2-methoxyestradiol restrains non-small cell lung cancer tumorigenesis through regulating circ_0010235/miR-34a-5p/NFAT5 axis

Abstract

Background: Non-small cell lung cancer (NSCLC) is one of the most prevalent and severe malignant tumors in the world and its molecular mechanism is still unclear. In recent years, increasing evidence indicates the significant roles of circRNAs in NSCLC. It has been determined that 2-methoxyestradiol (2-MeOE2) exerts antitumor roles in many cancers. However, the molecular mechanism of 2-MeOE2 in regulating the development of lung cancer needs further elucidation.

Methods: The expression levels of circ_0010235, microRNA-34a-5p (miR-34a-5p), and nuclear factor of activated T cells 5 (NFAT5) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation, apoptosis and invasion were detected by cell counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU) assay, flow cytometry and transwell assays, respectively. The interaction between miR-34a-5p and circ_0010235 or NFAT5 was predicted by bioinformatic software and confirmed by dual-luciferase reporter assay.

Results: Our data showed 2-MeOE2 hindered cell proliferation, invasion and induced apoptosis in NSCLC, which could be reversed by upregulation of circ_0010235 and NFAT5 or miR-34a-5p knockdown. Circ_0010235 and NFAT5 expression levels were increased, and miR-34a-5p expression level was decreased in NSCLC tissues and cells. In addition, 2-MeOE2 treatment suppressed the expression of circ_0010235 and NFAT5 while promoted the expression of miR-34a-5p. Furthermore, circ_0010235 functioned as a molecular sponge of miR-34a-5p to regulate NFAT5 expression. Knockdown of circ_0010235 or 2-MeOE2 treatment constrained tumor growth in vivo, and circ_0010235 depletion enhanced the inhibitory effect of 2-MeOE2 on tumor growth in vivo.

Conclusion: These findings demonstrated that 2-MeOE2 retarded NSCLC progression by modulating the circ_0010235/miR-34a-5p/NFAT5 axis, thus providing a new perspective for 2-MeOE2 treatment in NSCLC.

Keywords: 2-MeOE2; NFAT5; circ_0010235; miR-34a-5p; non-small cell lung cancer.

FIGURE 1

2‐MeOE2 played a repressive role on the malignancy of non‐small cell lung cancer (NSCLC) cells. Human bronchial epithelioid cells (HBE) cell and NSCLC cells were exposed to different concentrations of 2‐MeOE2 (5, 10 and 15 μM). (a–c) Cell viability was determined by cell counting kit‐8 (CCK‐8) assay. (d) 5‐ethynyl‐2'‐deoxyuridine (EdU) assay was utilized to determine the number of EdU‐positive cells. (e) Cell apoptosis was examined by flow cytometry analysis. (f) Cell invasion was evaluated by transwell assay. (g, h) The protein levels of cyclin D1 and MMP2 were measured by western blot assay. **p < 0.01, ***p < 0.001 and ****p < 0.0001.

FIGURE 2

2‐MeOE2 exerted its function through regulating circ_0010235 in non‐small cell lung cancer (NSCLC) cells. (a) The expression of circ_0010235 in normal tissues and HCC tissues was detected by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). (b) Circ_0010235 expression level was identified by qRT‐PCR in human bronchial epithelioid (HBE) and NSCLC cells (H1299 and A549). (c, d) The levels of circ_0010235 and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) in H1299 and A549 cells treated with or without RNase R were examined by qRT‐PCR assay. (e) The expression of circ_0010235 was detected by qRT‐PCR in NSCLC cells treated with different concentrations of 2‐MeOE2 (5, 10 and 15 μM). (f–l) H1299 and A549 cells were divided into four groups: Control, 2‐MeOE2, 2‐MeOE2 + pcD5‐ciR and 2‐MeOE2 + circ_0010235. (f) The expression level of circ_0010235 was quantified by qRT‐PCR assay. (g) Cell counting kit‐8 (CCK‐8) assay was conducted to evaluate cell viability in H1299 and A549 cells. (h) The number of 5‐ethynyl‐2'‐deoxyuridine (EdU)‐positive cells was measured by EdU assay in treated H1299 and A549 cells. (i) Cell apoptosis was determined using flow cytometry analysis. (j) Transwell assay was conducted to evaluate cell invasion ability. (k, l) Western blot assay was used to measure the protein levels of cyclin D1 and MMP2 in H1299 and A549 cells. **p < 0.01, ***p < 0.001 and ****p < 0.0001.

FIGURE 3

Circ_0010235 interacted with miR‐34a‐5p. (a) The binding sites between circ_0010235 and miR‐34a‐5p are shown. (b, c) Dual‐luciferase reporter assay was conducted to determine the interaction between circ_0010235 and miR‐34a‐5p. (d) The expression of miR‐34a‐5p in non‐small cell lung cancer (NSCLC) tissues and adjacent normal tissues was measured by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). (e) Correlation between the expression of circ_0010235 and miR‐34a‐5p was assessed in NSCLC tissues by Pearson's correlation analysis. (f) QRT‐PCR was used to determine miR‐34a‐5p expression in human bronchial epithelioid (HBE) and NSCLC cells. (g) The circ_0010235 expression level in NSCLC cells transfected with si‐NC or si‐circ_0010235. (h) NSCLC cells were transfected with si‐NC, si‐circ_0010235, pcD5‐ciR or circ_0010235. The expression of miR‐34a‐5p was examined by qRT‐PCR. **p < 0.01, ***p < 0.001 and ****p < 0.0001.

FIGURE 4

MiR‐34a‐5p mediated the regulatory effect of 2‐MeOE2 on the development of non‐small cell lung cancer (NSCLC). (a) The expression of miR‐34a‐5p was detected by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) in H1299 and A549 cells treated with different concentrations of 2‐MeOE2. (b–h) H1299 and A549 cells were divided into four groups: Control, 2‐MeOE2, 2‐MeOE2 + anti‐miR‐NC, and 2‐MeOE2 + anti‐miR‐34a‐5p. (b) The expression of miR‐34a‐5p was determined by qRT‐PCR. (c–f) The viability, proliferation, apoptosis and invasion of H1299 and A549 cells were evaluated by cell counting kit‐8 (CCK‐8) assay, 5‐ethynyl‐2'‐deoxyuridine (EdU) assay, flow cytometry analysis and transwell assay, respectively. (g, h) The protein levels of cyclin D1 and MMP2 were tested by western blot assay. **p < 0.01, ***p < 0.001 and ****p < 0.0001.

FIGURE 5

MiR‐34a‐5p directly interacted with NFAT5. (a) Shows the binding sites between miR‐34a‐5p and NFAT5. (b, c) The combination between miR‐34a‐5p and NFAT5 was analyzed by dual‐luciferase reporter assay. (d) NFAT5 mRNA level was assessed by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) in non‐small cell lung cancer (NSCLC) tissues and matched normal tissues. (e) The correlation between the levels of NFAT5 mRNA and miR‐34a‐5p in NSCLC tissues was estimated by Pearson's correlation coefficient. (f, g) NFAT5 protein expression was detected by western blot in NSCLC tissues and cells. (h) The transfection efficiencies of miR‐34a‐5p mimic were evaluated by qRT‐PCR. (i) The protein level of NFAT5 in H1299 and A549 cells transfected with miR‐NC, miR‐34a‐5p, anti‐miR‐NC or anti‐miR‐34a‐5p was quantified via western blot assay. **p < 0.01, ***p < 0.001 and ****p < 0.0001.

FIGURE 6

Circ_0010235 affected non‐small cell lung cancer (NSCLC) cell progression by regulating NFAT5. NSCLC cells were transfected with si‐NC, si‐circ_0010235, si‐circ_0010235 + pcDNA or si‐circ_0010235 + NFAT5. (a) NFAT5 expression was determined using western blot assay. The viability, proliferation, apoptosis and invasion of cells were detected using cell counting kit‐8 (CCK‐8) assay (b), 5‐ethynyl‐2'‐deoxyuridine (EdU) assay (c), flow cytometry (d), and transwell assay (e). (f, g) The protein levels of cyclin D1 and MMP2 were examined using western blot assay. **p < 0.01, ***p < 0.001 and ****p < 0.0001.

FIGURE 7

2‐MeOE2 regulated the malignancy of non‐small cell lung cancer (NSCLC) cells through NFAT5. (a) Western blot assay was used to detect the effect of 2‐MeOE2 treatment on ITGB1 protein level. (b–h) Cells were divided into four groups: Control, 2‐MeOE2, 2‐MeOE2 + pcDNA, and 2‐MeOE2 + NFAT5. (b) The level of NFAT5 protein was determined by western blot assay. (c–f) The viability, proliferation, apoptosis and invasion of H1299 and A549 cells were tested by cell counting kit‐8 (CCK‐8) assay, 5‐ethynyl‐2'‐deoxyuridine (EdU) assay, flow cytometry analysis and transwell assay, respectively. (g, h) The protein levels of cyclin D1 and MMP2 in H1299 and A549 cells were measured by western blot assay. **p < 0.01, ***p < 0.001 and ****p < 0.0001.

FIGURE 8

Circ_0010235 silencing augmented the anticancer activity of 2‐MeOE2 in vivo. (a) Tumor volume was monitored. (b) Tumor weight was examined after 23 days inoculation. (c, d) The expression levels of circ_0010235 and miR‐34a‐5p were measured by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) in resected xenograft tumor tissues. (e) The protein levels of NFAT5 were measured by western blot assay in resected xenograft tumor tissues. (f) The expression levels of NFAT5, Ki67 and MMP2 in the collected xenograft tumors were estimated by immunohistochemistry (IHC) assay. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.