Overexpression of lncRNA FEZF1-AS1 promotes progression of non-small cell lung cancer via the miR-130a-5p/CCND1 axis

doi:10.12122/j.issn.1673-4254.2024.05.05

Abstract

Abstract:

Objective To explore the molecular mechanism by which FEZF1-AS1 overexpression promotes progression of non-small cell lung cancer (NSCLC) via the miR-130a-5p/CCND1 axis. Methods TCGA database was used to analyze FEZF1-AS1 expression levels in NSCLC. FEZF1-AS1 expression was detected by qRT-PCR in clinical specimens of NSCLC tissues and NSCLC cell lines, and its correlation with clinical features of the patients were analyzed. The binding sites of FEZF1-AS1 with hsa-miR-130a-5p and those of hsa-miR-130a-5p with CCND1 were predicted. CCK8 assay, clone formation assay, scratch assay, and Transwell assay were employed to examine the effects of FEZF1-AS1 knockdown and hsa-miR-130a-5p inhibitor on proliferation, invasion, and migration abilities of lung cancer cell lines. Dual luciferase assay was used to verify the binding of FEZF1-AS1 with hsa-miR-130a-5p and the binding of hsa-miR-130a-5p with CCND1. Western blotting was performed to detect the changes in CCND1 protein expression level in H1299 and H358 cells following FEZF1-AS1 knockdown and treatment with hsa-miR-130a-5p inhibitor. Results FEZF1-AS1 was highly expressed in NSCLC tissues in close correlation with lymph node metastasis and also in H1299 and H358 cell lines (all P<0.05). FEZF1-AS1 knockdown obviously reduced proliferation, migration, and invasion abilities of NSCLC cells (P<0.05). Dual luciferase assay confirmed the binding of hsa-miR-130a-5p with FEZF1-AS1 and CCND1 (P<0.05), and hsa-miR-130a-5p inhibitor significantly inhibited proliferation, migration, and invasion of NSCLC cells (P<0.05). FEZF1-AS1 knockdown significantly reduced CCND1 protein expression in NSCLC cells, and this effect was strongly inhibited by treatment with hsa-miR-130a-5p inhibitor (P<0.05). Conclusion FEZF1-AS1 is highly expressed in NSCLC tissue in close correlation with lymph node metastasis to promote cancer progression through the miR-130a-5p/CCND1 axis.

Key words: non-small cell lung cancer, lncRNA, FEZF1-AS1, miR-130a-5p, CCND1

Feifan LI, Junxin XIANG, Jiahui LIU, Xiaojing WANG, Hao JIANG. Overexpression of lncRNA FEZF1-AS1 promotes progression of non-small cell lung cancer via the miR-130a-5p/CCND1 axis[J]. Journal of Southern Medical University, 2024, 44(5): 841-850.

Figures/Tables 7

Fig.1 Expression of FEZF1-AS1 in non-small cell lung cancer (NSCLC) tissues and cell lines. A: Expression of FEZF1-AS1 in pan-cancer in TCGA database. B: Expression of FEZF1-AS1 in unpaired (left) and paired (right) human lung cancer tissues in the TCGA database. C: Expression of FEZF1-AS1 in human NSCLC tissue. D: Expression of FEZF1-AS1 in human lung cancer cell lines. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Tab.1 Relationship between FEZF1-AS1 expression in NSCLC and clinical features of the patients （n）

Clinical features	Expression of FEZF1-AS1		χ²	P
Clinical features	High expression group (n=16)	Low expression group (n=17)	χ²	P
Age (year)
<60	5	7	0.351	0.554
≥60	11	10	0.351	0.554
Gender
Male	2	9	6.066	0.014
Female	14	8	6.066	0.014
Smoking history
Yes	1	6	4.160	0.041
No	15	11	4.160	0.041
Tumor size (cm)
≤5	15	16	0.002	0.965
>5	1	1	0.002	0.965
Lymph node metastasis
Yes	13	6	7.127	0.008
No	3	11	7.127	0.008
TNM stage
Ⅰ-Ⅱ	13	14	0.000	1.000
Ⅲ-Ⅳ	3	3	0.000	1.000

Fig.2 Effect of FEZF1-AS1 on proliferation, migration, and invasion of H1299 and H358 cells. A: Fluorescence microscopy of the cells transfected with si-FEZF1-AS1. B: Knockdown efficiency of si-FEZF1-AS1 in H1299 and H358 cells. C: CCK-8 assay of proliferation of H1299 and H358 cells after transfection with si-FEZF1-AS1 and si-NC. D: Formation of H1299 and H358 clones after transfection. E: Healing of H1299 and H358 cell scratches after transfection. F: Transwell migration assay of H1299 and H358 cells after transfection. G: Transwell invasion assay of H1299 and H358 cells after transfection. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig.3 Binding of FEZF1-AS1 to hsa-miR-130a-5p. A: Prediction of binding sites between FEZF1-AS1 and hsa-miR-130a-5p. B: Dual luciferase reporter assay of the binding between FEZF1-AS1 and hsa-miR-130a-5p. ***P<0.001.

Fig.4 Effect of hsa-miR-130a-5p expression level on proliferation, migration, and invasion of H1299 and H358 cells. A: Expression of hsa-miR-130a-5p in the cells after transfection with si-FEZF1-AS1+NC inhibitor and with si-FEZF1-AS1+hsa-miR-130a-5p inhibitor. B: Effect of si-FEZF1-AS1+NC inhibitor and si-FEZF1-AS1+hsa miR-130a-5p inhibitor on cell proliferation. C: Effect of si-FEZF1-AS1+NC inhibitor and si-FEZF1-AS1+hsa miR-130a-5p inhibitor on cell migration. D: Effect of si-FEZF1-AS1+NC inhibitor and si-FEZF1-AS1+hsa-miR-130a-5p inhibitor on cell invasion. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig.5 Binding status of hsa-miR-130a-5p with CCND1. A: Volcano map of differential gene expression between si-FEZF1-AS1 (group 2) and si-NC groups (group 1) by transcriptome sequencing. B: Database prediction of downstream target proteins and transcriptome sequencing results shown in Venn diagram. C: Knockdown of FEZF1-AS1 and expression of CCND1 in H1299 cells. D: Knockdown of FEZF1-AS1 and expression of CCND1 in H358 cells. E: Prediction of binding sites between hsa-miR-130a-5p and CCND1. F: Dual luciferase reporter assay of binding between hsa-miR-130a-5p and CCND1. ***P<0.001, ****P<0.0001.

Fig.6 FEZF1-AS1/hsa miR-130a-5p regulates CCND1 expression through ceRNA mechanism. A: Expression levels of CCND1 in H1299 cells transfected with si-FEZF1-AS1+NC inhibitor and si-FEZF1-AS1+hsa-miR-130a-5p inhibitor. B: Expression levels of CCND1 in H358 cell transfected with si-FEZF1-AS1+NC inhibitor and si-FEZF1-AS1+hsa-miR-130a-5p inhibitor. C: Protein expression of CCND1 in H1299 cells transfected with si-NC, si-FEZF1-AS1, si-FEZF1-AS1+NC inhibitor and si-FEZF1-AS1+hsa-miR-130a-5p inhibitor. D: Protein expression of CCND1 in H358 cells transfected with si-NC, si-FEZF1-AS1, si-FEZF1-AS1+NC inhibitor and si-FEZF1-AS1+hsa-miR-130a-5p inhibitor. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

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