Biosci. Rep. (2016) / 36 / art:e00354 / doi 10.1042/BSR20150300

miRNA-148b regulates radioresistance in non-small lung cancer cells via regulation of MutL homologue 1

Guangsheng Zhai*†, Gaozhong Li‡, Bo Xu†, Tongfu Jia§, Yinping Sun, Jianbo Zheng¶ and Jianbin Li*1

*Department of Radiotherapy, The Tumor Hospital of Shandong Province, No. 440 of Jiyan Road, Ji’nan 250000, Shandong Province, China †Department of Radiotherapy, The Central Hospital of Zibo, No. 54 of Gongqingtuan West Road 54, Zhangdian District, Zibo 255000,

Shandong Province, China Downloaded from http://portlandpress.com/bioscirep/article-pdf/36/3/e00354/807680/bsr036e354.pdf by guest on 30 September 2021 ‡Division of Gastroenterology, Department of Medicine, The Second Hospital of Shandong University, No. 54 of Gongqingtuan West Road 54, Zhangdian District, Zibo 255000, Shandong Province, China §No. 1 Oncology Department, The Central Hospital of Zibo, No. 54 of Gongqingtuan West Road 54, Zhangdian District, Zibo 255000, Shandong Province, China No. 3 Oncology Department, The Central Hospital of Zibo, No. 54 of Gongqingtuan West Road 54, Zhangdian District, Zibo 255000, Shandong Province, China ¶Department of Urology Surgery, The Central Hospital of Zibo, No. 54 of Gongqingtuan West Road 54, Zhangdian District, Zibo 255000, Shandong Province, China

Synopsis Radioresistance represents a major obstacle in cancer treatment, the underlying mechanism of which is complex and not well understood. miR-148b has been reported to be implicated regulating radioresistance in lymphoma cells. However, this function has not been investigated in lung cancer cells. Microarray analysis was performed in A549 cells 48 h after exposure to 8 Gy of γ -irradiation or sham irradiation to identify differentially expressed miRNAs. miR-148b mimic and inhibitor were transfected, followed by clonogenic survival assay to examine response to irradiation in A549 cells. Western Blot and luciferase assay were performed to investigate the direct target of miR-148b. Xenograft mouse models were used to examine in vivo function of miR-148b. Our data showed that expression of miR-148b was significantly down-regulated in both serum and cancerous tissues of radioresistant lung cancer patients compared with radiosensitive patients. Overexpression of miR-148b reversed radioresistance in A549 cells. MutL homologue 1 (MLH1) is the direct target of miR-148b which is required for the regulatory role of miR-148b in radioresistance. miR-148b mimic sensitized A549 xenografts to irradiation in vivo. Our study demonstrated that miR-148b regulates radioresistance of lung cancer cells by modulating MLH1 expression level. miR-148b may represent a new therapeutic target for the intervention of lung cancer. Key words: lung cancer, miR-148b, MutL homologue 1 (MLH1), radioresistance.

Cite this article as: Bioscience Reports (2016) 36, e00354, doi:10.1042/BSR20150300

INTRODUCTION Radiotherapy has been regarded as one of the main treatment strategies for NSCLC. However, a large proportion of NSCLC patients are resistant to radiotherapy, which significantly limits Lung cancer is one of the leading causes of cancer-related deaths its therapeutic effects and induces the local recurrence of NSCLC worldwide [1]. Non-small cell lung cancer (NSCLC) represents [6,7]. Although, it is generally thought to be due to tumour het- the most common type of lung cancer and accounts for approx- erogeneity related to cell of origins, pathology, aetiology and imately 80% of all lung cancer cases [2,3]. The 5-year overall molecular/genetic pathogenesis [8], the underlying mechanism survival rate of NSCLC cases is lower than 15% [3,4]. Approxim- remains unclear. Therefore, there is an urgent need to develop ately 40% of NSCLC patients have unresectable stage III disease novel approaches for the intervention of NSCLC, such as tar- or medically inoperable disease [5]. geted gene treatment as a radiosensitizer.

...... Abbreviations: DDR, DNA damage repair; DNMT1, DNA (cytosine-5)-methyltransferase 1; DSB, double-strand break; IHC, immunohistochemistry; MLH1, MutL homologue 1; MMR, mismatch repair; MSI, microsatellite instability; NSCLC, non-small cell lung cancer. 1 To whom correspondence should be addressed (email [email protected]).

c 2016 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. 1 G. Zhai and others

miRNAs is an abundant class of conservative and small non- sequencing platform single-end sequencing mode to conduct coding RNAs which post-transcriptionally regulate gene by tar- high-throughput sequencing for the four samples (Genenergy); geting mRNA for degradation or inhibition of translation [9]. each sample had three parallel groups. Differences between the miRNAs are implicated in regulating diverse tumorigenic pro- groups were determined using the DESeq statistical tests of R cesses, such as in cell apoptosis, cell proliferation, stress response software (genetic screening criteria: P  0.05 and fold differ- and metabolism, etc., and have been found to be dysregulated in ence in expression  1.5). Differential expression of miRNA a wide variety of cancer types, including lung cancer [10–12]. between irradiated and nonirradiated cells was analysed after Previous studies have shown that some miRNAs may be in- the sequencing was proofread using the genome and miRBase volved in modulating the sensitivity of cancer cells to radio- databases. therapy and dysregulation of miRNA function might contribute to the acquisition of radioresistance, particularly in lung cancer

[13–15]. Plasmids and cell transfection Downloaded from http://portlandpress.com/bioscirep/article-pdf/36/3/e00354/807680/bsr036e354.pdf by guest on 30 September 2021 Aberrant expression of miR-148b has been found in many Cells in exponential growth phase were prepared for cell trans- cancer types, including breast cancer, prostate cancer, lung fection. Transfection of miR-148b inhibitor, miR-148b mimic and cancer, etc. [16–18]. In particular, miR-148b has been repor- its non-specific control (Invitrogen) were performed according ted to be down-regulated in NSCLC and associated with poor to the manual provided with the siPORTM NeoFXTM Trans- survival [19] and it may function as a tumour suppressor in fection Agent (Ambion). pLenti-C-Myc-DDK MLH1 cDNA NSCLC by targeting carcinoembryonic antigen (CEA) [18]. Re- (RC201607L1) and shRNA plasmid (TL320419) targeting MutL cently, it has been shown that miR-148b can reverse cisplatin- homologue 1 (MLH1) were obtained from Origene. cDNA trans- resistance in NSCLC via down-regulating DNA (cytosine-5)- fections were performed with Lipofectamine LTX reagent (Invit- methyltransferase 1 (DNMT1) expression [20]. Up-regulation of rogen) as per manufacturer’s protocol. miR-148b has been observed in human endothelial cells after ionized radiation [21]. In the present study, we investigated the potential role of Viral transductions and stable selections miR-148b in regulating radioresistance in NSCLC, the underly- For lentivirus production, 1 μg of pLenti-C-Myc-DDK cDNA ing mechanism and the potential clinical values using xenograft or shMLH1 plasmid together with 1 μg of helper plasmids mouse models. (0.4 μg of pMD2G and 0.6 μg of psPAX2) were transfected into 293FT cells (A.T.C.C.) with Effectene reagent (Qiagen). Viral supernatants were collected 48 h after transfections and cleared through a 0.45 μm filter. Cells were infected with viral MATERIALS AND METHODS supernatants containing 4 μg/ml Polybrene (Sigma–Aldrich) and selected with puromycin for 7 days.

Cell culture and treatment Human lung cancer cell line A549 (A.T.C.C.) was cultured in Real-time PCR basal medium supplemented with 10% serum at 37 ◦Cand5% The cells or spheroids were harvested after the transfection and CO2. These cells were tested mycoplasma free. the RNA was isolated using TRI reagent (Sigma–Aldrich). Ten nanograms of RNA were used for reverse transcription using the TaqMan MicroRNA RT Kit (Applied Biosystems, Life Techno- Human samples logies). Briefly, 5 μl of the RNA was added to 10 μl of the master All the cancer samples and normal tissues were retrieved from mix containing 0.15 μl of dNTP (100 nM), 1 μl of multiscribe The Tumor Hospital of Shandong Province. All tissues were im- enzyme (50 units/μl), 1.5 μlof10× RT-puffer, 0.19 μlofRNAse ◦ mediately snap-frozen in liquid nitrogen and stored at − 80 C inhibitor (20 units/μl), 4.16 μl of RNAse free H2Oand3μlof until use. In addition, the patients with any other tumour were primers ( miR-200a/b/c, miR-141). The conditions for the reverse excluded from the study. Serum samples were extracted from transcription were 30 min at 16 ◦C, 30 min at 42 ◦Cand5min whole blood after centrifugation (2800 g, 10 min) and stored at at 85 ◦C. Quantitative real-time PCR of the individual miRNAs − 80 ◦C until further processing. The present study was approved was performed in a total volume of 10 μl containing 5 μlofTaq-

by The Tumor Hospital of Shandong Province and written in- Man master mix, 3.17 μl of RNAse free H2O, 0.5 μlofTaqMan formed consent was obtained from all patients. primer and 1.33 μl of cDNA. The PCR was performed in quad- ruple in a Rotor-Gene Corbett 6000 Q PCR (Life Science). The conditions for the reaction were: 2 min at 50 ◦C, 10 min at 95 ◦C, miRNA sequencing cycling(50repeats):step1–15sat95◦C,step2–60sat A549 cells were seeded in six-well plates and exposed to 8 Gy 60 ◦C. The data were collected and analysed using the quantitat- of γ -irradiation or sham irradiation. After 48 h, these samples ive Rotor-Gene software. The miRNA levels were normalized to were lysed using Trizol reagent for total RNA collection. Sub- the stable internal control miRNA RNU 6B using the algorithm of sequently, miRNA sequencing was performed using the Illumina Pfaffl.

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Colony formation assay corresponding antibodies at 4 ◦C overnight. After secondary an- Cells were trypsinized and seeded in six-well plates at density tibody incubation at room temperature for 1 h, the slides were of 1000 cells/well in triplicates immediately after exposure to γ - developed in 0.05% diaminobenzidine containing 0.01% hydro- radiation. The cells were incubated for 1 week and then washed gen peroxidase. with PBS for three times, fixed with 4% paraformaldehyde for 15 min, and stained with 0.1% crystal violet (Sigma–Aldrich) Xenograft experiments solution for 30 min. After these, the colonies were carefully All animal experiments were approved by Institutional Animal washed with PBS until the background is clear. Colony formation Care and Use Committee of National Cancer Center. Control or efficiency was calculated as colony number divided by 1000 and miR-148b mimic transfected A549 cells (2 × 106 cells/injection) normalized to scramble or control vector cells. were subcutaneously injected into both flanks of 5 weeks old fe- male nude mice group. Tumour volumes were measured 3 weeks γ Downloaded from http://portlandpress.com/bioscirep/article-pdf/36/3/e00354/807680/bsr036e354.pdf by guest on 30 September 2021 Immunoblot analysis after exposure to 8 Gy of -radiation using calliper and determ- ined by a formula [volume = (length × width2)/2] from day 3 Total cell lysates were prepared by harvesting cells in Laemmli to day 21 post implantation. The results were expressed as mean SDS reducing buffer [50 mM Tris/HCl (pH 6.8), 2% SDS and tumour volumes with S.D. 10% glycerol], boiled and resolved on an 8–10% polyacryl- amide gel, and transferred to polyvinylidinefluoride. Antibod- ies against MLH1 (Abcam) and β-actin (Sigma–Aldrich) were Statistical analysis used. The blots were incubated with horseradish-peroxidase- Quantitative data are expressed as mean −+ S.D. Statistical sig- conjugated donkey anti-rabbit or anti-mouse IgG (Santa Cruz nificance was assessed by the Student’s t test. Differences were Biotechnology) at a dilution of 1:5000 and detected with Su- considered to be significant when P < 0.05. perSignal West Pico or Femto Chemiluminescent Substrate Kit (Thermo Scientific). RESULTS γ -H2AX assay Cells were exposed to 2 Gy of γ -radiation and were fixed using miR-148b expression is negatively related to 3.7% paraformaldehyde for 10 min, permeabilized with 0.5% radioresistance in lung cancer Triton X-100 in PBS on ice for 5 min and incubated with blocking In order to identify the miRNAs implicated in lung cancer radi- solution (10% goat serum and 3% BSA in PBS containing 0.1% osensitivity, miRNA sequencing was used to monitor their ex- Triton X-100) for 30 min. Cells were then incubated with primary pression in A549 cells 48 h after radiation treatment. A549 has antibodies diluted in blocking solution for an hour. Anti-γ -H2AX been demonstrated to be a radioresistant cell line before [22].The antibody (1:100, Abcam, ab18311) was used. After incubation results showed that radiation affected the expression of various with secondary antibodies labelled with Alexa Fluor-568 (1:400, miRNAs in A549 cells. Supplementary Figure S1(A) lists the Invitrogen), slides were mounted with ProLong Gold Antifade top nine miRNAs that showed considerable up-regulation/down- Reagent with DAPI (Life Technologies). Cells were observed regulation after irradiation. miR-148b was selected as the target under a fluorescence microscope, Axiovert 200 M (Carl Zeiss). miRNA as it showed greatest fold change in expression before To quantify the number of γ -H2AX foci, at least 100 nuclei were and after irradiation. In addition, the difference in this expression analysed from each sample. was statistically significant before and after irradiation in A549 cells (P = 0.0003) as confirmed by quantitative real-time PCR (Supplementary Figure S1B). miR-148b expression was analysed Luciferase assay in lung cancerous tissues and matched non-cancerous tissues as Two hundred and fifty nanograms of pGL3 reporter vector carry- well as serum from 20 CRC patients. The results showed lower ing the WT or mutant miR-148b-binding site (see plasmid con- expression of miR-148b in tumour tissues than in normal tissues struct, Figure 3A), 25 ng of the phRL-SV40 control vector (Pro- (Figures 1Aand1C). Additionally, miR-25 expression was also mega) and 100 nM miRNA precursors or scrambled sequence related to radiotherapy sensitivity. As shown in Figures 1(B) and miRNA control (Ambion) were co-transfected into HEK293 cells 1(D), its expression was significantly lower in both tumour and in 24-well plates. Firefly luciferase activity was measured with a serum samples of radioresistant patients than in radiosensitive Dual Luciferase Assay Kit (Promega) 24 h after transfection and counterparts. Therefore, dysregulation of miR-148b expression normalized with a Renilla luciferase reference plasmid. may be associated with radioresistance in lung cancer.

Immunohistochemistry staining miR-148b modulates radiosensitivity in lung cancer The paraffin-embedded sections were subjected to antigen re- cells trieval by heating the slides in a microwave at 100 ◦C for 10 min To investigate whether miR-148b is involved in regulating ra- in 0.1 M citric acid buffer (pH 6.0), and then incubated with diosensitivity in lung cancer cells, we transfected miR-148b

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Figure 1 miR-148b expression is negatively related to radioresistance in lung cancer (A and C) Quantitative real-time PCR analysis of relative miR-148b expression in serum (A) and tissue (C) of lung cancer patients (n=20) and healthy controls (n=20). (B and D) Quantitative real-time PCR analysis of relative miR-148b expression in serum (B) and tissue (D) of radiosensitive patients (n=15) and radioresistant patients (n=15). (A–D) Data represent + mean − S.D. ****, P < 0.0001.

mimic or inhibitor into A549 cells which led to a signific- miR-148b directly targets the 3’-UTR of MLH1 and ant increase or decrease in miR-148b expression (Figure 2A). regulates its expression level in lung cancer cells Clonogenic survival assay showed that effective up-regulation To explore potential gene targets of miR-148b that may be in- or down-regulation of miR-148b significantly inhibited or pro- volved in this effect, we examined for putative targets using moted cell survival in A549 cells compared with control cells miRNA target prediction programmes and MLH1 showed up after exposure to 4 Gy of γ -radiation. To further elucidate the as one of the hits that are most prevalent and this protein has regulatory role of miR-148b in radioresistance, DNA double- been reported to play a role in regulating radioresistance in colon strand breaks (DSBs) were measured and quantified using γ - cancer cells [23]. To verify MLH1 is a direct target of miR-148b, H2AX foci staining. Compared with control cells, significantly we cloned the 3’-UTR of MLH1 containing the single putat- more γ -H2AX foci persisted 6 h after irradiation in cells trans- ive miR-148b-binding site downstream of the Renilla luciferase fected with miR-148b mimic. On the contrary, significantly fewer open reading frame. Both wild-type 3’-UTR and a mutant form, foci persisted 2 and 6 h after irradiation in cells transfected with in which the putative seed-binding site was mutated, were evalu- miR-148b inhibitor (Figure 2C). This suggests that miR-148b ated using luciferase assay (Figure 3A). As shown in Figure 3(B), regulates radioresistance through mediating DNA damage repair co-transfection of miR-148b precursor with wild-type MLH1 3’- (DDR). UTR reporter construct significantly repressed relative luciferase

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Figure 2 miR-148b modulates radiosensitivity in lung cancer cells (A) Quantitative real-time PCR analysis of relative miR-148b expression in A549 cells transfected with miR-148b mimic or + miR-148b inhibitor. Data represent mean − S.D., n=3. **, P < 0.01. (B) Clonogenic survival in control, mimic or inhibitor transfected cells after exposure to 4 Gy of γ -radiation. Survival was normalized to unirradiated control cells. Data represent + mean − S.D., n=3. **, P < 0.01; ***, P < 0.001. (C). DNA damage was assessed by γ -H2AX foci staining. A549 cells transfected with miR-148b mimic or inhibitor were irradiated with 2 Gy of γ -radiation, stained with γ -H2AX antibody at subsequent time intervals, and γ -H2AX foci were counted. *, P < 0.05 compared with control transfected cells. To quantify the number of γ -H2AX foci, at least 100 nuclei were analysed from each sample.

activity whereas mutation of the miR-148b-binding site elimin- miR-148b mediates radiosensitivity through ated this effect in A549 cells (Figure 3B). On the other hand, regulating MLH1 protein expression co-transfection of miR-148b inhibitor with wild-type MLH1 3’- To determine the importance of MLH1 in the regulatory role of UTR reporter construct into A549 cells significantly induced rel- miR-148b in radioresistance, we ectopically express MLH1 in ative luciferase activity which was reversed when the miR-148b- A549 cells transfected with miR-148b mimic (Figure 4A). Al- binding site was mutated (Figure 3C). In addition, transfection though transfection of miR-148b mimic reversed radioresistance, of miR-148b mimic or inhibitor decreased and increased MLH1 overexpression of MLH1 eliminated this sensitizing effect (Fig- protein level in A549 cells (Figure 3D). Taken together, these ure 4B). Additionally, stable knockdown of MLH1 in A549 cells results demonstrate that miR-148b can directly regulate MLH1 phenocopied the effect of miR-148b overexpression by sensitiz- expression level. ing these cells to irradiation (Figures 4Cand4D). These results

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Figure 3 miR-148b directly targets the 3’-UTR of MLH1 and regulate its expression level in lung cancer cells (A) The wild-type 3’-UTR of mammalian MLH1 mRNA contains a putative miR-148b-binding site. The mutant form was shown below. (B and C) Luciferase reporter assays. A549 cells were transfected with reporters containing the wild-type or mutant form after transfection with control miRNA as well as miR-148b mimic or inhibitor respectively. Data represent the + mean − S.D. *, P < 0.05; **, P < 0.01. (D) Western Blot analysis of MLH1 protein level in A549 cells transfected with control, miR-148b mimic or inhibitor. β-Actin was used as loading control.

suggest that MLH1 is required for the regulatory role of miR-148b antly increase the morbidity and mortality of NSCLC patients. in radioresistance. Therefore, identification of effective therapeutic strategies which can sensitize the NSCLC to radiotherapy is urgently needed to enhance survival rate in NSCLC. In the present study, we iden- Up-regulation of miR-148b decreases A549 cell tified a panel of altered miRNAs in a radioresistant NSCLC radioresistance in vivo cell line A549, after IR exposure and analysed the expression To validate the regulatory role of miR-148b in radioresistance level of one of the top candidates, miR-148b in clinical human in vivo, we examined the effects of miR-148b inhibition on ra- samples (Supplementary Figure S1). We observed that miR-148b dioresistance of A549 cells using xenograft mouse model. As was down-regulated in both serum and tissues from NSCLC pa- shown in Figures 5(A)–5(C), transfection with miR-148b mimic tients compared with healthy controls. It is also down-regulated significantly reduced tumour size, inhibited tumour growth and in radioresistant NSCLC patient samples compared with radio- reduced tumour weight. Immunohistochemistry (IHC) analysis sensitive patient samples (Figure 1). These observations suggest showed that the miR-148b mimic transfected xenografts showed that miR-148b may serve as a potential predictive biomarker for significantly increased cleaved caspase 3 compared with the con- radiotherapy in NSCLC. trol xenografts (Figure 5D). Taken together, these results suggest A growing body of evidence has suggested that there is a that miR-148b can regulate radioresistance in vivo. link between a collection of miRNAs and radioresistance and those miRNAs function as either radiosensitizers or radiopro- tectors through various mechanisms. For example, miR-23 and DISCUSSION miR-203 have been shown to enhance radiosensitivity by target- ing IL8/Stat3 and IL8/AKT signalling pathway, respectively in nasopharyngeal carcinoma [24,25]; miR-205 has been reported Acquisition of resistance to radiotherapy represents a major to function as a tumour radiosensitizer by inhibiting DNA re- obstacle in the treatment of NSCLC. It is known to signific- pair pathway via down-regulation of ZEB1 and Ubc13 in breast

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Figure 4 miR-148b mediates radiosensitivity through regulating MLH1 protein expression (A) Western Blot analysis of MLH1 protein level in A549 cells transfected with control or miR-148b mimic or miR-148b plus MLH1 cDNA. β-Actin was used as loading control. The MLH1 to β-actin ratio is 1:0.12:1.8. (B) Clonogenic survival in cells described in (A) after exposure to 2, 4 or 8 Gy of γ -radiation. Survival was normalized to unirradiated control cells. Student’s t test was used to compare the difference between control and miR-148b mimic group. (C) Western Blot analysis of MLH1 protein level in A549 cells expressing control or MLH1 shRNA. β-Actin was used as loading control. The MLH1 to β-actin ratio is 1:0.23. (D) Clonogenic survival in cells described in (C) after exposure to 2, 4 or 8 Gy of γ -radiation. + Survival was normalized to unirradiated control cells. (B and D), data represent mean − S.D., n=3. *, P < 0.05; **, P < 0.01.

cancer cells [26]; miR-15a/16 can enhance radiation sensitivity Several reports have supported that miR-148b is down- of NSCLC cells by targeting the TLR1/NF-κB signalling path- regulated in many cancer types, including NSCLC and may way [27]. On the other hand, it has been reported that miR-106b function as a tumour suppressor [16–18]. Additionally, miR- can induce cell radioresistance via the PTEN/phosphoinositide 3- 148b has been shown to reverse cisplatin resistance in NSCLC kinase (PI3K)/AKT pathways and p21 in colorectal cancer [28]; and is up-regulated after IR exposure in human endothelial cells miR-20a has been recently shown to induce cell radioresistance [20,21]. However, its role in regulating radioresistance has not by activating the PTEN/PI3K/AKT signalling pathway in hep- been investigated. In the present study, we found that overex- atocellular carcinoma. pression or inhibition of miR-148b could increase or decrease

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Figure 5 Up-regulation of miR-148b decreases A549 cell radioresistance in vivo (A) Image of representative tumours from control or miR-148b mimic transfected A549 xenografts 3 weeks after exposure to 8 Gy of γ -radiation. (B) Tumour growth curve of control or miR-148b mimic injected A549-IR tumours. n=5 for each group. (C) Weights of tumours from mice described above at sacrifice. (D) Representative cleaved caspase 3 IHC staining of xenografts from the two groups in (C). The bar graph presents the quantification of IHC staining. The scale bar indicates + 100 μm. Data represent mean − S.D. **, P < 0.01; ***, P < 0.001.

radiosensitivity in NSCLC cells by regulating DNA repair path- apy [35]. Our results showed that miR-148b can directly bind to way (Figure 2). Importantly, expression of miR-148b mimic sig- the 3’-UTR of MLH1 and manipulation of miR-148b expression nificantly inhibited tumour growth after IR exposure in A549 affected MLH1 protein level (Figure 3). Additionally, knockdown xenograft with resultant increased apoptotic cell death (Figure 5). of MLH1 sensitized A549 cells to irradiation, phenocopying the Radiotherapy leads to the production of a variety of ionizing effect of miR-148b down-regulation (Figure 4A). Overexpres- radiation-induced lesions in DNA, which are required to be re- sion of MLH1 reversed the radiosensitizing effects of miR-148b paired by DDR pathways, including DNA single-strand breaks mimic (Figure 4B). These results suggest that miR-148b exerts (SSBs), DNA DSBs, DNA base alterations and DNA–DNA or its radiosensitizing effects primarily through regulating MLH1 DNA–protein cross-links [29]. Over the past decade, a variety of expression. approaches have been employed to target DDR components for In the present study, we found that miR-148b was down- radiosensitization, including small interfering RNA, aptamers, regulated in human NSCLC and radioresistant patient samples. antisense and small molecule inhibitors [30–33]. MLH1 is a pro- Up-regulation of miR-148b sensitized radioresistant NSCLC tein involved in mismatch repair (MMR) pathway and genetic or A549 cells to IR by interfering DDR through down-regulating epigenetic modification in this gene results in microsatellite in- MLH1 protein level. These observations provide a rationale for stability (MSI) [34]. Accumulating evidence suggests that DNA the development of new therapeutic strategies to improve radio- MMR proteins may influence and/or are directly involved in the therapy efficacy in lung cancer patients. miR-148b may repres- DDR following radiation induced DSBs and MSI cancers with ent a novel biomarker for the prediction of radiosensitivity in deficiency in those proteins may indicate sensitivity to radiother- NSCLC.

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15 Yan, D., Ng, W.L., Zhang, X., Wang, P., Zhang, Z., Mo, Y.Y., Mao, H., AUTHOR CONTRIBUTION Hao, C., Olson, J.J., Curran, W.J. and Wang, Y. (2010) Targeting Conception and design, acquisition of data, analysis and inter- DNA-PKcs and ATM with miR-101 sensitizes tumors to radiation. PLoS One 5, e11397 CrossRef PubMed pretation of data: Guangsheng Zhai, Gaozhong Li, Bo Xu, Tongfu 16 Cimino, D., De Pitta,` C., Orso, F., Zampini, M., Casara, S., Penna, Jia, Yinping Sun, Jianbo Zheng and Jianbin Li; drafting the article: E., Quaglino, E., Forni, M., Damasco, C., Pinatel, E. et al. (2013) Guangsheng Zhai and Jianbin Li. All authors approved the final miR148b is a major coordinator of breast cancer progression in a version to be published. relapse-associated microRNA signature by targeting ITGA5, ROCK1, PIK3CA, NRAS, and CSF1. FASEB J. 27, 1223–1235 CrossRef PubMed FUNDING 17 Zhang, J.G., Shi, Y., Hong, D.F., Song, M., Huang, D., Wang, C.Y. 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Received 4 December 2015/22 December 2015; accepted 4 January 2016 Accepted Manuscript online 7 March 2016, doi 10.1042/BSR20150300 Downloaded from http://portlandpress.com/bioscirep/article-pdf/36/3/e00354/807680/bsr036e354.pdf by guest on 30 September 2021

...... 10 c 2016 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0.