Circular RNA CircCDYL Regulates Proliferation and Apoptosis in Non-Small Cell Lung Cancer Cells by Sponging miR-185-5p and Upregulating TNRC6A

Abstract

Aim: A series of research reveal that circular RNA (circRNA) plays a vital role in regulating the development of tumor cells. In this research, we would explore the role and mechanism of circCDYL in non-small cell lung cancer (NSCLC).

Methods: RT-PCR was performed to detect the expression of circCDYL in NSCLC tissues, plasma, and cell lines. The tumor cell proliferation ability was evaluated by clone formation assay, and cell cycle determination. Flow cytometry was used to detect apoptosis in NSCLC cell lines. Western blot and RT-PCR were used to assess the expression of proteins and genes. Luciferase assay was performed to confirm the relationship of circRNA-miRNA-mRNA.

Results: The decreased level of circCDYL was observed in NSCLC patients' tissues and plasma, which was also downregulated in NSCLC cell lines. Forced expression of circCDYL inhibited cell viability, proliferation and induced apoptosis in A549 cells. Luciferase assay verified that circCDYL could bind with miR-185-5p and confirmed that TNRC6A was a downstream target of miR-185-5p. Overexpression of miR-185-5p or silencing of TNRC6A could inhibit the anti-tumor effect of circCDYL in A549 cells via regulating the ERK1/2 signal.

Conclusion: Here, we revealed that circCDYL inhibited proliferation and induced apoptosis in NSCLC cell lines via regulating ERK1/2 signal, and the mechanism of this progression may target miR-185-5p/TNRC6A, which provided a theoretical basis for clinical therapy.

Keywords: apoptosis; circular RNA; non-small cell lung cancer; proliferation.

Figure 1

CircCDYL is downregulated in NSCLC tissues and cell lines. (A) The expression of circCDYL was detected by RT-PCR in tumor tissue and adjacent normal tissues from NSCLC patients. n=30, *P<0.05. (B) The expression of circCDYL was detected by RT-PCR in plasma from NSCLC patients and healthy volunteers. n=30, **P<0.01. (C) The expression of circCDYL in NSCLC cell lines (A549, H2170, PG49, SPC-A-1), and BEAS-2B was indicated as control. n=30, *P<0.05, **P<0.01.

Figure 2

CircCDYL inhibits apoptosis and induces apoptosis in A549 cells. (A) The transfection efficiency of circCDYL was detected by RT-PCR. n=5, *P<0.05. (B) CCK8 assay determined the cell viability of A549 cells. n=6, *P<0.05. (C) Cell cycle was confirmed by flow cytometry. n=4, *P<0.05. (D) Clone formation assay was performed in A549 cells. n=3, **P<0.01. (E) Flow cytometry was used to explore the apoptosis level in A549 cells. n=4, *P<0.05.

Figure 3

CircCDYL could interact with miR-185-5p. (A) The prediction binding sites between circCDYL and miR-185-5p. (B) Luciferase assay was performed to verify the relationship between circCDYL and miR-185-5p. n=4, *P<0.05. (C) RNA pull-down assay performed the enrichment of miR-185-5p by circCDYL probe. n=4, **P<0.01. (D) The expression level of miR-185-5p in A549 cells after sh-circCDYL/sh-NC or circCDYL/vector transfection. n=4, *P<0.05. (E) The level of miR-185-5p in NSCLC tumor tissues and adjacent normal tissues. n=30, *P<0.05.

Figure 4

TNRC6A is a downstream target of miR-185-5p. (A) Four bioinformatics sites (miRmap, PITA, miRanda, microT) were used to predict the downstream of miR-185-5p. (B) Luciferase assay was performed to verify the relationship between TNRC6A and miR-185-5p. n=4, *P<0.05. (C and D) The mRNA and apoptosis level of TNRC6A in A549 cells after miR-185-5p transfection. n=5, *P<0.05. (E and F) The mRNA and apoptosis level of TNRC6A in NSCLC tumor tissues and adjacent tissues. *P<0.05.

Figure 5

CircCDYL inhibits apoptosis and induces apoptosis via the EKR1/2 signal by targeting miR-185-5p/YNRC6A. (A) The level of TNRC6A was detected by RT-PCR. n=5, *P<0.05, ^#P<0.05. (B) CCK8 assay determined the cell viability of A549 cells. n=6, *P<0.05, ^#P<0.05. (C) Cell cycle was confirmed by flow cytometry. n=4, *P<0.05, ^#P<0.05. (D) Clone formation assay was performed in A549 cells. n=3, **P<0.01, ^#P<0.05. (E) The protein level of p-ERK1/2 and ERK1/2 in A549 cells. n=4, *P<0.05, ^#P<0.05.

Figure 6

Silencing of circCDYL prevents tumor growth in vivo. (A) A549-sh-circCDYL and A549-sh-NC cells were subcutaneously injected into nude mice, and tumor growth curves were plotted (n = 4). (B and C) Tumor volume and weight was determined for each group. n=4, *P<0.05. (D) The expression of circCDYL, miR-185-5p, and TNRC6A in collected tumor. n=4, *P<0.05. (E) Immunohistochemical staining for Ki-67, ERK, p-ERK, Caspase3 in the xenograft tumor tissues.