Inhibition of miR-191 contributes to radiation-resistance of two lung cancer cell lines by altering autophagy activity
© Liu and Huang; licensee BioMed Central. 2015
Received: 28 November 2014
Accepted: 20 January 2015
Published: 4 February 2015
Lung cancer is the leading cause of cancer-related morbidity and mortality all over the world. Surgery resection, radiotherapy, chemotherapy, immunotherapy and combined treatments have been discovered and well established for treatments. However, low survival rate of five years after clinical treatments mainly due to recurrence of stress-resistant cancer cells calls for better understanding and new ideas. Our project aimed to understand the forming process of stress resistant lung cancer cells after radiotherapy.
Two classic non-small cell lung cancer (NSCLC) cell lines A549 and H1299 initially were radiated with a 137Cs gamma-ray source with doses ranging from 0 to 12 Gy to generate radiation-resistant cancer cells. 8 Gy of radiation was regard as a standard dosage since it provides effective killing as well as good amount of survivals. The expression levels of autophagy-related proteins including Beclin-1, LC3-II and p62 were studied and measured by both western blot and quantitative real-time polymerase chain reaction (real-time RT-PCR).
Increased Beclin-1, LC3-II and decreased p62 have been observed in radiation-resistant cells indicating elevated autophagy level. Decreased miR-191 in radiation-resistant cells performed by Taqman qRT-PCR also has been seen. Two binding sites between Beclin-1 and miR-191 suggest potential association between.
It is reasonable to speculate that inhibition of miR-191 expression in lung cancer cells would contribute to the establishment of radiation-resistant cells via mediating cellular autophagy. Therefore, miR-191 is a potential target for therapy in treating radiation-resistant lung cancer.
Lung cancer is the leading cause of cancer-related mortality and morbidity and it is one of the predominant life-threatening conditions among cancers. For 50 years both the morbidity and mortality rates from many countries have increased significantly . However, the exact cause underlying lung cancer is still unrevealed. Improvements in long term survival rate have been achieved by early diagnosis and combinations of chemo/radiotherapy and surgery. However, the recurrence rate is still high and five years survival for NSCLC patients remains as low as 15% . One main factor could be the resistant cancer cells after certain therapies.
For now, there is no medicine shown to be significantly effective and consistent to treat lung cancer. When feasible, surgical resection is still the single most effective and successful option especially for early-stage patients [3-5]. There are two main types of lung cancer, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC accounts for around 84% of all the cases. NSCLC patients are more likely to be cured with surgery resection if discovered at an early stage . Radiotherapy, chemotherapy, immunotherapy and combined treatments would be chosen based on tumour stage and histologic type based on accurate diagnosis. Radiotherapy is the most important localised treatment across every clinical phase of NSCLC patients. However, the records of the past two decades shown that only 5-10% of patients survived 5 years after clinical therapy . Malissard et al. has shown that there is a tight correlation between recurrence and distant metastasis , therefore recurrence is the main cause responsible for failed treatment and the occurrence of metastasis of NSCLC. The recurrence is frequently associated with stress-resistant cancer cells after therapies . A study focusing on 598 stage I NSCLC patients who underwent surgery resection illustrated around 27% of overall recurrence incidences . Our project aimed to understand the forming process of stress-resistant lung cancer cells after radiotherapy. In our experiment, two classic NSCLC cell lines A549 and H1299 were chosen. Survived cells with three week’s radiation were used as the cell model after radiotherapy for further experiments.
Autophagy is a catabolic process to breakdown and recycles dysfunctional cellular components such as organelles and proteins by isolating these selected components inside a double membrane-bound vesicle. This function is used as a survival mechanism by starving cells [11,12]. When it comes to cancer cells, it could maintain the metabolism of mitochondrial, improve cell proliferation and increase stress tolerance. All of these effects together would eventually contribute to the level of malignance. Additionally, it may interfere with chemotherapy and radiation-induced apoptosis through removal of damaged organelles . These evidences suggest the role autophagy might play in tumor development . It hints that inhibition of autophagy activity is the effective way to suppress the development of cancer. MicroRNA is a single-stranded small sequence of non-coding RNA molecule with length of 20–24 nucleotides which accounts for post-transcriptional regulation of gene expression [15,16]. MicroRNA could silence gene expression by specific binding through base pairing with complementary mRNA sequence followed by degradation of the target mRNA . Therefore it plays a crucial role in regulation of gene expression, cell cycling and developmental timing . It is estimated that miRNAs modulates over one-third of human genes and various biological pathways . Some studies illustrated degradation of miRNA might contribute to the developments of various diseases including cancer . The link established between autophagy and miRNA is the possible regulation function of miRNA upon autophagy . Dysfunctional miRNAs are frequently found in malignancies where they function as either oncogenes or tumor suppressors . MiRNA has been suggested to prevent the development of lung cancer by inhibiting some specific function proteins involved in autophagy pathway. So far some researches have pointed out the potential correlation between dysfunctions of miRNA-186, 143 , 17–92 cluster [23,24] and the progress of lung cancer. In this report, we also found the reduced expression level of miRNA-191 in two types of NSCLC based on miRNA microarray techniques. However, the link between miRNA-191 and autophagy has not been reported in other studies. Taken together, we propose that the inhibition mechanism to lung cancer by miRNA-191 is conducted by targeting specific proteins of autophagy pathway, which eventually inhibit the growth and proliferation of lung cancer cells. The phenomenon could be helpful for therapy of lung cancer in the future.
Results and discussion
Various studies have illustrated the significant role autophagy plays during the progress of tumor cells, including representing cell survival mechanism as well as tumor suppression. In our experiments, autophagy related proteins such as Beclin-1 and LC3-II were found to be upregulated in two NSCLC radiation-resistant cell lines, indicating enhanced level of autophagy in radiation-resistant cancer. Increasing amount of studies were performed to understand tumor biology from microRNA profile perspective.
Establishment of radiation-resistant cancer cells
Autophagy-associated protein profile
Association between miR-191 and autophagic marker
MiR-191 modulated Beclin-1 expression
To summarize our study, we performed high-throughput microarray assay and found significantly decreased level of miR-191 in radiation-resistant cells than normal non-treatment cancer cells. Base-paired sequences between miR-191 and Beclin-1 reveal certain correlation between them. Taken together we speculate that miR-191 level reduces in survived radiation-resistant lung cancer cells, leading to increased autophagy level. Our study provides evidence supporting the role of miR-191 as potential therapeutic target in treating of lung cancer, especially in radiation-resistant cases in the future.
Cell culturing and proliferation
Cell culturing and established models of radiation-resistant cell lines A549 and H1299 cells were harvested by exposing to trypsin. Cell suspensions were irradiated using a 137Cs gamma-ray source with doses ranging from 0 to 12 Gy and plated in 6 well plates, 500 cells per well. Cultures were incubated at 37°C in 5% CO2. Optical density of each group with different dosages of radiation was detected at 7 times by using CCK-8 (Day 2, 4, 7, 10, 14, 18 and 21). The 8 Gy was chosen as a standard radiation dosage for the next test. Both normal and 8 Gy radiation-treated A549 and H1299 after 3 weeks were stained with DAPI (blue) for cell viability. Images were acquired under fluorescent microscopy.
CCK-8 staining was used to detect cell growth conditions. 100 μl of cell suspension (5000 cells/well) obtained from 2, 4, 7, 10, 14, 18 and 21 days after radiation treatment were dispensed into a 96-well plate. This plate was pre-incubated for 24 hours in a humidified incubator (e.g., at 37°C, 5% CO2). 10 μl of various concentrations of substances were added to the plate to be tested. The plate was then incubated for an appropriate length of time (e.g. 6, 12, 24 or 48 hours) in the incubator. 10 μl of CCK-8 solution was added to each well of the plate. Incubate the plate for 1–4 hours in the incubator. Measurement of the absorbance at 450 nm was performed by a microplate reader.
Expression levels of Beclin-1, LC3-II and p62 were determined by Western blot. Cells were harvested and lysed with lysis buffer (RIPA, Abcam). 20 μl 2 × sample loading buffer was added into each sample (0.125 M of 5 M Tris–HCl, amresco; 20% glycerol, usb; 4% of 10% sodium dodecyl sulfate, amresco; 1% β-mercaptoethanol, amresco; 0.2% of 0.05% (w/v) bromophenol blue, sigma). GAPDH was used as a loading control. 10% running gel (25% of 40% acrylamide stock, Beyotime; 0.375 M of 1.5 M Tris–HCl, pH 8.8; 1% of 10% sodium dodecyl sulfate; 1% of 10% ammonium persulfate; 0.1% tetramethylethylenediamine) was utilized for samples that have been boiled for 5 minutes. The gel was transferred to a same size membrane (Nitrocellulose transfer membrane, Protian) within transfer buffer (25 mM Tris base, 192 mM glycine, 0.037% sodium dodecyl sulfate, and 20% methanol) under 45 V for 40 min. The membrane was then incubated in primary antibody (Beclin-1, GAPDH, Abcam) with a 1/1000 dilution in blocking buffer (50 mM Tris base; 100 mM NaCl; 0.02% Tween 20; and 3% BSA) overnight. The membrane was rinsed by TTBS (0.1% Tween 20, 10 mM Tris base, 100 mM NaCl, pH 7.5) for three times before adding secondary antibody (Abcam) with 1/5000 dilution in blocking buffer for 2 hours. Background noise was reduced by careful wash. The results were visualized using ECL kit (Abcam) and observed by GeneGnome mechine (Syngene).
mRNA of Beclin-1, LC3-II and p62 from cells were extracted by RNeasy Mini Kit (Qiagen). NanoDrop 8000 spectrophotometer (Thermo Scientific) was used to determine the quality and quantity of extracted RNA. 1000 ng RNA was used for each reaction to produce cDNA using high capacity cDNA reverse transcription kit (Applied biosystems) following manufacturer’s instructions. The reaction was initiated at 25°C for 5 min followed by annealing at 50°C and subsequent elongation at 70°C. cDNA products were then diluted by adding DNase and RNase free water to 250 μl and frozen at −20°C for further gene expression assay. 10 μl 1 × PCR master mix, 5 μl diluted cDNA, 4 μl water and 1 μl probe were mixed together in every reaction. Gene expression was measured with quantitative real-time RT-PCR system. Taqman real-time RT-PCR was performed to specifically detect the relative levels of miR-191 in radiation resistant and no radiation resistant H1299 for higher sensitivity.
Micro-RNA profiles in radiation-resistant cells
Overexpressed and downexpressed miRNAs in both A549 and H1299 were screened and selected based on miRNA microarray assay. Taqman real-time qRT-PCR was performed to detect the relative levels of miR-191 in radiation resistant and non-treatment cells. miR-191 was normalized against U6 spliceosomal RNA (U6 snRNA).
Cell viability evaluation with additional miR-191
Radiation resistant A549 and H1299 cells were infected with Lenti-virus-miR-191 for 48 h. The cells with or without treatment of Lenti-virus-miR-191 were subjected to FACS analysis after being stained by Trypan blue for the measurement of cell viability (n = 3).
The authors thank Jieping Zhang and Wenjuan Wu for the discussion. This work was supported by Tianjin Talented Graduate Fund.
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