CircRNA_100395 Carried by Exosomes From Adipose-Derived Mesenchymal Stem Cells Inhibits the Malignant Transformation of Non-Small Cell Lung Carcinoma Through the miR-141-3p-LATS2 Axis

Abstract

Objective: The specific purpose of this study is to investigate the impact exosomes from adipose-derived mesenchymal stem cell (AMSC) has on non-small cell lung carcinoma (NSCLC) and the relative applications.

Methods: circ_100395, miR-141-3p, and LATS2 were expressed and detected in NSCLC and paracancerous tissues as well as NSCLC cell lines. Pearson correlation analysis, Dual-Luciferase Reporter Assay and RNA pull-down assay were used to validate their expression and interaction, respectively. After isolation and culture of AMSCs, exosomes were extracted and identified. EdU, epithelial-mesenchymal transition (EMT), and cell colony formation assay were used to distinguish the biological activity of the cells. Expression Hippo/YAP signalling pathway-related proteins were measured by western blotting. Subsequently, tumour volume and weight were confirmed based on xenograft nude mice models, Ki-67 and LATS2 expression was observed by immunohistochemistry.

Results: circ_100395 was lowly expressed in NSCLC tissues or cells. The negative correlations and interactions were confirmed between circ_100395 and miR-141-3p, miR-141-3p, and LATS2. AMSC-derived exosomes with overexpression of circ_100395 (exo-circ_100395) significantly inhibited the biological activity as well as EMT of H1650 cells and Hippo/YAP signalling pathway activity. In addition, exo-circ_100395 markedly reduced tumour volume and weight as well as Ki-67 and LASP1 expression in vivo. However, overexpressed miR-141-3p or knocked down LATS2 alleviated the above effects.

Conclusion: Exo-circ_100395 can increase LATS2 expression by sponging miR-141-3p to regulate Hippo/YAP signalling pathway, thereby inhibiting NSCLC malignant transformation.

Keywords: Hippo/YAP signalling pathway; circRNA_100395; exosomes from adipose-derived mesenchymal stem cells; miR-141-3p; non-small cell lung carcinoma (NSCLC).

FIGURE 1

Low circ_100395 expressed in NSCLC tissues and cells. (A) Evaluation of circ_100395 expressed in paracancerous and NSCLC tissues using qRT-PCR. ***p < 0.001 vs Normal group. (B) Evaluation of circ_100395 expressed in human bronchial epithelial cell line 16 HBE-T and NSCLC cell lines using qRT-PCR. **p < 0.01 and ***p < 0.001 vs HBE; (C) Evaluation of circ_100395 expressed in H1650 cells of each treatment group using qRT-PCR. ***p < 0.001 vs NC group and ^###p < 0.001 vs si-NC group.

FIGURE 2

Outcomes circ_100395 has regarding the progression of NSCLC cells and Hippo/YAP signalling pathway. (A) Cell proliferation identified by using an EdU assay-base; (B) Cell viability identified by using a cell colony formation assay-base; (C) Flow cytometry-based detection of cell apoptosis; (D) Cell invasion identified by using a Transwell assay-base; (E) Wound healing assay-assessment of cell migration; (F) E-cadherin, vimentin, and N-cadherin proteins identified by using Western blot of; (G) LATS2, p-YAP, and YAP proteins identified by Western blot. **p < 0.01 vs NC group; ^##p < 0.01 vs si-NC group.

FIGURE 3

circ_100395 can as a sponge to adsorb miR-141-3p. (A) The predicted targeting sequence of circ_100395 and miR-141-3p. (B) Dual-luciferase reporter assay-based validation that circ_100395 targets miR-141-3p. **p < 0.01 vs NC group. (C) RNA pull down assay-based validation that circ_100395 targets miR-141-3p. ***p < 0.001 vs NC group. (D) MiR-141-3p expression in non-small cell lung cancer (NSCLC) tissues in StarBase3.0 database; (E) qRT-PCR detection of miR-141-3p expression in NSCLC tissues and paracancerous tissues. ***p < 0.001 vs Normal group. (F) qRT-PCR detection of miR-141-3p expression in NSCLC cell lines and human bronchial epithelial cell line 16 HBE-T. **p < 0.01 and ***p < 0.001 vs HBE. (G) qRT-PCR detection of miR-141-3p expression in H1650 cells after overexpression and interference of circ_100395. **p < 0.01 vs NC group, ^##p < 0.01 vs si-NC group. (H) A negative correlation between circ_100395 and miR-141-3p expression in NSCLC tissues.

FIGURE 4

Circ_100395 up-regulates LATS2 expression by competitively binding to miR-141-3p. (A) Confirmation of miR-141-3p targeting LATS2 gene via Dual-luciferase reporter assay. (B) LATS2 expression in NSCLC tissues in the StarBase3.0 database; (C) LATS2 expressed in paracancerous and NSCLC tissues qRT-PCR analysis. (D) LATS2 protein expressed in paracancerous and NSCLC tissues detected via western blot; *p < 0.05, **p < 0.01, and ***p < 0.001 vs Normal group. (E) Expressed LATS2 in human bronchial epithelial cell line 16 HBE-T and NSCLC cell lines via qRT-PCR analysis, *p < 0.05, **p < 0.01, ***p < 0.001 vs HBE. (F) qRT-PCR analysis of LATS2 expression in H1650 cells after overexpression and interference of circ_100395; **p < 0.01 vs NC group, ^##p < 0.01 vs si-NC group. (G) A beneficial relationship was found amidst expressed circ_100395 and LATS2 within tissues of NSCLC; (H) Expressed LATS2 post overexpression and interference of miR-141-3p discovered using qRT-PCR. **p < 0.01 vs NC group, ^##p < 0.01 vs si-NC group. (I,J) Expressed LATS2 protein in H1650 cells post overexpression and interference of miR-141-3p discovered using Western blot. **p < 0.01 vs NC group, ^##p < 0.01 vs si-NC group; (K) A contrary relationship was found amidst expressed miR-141-3p and LATS2 within tissues of NSCLC.

FIGURE 5

AMSCs exosomes transport circ_100395 into NSCLC cells. (A) Morphology of AMSC observed using transmission electron microscopy (100 nm); (B) AMSC size analysed using nanoparticle tracking**; (C) Expressed HSP70, CD63, and TSG101 detected via western blot; (D) Exosomes in circ_100395 analysed via qRT-PCR; (E–G) Expressed circ_100395, miR-141-3p, LATS2 in H1650 cells post exo-NC or exo-circ_100395 treatment analysed via qRT-PCR. **p < 0.01 and ***p < 0.001 vs exo-NC group.

FIGURE 6

Circ_100395 carried by exosomes from AMSCs inhibiting NSCLC progression through the miR-141-3p/LATS2 axis. (A,B) EdU assay-based observation of cell proliferation; (C,D) Colony formation assay-based observation of cell viability; (E,F) Flow cytometry-based detection of cell apoptosis; (G,H) Cell invasion exposed via transwell assay; (I,J) Wound healing assay-based detection of cell migration; (K,L) Detecting expressed E-cadherin, vimentin, and N-cadherin protein in individual treatment groups via western blot; (M,N) Detecting expressed LATS2, p-YAP, and YAP protein in individual treatment groups via western blot. **p < 0.01 vs exo-NC group, ^##p < 0.01 vs exo-circ_100395 group.

FIGURE 7

Circ_100395 carried by exosomes from AMSCs inhibiting NSCLC in vivo growth. (A) Tumors of nude mice; (B) Line graph of the change of tumour volume; (C) Statistical graph of tumour weight; (D) Expressed LAST2 in tumour tissue analysed via qRT-PCR; (E) Expressed E-cadherin, vimentin, and N-cadherin protein detected via western blot; (F) Expressed LATS2, p-YAP and YAP protein in tumour tissues detected by western blot; (G) Expressed Ki-67 and LATS2 in tumour tissues determined by immunohistochemistry. **p < 0.01 vs exo-NC group, ^##p < 0.01 vs exo-circ_100395 group.