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

Abstract

Objective: To explore the molecular mechanism by which FEZF1-AS1 overexpression promotes progression of nonsmall 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.

Keywords: CCND1; FEZF1-AS1; lncRNA; miR-130a-5p; non-small cell lung cancer.

图1

FEZF1-AS1在NSCLC中的表达情况 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.

图2

FEZF1-AS1对H1299与H358的增殖、迁移、侵袭功能的影响 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.

图3

FEZF1-AS1与hsa-miR-130a-5p的结合情况 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.

图4

hsa-miR-130a-5p对H1299与H358的增殖、迁移、侵袭功能的影响 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.

图5

hsa-miR-130a-5p与CCND1的结合情况 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.

图6

FEZF1-AS1/hsa-miR-130a-5p通过ceRNA机制调控CCND1的表达 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.