Dataset Information

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.

SUBMITTER: Wang X

PROVIDER: S-EPMC7335273 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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

Wang Xiyong X Xiao Huaiqing H Wu Dongqiang D Zhang Dongliang D Zhang Zhihao Z

OncoTargets and therapy 20200630

<h4>Background</h4>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).<h4>Methods</h4>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 detec ...[more]

PMID: 32636645

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Project description:ObjectiveThis study aimed to describe the mechanism of exosome-derived miR-486-5p underlying the cell cycle and progression in lung adenocarcinoma (LUAD).MethodsBioinformatics methods were applied for identifying the differentially expressed genes (DEGs) in the GEO-LUAD dataset, predicting where the potential target miRNA was expressed and exploring the corresponding downstream target mRNA. qRT-PCR was conducted to detect the levels of the target genes in cancer cells. Thereafter, a series of in vitro experiments were performed for cell activities evaluation, including CCK-8, EdU, colony formation assay and transwell. Besides, Western blot was applied to determine the protein levels of the migration and invasion-related factors (NEK2, E-cadherin, N-cadherin, Vimentin, MMP-2, and MMP-9). Dual-luciferase reporter gene assay was employed for validating the targeted relationship between the target genes. Furthermore, nude mouse transplantation tumor experiment was conducted to further validate the role of the target miRNA in tumor development, and immunohistochemistry was used for Ki67 detection and TUNEL was applied for cell apoptosis assay.ResultsmiR-486-5p was observed to be enriched in serum exosomes, and seen to be significantly down-regulated in cancer tissues as well as in cancer serum exosomes. It was proven that exosomes could release miR-486-5p, thus regulating LUAD progression and affecting cell cycle. Moreover, NEK2 was identified as a target of miR-486-5p both in vivo and in vitro. Enrichment analysis revealed that NEK2 was mainly activated in cell cycle and mitosis-related pathways. Meanwhile, NEK2 was found to present significant difference in different TNM stages. Furthermore, rescue experiments indicated that the inhibitory effect of miR-486-5p overexpression on LUAD progression could be abrogated when miR-486-5p and NEK2 were simultaneously up-regulated.ConclusionExosome-derived miR-486-5p is responsible for cell cycle arrest as well as the inhibition of cell proliferation and metastasis in LUAD via targeting NEK2.

Project description:BackgroundLung adenocarcinoma (LUAD) is currently the leading cause of cancer-related death worldwide. Long noncoding RNAs (lncRNAs) play key roles in tumor occurrence and development as crucial cancer regulators. The present study aimed to explore the molecular mechanism and regulatory network of Linc00511 in LUAD and to identify new potential therapeutic targets for LUAD.MethodsReal-time quantitative polymerase chain reaction (RT-qPCR) was performed to determine the relative Linc00511 levels in LUAD tissues and cells. The proliferation, apoptosis, migration, and invasion abilities of LUAD cells were assessed by a Cell Counting Kit-8 (CCK-8) assay, a colony formation assay, flow cytometry, and a Transwell assay. Changes in hsa_miR-126-5p, hsa_miR-218-5p, and COL1A1 expression were analyzed using western blotting and RT-qPCR. Targeted binding between miR-126-5p/miR-218-5p and Linc00511 or COL1A1 was verified with a luciferase reporter system and confirmed by an RNA pulldown assay. The participation of the PI3K/AKT signaling pathway was confirmed via western blotting. Xenograft animal experiments were performed to detect the impact of Linc00511 on LUAD tumor growth in vivo.ResultsIn the present work, we observed that Linc00511 was upregulated in LUAD tissues and cells. Loss/gain-of-function experiments indicated that knockdown of Linc00511 significantly inhibited LUAD cell proliferation, migration and invasion and promoted LUAD cell apoptosis, whereas overexpression of Linc00511 showed the opposite effects. In addition, we determined that Linc00511 promoted COL1A1-mediated cell proliferation and cell motility by sponging miR-126-5p and miR-218-5p. Moreover, Linc00511 activated the PI3K/AKT signaling pathway through upregulation of COL1A1. Finally, silencing of Linc00511 inhibited LUAD tumor growth in vivo.ConclusionsLinc00511 acts as a competing endogenous RNA to regulate COL1A1 by targeting miR-126-5p and miR-218-5p, thereby promoting the proliferation and invasion of LUAD cells.

Dataset Information

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

Publications

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

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