LncRNA TTN‑AS1 promotes endometrial cancer by sponging miR‑376a‑3p

Abstract

Increasing research has demonstrated that lncRNAs participate in the development of multiple cancer types. However, the role of TTN‑AS1 in endometrial cancer (EC) remains unknown. The present study aimed to explore the function of titin‑antisense RNA1 (TTN‑AS1) in EC progression and the underlying mechanisms. qRT‑PCR was performed to assess the TTN‑AS1 expression patterns in EC tissues and cell lines. Loss of function experiments were carried out to estimate the effects of TTN‑AS1 on EC cell proliferation, migration and invasion. To reveal the underlying mechanisms, informatics tools were used to predict the targets. Rescue experiments were performed to investigate the TTN‑AS1‑regulated miR‑376a‑3p/pumilio homolog 2 (PUM2) axis involved. The results of the present study revealed that TTN‑AS1 was highly expressed in both EC tissues and cell lines, and TTN‑AS1 knockdown inhibited EC cell proliferation, migration and invasion. With respect to the mechanisms, miR‑376a‑3p was revealed to be targeted by TTN‑AS1, and reversed the effects on EC development induced by TTN‑AS1. In addition, PUM2 was positively regulated by TTN‑AS1, and miR‑376a‑3p mediated the regulation between them. Furtherly, in vivo experiments confirmed the results. Collectively, TTN‑AS1 enhanced EC cell proliferation and metastasis by targeting the miR‑376a‑3p/PUM2 axis, which may shed light on EC diagnosis and treatment.

Keywords: lncRNA TTN-AS1; endometrial cancer; miR-376a-3p; PUM2; cell proliferation.

Figure 1.

Upregulation of TTN-AS1 in EC tissues and cell lines. (A) TTN-AS1 expression levels in EC tissues (n=45) and corresponding normal tissues (n=45) were determined by qRT-PCR. (B) The expression levels of TTN-AS1 in EC cell lines (HEC1B, KLE, HEC1A and Ishikawa) and ESC were determined by qRT-PCR. (C) Association between TTN-AS1 expression and overall survival of EC patients was analyzed by Kaplan-Meier curve test. **P<0.01. TTN-AS1, titin-antisense RNA1; EC, endometrial cancer; ESC, endometrial stromal cells.

Figure 2.

Depletion of TTN-AS1 restrains EC cell proliferation, migration and invasion. (A) TTN-AS1 expression was detected by qRT-PCR with transfection of si-RNAs (si-TTN-AS1#1 and si-TTN-AS1#2). After transfection with si-TTN-AS1 or negative control (si-NC), (B) a CCK-8 assay was performed to assess the cell viability, (C) flow cytometry was used to evaluate the apoptotic rate and (D) western blotting was performed to detect the protein expression levels of cleaved caspase-3 and cleaved PARP. (E and F) Transwell assays were performed to assess the cell migration and invasion abilities. **P<0.01. TTN-AS1, titin-antisense RNA1; EC, endometrial cancer.

Figure 3.

TTN-AS1 sponges miR-376a-3p. (A) Starbase 2.0 revealed the interacting sequences of TTN-AS1 and miR-376a-3p. (B) A luciferase reporter assay was carried out to validate the binding sites. (C) miR-376a-3p expression was assessed by qRT-PCR with si-TTN-AS1 transfection. (D) TTN-AS1 expression was assessed by qRT-PCR with miR-376a-3p overexpression. (E) Anti-Ago2 RIP was used to detected the direct interactions between TTN-AS1 and miR-376a-3p. (F) miR-376a expression in EC tissues and normal tissues was assessed by qRT-PCR. (G) Correlation analysis of the relationship between miR-376a-3p and TTN-AS1 expression in EC tissues. **P<0.01. TTN-AS1, titin-antisense RNA1.

Figure 4.

miR-376a-3p mediates the suppressive effects on EC cells induced by si-TTN-AS1. (A) qRT-PCR was used to assess miR-376a-3p expression with miR-376a inhibitor transfection. si-TTN-AS1 was co-transfected with miR-376a-3p inhibitor or negative control (miR-NC), and then (B) a CCK-8 assay was performed to assess the cell viability, (C) flow cytometry was used to evaluate the apoptotic rate and (D) western blotting was performed to detect the protein expression levels of cleaved caspase-3 and cleaved PARP. (E and F) A Transwell assay was carried out to assess the cell migration and invasion abilities. (G) Quantitative analysis of C-F, respectively. **P<0.01 vs. the si-NC+miR-NC group; ^##P<0.01 vs. the si-TTN-AS1+miR-NC group. TTN-AS1, titin-antisense RNA1.

Figure 5.

PUM2 is targeted by miR-376a-3p. (A) TargetScan revealed the interacting sequences of miR-376a-3p and PUM2. (B) A luciferase reporter assay was carried out to validate the binding sites. PUM2 expression was assessed by (C) qRT-PCR and (D) western blotting with miR-376a-3p overexpression. (E) PUM2 expression in EC tissues and normal tissues was assessed by qRT-PCR. (F) Correlation analysis of the relationship between PUM2 and miR-376a-3p expression in EC tissues. **P<0.01. PUM2, pumilio homolog 2; EC, endometrial cancer.

Figure 6.

TTN-AS1 regulates PUM2 by targeting miR-376a-3p. si-TTN-AS1 was co-transfected with miR-NC or miR-376a-3p inhibitor, and then (A) qPCR was performed to determine the mRNA expression level of PUM2. (B and C) Western blotting was used to determine the protein expression level of PUM2. (D) Correlation analysis of relationship between PUM2 and TTN-AS1 expression in EC tissues. **P<0.01 vs. si-NC+miR-NC group; ^##P<0.01 vs. the si-TTN-AS1+miR-NC group. TTN-AS1, titin-antisense RNA1; PUM2, pumilio homolog 2; EC, endometrial cancer.

Figure 7.

Depletion of TTN-AS1 restrains tumor growth via the miR-376a-3p/PUM2 axis in vivo. sh-TTN-AS1 or sh-NC transfected HEC1A cells were injected into nude mice, and (A) the tumor size was assessed weekly. (B) The tumor weight was assessed 5 weeks after treatment. (C) The protein expression levels of cleaved caspase-3 and cleaved PARP were determined by western blotting. (D) miR-376a-3p was assessed by qRT-PCR. (E) PUM2 protein level was assessed by western blotting. (F) Ki-67 was detected by immunohistochemical staining. **P<0.01. TTN-AS1, titin-antisense RNA1; PUM2, pumilio homolog 2.