circ_0114866 promotes the progression and EMT of non-small cell lung cancer via miR-653-5p/MYL6B axis

Abstract

Background: Non-small-cell lung cancer (NSCLC) is the most prevalent form of lung cancer. Circular RNA (circRNA) has emerged as a key player in the development of NSCLC by acting as miRNA sponges. However, the precise role of circ_0114866 in regulating NSCLC process is yet to be elucidated.

Methods: The expression of circ_0114866, miR-653-5p, and MYL6B were assessed by qPCR. Cell viability, proliferation, invasion, and migration were investigated using CCK-8, colony formation, Transwell, and wound healing assays. The protein levels of MYL6B, MMP-2, N-cadherin, E-cadherin, and vimentin were evaluated through Western blot analysis. Xenograft tumor model were selected to analyze the impact of circ_0114866 on NSCLC tumor growth. Through circBank or Starbase databases, the binding interactions between miR-653-5p and circ_0114866 or MYL6B were predicted. Subsequently, these interactions were verified by dual-luciferase reporter assay.

Results: The expression of circ_0114866 and MYL6B were clearly elevated, while miR-653-5p expression was notably reduced in NSCLC tissues and cells. Notably, circ_0114866 knockdown obviously suppressed the proliferation, metastasis, and EMT process in NSCLC cells. Additionally, circ_0114866 functioned as a sponge for miR-653-5p, leading to an increase in MYL6B expression by absorbing miR-653-5p. Furthermore, the inhibitory effects on biological behaviors and EMT process of NSCLC cells induced by circ_0114866 knockdown were reversed by miR-653-5p inhibitor. Moreover, in vivo experiments demonstrated that silencing circ_0114866 resulted in a repression of tumor growth.

Conclusion: Our findings indicate that circ_0114866 knockdown upregulated MYL6B transcription by sponging miR-653-5p, leading to hinder the progression and EMT process of NSCLC.

Fig. 1

The expression of circ_0114866 in NSCLC tissues and cell lines. (A) Circ_0114866 expression in NSCLC tissues. (B) Circ_0114866 expression in BSAE-2B, H1299, PC9, H358 and A549 cells. (C–D) The stability of circ_0114866 in H358 and A549 cells. (E–F) The subcellular localization for circ_0114866 in H358 and A549 cells. **P < 0.01; ***P < 0.001.

Fig. 2

Inhibition of circ_0114866 suppressed the proliferation, migration, invasion and EMT process of NSCLC cells. (A) Circ_0114866 expression in H358 and A549 cells. (B) Cell viability in H358 and A549 cells in each group. (C) Cell proliferation in H358 and A549 cells in each group. (D) Cell migration in H358 and A549 cells in each group. (E) Cell invasion in H358 and A549 cells in each group. (F–G) The protein levels of MMP-2, N-cadherin, E-cadherin and vimentin in H358 and A549 cells. **P < 0.01; ***P < 0.001. Full Western blot images can be found at Supplementary Materials.

Fig. 3

Inhibition of circ_0114866 inhibited NSCLC tumor growth in vivo. (A) Circ_0114866 expression in A549 cells. (B) Representative pictures of tumor tissues in each group. (C) Tumor volumes at different days after injection in each group. (D) Tumor weights in each group. (E) Circ_0114866 expression in tumor tissues. **P < 0.01; ***P < 0.001.

Fig. 4

Circ_0114866 acted as the sponge of miR-653-5p. (A) The binding sites between circ_0114866 and miR-653-5p. (B) MiR-653-5p expression in H358 and A549 cells. (C–D) Fluorescence intensity of circ_0114866 3′UTR in H358 and A549 cells in each group. (E) MiR-653-5p expression in H358 and A549 cells. (F) MiR-653-5p expression in NSCLC tissues. (G) The correlation between circ_0114866 expression and miR-653-5p expression in NSCLC tissues. (H) MiR-653-5p expression in BSAE-2B, H358 and A549 cells. ***P < 0.001.

Fig. 5

Circ_0114866 knockdown suppressed the proliferation, migration, invasion and EMT process of NSCLC cells by absorbing miR-653-5p. (A) MiR-653-5p expression in H358 and A549 cells. (B) MiR-653-5p expression in H358 and A549 cells in each group. (C) Cell proliferation in H358 and A549 cells in each group. (D) Cell proliferation in H358 and A549 cells in each group. (E–F) Cell invasion in H358 and A549 cells in each group. (G–H) Cell migration in H358 and A549 cells in each group. (I–J) The protein levels of MMP-2, N-cadherin, E-cadherin and vimentin in H358 and A549 cells. *P < 0.0; **P < 0.01; ***P < 0.001. Full Western blot images can be found at Supplementary Materials.

Fig. 6

MiR-653-5p was bound to MYL6B and suppressed its expression. (A) The binding sites between miR-653-5p and MYL6B. (B–C) Fluorescence intensity of MYL6B 3′UTR in H358 and A549 cells in each group. (D) The protein level of MYL6B in H358 and A549. (E) The mRNA level of MYL6B in NSCLC tissues. (F) The correlation between miR-653-5p expression and MYL6B expression in NSCLC tissues. (G) The protein level of MYL6B in NSCLC tissues. (H) e protein level of MYL6B in BSAE-2B, H358 and A549 cells. **P < 0.01; ***P < 0.001. Full Western blot images can be found at Supplementary Materials.

Fig. 7

miR-653-5p suppressed the proliferation, migration, invasion and EMT process of NSCLC cells by binding to MYL6B. (A) The protein level of MYL6B in H358 and A549 cells. (B) The protein level of MYL6B in H358 and A549 cells in each group. (C) Cell proliferation in H358 and A549 cells in each group. (D) Cell proliferation in H358 and A549 cells in each group. (E–F) Cell invasion in H358 and A549 cells in each group. (G–H) Cell migration in H358 and A549 cells in each group. (I–J) The protein levels of MMP-2, N-cadherin, E-cadherin and vimentin in H358 and A549 cells. *P < 0.0; **P < 0.01; ***P < 0.001. Full Western blot images can be found at Supplementary Materials.

Fig. 8

Circ-0114866 silencing decreased MYL6B expression by sponging miR-653-5p. (A–B) The mRNA level of MYL6B in H358 and A549 cells in each group. (C–D) The protein level of MYL6B in H358 and A549 cells in each group. **P < 0.01; ***P < 0.001. Full Western blot images can be found at Supplementary Materials.