miR-335-5p Regulates Cell Cycle and Metastasis in Lung Adenocarcinoma by Targeting CCNB2

Abstract

Background: Lots of studies have shown that cyclin disorders can promote tumor development. This study aims to investigate the biological function and molecular mechanism of CCNB2 in lung adenocarcinoma (LUAD).

Methods: LUAD data were downloaded from GEO database and TCGA-LUAD database. Differential analysis was conducted to find the differentially expressed miRNAs and mRNAs, while targeted prediction was done for the access of potential target mRNAs. Gene expression level was detected by qRT-PCR and Western blot in human LUAD cell lines A-427, A549, Calu-3, PC-9 and human bronchial epithelial cell line BEAS-2B. MTT, colony formation, Transwell and flow cytometry assays were used to detect cell proliferation, metastasis, and cell cycle changes of PC-9 cell line. The dual-luciferase reporter gene was used to detect the targeted binding relationship of the target miRNA and mRNA.

Results: CCNB2 was highly expressed and served as a biomarker indicating poor prognosis in LUAD patients. Cell function experiments confirmed the inhibitory effects of silencing CCNB2 on the proliferation, migration and invasion of LUAD cells and cell cycle was blocked in the G0/G1 phase. In addition, with regard to the regulatory mechanism, we demonstrated that miR-335-5p had binding sites with 3'-UTR of CCNB2, indicating that miR-335-5p could target the regulation expression of CCNB2. In subsequent cell function tests, overexpression of miR-335-5p inhibited the proliferation and metastasis of cancer cells, and the rescue experiments also verified that miR-335-5p could reverse the promotion of CCNB2 overexpression on the progress of cancer cells.

Conclusion: In summary, our results revealed that miR-335-5p could target the down-regulation of CCNB2 to inhibit the occurrence and development of LUAD.

Keywords: CCNB2; LUAD; metastasis; miR-335-5p; proliferation.

Figure 1

The expression and prognosis of CCNB2 in LUAD. (A and B) mRNA and protein expressions of CCNB2 in BEAS-2B and LUAD cell lines (A-427, A549, Calu-3, PC-9); (C) The expression of CCNB2 was low in normal samples (green) and high in tumor samples (red); (D) Survival curves of CCNB2 expression for prognosis in the TCGA-LUAD dataset. Red indicates high expression group and blue indicates low expression group; (E–G) Box plots of CCNB2 expression in different clinical stages, N stages and T stages of LUAD. Note: *Indicates P<0.05.

Figure 2

Low expression of CCNB2 inhibits the proliferation, metastasis and cell cycle of LUAD cells. (A and B) mRNA and protein levels in CCNB2-silenced LUAD cells; (C) The MTT assay was used to measure the cell viability of LUAD cells at 24 h, 48 h and 72 h, respectively; (D) The proliferation of LUAD cells was determined by colony formation assay; (E and F) Transwell assay was used to determine the migration and invasion abilities of LUAD cells; (G) GSEA pathway enrichment analysis results of CCNB2; (H) FCM was used to analyze the proportion of cells in each cell cycle phase of LUAD cells (CCNB2-silenced cells and NC cells). Note: *Indicates P<0.05.

Figure 3

miR-335-5p targeted down-regulates CCNB2. (A) DEmiRNA volcano map of normal group and tumor group in TCGA-LUAD dataset; (B) Venn diagram of predicted upstream down-regulated DEmiRNAs for CCNB2; (C) Pearson correlation analysis of CCNB2 and miR-335-5p; (D) The miR-335-5p expression was highly expressed in normal samples (green) and lowly expressed in tumor samples (red); (E) Expression of miR-335-5p in BEAS-2B cell line and LUAD cell lines (A-427, A549, Calu-3, PC-9); (F) Survival curves of miR-335-5p expression for prognosis. Red indicates high expression group and blue indicates low expression group; (G) Binding sites of miR-335-5p and 3 ‘UTR of CCNB2; (H and I) qRT-PCR and WB were used to detect the effects of miR-335-5p expression on CCNB2 mRNA and protein levels; (J) Dual-luciferase reporter gene assay was used to determine the targeted binding of miR-335-5p and CCNB2. Note: *Indicates P<0.05.

Figure 4

Overexpression of miR-335-5p inhibits LUAD development which can be reversed by CCNB2. (A) Expression of miR-335-5p and CCNB in transfected cells was detected by qRT-PCR; (B) MTT assay was used to determine the cell viability at 24 h, 48 h, and 72 h, respectively; (C) The proliferation of LUAD cells was determined by colony formation assay; (D and E) Transwell assay was used to determine the migration and invasion ability of LUAD cells; (F) The proportion of cells in each cell cycle stage of LUAD cells was analyzed by FCM. Note: *Indicates P<0.05.