX1 to enhance glioma progression via sponging miR‑27b‑3p

Abstract

Long non‑coding RNAs (lncRNAs) contribute to the tumorigeneses of numerous types of cancer, including glioma. The present study was designed to unveil a novel lncRNA functioning in glioma and explore the underlying mechanisms. lncRNA titin‑antisense RNA1 (TTN‑AS1), miR‑27b‑3p and Runt‑related transcription factor 1 (RUNX1) expression in glioma tissues and cell lines was estimated by RT‑qPCR. Si‑TTN‑AS1 was transfected into glioma cell lines (U251 and LN229), and CCK‑8 assay, flow cytometry, wound healing and Transwell assays were applied to estimate the function of TTN‑AS1 in glioma cells. miR‑27b‑3p inhibitor was used to explore the mechanisms. The results revealed that TTN‑AS1 was highly expressed in glioma specimens and cell lines. Downregulation of TTN‑AS1 inhibited the proliferation, migration and invasion of the glioma cells, as well as increased the rate of apoptosis. In vivo, the tumor growth was also inhibited by TTN‑AS1 depletion in nude mice. Furthermore, we revealed that TTN‑AS1 exerted oncogenic effects via sponging miR‑27b‑3p and thereby positively regulating RUNX1 expression. In conclusion, the present study supported that TTN‑AS1 acts as an oncogene in glioma by targeting miR‑27b‑3p to release RUNX1. This finding may contribute to gene therapy of glioma.

Keywords: glioma; TTN-AS1; miR-27b-3p; RUNX1.

Figure 1.

lncRNA TTN-AS1 is highly expressed in glioma specimens and cells. (A) TTN-AS1 expression in glioma specimens in comparison with normal controls as detected by qPCR. **P<0.01, as compared with normal tissues. (B) TTN-AS1 expression levels in different tumor stages of glioma patient tissues were assessed by qPCR. (C) TTN-AS1 expression levels in different tumor stages and corresponding nearby normal tissues were measured by qPCR. **P<0.01, as compared with normal tissue. (D) TTN-AS1 expression in glioma cell lines (U87, A172, LN229 and U251) and normal astrocytes (NHA) were estimated by qPCR. **P<0.01, compared with normal astrocytes (NHA). (E) Survival curves of glioma patients with high or low TTN-AS1 levels as analyzed by Kaplan-Meier method. TTN-AS1, long non-coding (lnc)RNA titin-antisense RNA1.

Figure 2.

TTN-AS1 accelerates glioma cell viability, migration and invasion, as well as inhibits apoptosis in vitro. (A) Glioma U251 and LN229 cells were transfected with TTN-AS1 knockdown oligonucleotides (si-TTN-AS1-1 and si-TTN-AS1-2) or control (sh-ctrl) and the relative TTN-AS1 level was assessed. (B) CCK-8 assays displayed the viability of glioma cells following silencing. (C) Flow cytometry assays demonstrated the apoptotic state of the glioma cells following silencing of TTN-AS1. (D) Wound healing assays presented the migration ability of the glioma cells following silencing of TTN-AS1. (E) Transwell assays presented the invasion ability of the glioma cells following silencing of TTN-AS1. **P<0.01, compared with the si-ctrl group. TTN-AS1, long non-coding (lnc)RNA titin-antisense RNA1.

Figure 3.

TTN-AS1 acts as ceRNA for miR-27b.3p. (A) Schematic diagram of the interacting sites. (B) Wild type (wt)/mutant (mut) of TTN-AS1-AS1 was co-transfected with miR-27b-3p, then luciferase reporter assay was performed. **P<0.01, compared with the ctrl mimic group. (C) miR-27b-3p expression was assessed following si-TTN-AS1 transfection in the U251 and LN229 cells. **P<0.01, compared with the si-ctrl group. (D) TTN-AS1 expression was assessed following miR-27b-3p overexpression. **P<0.01, compared with the ctrl mimic group. (E) RIP experiment followed by qPCR to determine the immunoprecipitation of TTN-AS1 and miR-27b-3p. **P<0.01, compared with IgG. (F) Expression profile of miR-27b-3p in glioma specimens and controls analyzed by qPCR. (G) Correlation between TTN-AS1 and miR-27b-3p estimated by Pearson's analysis. TTN-AS1, long non-coding (lnc)RNA titin-antisense RNA1; ceRNA, competitive endogenous RNA.

Figure 4.

miR-27b-3p reverses the TTN-AS1 inhibited effects on tumor progression of glioma cells. (A) Glioma cells were transfected with miR-27b-3p knockdown oligonucleotides (miR-27b-3p inhibitor) or control (ctrl inhibitor). Then, miR-27b-3p inhibitor or control was co-transfected with sh-TTN-AS1. **P<0.01, compared with the ctrl inhibitor. (B) CCK-8 assays showing the viability of glioma cells. **P<0.01 vs. si-ctrl+ctrl inhibitor, ^##P<0.01 vs. si-TTN-AS1+ ctrl inhibitor. (C) Flow cytometry assays demonstrated the apoptosis state of glioma cells. (D) Wound healing assays presented the migration ability of glioma cells. (E) Transwell assays presented the invasion ability of the glioma cells. (F) Data are presented in bar graphs. **P<0.01 vs. si-ctrl+ctrl inhibitor, ^##P<0.01 vs. si-TTN-AS1+ ctrl inhibitor. TTN-AS1, long non-coding (lnc)RNA titin-antisense RNA1.

Figure 5.

(A) Schematic diagram of the interacting sites. (B) Wild-type (wt)/mutant (mut) of RUNX1 was co-transfected with miR-27b-3p, and then luciferase reporter assay was performed. **P<0.01, compared with ctrl mimics. (C and D) RUNX1 expression was assessed following miR-27b-3p overexpression in U251 and LN229 cells by qPCR (C) and western blot analysis (D). **P<0.01, compared with ctrl mimics. (E) Expression profiles of RUNX1 in glioma specimens and controls as analyzed by qPCR. **P<0.01, compared with normal tissue. (F) Correlation between RUNX1 and miR-27b-3p estimated by Pearson's analysis. RUNX1, runt-related transcription factor 1.

Figure 6.

TTN-AS1 absorbs miR-27b-3p to upregulated RUNX1. miR-27b-3p inhibitor or control was co-transfected with si-TTN-AS1 in the U251 and LN229 cell lines and (A) qPCR was applied to assess the mRNA level of RUNX1, and (B and C) western blot analysis was applied to assess the protein level of RUNX1. **P<0.01 vs. si-ctrl+ctrl inhibitor, ^##P<0.01 vs. si-TTN-AS1+ ctrl inhibitor. (D) Correlation between RUNX1 and TTN-AS1 estimated by Pearson's analysis. TTN-AS1, long non-coding (lnc)RNA titin-antisense RNA1; RUNX1, runt-related transcription factor 1.

Figure 7.

TTN-AS1 accelerates glioma growth in vivo. Nude mice were injected with U251 cells stably transfected with sh-TTN-AS1 or sh-ctrl. (A) Every week, tumor volume was recorded. **P<0.01, compared with the sh-ctrl group. (B) Five weeks later, mice were sacrificed and tumor weights were detected. **P<0.01, compared with the sh-ctrl group. (C) miR-27b-3p expression in tumor tissues was analyzed. **P<0.01, compared with the sh-ctrl group. (D and E) RUNX1 expression in tumor tissues was detected by western blot analysis (D) and immunohistochemistry (IHC) (E). **P<0.01, compared with the sh-ctrl group. TTN-AS1, long non-coding (lnc)RNA titin-antisense RNA1; RUNX1, runt-related transcription factor 1.