MicroRNAs (miRNAs) represent a group of novel therapeutic small molecules involved in the management of lung cancer treatment. Our study aims to investigate the potential role of miRNAs in the treatment of non-small cell lung cancer (NSCLC). Human miRNA microarray was performed in 60 recurrent NSCLC patient tissue samples following radiotherapy and their adjacent normal tissues. miRNA profiling results were validated using quantitative real-time PCR. Inner cell radiosensitivity and endogenous miRNA expression was determined by colony-formation assay and RT-PCR. We determined the effect of miRNA on cell proliferation, survival and metastasis; tumor xenografts were taken to identify the presence of miRNA in vivo. miRNA panel results indicated that a total of 14 miRNAs were differentially expressed in the recurrent NSCLC samples. In our study, miRNA-95 was highly overexpressed in recurrent NSCLC cells. Knockdown of miRNA-95 expression increased the radiosensitivity of NSCLC, promoted tumor cell apoptosis and decreased cellular proliferation. In vivo assays demonstrated reduced tumor growth and resistance to radiation in tumor xenografts by downregulating miRNA-95. Our study demonstrated a potential therapeutic measure of miRNA-95 as a radiosensitive marker for the treatment of non-small lung cancer.
Despite the research conducted, lung cancer is still highly prevalent across the globe.1, 2 Non-small cell lung cancer (NSCLC) represents the major pathological type of lung cancer and nearly half of NSCLC patients are in unresectable advanced clinical stage when they are initially diagnosed.3, 4, 5 Radiotherapy is a routine treatment measure offered to lung cancer patients;6 however, NSCLC cells become resistant to radiotherapy causing a local recurrence and a reduced survival rate in lung cancer patients.7, 8, 9 Because of the high prevalence and limited efficacy towards radiotherapy in NSCLC,10 it is imperative to analyze effective approaches for NSCLC treatment for long-term survival of lung cancer patients.
In addition, miRNAs are short, small non-coding RNAs that regulate gene expression and participate in radiotherapy.11, 12, 13 Certain studies have shown a close association between miRNA expression and radiosensitivity in lung cancer.14, 15 Many reports have suggested that miRNA can serve as a novel biomarker;16, 17, 18 however, there are limited clinical studies that have determined the efficacy of miRNA in NSCLC.
The aim of our study is to determine the role of a specific miRNA in the treatment of NSCLC using radiotherapy. We collected patient tissue samples to identify specific miRNAs. Afterwards, NSCLC and xenografts were analyzed to test their radiosensitivity following miRNA treatment. Finally, we investigated the clinical implication of miRNA as a potential therapeutic marker for lung cancer.
Materials and methods
For our study, 60 normal and cancer tissue samples were taken from Huaihe Hospital of Henan University between 2010 and 2012. The study protocol was approved by the Ethical Committee of Henan University. The signed informed consents were collected from all patients. All patients were newly diagnosed with NSCLC who underwent radiotherapy with a dose of 60–66 Gy in 2-Gy fractions over 6 weeks combined with adjuvant cisplatin chemotherapy. Patients were reassessed for tumor condition and treatment response after 12 weeks with regular laboratory test, chest X-ray, CT scan and bronchoscopy along with physical exam. Lastly, the histological features of the NSCLC specimens were evaluated by two senior pathologists in our hospital according to the classification criteria from the World Health Organization (WHO). Samples was stored in −80°C for future use. All patients had no other serious diseases and no experience of chemotherapy or radiotherapy. The lab work investigator was blind to tissue resources while clinic coordinators only collected patient data and samples and were not involved in other experimental process.
Four non-small lung cell lines, A549, H23, H460 and H1299 were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured with Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum, 100 Uml−1 penicillin and 100 μgml−1 streptomycin (Sigma, St Louis, MO, USA) in a humidified atmosphere at 37 °C supplied with 5% CO2. All cell lines were tested for mycoplasma contamination and short tandem repeats each month before further experiments.
Western blot analysis
Protein samples was extracted with commercial kit (Thermo Life Science, Hercules, CA, USA) and separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel. The gel was transferred to polyvinylidene difluoride membrane under semi-dry condition and incubated with mouse monoclonal primary antibodies (# sc-7148 Casp 3, # sc-56073 Casp 9, # sc-7382 Bcl-2, # sc-372 NF-kB and # sc-47778 β-actin) for 1 h at room temperature, which was followed by an extra 1-h incubation with second goat anti-mouse antibodies. All protein antibodies were purchased from Santa Cruz (Santa Cruz, CA, USA). Western blot bands was observed and analyzed with enhanced chemiluminescence solution (Thermo Fisher Science, Boston, MA, USA).
MicroRNA panel with real-time PCR
Total RNAs were extracted using miRCURY RNA isolation kits according to instructions (Exiqon, Vedbaek, Denmark) and was assessed with Agilent 2100 bioanalyzer (Agilent, Santa Clara, CA, USA) for RNA integrity and concentration. Different miRNA expression profiling was detected with the commercial cancer focus miRNA PCR panels (miRCURY LNA Universal RT microRNA PCR, Exiqon) which comprised 84 miRNA primer sets focusing on cancer-relevant human miRNAs. The panel was tested for quantitative PCR reaction on the ABI-Prism 7900 (Applied Biosystems, Carlsbad, CA, USA) in a 384-well plate according to the manufacturer's instruction. RNU6 was chosen as an internal control. Relative expression of miRNA-95 was calculated with 2−ΔΔCT method.
Cells (1 × 105 per well) were seeded in a 24-well plate for transfection by using LipofectamineTM2000 (Invitrogen) following the manufacturer's protocol. Anti-miR-95 (5'-IndexTermUCAACAUCAGUCUGAUAAGCUA-3') and the mock control oligonucleotides (5'-IndexTermAUGCCUGAUGUUUGUACAA-3') (Abcam, Cambridge, UK) were transfected into vector at a final concentration of 20 nmoll−1.
The protocol for animal experiments was approved by the animal research committee of Henan University, and laboratory animal care and user guide were followed. A total of 40 male Sprague Dawley rats (250 g average weight, randomly 10 for each groups) were obtained from the animal center of Henan University. Cells (1 × 105; control or miR-95 knockdown cells) were injected subcutaneously into the right flanks of rats. Tumor volume was determined by caliper measurements performed every 2 days and calculated by using the modified ellipse formula: (volume=length × width2/2).19 Mean tumor volumes were normalized to starting volume. When the average tumor volume reached approximately 200 mm3, the tumors were irradiated with a single 8 Gy dose of ionizing radiation. Twenty-eight days after tumor initiation, the rats were killed by cervical dislocation, and tumor tissues were collected and measured.
Radiosensitivity of the innate cells was assessed by using the colony-forming assay. After 48 h of transfection, the cells were given different dosage of ionizing radiation and were seeded in fresh six-well plates (1000 per well). Visible colonies were fixed and stained with 2% crystal violet (Sigma). One colony was defined as a population of more than 50 cells. The number of colonies were examined using macroscopic observation (Nikon, Tokyo, Japan).
After 48 h irradiation, 10 μl per well of MTT reagent (Sigma) was added to 96-well microplate to conduct cell proliferation test following the manufacturer's instruction. The reaction was terminated with 100 μl DMSO and measured on a microplate reader (Bio-Rad, Hercules, CA, USA) at 490 nm absorbance.
Cell apoptosis was detected using the annexin V-FITC apoptosis detection kit (Sigma). Briefly, 1 × 106 cells were suspended in 1 × binding buffer and mixed with 5 μl of annexin V-FITC conjugate and 10 μl of propidium iodide solution at the final 500 μl volume in a plastic 12 mm × 75 mm test tube, which was followed by incubation at room temperature for 15 min and protection from light. The fluorescence of the cells was determined immediately with a flow cytometery (Bio-Rad).
SPSS 19.0 (SPSS Inc., Chicago, IL, USA) was applied to analyze all clinical and experimental data. F test reported no variance difference in each groups. The results of the quantitative data in this study are expressed as the mean±s.d. with analysis of variance test for multiple groups and categories are presented as percentage with chi-square test. A P value of less than 0.05 was considered significant.
Comparison of miRNA expression between recurrent NSCLC and corresponding non-cancerous tissue and its association with clinical features
miRNA panel PCR results identified eight upregulated and six downregulated miRNAs in tissue samples compared with paired adjacent normal tissues (fold change >1.5) (Table 1). In our study, miR-95 was highly upregulated in primary NSCLC samples. All tissue samples were used to validate the expression of miR-95. As expected, significant upregulation miR-95 was observed in NSCLC tissue compared with corresponding non-cancerous tissues (Figure 1). Further statistical analysis revealed an association of overexpressed miR-95 with enhanced lymph node metastasis and advanced clinical stage of NSCLC (Table 2).
Inner radiosensitivity and expression level of miR-95 in NSCLC cells
Our study used four commonly used NSCLC cell lines to examine their biological function. Radiosensitivity of cell lines was evaluated with colony-formation assay by exposing them to different ranges of doses of irradiation (0, 2, 4, 6, 8 Gy). Our results demonstrated that H460 cells had the highest radiosensitivity, while A549 carried more radiotherapy tolerance compared with other cell lines. (Figures 2a and b, P<0.05). The fraction of surviving cells decreased with increasing irradiation dosages. Endogenous miR-95 expression levels was correspondent with the trend of radiosensitivity within four NSCLC cell lines, and the expression of miRNA-95 increased with respect to cells that were radiotherapy-resistant. The most radio-resistant A549 cells exhibited approximately 2.5 fold changes of miRNA-95 expression level (Figure 2c, P<0.05) compared with the most radiosensitive H460 cells. To examine the influence of miR-95 on radiation therapy, miR-95 was knocked down in A549 cells and the knocked down efficiency was evaluated by RT-PCR (Figure 2d).
Downregulation of miR-95 leads to enhanced apoptosis and increased radiosensitivity
Our study examined the role of miR-95 with respect to irradiation. A549 cells were transfected with anti-miR-95 and a negative control vector for 48 h and exposed to a range of irradiation doses (0, 2, 4, 6 and 8 Gy). miR-95 expression was examined by RT-PCR to verify transfection efficiency (Figure 2d). Colony-formation assay showed that miR-95 downregulation cells were more sensitive to radiation as compared with the control groups (Figure 3a). Similarly, cell proliferation was inhibited in miR-95 knockdown cells (Figure 3b). As shown in Figure 3c, miR-95 knockdown increased the expression of pro-apoptotic proteins, such as Caspase-3 and Caspase-9, and decreased the expression of anti-apoptotic proteins such as Bcl-2 and NF-κB regardless of irradiation. miR-95 significantly increased X-ray-induced apoptosis of A549 cells compared with non-transfected control (Figure 3d). Hence, our study exhibited that downregulatory cells were likely to suffer from X-ray-induced apoptosis than normal cells.
Downregulation of miRNA-95 increases radiation sensitivity in vivo
Subcutaneous tumors models in rat were established by using the miRNA-95 knockdown A549 cell line and negative control cells. The models were exposed to 8Gy irradiation for a period of 21 days after tumor growth to 200 mm3. Tumor volume started to significantly decrease from day 12 in models with irradiation treatment. miR-95 knockdown cells exhibited irradiation-induced cell death from day 21 compared with parental cells (Figure 4a). Tumors were removed at the end of the study and tumor weight was measured. As shown in Figures 4b and c, downregulation of miR-95 promoted irradiation induced tumor growth inhibition effect.
NSCLC poses a significant problem in patients because of its resistance to radiotherapy; hence, treatment strategies that can eliminate the radiosensitivity of NSCLC cells can provide immense benefit and could reduce morbidity among patients. miRNAs are involved in multiple oncogenic pathways, and thus analyzing their part in radiotherapy resistance could be advantageous in the long run.20, 21, 22 Our data showed that the expression of some miRNAs dramatically changed during the radiotherapy process, but with different directions. Downregulation of miR-95 recovered the sensitive A549 cells with respect to varying doses of irradiation and led to apoptosis effect. Furthermore, anti-95 could significantly block NSCLC tumor growth progression and achieve optimal radiation therapy outcome in vivo. Our results indicated that the downregulation of miRNA sensitizes NSCLC cells to radiotherapy.
Several studies have shown evidence regarding the ability of miRNAs to alter radiosensitivity in lung cancer treatment. Some specific miRNA expression levels in human lung carcinoma cell lines were heavily changed in response to different irradiation dosage.23 These specific miRNAs were found to achieve their regulatory role in response to irradiation mainly through influencing lung cancer cell DNA damage and repair process, cell cycle distribution and apoptosis.24, 25 In addition, miRNAs interacted with multiple pathways that participated in irradiation response, such as K-Ras.26, 27 It is important to recognize the endogenous levels of miRNA before applying them as a biomarker because baseline levels of miRNA could potentially imbalance the effect of radiotherapy.27 Despite the plethora of data available, there have been only few research studies on this subject. However, Wang et al.28 conducted a study by enrolling 30 radiosensitive and resistant NSCLC patients based on the overall survival and recurrence rates following post-operation irradiation therapy. They also showed five upregulated miRNAs and seven downregulated miRNAs in the sensitive group compared with the resistant group. In our study, we found nine upregulated miRNAs and seven downregulated miRNAs suggesting the multidirectional role of miRNAs toward irradiation therapy. In this study, we found an upregulation of miR-95 in recurrence tumor tissue and association between upregulated miR-95 with worse clinical features including advanced clinical stage. Similarly, this effect was observed in tested A549 cells that showed a significant miRNA expression level and radiosensitivity. Generally, irradiation is used to speed up the tumor cell death process; miRNAs can potentially bind to apoptotic pathways and could alter the outcome of irradiation. Previous studies showed anti-apoptotic function in miRNAs and this could be linked to the effect observed in NSCLC patients.29 On the basis of our study, it can be argued that miR-95 knockdown can lead to apoptosis of A549 cells and affects the radiosensitivity of NSCLC cells caused by irradiation. As expected, we found that A549 cells transfected with anti-miR-95 had a significantly higher percentage of apoptotic cells than the negative control-transfected group following irradiation. In addition, anti-miR-95 had an increased expression of pro-apoptotic proteins, such as caspase-3 and caspase-9 and decreased the expression of the anti-apoptotic proteins Bcl-2 and NF-κB. Various signaling molecules are involved in apoptosis signaling pathways and abnormal regulation of apoptosis signaling pathways occurs often in tumor cells, which eventually leads to radioresistance.30, 31, 32 Apoptosis may be one of the mechanisms of ionizing radiation-induced cell death,33 and it is believed that miRNAs regulate radiation-induced apoptosis. Previous studies have shown that miR-148b increases the radiosensitivity of non-Hodgkin’s lymphoma cells by promoting radiation-induced apoptosis.34 Some studies have shown that miR-185 enhances radiation-induced apoptosis in gastric cancer cells by repressing the apoptosis-related ATR (ATM- and Rad3-related) pathway. In addition, miRNA-21 contributes to the radioresistance of tumors by blocking the expression of critical apoptosis-related genes such as caspase-3 and PARP-1.35, 36 Furthermore, our results demonstrated significant suppression of tumor growth by downregulation of miR-95 in vivo and the activation of induced cell death mechanism.
We summarized the potential targets of miR-95 in Supplementary Table S1 by searching web-source database (TargetScan, DIANA Tools, miRDB and PicTar) and literature reports (Supplementary Table S2). Most of predicted target genes of miR-95 were found to be involved in multiple cell regulation processes, cancer initiation or progression. Previous studies provide the evidence that upregulation of miR-95 could significantly enhance the resistance of NSCLC cells to chemotherapy or radiotherapy by directly targeting SNX1.37 Targeting inhibition of miR-95 can inhibit lung cancer cell growth through elevating PTEN, SNX1 and SGPP1 expression.38 The same binding effect was also reported in colorectal cancer study and prostate cancer cells.19, 39 Inhibition of miR-95 can also inhibit brain metastasis of lung adenocarcinoma cells by targeting CCND1 and CELF2.40, 41 Taking all the findings of our study into conclusion, we can conclude that anti-miR-95 promotes apoptosis in cells by inducing radiosensitivity in cancer cells.
To conclude, our study provided a thorough clinical evidence for miRNA-95 as a radiotherapy promoter in non-small lung cancer. This knowledge can be utilized to develop methods to combat radiotherapy resistance and could be beneficial for specific NSCLC patients.
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We want to acknowledge the evaluators, research assistants and particularly the adolescents and families who participated in this study.
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on Cancer Gene Therapy website
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Ma, W., Ma, Cn., Li, Xd. et al. Examining the effect of gene reduction in miR-95 and enhanced radiosensitivity in non-small cell lung cancer. Cancer Gene Ther 23, 66–71 (2016). https://doi.org/10.1038/cgt.2016.2
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