MicroRNA‑21 promotes the proliferation, migration and invasion of non‑small cell lung cancer A549 cells by regulating autophagy activity via AMPK/ULK1 signaling pathway

Abstract

Abstract. The present study investigated the expression of microRNA (miR)-21 in non-small cell lung cancer (NSCLC) tissues, its biological functions and mechanism of autophagy regulation. A total of 46 patients with NSCLC were enrolled in the present study. To measure the expression of miR-21, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was employed. NSCLC A549 cells were transfected with miR-negative control (NC), miR-21 mimics or inhibitor. The CCK-8 assay was used to investigate the proliferation of A549 cells. To study migration and invasion abilities of A549 cells, The Transwell assay was performed. In addition, to determine the expression levels of ULK1, LC3B, AMPKα, p-AMPKα and p62 proteins, western blotting was conducted and laser confocal microscopy was performed to observe the formation of autophagosomes in A549 cells. To explore whether miR-21 regulates the biological functions of A549 cells via autophagy, an autophagy inhibitor, 3-MA, or agonist, rapamycin, were used in a rescue assay. Results indicated that miR-21 expression in NSCLC tissues was enhanced, and closely correlated with the occurrence and development of NSCLC. In vitro experiments showed that miR-21 mimics promoted the proliferation, migration and invasion of A549 cells, while miR-21 inhibitor inhibited these biological functions. Western blotting indicated that miR-21 upregulated autophagy marker LC3BⅡ protein, but downregulated p62 protein. Laser confocal microscopy showed that miR-21 activated autophagy of A549. Rescue experiments indicated that autophagy reversed the effect of miR-21 on the proliferation, migration and invasion of A549 cells. Western blotting data suggested that autophagy-related AMPK/ULK1 signaling pathway was activated by miR-21, and interference or overexpression of ULK1 reversed the biological functions of miR-21. The present study demonstrated that miR-21 expression in NSCLC tissues was upregulated and positively correlated with lymphatic metastasis and clinical staging. In addition, miR-21 regulated autophagy activity of NSCLC A549 cells via AMPK/ULK1 signaling pathway, and promoted the proliferation, migration and invasion of NSCLC A549 cells.

The Effect of NNK, A Tobacco Smoke Carcinogen, on the miRNA and Mismatch DNA Repair Expression Profiles in Lung and Head and Neck Squamous Cancer Cells

“…In general, the deregulation of microRNA expression has been associated with gene alterations that are commonly linked to smoke-related cancers [67][68][69]. The "oncomirs" miR-21 and miR-155 have previously been associated with cancers of the upper aerodigestive tract [48,49,[51][52][53][54]. In particular, miR-21 has been considered as a marker of poor prognosis in lung and head and neck cancer [70,71].…”

“…Hypo-or hyper-methylation of miRNA was considered to represent a new level of complexity in gene regulation in human cancers [81], suggesting miR-21 or miR-155 promoter hypo-methylation [81][82][83][84] and miR-422a hyper-methylation, as previously reported for miR-373 [81,85], as potential epigenetic modifications caused by tobacco carcinogenic effects on MMR. On the other hand, alkylating agents, such as NNK can also directly or after biological activation react and form covalent bonds with nucleophilic centers found in DNA and RNA and proteins [86], supporting possible direct interference of NNK with levels of miRNAs, thereby causing their deregulation [48,49,[51][52][53][54]. Subsequently, NNK-induced miRNA deregulations can affect MMR gene expression, either thought post-transcriptional modifications or through DNA methylation by targeting DNA methyltransferases or methylation-related proteins [81].…”

“…Some reports have also shown that oncomir miR-21 downregulates hMSH2 gene expression by targeting the 3 untranslated region of its mRNA [45], and that miR-155 can significantly downregulate hMSH2, hMSH6, and hMLH1 [46], while others have suggested that miRNAs play an important role in modulating cell cycle progression by targeting hMSH2 in lung cancer [42]. Although there are reports suggesting a relationship between the MMR mechanism and miRNA profiles [41,43,44,46], the underlying molecular mechanism by which tobacco smoke carcinogens induce miRNA deregulation and affect the expression profiles of mismatch repair genes, particularly in lung and head and neck cancer, is not yet known.Here, we attempt to explore whether NNK affects the expression of small regulatory molecules, such as known miRNA markers, previously associated with upper aerodigestive tract malignancies [47][48][49][50][51][52][53][54] that may directly or indirectly be involved in the regulation for MMR expression phenotypes. Understanding the molecular changes induced by various risk factors, such as tobacco smoke, which promote the development and progression of cancer, will help to develop new diagnostic and therapeutic approaches [55,56], leading to optimization of their management.…”

“…Here, we attempt to explore whether NNK affects the expression of small regulatory molecules, such as known miRNA markers, previously associated with upper aerodigestive tract malignancies [47][48][49][50][51][52][53][54] that may directly or indirectly be involved in the regulation for MMR expression phenotypes. Understanding the molecular changes induced by various risk factors, such as tobacco smoke, which promote the development and progression of cancer, will help to develop new diagnostic and therapeutic approaches [55,56], leading to optimization of their management.…”

MicroRNA‐374b mediates the initiation of non‐small cell lung cancer by regulating ITGB1 and p53 expressions

“…For example, miR‐577 inhibits cell proliferation and epithelial‐mesenchymal transition (EMT) in NSCLC . Conversely, miR‐21 promotes cell proliferation, migration and invasion in NSCLC . Now, due to the dysregulation of miR‐374b function in human cancer, it has attracted our attention.…”