Examining the effect of gene reduction in miR-95 and enhanced radiosensitivity in non-small cell lung cancer



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

Clinical specimens

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.

Cell culture

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.

Cell transfection

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.

Mouse model

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.

Colony-forming assay

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).

MTT assay

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.

Apoptosis assay

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).

Statistical analysis

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).

Table 1 miRNA differentially expressed in recurrent NSCLC compared with corresponding non-cancerous tissue
Figure 1

Differential miR-95 expression levels from NSCLC primary tumor and paired normal tumor tissue. miR-95 expression in all selected recurrent tumors and their paired normal tissues were detected by real-time RT-PCR. RNU6 snRNA was used as an internal control. *P<0.05 vs normal tissue.

Table 2 Relationship between miR-95 expression and tumor clinicopathologic features

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).

Figure 2

Innate radiosensitivity and miR-95 expression in four NSCLC cell lines. (a) The fraction of four NSCLC cell lines A549, H1299, H23 and H460 following a range of irradiation with 0, 2, 4, 6 or 8 Gy. (b) The indices including 40% dose slope (D0), corresponding to radioresistance were lower in H460 cells and higher in A549 cells. (c) miRNA-95 expression in four NSCLC cell lines was detected using qRT-PCR. U6 served as an internal control. (d) miR-95 knockdown efficiency evaluation. The results are presented as the means±s.d. of values obtained in three independent experiments and underwent analysis of variance test. *P<0.05.

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.

Figure 3

miR-95 overexpression increases radiosensitivity and promotes cell apoptosis of A549 cells. (a) A549 parental, negative control and knockdown miR-95 cells were irradiated with 0, 4 or 8 Gy and subjected to colony-formation assay. (b) A549 cells treated with anti-miR-95 and negative control. After 24 h, the cells were subjected to 8-Gy irradiation. After 48 h, MTT was used to detect cell viabilities *P<0.05. These results are representative of at least three separate experiments. miR-95 promoted X-ray-induced apoptosis of A549 cells. (c) Western blot analysis of apoptotic marker proteins Caspase-3, Caspase-9, Bcl-2 or NF-κB in A549 cells with anti-miR-95 in transfected and non-transfected cells before and after exposure to 8-Gy X-ray irradiation. β-Actin was used as an internal control. (d) Flow cytometry assay to determine the apoptosis of A549 cells transfected with anti-miR-95 or negative control and non-transfected cells. Percentage of apoptotic cells in non-transfected and transfected cells subjected to 0- and 8-Gy irradiation. Data are means±s.d. of three independent experiments.

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.

Figure 4

miR-95 promotes irradiation-induced tumor death in vivo. (a) Nude rat (n=10 each group) were subcutaneously injected with A549 cells infected with a control vector or miR-95 knockdown vector. (b) Representative images of the tumors from rat at the end of the day of experiment (28 days). (c) A quantitative summary of the tumor weights. Data are means±s.d. of three independent experiments. *P<0.05.


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.


  1. 1

    Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T et al. Cancer statistics, 2008. CA Cancer J Clin 2008; 58: 71–96.

    Article  Google Scholar 

  2. 2

    Siegel R, Naishadham D, Jemal A . Cancer statistics, 2012. CA Cancer J Clin 2012; 62: 10–29.

    Article  Google Scholar 

  3. 3

    Fidias P, Novello S . Strategies for prolonged therapy in patients with advanced non-small-cell lung cancer. J Clin Oncol 2010; 28: 5116–5123.

    Article  Google Scholar 

  4. 4

    Fuld AD, Dragnev KH, Rigas JR . Pemetrexed in advanced non-small-cell lung cancer. Expert Opin Pharmacother 2010; 11: 1387–1402.

    CAS  Article  Google Scholar 

  5. 5

    Whitehurst AW, Bodemann BO, Cardenas J, Ferguson D, Girard L, Peyton M et al. Sythetic lethal screen identification of chemosensitizer loci in cancer cells. Nature 2007; 446: 815–819.

    CAS  Article  Google Scholar 

  6. 6

    le Péchoux C . Role of postoperative radiotherapy in resected non-small cell lung cancer: a reassessment based on new data. Oncologist 2011; 16: 672–681.

    Article  Google Scholar 

  7. 7

    Danesi R, Pasqualetti G, Giovannetti E, Crea F, Altavilla G, Del Tacca M et al. Pharmacogenomics in non-small-cell lung cancer chemotherapy. Adv Drug Deliv Rev 2009; 61: 408–417.

    CAS  Article  Google Scholar 

  8. 8

    Rödel F, Hoffmann J, Distel L, Herrmann M, Noisternig T, Papadopoulos T et al. Survivin as a radioresistance factor, and prognostic and therapeutic target for radiotherapy in rectal cancer. Cancer Res 2005; 65: 4881–4887.

    Article  Google Scholar 

  9. 9

    Lee S, Lim MJ, Kim MH, Yu CH, Yun YS, Ahn J et al. An effective strategy for increasing the radiosensitivity of Human lung Cancer cells by blocking Nrf2-dependent antioxidant responses. Free Radic Biol Med 2012; 53: 807–816.

    Article  Google Scholar 

  10. 10

    Provencio M, Sánchez A, Garrido P, Valcárce F . New molecular targeted therapies integrated with radiation therapy in lung cancer. Clin Lung Cancer 2010; 11: 91–97.

    CAS  Article  Google Scholar 

  11. 11

    Del Vescovo V, Grasso M, Barbareschi M, Denti MA . MicroRNAs as lung cancer biomarkers. World J Clin Oncol 2014; 5: 604–620.

    Article  Google Scholar 

  12. 12

    Zhang Y, Yang Q, Wang S . MicroRNAs: a new key in lung cancer. Cancer Chemother Pharmacol 2014; 74: 1105–1111.

    CAS  Article  Google Scholar 

  13. 13

    Xu YM, Liao XY, Chen XW, Li DZ, Sun JG, Liao RX . Regulation of miRNAs affects radiobiological response of lung cancer stem cells. Biomed Res Int 2015; 2015: 851–841.

    Google Scholar 

  14. 14

    Cortez MA, Valdecanas D, Zhang X, Zhan Y, Bhardwaj V, Calin GA et al. Therapeutic delivery of miR-200c enhances radiosensitivity in lung cancer. Mol Ther 2014; 22: 1494–1503.

    CAS  Article  Google Scholar 

  15. 15

    Korpela E, Vesprini D, Liu SK . MicroRNA in radiotherapy: miRage or miRador? Br J Cancer 2015; 112: 777–782.

    CAS  Article  Google Scholar 

  16. 16

    Oh J-S, Kim J-J, Byun J-Y, Kim I-A . Lin28-let7 modulates radiosensitivity of human cancer cells with activation of K-ras. Int J Radiat Oncol Biol Phys 2010; 76: 5–8.

    CAS  Article  Google Scholar 

  17. 17

    Salim H, Akbar NS, Zong D, Vaculova AH, Lewensohn R, Moshfegh A et al. miRNA-214 modulates radiotherapy response of non-small cell lung cancer cells through regulation of p38MAPK, apoptosis and senescence. Br J Cancer 2012; 107: 1361–1373.

    CAS  Article  Google Scholar 

  18. 18

    Zhao Z, Zhang L, Yao Q, Tao Z . miR-15b regulates cisplatin resistance and metastasis by targeting PEBP4 in human lung adenocarcinoma cells. Cancer Gene Ther 2015; 22: 108–114.

    CAS  Article  Google Scholar 

  19. 19

    Huang X, Taeb S, Jahangiri S, Emmenegger U, Tran E, Bruce J et al. miRNA-95 mediates radioresistance in tumours by targeting the sphingolipid phosphatase SGPP1. Cancer Res 2013; 73: 6972–6986.

    CAS  Article  Google Scholar 

  20. 20

    Shen Z, Wu X, Wang Z, Li B, Zhu X . Effect of miR-18a overexpression on the radiosensitivity of non-small cell lung cancer. Int J Clin Exp Pathol 2015; 8: 643–648.

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Babar IA, Czochor J, Steinmetz A, Weidhaas JB, Glazer PM, Slack FJ . Inhibition of hypoxia-induced miR-155 radiosensitizes hypoxic lung cancer cells. Cancer Biol Ther 2011; 12: 908–914.

    CAS  Article  Google Scholar 

  22. 22

    Ma Y, Xia H, Liu Y, Li M . Silencing miR-21 sensitizes non-small cell lung cancer A549 cells to ionizing radiation through inhibition of PI3K/Akt. Biomed Res Int 2014; 2014: 617868.

    PubMed  PubMed Central  Google Scholar 

  23. 23

    Shin S, Cha HJ, Lee EM, Lee SJ, Seo SK, Jin HO et al. Alteration of miRNA profiles by ionizing radiation in A549 human non-small cell lung cancer cells. Int J Oncol 2009; 35: 81–86.

    CAS  PubMed  Google Scholar 

  24. 24

    Liu YJ, Lin YF, Chen YF, Luo EC, Sher YP, Tsai MH et al. MicroRNA-449a enhances radiosensitivity in CL1–0 lung adenocarcinoma cells. PLoS One 2013; 8: e62383.

    CAS  Article  Google Scholar 

  25. 25

    Di Francesco A, de Pitta C, Moret F, Barbieri V, Celotti L, Mognato M . The DNA-damage response to gamma-radiation is affected by miR-27a in A549 cells. Int J Mol Sci 2013; 14: 17881–17896.

    Article  Google Scholar 

  26. 26

    Weidhaas JB, Babar I, Nallur SM, Trang P, Roush S, Boehm M et al. MicroRNAs as potential agents to alter resistance to cytotoxic anticancer therapy. Cancer Res 2007; 67: 11111–11116.

    CAS  Article  Google Scholar 

  27. 27

    Arora H, Qureshi R, Jin S, Park AK, Park WY . miR-9 and let-7g enhance the sensitivity to ionizing radiation by suppression of NFkappaB1. Exp Mol Med 2011; 43: 298–304.

    CAS  Article  Google Scholar 

  28. 28

    Wang XC, Du LQ, Tian LL, Wu HL, Jiang XY, Zhang H et al. Expression and function of miRNA in postoperative radiotherapy sensitive and resistant patients of non-small cell lung cancer. Lung Cancer 2011; 72: 92–99.

    Article  Google Scholar 

  29. 29

    Ranade AR, Weiss GJ . Methods for microRNA microarray profiling. Methods Mol Biol 2011; 700: 145–152.

    CAS  Article  Google Scholar 

  30. 30

    Schulze-Bergkamen H, Krammer PH . Apoptosis in cancer—implications for therapy. Semin Oncol 2004; 31: 90–119.

    CAS  Article  Google Scholar 

  31. 31

    Mirkovic N, Voehringer DW, Story MD, McConkey DJ, McDonnell TJ, Meyn RE . Resistance to radiation-induced apoptosis in Bcl-2-expressing cells is reversed by depleting cellular thiols. Oncogene 1997; 15: 1461–1470.

    CAS  Article  Google Scholar 

  32. 32

    Raffoul JJ, Wang Y, Kucuk O, Forman JD, Sarkar FH, Hillman GG . Genistein inhibits radiation-induced activation of NF-kappaB in prostate cancer cells promoting apoptosis and G2/M cell cycle arrest. BMC Cancer 2006; 6: 107.

    Article  Google Scholar 

  33. 33

    Chen S, Wang H, Ng WL, Curran WJ, Wang Y . Radiosensitizing effects of ectopic miR-101 on non-small-cell lung cancer cells depend on the endogenous miR-101 level. Int J Radiat Oncol Biol Phys 2011; 81: 1524–1529.

    CAS  Article  Google Scholar 

  34. 34

    Wu Y, Liu GL, Liu SH, Wang CX, Xu YL, Ying Y et al. MicroRNA-148b enhances the radiosensitivity of non-Hodgkin’s Lymphoma cells by promoting radiation-induced apoptosis. J Radiat Res 2012; 53: 516–525.

    CAS  Article  Google Scholar 

  35. 35

    Wang J, He J, Su F, Ding N, Hu W, Yao B et al. Repression of ATR pathway by miR-185 enhances radiation-induced apoptosis and proliferation inhibition. Cell Death Dis 2013; 4: e699.

    CAS  Article  Google Scholar 

  36. 36

    Wang XC, Wang W, Zhang ZB, Zhao J, Tan XG, Luo JC . Overexpression of miRNA-21 promotes radiation-resistance of non-small cell lung cancer. Radiat Oncol 2013; 8: 146.

    CAS  Article  Google Scholar 

  37. 37

    Chen X, Chen S, Hang W, Huang H, Ma H . MiR-95 induces proliferation and chemo- or radioresistance through directly targeting sorting nexin1 (SNX1) in non-small cell lung cancer. Biomed Pharmacother 2014; 68: 589–595.

    CAS  Article  Google Scholar 

  38. 38

    Zhang J, Zhang C, Hu L, He Y, Shi Z, Tang S et al. Abnormal expression of miR-21 and miR-95 in cancer stem-like cells is associated with radioresistance of lung cancer. Cancer Invest 2015; 33: 165–171.

    CAS  Article  Google Scholar 

  39. 39

    Huang Z, Huang S, Wang Q, Liang L, Ni S, Wang L et al. MicroRNA-95 promotes cell proliferation and targets sorting Nexin 1 in human colorectal carcinoma. Cancer Res 2011; 71: 2582–2589.

    CAS  Article  Google Scholar 

  40. 40

    Hwang SJ, Lee HW, Kim HR, Song HJ, Lee DH, Lee H et al. Overexpression of microRNA-95-3p suppresses brain metastasis of lung adenocarcinoma through downregulation of cyclin D1. Oncotarget 2015; 6: 20434–20448.

    PubMed  PubMed Central  Google Scholar 

  41. 41

    Fan B, Jiao BH, Fan FS, Lu SK, Song J, Guo CY et al. Downregulation of miR-95-3p inhibits proliferation, and invasion promoting apoptosis of glioma cells by targeting CELF2. Int J Oncol 2015; 47: 1025–1033.

    CAS  Article  Google Scholar 

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We want to acknowledge the evaluators, research assistants and particularly the adolescents and families who participated in this study.

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Correspondence to Y-j Zhang.

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The authors declare no conflict of interest.

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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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