microRNA-145-3p inhibits non-small cell lung cancer cell migration and invasion by targeting PDK1 via the mTOR signaling pathway.
The mammalian target of rapamycin (mTOR) pathway is dysregulated in more than 50% of all human malignancies and is a major target in cancer treatment. In this study, we explored the underlying mechanism involving microRNA-145-3p (miR-145-3p) in the development and progression of non-small cell lung cancer (NSCLC) by targeting PDK1 via the mTOR signaling pathway. NSCLC tissues and adjacent normal tissues were obtained from 83 NSCLC patients. miR-145-3p, PDK1, and mTOR levels were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry. Human NSCLC cell lines A549 and H1299 were transfected with miR-145-3p and siPDK1 to confirm the effect of miR-145-3p and PDK1 on NSCLC cells in vitro. Cell growth was evaluated by a CCK8 assay. Cell motility and chemotaxis analysis were determined by the scratch test and chemotaxis assay, respectively. The protein levels of PDK1 and mTOR were measured using the western blotting. Results showed lower level of miR-145-3p and higher levels of PDK1 and mTOR in NSCLC tissues compared to the adjacent normal tissues. In vitro results showed that cell growth, cell motility, and chemotaxis were all inhibited in cells transfected with miR-145-3p and those transfected with siPDK. Additionally, dual luciferase reporter gene assay helped confirmed that PDK1 is a target of miR-145. Finally, levels of PDK1, mTOR, and phosphorylated-mTOR were lower in cells transfected with miR-145-3p as well as those with siPDK1. These findings indicate that miR-145-3p may inhibit cell growth, motility, and chemotaxis in NSCLC by targeting PDK1 through suppressing the mTOR pathway.
Chen GM
，Zheng AJ
，Cai J
，Han P
，Ji HB
，Wang LL
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Effect of microRNA-135a on Cell Proliferation, Migration, Invasion, Apoptosis and Tumor Angiogenesis Through the IGF-1/PI3K/Akt Signaling Pathway in Non-Small Cell Lung Cancer.
This study explored the ability of microRNA-135a (miR-135a) to influence cell proliferation, migration, invasion, apoptosis and tumor angiogenesis through the IGF-1/PI3K/Akt signaling pathway in non-small cell lung cancer (NSCLC).
NSCLC tissues and adjacent normal tissues were collected from 138 NSCLC patients. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression levels of miR-135a and IGF-1, PI3K, Akt, VEGF, bFGF and IL-8 mRNA; western blotting was used to determine the expression levels of IGF-1, PI3K and Akt protein; and enzyme-linked immunosorbent assay (ELISA) was used to analyze the expression levels of VEGF, bFGF and IL-8 protein. Human NSCLC cell lines (A549, H460, and H1299) and the human bronchial epithelial cell line (HBE) were selected. A549 cells were assigned to blank, negative control (NC), miR-135a mimics, miR-135a inhibitors, IGF-1 siRNA and miR-135a inhibitors + IGF-1 siRNA groups. The following were performed: an MTT assay to assess cell proliferation, a scratch test to detect cell migration, a Transwell assay to measure cell invasion, and a flow cytometry to analyze cell apoptosis.
The expression level of miR-135a was lower while those of IGF-1, PI3K and Akt mRNA were higher in NSCLC tissues than in the adjacent normal tissues. Dual-luciferase reporter assay indicated IGF-1 as a target of miR-135a. The in vitro results showed that compared with the blank group, cell proliferation, migration and invasion were suppressed, mRNA and protein levels of IGF-1, PI3K, Akt, VEGF, bFGF and IL-8 were reduced, and cell apoptosis was enhanced in the miR-135a mimics and IGF-1 siRNA groups. Compared with the IGF-1 siRNA group, cells in the miR-135a inhibitors + IGF-1 siRNA group demonstrated increased cell proliferation, migration and invasion, elevated mRNA and protein levels of IGF-1, PI3K, Akt, VEGF, bFGF and IL-8 and reduced cell apoptosis.
These findings indicated that miR-135a promotes cell apoptosis and inhibits cell proliferation, migration, invasion and tumor angiogenesis by targeting IGF-1 gene through the IGF-1/PI3K/Akt signaling pathway in NSCLC.
Zhou Y
，Li S
，Li J
，Wang D
，Li Q
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MiroRNA-31-3p Promotes the Invasion and Metastasis of Non-Small-Cell Lung Cancer Cells by Targeting Forkhead Box 1 (FOXO1).
To explore the possibility of microRNA miR-31-3p as a biomarker for bone metastasis of non-small-cell lung cancer (NSCLC) and its molecular mechanism to the invasion and metastasis of NSCLC cells.
Real-time quantitative PCR (RT-qPCR) was used to detect the expression levels of miR-31-3p and forkhead box 1 (FOXO1) in NSCLC tissues, serum, and cells to analyze the correlation between the expression levels of miR-31-3p and the clinicopathology of NSCLC. After interference with or overexpressing miR-31-3p, NSCLC cell proliferation, apoptosis, invasion ability, and migration ability were detected by MTT, flow cytometry, Transwell, and scratch experiment, respectively. The interaction between miR-31-3p and FOXO1 was further verified by the dual-luciferase reporter experiment. Western blot was performed to detect the protein expression of FOXO1 in tissues and FOXO1, RhoA, p-RhoA, ROCK-2, and p-ROCK-2 in cells.
In tissues, serum, and NSCLC cell line A549 of the NSCLC patients, the expression of FOXO1 was notably lower, and the miR-31-3p expression was significantly higher. Overexpression of miR-31-3p could distinctly improve the proliferation, invasion, and migration of A549 cells, meanwhile inhibit cell apoptosis, and activate the RhoA/ROCK-2 signaling pathway, while interfering with the expression of miR-31-3p has the opposite function. Besides, bioinformatics analysis and luciferase reporter assay confirmed that FOXO1 was a target gene of miR-31-3p. Overexpressing FOXO1 could inhibit the proliferation and metastasis of A549 cells, but overexpressing miR-31-3p reverses the results.
This study confirmed that miR-31-3p promotes the proliferation, invasion, and migration of NSCLC cells and inhibits apoptosis through targeted regulating FOXO1 and be a potential therapeutic targets for the treatment of NSCLC.
Zeng X
，Liu D
，Peng G
，Liu J
，Yang H
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ResearchGate
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