circRNA Hsa_circ_0020850 Silence Represses the Development of Lung Adenocarcinoma via Regulating miR-195-5p/IRS2 Axis

Abstract

Background: The dysregulated circular RNAs (circRNAs) are relevant to lung adenocarcinoma development. Nevertheless, the function and mechanism of hsa_circ_0020850 (circ_0020850) in lung adenocarcinoma development are uncertain.

Methods: A total of 35 lung adenocarcinoma patients were recruited, and the tumor and normal tissue samples were harvested. A549 and PC-9 cells were exhibited for the experiments in vitro. circ_0020850, microRNA-195-5p (miR-195-5p) and insulin receptor substrate 2 (IRS2) abundances were detected via quantitative reverse transcription-polymerase chain reaction or Western blot. Cell proliferation, apoptosis, migration and invasion were measured via cell counting kit-8 (CCK8) assay, colony formation, flow cytometry, transwell and Western blot. The relationship between miR-195-5p and circ_0020850 or IRS2 was tested via dual-luciferase reporter analysis. The function of circ_0020850 on cell growth in vivo was measured via xenograft model.

Results: circ_0020850 expression was enhanced in lung adenocarcinoma tissues and cells. circ_0020850 silence suppressed cell proliferation, migration and invasion and facilitated apoptosis. miR-195-5p was targeted via circ_0020850, and its knockdown reversed the inhibitive effect of circ_0020850 silence on lung adenocarcinoma development. IRS2 was targeted via miR-195-5p, and miR-195-5p inhibited cell proliferation, migration and invasion and induced apoptosis via decreasing IRS2. circ_0020850 knockdown decreased IRS2 expression via regulating miR-195-5p. circ_0020850 down-regulation decreased lung adenocarcinoma xenograft tumor growth.

Conclusion: circ_0020850 knockdown repressed lung adenocarcinoma cell proliferation, migration and invasion and promoted apoptosis via regulating miR-195-5p and IRS2.

Keywords: IRS2; circ_0020850; lung adenocarcinoma; miR-195-5p.

Figure 1

Circ_0020850 level in lung adenocarcinoma. (A) The common dysregulated circRNAs in lung adenocarcinoma predicted via GSE101684. (B) circ_0020850 level was detected in lung adenocarcinoma tissues and normal tissues. n = 35. (C) circ_0020850 abundance was measured in A549, PC-9 and BEAS-2B cells. *P<0.05.

Figure 2

The influence of circ_0020850 on lung adenocarcinoma development. (A) circ_0020850 abundance was examined in cells with transfection of si-circ_0020850 or si-NC. (B and C) Cell viability was detected in cells with transfection of si-circ_0020850 or si-NC at 0, 24, 48 or 72 h. (D) The colony formation was analyzed in cells with transfection of si-circ_0020850 or si-NC. (E) Cell apoptotic rate was measured in cells with transfection of si-circ_0020850 or si-NC. (F and G) Cell migration and invasion were examined in cells with transfection of si-circ_0020850 or si-NC. (H–K) CyclinD1, Bcl-2, Cleaved-casp-3, Vimentin, N-cadherin and E-cadherin protein abundances were detected in cells with transfection of si-circ_0020850 or si-NC. *P<0.05.

Figure 3

The relationship of circ_0020850 and miR-195-5p. (A) The binding sequence of circ_0020850 and miR-195-5p. (B and C) Luciferase activity was detected in A549 and PC-9 cells with transfection of circ_0020850-WT or circ_0020850-MUT and miR-195-5p mimic or miR-NC. (D and E) miR-195-5p abundance was measured in lung adenocarcinoma tissues and cells. (F) The association between circ_0020850 and miR-195-5p in lung adenocarcinoma tissues. (G) miR-195-5p level was examined in A549 and PC-9 cells with transfection of si-NC, si-circ_0020850, si-circ_0020850 + anti-miR-NC or anti-miR-195-5p. *P<0.05.

Figure 4

The influence of miR-195-5p on circ_0020850-mediated lung adenocarcinoma development. Cell viability (A and B), colony formation (C), apoptotic rate (D), migration and invasion (E and F), and CyclinD1, Bcl-2, Cleaved-casp-3, Vimentin, N-cadherin and E-cadherin protein levels (G–J) were examined in cells with transfection of si-NC, si-circ_0020850, si-circ_0020850 + anti-miR-NC or anti-miR-195-5p. *P<0.05.

Figure 5

The relationship of miR-195-5p and IRS2. (A) The binding sequence of miR-195-5p and IRS2. (B and C) Luciferase activity was tested in cells with transfection of IRS2 3ʹUTR-WT or IRS2 3ʹUTR-MUT and miR-195-5p mimic or miR-NC. (D–G) IRS2 expression was detected in lung adenocarcinoma tissues and cells. (H) The correlation between miR-195-5p and IRS2 in lung adenocarcinoma tissues. (I and J) IRS2 abundance was examined in A549 and PC-9 cells with transfection of miR-NC, miR-195-5p mimic, miR-195-5p mimic + pcDNA-NC or pcDNA-IRS2. *P<0.05.

Figure 6

The influence of miR-195-5p and IRS2 on lung adenocarcinoma development. Cell viability (A and B), colony formation (C), apoptotic rate (D), migration and invasion (E and F), and CyclinD1, Bcl-2, Cleaved-casp-3, Vimentin, N-cadherin and E-cadherin protein levels (G–J) were detected in cells with transfection of miR-NC, miR-195-5p mimic, miR-195-5p mimic + pcDNA-NC or pcDNA-IRS2. *P<0.05.

Figure 7

The influence of circ_0020850 and miR-195-5p on IRS2 expression in lung adenocarcinoma. (A and B) IRS2 expression was detected in A549 and PC-9 cells with transfection of si-NC, si-circ_0020850, si-circ_0020850 + anti-miR-NC or anti-miR-195-5p. (C) The correlation between circ_0020850 and IRS2 in lung adenocarcinoma tissues. *P<0.05.

Figure 8

The influence of circ_0020850 on lung adenocarcinoma growth in xenograft model. (A) Tumor volume was detected every 4 days. (B) Tumor weight was measured at the end point. (C–F) circ_0020850, miR-195-5p and IRS2 abundances were examined in tumor tissues of each group. *P<0.05.