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Effects of miR-218-1-3p and miR-149 on proliferation and apoptosis of non-small cell lung cancer cells

ABSTRACT: The aim of the present study was to explore the effects of miR-218-1-3p and miR-149 on the biological function of non-small cell lung cancer (NSCLC) cells A549. Paired NSCLC and adjacent tissues were obtained from 50 NSCLC patients admitted to Shandong Provincial Chest Hospital Affiliated to Shandong University (Jinan, China) from April 2015 to May 2018. The expression levels of miR-218-1-3p and miR-149 were detected by reverse transcription-quantitative PCR (RT-qPCR). The lung adenocarcinoma A549 cells were assigned into the blank group (without transfection), negative control (NC) group (transfected with miRNA NC), and the transfected groups miR-218-1-3p mimic and miR-149 mimic groups. Proliferation and cell growth were determined by CCK-8 assay and cell invasion ability in vitro was assessed by Transwell assay. Flow cytometry was carried out for the detection of cell apoptosis. RT-qPCR results showed that the expression levels of miR-218-1-3p and miR-149 in NSCLC tissues were significantly lower than those in adjacent tissues (P<0.001). At 48 and 72 h, the cell growth of the A549 cells in the miR-218-1-3p mimic and miR-149 mimic groups was significantly lower than that in the NC and blank groups (P<0.05). The number of invasive cells in the miR-218-1-3p mimic and miR-149 mimic groups was significantly lower than that in the NC and blank groups (P<0.05). The apoptotic rate of A549 cells in the miR-218-1-3p mimic and miR-149 mimic groups was significantly higher than that in the NC and blank groups (P<0.05). In conclusion, upregulation of miR-218-1-3p and miR-149 can inhibit the proliferation, invasion and migration of A549 cells in NSCLC, thereby promoting the apoptosis of A549 cells. Thus, miR-218-1-3p and miR-149 can be used as new molecular targets for the diagnosis and treatment of NSCLC.

SUBMITTER: Guo P

PROVIDER: S-EPMC7439122 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Project description:Purpose:Non-small cell lung cancer (NSCLC) is the largest type of lung cancer (LC) with a higher mortality rate. Circular RNAs (circRNAs) have been shown to play an important role in cancer progression. Therefore, this study was to explore the function of hsa_circ_0043265 in NSCLC. Methods:The expression levels of hsa_circ_0043265, microRNA-25-3p (miR-25-3p) and forkhead box P2 (FOXP2) were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Ribonuclease R (RNase R) and Actinomycin D (ActD) were used to verify the authenticity and stability of hsa_circ_0043265. Cell counting kit-8 (CCK-8), flow cytometry and transwell assays were used to evaluate the abilities of proliferation, apoptosis, migration and invasion of NSCLC cells. Also, Western blot (WB) analysis was performed to assess the levels of apoptosis, epithelial-mesenchymal transition (EMT) and proliferation-related proteins and FOXP2 protein. RNA immunoprecipitation (RIP) and dual-luciferase reporter assays were used to verify the interaction between miR-25-3p and hsa_circ_0043265 or FOXP2. Besides, mice xenograft models were constructed to confirm the effect of hsa_circ_0043265 on NSCLC tumor growth in vivo. Results:Hsa_circ_0043265 was lowly expressed in NSCLC tissues and cells, and its overexpression inhibited the proliferation, migration, invasion and EMT process, while improved the apoptosis of NSCLC cells. MiR-25-3p could be sponged by hsa_circ_0043265, and its overexpression could invert the suppression effect of overexpressed-hsa_circ_0043265 on NSCLC progression. Moreover, FOXP2 was a target of miR-25-3p, and its silencing also could reverse the inhibition effect of overexpressed-hsa_circ_0043265 on NSCLC progression. In addition, hsa_circ_0043265 overexpression reduced the tumor growth of NSCLC in vivo. Conclusion:Hsa_circ_0043265 could sponge miR-25-3p to improve FOXP2 expression, thereby inhibiting NSCLC progression. This study showed that hsa_circ_0043265 could be a potential biomarker for early diagnosis of NSCLC.

Project description:Chronic obstructive pulmonary disease (COPD) has become a significant public health risk. Long non-coding RNAs (lncRNAs) have been identified as important factors involved in the proliferation, apoptosis and inflammatory cytokine expression of lung cells. Peripheral blood samples from 66 subjects (18 non-smokers, 24 smokers without COPD and 28 smokers with COPD) and HBE135-E6E7 cell treated with cigarette smoke extract (CSE) or not were used as the research object. The aim of the present study was to investigate the underlying mechanism of lncRNA maternally expressed gene 3 (MEG3) in COPD. Following transfection with microRNA (miR)-149-3p mimics, miR-negative control mimics, miR-149-3p inhibitor, miR-negative control inhibitor, small interfering (si)RNA targeting MEG3 (si-MEG3) and si-negative control (si-NC), levels of MEG3 and microRNA (miR)-149-3p were detected using reverse transcription-quantitative PCR, Proliferation and apoptosis were examined using the Cell Counting Kit-8 and flow cytometry assays, respectively. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the expression of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Protein levels of B-cell lymphoma-2 (Bcl-2), cleaved-caspase-3, cleaved-caspase-9, phosphorylated (p)-p65, total (t)-p65, p-lkBα and t-lkBα were measured by western blotting. Luciferase assay was conducted to examine the relationship between MEG3 and miR-149-3p. LncRNA MEG3 was highly expressed, whereas miR-149-3p expression was downregulated in smokers with COPD peripheral blood samples, compared with non-smokers and smokers without COPD samples. Compared with untreated human bronchial epithelial (HBE) cells, MEG3 expression was increased in cigarette smoke extract (CSE)-treated HBE cells. Compared with CSE-treated HBE cells transfected with si-NC, MEG3 knockdown promoted cell proliferation and inhibited apoptosis in CSE-treated HBE cells transfected with si-MEG3, and it also decreased the levels of IL-6, TNF-α, Bcl-2 and increased cleaved-caspase-3 and cleaved-caspase-9 in CSE-treated HBE cells transfected with si-MEG3. The luciferase assay demonstrated that miR-149-3p has target sites for MEG3. MEG3 was demonstrated to regulate the NF-κB signaling pathway by sponging miR-149-3p in CSE-treated HBE cells. In conclusion, these findings suggested that MEG3 promoted proliferation and inhibited apoptosis by regulating the NF-κB signal pathway via miR-149-3p in CSE-treated HBE cells. These results provide an insight for further verification and understanding of the molecular basis of COPD.

Project description:Objective:Mounting research has established the role of microRNAs (miRNAs) as oncogenes or anti-oncogenes (tumor suppressors) in the development and progression of several cancers. The purpose of our current study is to delineate the roles and functional mechanisms of miR-331-3p and MLLT10 in non-small cell lung cancer (NSCLC) tumorigenesis. Patients and Methods:Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was employed to measure miR-331-3p expression levels in twenty-six matched tumor tissues and non-cancerous tissues collected from patients suffering from NSCLC, and from six NSCLC cell lines separately: A549, H1650, H292, H1299, H1944 and BEAS-2b. We employed the dual-luciferase activity assay to check whether the putative gene, MLLT10, was a downstream target of miR-331-3p in NSCLC pathogenesis and development. Western blot was conducted to analyze the protein expression levels of MLLT10 (AF10), E-cadherin, Vimentin, and GAPDH. CCK-8 assay, transwell migration assay, and transwell invasion assay were carried out to observe the functions of miR-331-3p and MLLT10 on NSCLC tumor cell proliferation, metastasis, and invasion, respectively. To identify whether the metastasis of NSCLC tumor cells was EMT-mediated, supplementary experiments involving E-cadherin and Vimentin were implemented. Results:miR-331-3p was downregulated in NSCLC, which promoted tumor cell proliferation, whereas the overexpression of miR-331-3p inhibited tumor cell proliferation. Being a direct target of miR-331-3p, MLLT10 was negatively modulated by miR-331-3p, which suppressed tumor cell proliferation, migration, and invasion in NSCLC. However, MLLT10 overexpression alleviated the above inhibitory effects. Furthermore, EMT-mediated metastasis was proved to be present in NSCLC. Conclusion:miR-331-3p played a suppressor role in NSCLC tumor cell proliferation, EMT-mediated metastasis, and invasion by targeting MLLT10. Our findings highlighted that miR-331-3p/MLLT10 axis could be useful as a clinical diagnostic marker and therapeutic target in NSCLC patients.

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Effects of miR-218-1-3p and miR-149 on proliferation and apoptosis of non-small cell lung cancer cells

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