Lung cancer is the most common cause of cancer-related death in the world, threatening severely human health. During the last 10 years, over one million persons died from lung cancer all over the world (Parkin et al.,1999; Spira and Ettinger,2003; Parkin et al.,2005). Five-year survival rate of lung cancer was approximately lower than 15% and ∼90% of deaths from tumors are caused by metastasis. Metastasis is a complex, multistep process by which primary tumor cells invade adjacent tissue, enter the systemic circulation, translocate through the vasculature, arrest in distant capillaries, extravasate into the surrounding tissue parenchyma, and finally, proliferate from microscopic growths into macroscopic secondary tumors (Fidler,2003; Weigelt et al.,2005; Gupta and Massague,2006). In recent years, more studies have been carried out to investigate the genes and gene products that drive the metastatic process (Sinha et al.,1998).
MicroRNAs (miRNAs) are a class of 19- to 30-nucleotide-long, noncoding RNAs widely expressed in eukaryotes and predominantly inhibit gene expression at the post-transcriptional level (Bartel,2004; Du and Zamore,2005; Engels and Hutvagner,2006; Valencia-Sanchez et al.,2006). Up to now, a large body of studies have documented many abnormal miRNAs expression patterns in diverse human malignancies, some of them act as oncogenes while others as tumor suppressor genes. This suggests that the role of different miRNAs may vary in carcinogenesis that requires careful examination.
More recently, miR-126, miR-335, and miR-206 may function as metastasis suppressor miRNAs in human breast cancer (Tavazoie et al., 2008), and miR-126 has been proved to have effects on the invasion breast cancer and lung cancer (Musiyenko et al.,2008; Negrini and Calin,2008). However, the expression and function of miR-206 in lung cancer were poorly understood. Here, we examined the expression of miR-206 in lung tissues and lung cancer cell lines (LCCLs), including high metastasis LCCLs.
MATERIALS AND METHODS
Cell Lines and Tissue Samples
Human LCCLs (SPC-A-1, A549, 95D, LTEP-Sm1, NCI-H226, and NCI-H520) and human normal bronchial epithelial cell line were purchased from Chinese Academy of Sciences Cell Bank. Fresh frozen human normal lung tissues and tumors were obtained after informed consent from the patients in Department of Thoraco-Cardiac Surgery, the First Affiliated Hospital of Soochow University.
Cell Culture and Materials
The seven cell lines were maintained in a 37°C, 5% CO2 incubator (Thermo) in DMEM supplemented with 10% fetal bovine serum (Hyclone). 2′-O-methyl (2′-O-Me)-hsa-miR-206 mimics were chemically synthesized by Shanghai GenePharma Company (Shanghai, China). Sequences were as follows: 2′-O-Me-hsa-miR-206: 5′-TGGAATGTAAG GAAGTGTGTGG-3′ and nonspecific control miRNA: 5′-UUCUCCGAACGUGUCACGU-3′.
MiRNA mimics were transfected by INTERFERin™ (Shanghai DAKEWEI) reagent according to the manufacturer's instructions. The cell lines were transfected with 50 nmol/L FITC-conjugated miR-206 and were analyzed by flow cytometry analysis 24 hr later.
RNA Isolation and Real-Time RT-PCR
Total RNA from tissues or cultured cells was isolated using mirVana™ miRNA Isolation Kit (Ambion) for miR-206 analyses. Synthesis of cDNA with reverse transcriptase was performed by NCode™ miRNA quantitative RT-PCR Kits (Invitrogen). For analysis of miRNA expression, real-time PCR analyses were carried out using SYBR® Green Reagents (Biosystems) according to the manual. Relative expression was calculated using the ΔΔCT method. All real-time RT-PCR were performed on a 7500 Real-Time PCR System. Sequences were as follows: human 18S rRNA: 5′-GTGAACCTGCGGAAGGATCA-3′, miR-206: 5′-CCACACACUUCCUUACAUUCCA-3′.
Detection of Apoptosis by Annexin-V and Propidium Iodide Staining
Cell lines were transfected with miR-206 or nonspecific control miRNA (N.C.) at 50% confluence. After 48 hr, the adherent cells were harvested by trypsinization. The cells were washed with PBS for once and resuspended in 500 μL of 1 × binding buffer (Annexin-V/FITC Kit; Sigma). Then, 10 μL of Annexin-V/FITC and 5 μL of propidium iodide were added to the binding buffer, and incubate the tubes at room temperature for exactly 10 min in dark. Thus, the fluorescence of the cells was determined immediately with a flow cytometer. Apoptosis cells can be stained with either the propidium iodide solution or Annexin-V/FITC (Tebar et al.,2001).
Cellular Proliferation Assay
Cell counts were measured using the Cell Counting Kit-8 (DOJINDO Japan). Two thousand cells per well were seeded in a 96-well plate and incubated for 24 hr, and the cells were transfected with miR-206 or nonspecific control miRNA at the final concentration of 50 nm/L. Ten microliters of Cell Counting Kit-8 was added to 100-μL cell culture media and incubated for 2 hr in CO2 incubator at 0, 24, 48, 72, 96, and 120 hr after transfection. Absorbance was measured at 450 nm. Three independent experiments were performed.
Biocoat Matrigel invasion chambers and wells (BD Biosciences, Bedford, MA) were rehydrated with 500-μL serum-free medium at 37°C for 2 hr. The 95D cells with miR-206 or nonspecific control miRNA were trypsinized, quenched with PBS plus 0.1% bovine serum albumin, and counted. After removing rehydration medium, we added 750-μL medium plus 10% fetal bovine serum to each well of the 24-well plate and then added immediately 1 × 105 cells in 500-μL serum-free medium plus the miR-206 (50 nM) or N.C. (50 nM) for each chamber, respectively. Plates were incubated for 24 hr at 37°C. Then the inserts were removed and the noninvading cells on the upper surface were removed with a cotton swab after 24 hr. The cells on the lower surface of the membrane were fixed in 100% methanol for 15 min, air-dried, and stained with gentian violet stain for 15 min. The cells were recorded with a digital camera.
Wound Healing and Cell Motility Assay
After 24 hr, 95D cells were transfected with miR-206 (50 nM) or nonspecific control miRNA (N.C., 50 nM) and were allowed to grow to confluence. A linear wound was created by scraping the wells with a 200-μL pipette tip. The floating cells were removed by gentle washes with culture medium. The healing process was examined dynamically and was recorded with a digital camera at 24 hr after the wound was created.
Data shown in the graphs represent the mean values ± SD of three independent experiments performed. Statistical analyses were performed using the student's t test. P values of less than 0.05 were considered statistically significant. All statistical analyses were performed by SigmaPlot 10 statistical analysis software (Systat Software).
Real-Time RT-PCR Analysis
We used real-time PCR to detect whether hsa-miR-206 was involved in LCCLs, including SPC-A-1, A549, 95D, LTEP-Sm1, NCI-H226, and NCI-H520, human normal bronchial epithelial cell line, and tissues. The expression of miR-206 was significantly lower in 95D than other LCCLs (Fig. 1A). MiR-206 expression levels were significantly reduced in high metastasis tumors compared with normal lung tissues and no transfer or only regional lymph node metastasis (low metastasis; Fig. 1B).
Cell Proliferation Rate and Cell Apoptosis Assessment After Transfection of miR-206
Cell proliferation was dramatically decreased in the cells after 24-hr transfection with miR-206 (Fig. 2A). To determine whether the poor cell proliferation was due to the increased apoptosis by overexpression of miR-206, we performed cell apoptosis assay. The results revealed that N.C. 95D cells transfected with miR-206 presented more apoptotic compared with the cells transfected with nonspecific control miRNA (Fig. 2B).
MiR-206 Slowed the Migration and Invasion Ability of Lung Cancer Cells In Vitro
Cell migration and invasion are two essential processes during cancer metastasis. Thus, we first examined the cell invasive capability to explore the potential role of miR-206 in lung cancer metastasis. Transwell Insert results showed that cells transfected with miR-206 invaded more decreasingly to the membrane than cells transfected with nonspecific control miRNA (Fig. 3). Then, we assayed the migration of 95D cells using scratch-wound model. As shown in Fig. 4, cells transfected with miR-206 closed the wound more slowly than untreated cells. Consisting with the migration results, expression of miR-206 in 95D cells inhibited significantly their migration. Taken together, the results demonstrated that miR-206 played an inhibitory role in 95D cells migration and invasion in vitro.
Metastasis is a complex and multistep process, which begins when cancer cells break away from their neighbor cells and invade through the basement membrane (Ma and Weinberg,2008). As the surprising discovery of miRNAs, a lot of studies focused on the relationship between cancers and dysregulated miRNAs. Indeed, each miRNA is predicted to regulate hundreds of mRNAs, suggesting that different miRNA may have different functions. In particular, miRNAs, acting as agents of the RNA interference pathway, can result in silencing of their cognate target genes and control a wide range of biological processes (Ambros,2004; Bartel,2004; Carthew,2006). MiRNAs have been implicated in the regulation of a variety of cellular processes, including apoptosis, hematopoietic differentiation, metabolism, skin morphogenesis, and neural development (Brennecke et al.,2003; Chen et al.,2004; Poy et al.,2004; Schratt et al.,2006; Yi et al.,2006). Furthermore, aberrant expression of miRNAs has been associated with human diseases, including lung cancer.
More recently, miR-126, miR-335, and miR-206 may function as metastasis suppressor miRNAs in human breast cancer (Tavazoie et al., 2008). Although miR-126 has been proved to have effects on the invasion of cancer, including lung cancer (Musiyenko et al.,2008; Negrini and Calin,2008), an association of expression and function of miR-206 with lung cancer was unclear. The miR-206 gene is located at chromosome 6p12.2, and it is conserved noncoding RNAs of ∼21 nucleotides that regulate translation and stability of target mRNAs based on sequence complementarity.
In the present study, quantitative real-time RT-PCR was used to detect the precise expression of miR-206 in the LCCLs and tumor tissues. The results showed that the level of miR-206 is remarkably lower in 95D cancer cells and high metastasis tumor tissues, suggesting that miR-206 might be involved in human lung cancer metastasis. We ectopically raised the miR-206 level in lung cancer cells to estimate its influence on cell growth, apoptosis, and invasion. Coincided with our expectation, increasing the level of miR-206 in lung cancer cells could obviously attenuate 95D cell growth and promote cell apoptosis. Transwell assay revealed that overexpression of miR-206 significantly diminished the invasion ability of 95D cells. Wound healing experiment demonstrated that miR-206 played an inhibitory role in 95D cells migration in vitro. Taken together, expression level of miR-206 was inversely correlated with the metastatic potential of lung cancer, which may contribute to inhibition of lung cancer metastasis. Interestingly, miR-206 expression is markedly decreased in ERalpha-positive human breast cancer tissues (Kondo et al.,2008), suggesting that miR-206 might pay a role in development and progression of lung cancer by regulating targeted ER-a gene (Adams et al.,2007).
In summary, we proved that miR-206 was markedly downregulated in human LCCLs and high metastasis tumor tissues. Increasing the expression of miR-206 may lead to cell proliferation arrest and decrease of lung cancer cells invasion ability. The findings suggest that the miR-206 might have a close relation with metastasis of lung cancer.
The authors appreciate sincerely the patients with lung cancer for their cooperation and participation.