Hsa_circ_0129047 regulates the miR-375/ACVRL1 axis to attenuate the progression of lung adenocarcinoma

Abstract

Background: Circular RNAs (circRNAs) are attractive candidates to be used as biomarkers of human cancers, including lung adenocarcinoma (LUAD). Our study aimed to investigate the functions and regulatory mechanisms of hsa_circ_0129047 in the tumorigenesis of LUAD.

Methods: Reverse transcription-quantitative polymerase chain reaction was performed to determine the circRNA, microRNA (miRNA), and mRNA expression levels in LUAD cell lines and tissues. Tumor xenografts were established in nude mice to evaluate whether hsa_circ_0129047 affected LUAD tumor development in vivo. Cell counting kit-8 and transwell assays were performed to assess the mechanisms by which hsa_circ_0129047 influenced the viability and migration of LUAD cells, respectively. Apoptosis was evaluated via determination of the levels of the apoptotic markers, B-cell lymphoma-2, and Bcl-2-associated X, via Western blotting. Dual-luciferase reporter assay, RNA immunoprecipitation assay, and Pearson's correlation analysis were performed to determine the relationships among miR-375 and hsa_circ_0129047 and activin A receptor-like type 1 (ACVRL1).

Results: Downregulation of hsa_circ_0129047 levels was observed in LUAD cell lines and tissues. Meanwhile, the upregulation of hsa_circ_0129047 levels repressed the proliferative, migratory, and survival capacities of LUAD cells in vitro. Hsa_circ_0129047 exerted antitumor effects during in vivo tumor development. Finally, we demonstrated that hsa_circ_0129047 sponged miR-375. This interaction facilitated the expression of the downstream target of miR-375, ACVRL1, whose upregulation inhibited the development and malignancy of LUAD.

Conclusion: These findings demonstrate that hsa_circ_0129047 functions as a tumor inhibitor in LUAD by modulating the miR-375/ACVRL1 axis. Hence, hsa_circ_0129047 may be a promising biomarker and gene target for LUAD treatment.

Keywords: ACVRL1; hsa_circ_0129047; lung adenocarcinoma; miR-375.

FIGURE 1

Hsa_circ_0129047 was downregulated in LUAD tissues and cell lines. (A) Real‐time quantitative reverse transcription‐polymerase chain reaction (RT‐qPCR) was used to measure hsa_circ_0129047 expression in tissues from 40 LUAD patients and normal lung tissues. (B) hsa_circ_0129047 expression level in LUAD cell lines (A549, Calu‐3, PC9 and H1975) and normal lung cell line (BEAS‐2B) was detected by RT‐qPCR. *p < 0.05, **p < 0.001 vs. BEAS‐2B. (C) Subcellular fractionation analysis was conducted to determine the subcellular localization of hsa_circ_0129047 in LUAD. (D) Hsa_circ_0129047 and circ_0129047 mRNA expression was detected by RT‐qPCR after RNase R treatment. **p < 0.001 vs. Control

FIGURE 2

Hsa_circ_0129047 overexpression inhibited LUAD cell viability, migration and promoted cell apoptosis. (A) Hsa_circ_0129047 overexpression efficiency verified by RT‐qPCR after transfection with oe‐circ_0129047 vector. **p < 0.001 vs. Oe‐NC. (B) CCK‐8 assay was adapted to evaluate the cell viability in A549 and Calu‐3 cells transfected with oe‐NC and oe‐circ_0129047. *p < 0.05, **p < 0.001 vs. Oe‐NC. (C) Western blot experiment showed the expression of anti‐apoptotic marker Bcl‐2 and pro‐apoptotic marker Bax in A549 and Calu‐3 cells transfected with oe‐NC and oe‐circ_0129047. **p < 0.001 vs. Oe‐NC. (D) Cell migration ability was determined by Transwell assay in A549 and Calu‐3 cells transfected with oe‐NC and oe‐circ_0129047. **p < 0.001 vs. oe‐NC

FIGURE 3

Hsa_circ_0129047 overexpression suppressed LUAD cell growth in vivo. (A) Tumors derived from nude mice injected with A549 cells stably transfected with either oe‐NC or oe‐circ 0129047 are shown. (B) Tumor volume (mm³) was recorded every week. *p < 0.05, **p < 0.001 vs. oe‐NC. (C) On the 35th day after injection, the nude mice were euthanized and the tumors were excised and weighed. **p < 0.001 vs. oe‐NC

FIGURE 4

Hsa_circ_0129047 acted as a sponge for miR‐375. (A) The binding site between hsa_circ_0129047 and miR‐375 was predicted by circInteractome (https://ngdc.cncb.ac.cn/databasecommons/database/id/4778). (B) Luciferase reporter experiment was performed to validate the interaction of miR‐375 and hsa_circ_0129047 in the A549 and Calu‐3 cells. **p < 0.001 vs. miR‐NC. (C) RIP assay was used to validate the interaction between hsa_circ_0129047 and miR‐375. **p < 0.001 vs. anti‐IgG. (D) MiR‐375 expression level in LUAD tissues and normal adjacent tissues were detected by RT‐qPCR. (E) RT‐qPCR was conducted to measure miR‐375 expression level in LUAD cell lines (A549 and Calu‐3) and normal lung cell line (BEAS‐2B). **p < 0.001 vs. BEAS‐2B. (F) Correlation between hsa_circ_0129047 and miR‐375 was determined via Pearson's correlation coefficient. (G) The expression of miR‐375 in A549 and Calu‐3 cells transfected with Oe‐NC, mimic‐NC, Oe‐circ, mimic, and Oe + mimic was detected by RT‐qPCR. *p < 0.05 and **p < 0.001 vs. Oe‐NC; ^# p < 0.05 and ^## p < 0.001 vs. miR‐NC; ^& p < 0.05 and ^&& p < 0.001 vs. miR‐375 mimic

FIGURE 5

Overexpression of hsa_circ_0129047 inhibited LUAD progression by sponging miR‐375. Five groups were established: oe‐NC, miR‐NC, miR‐375 mimic, oe‐ circ_0129047, and oe‐circ_0129047 + miR‐375 mimic were transfected into the A549 and Calu‐3 cells in five groups, respectively. (A) CCK‐8 experiment was carried out to assess the cell viability in different groups. (B) Bax and Bcl‐2 protein levels in transfected A549 and Calu‐3 cells were detected in different groups. (C) Transwell assay showed the cell ability of migration in different groups. *p < 0.05 and **p < 0.001 vs. OE‐NC; ^# p < 0.05 and ^## p < 0.001 vs. miR‐NC; ^& p < 0.05 and ^&& p < 0.001 vs. miR‐375 mimic

FIGURE 6

ACVRL1 was a target gene of miR‐375. (A) MiR‐375 targeted ACVRL1 3’UTR with a potential binding site by starBase. (B) Dual‐luciferase reporter assay was used to measure the relationship between miR‐375 and ACVRL1. **p < 0.001 vs. miR‐NC. (C) RT‐qPCR was performed to measure ACVRL1 expression in LUAD tissues and adjacent normal tissues. (D) RT‐qPCR was used to measure ACVRL1 expression in LUAD cell lines (A549 and Calu‐3) and normal cell line (BEAS‐2B). **p < 0.01 vs. BEAS‐2B. (E) Pearson's correlation analysis was carried out to evaluate the relationship between miR‐375 and ACVRL1 expression. (F) Western blotting was used to measure ACVRL1 expression in A549 and Calu‐3 cells in the presence of oe‐NC, mimic‐NC, mimic, oe‐ACVRL1, and oe‐ACVRL1 + mimic. *p < 0.05, **p < 0.001 vs. oe‐NC; ^# p < 0.05 and ^## p < 0.001 vs. miR‐NC; ^& p < 0.05 and ^&& p < 0.001 vs. miR‐375 mimic

FIGURE 7

MiR‐375 facilitated the proliferation and migration of LUAD cells and inhibited their apoptosis by reducing ACVRL1 expression. Five groups (oe‐NC, miR‐NC, miR‐375 mimic, oe‐ACVRL1, and oe‐ACVRL1 + miR‐375 mimic) were established in the following experiments. (A) CCK‐8 experiment was conducted to evaluate the viability of cells from the different groups. (B) Cell apoptosis was detected by analyzing the expression of Bax and Bcl‐2 protein in different groups. (C) Transwell assay was adapted to determine the cell migration in different groups. *p < 0.05 and **p < 0.001 vs. Oe‐NC; ^# p < 0.05 and ^## p < 0.001 vs. miR‐NC; ^& p < 0.05 and ^&& p < 0.001 vs. miR‐375 mimic