MiR-338-5p enhances the radiosensitivity of esophageal squamous cell carcinoma by inducing apoptosis through targeting survivin

Radioresistance is a challenge in the treatment of esophageal squamous cell carcinoma (ESCC). MicroRNAs (miRNAs) are known to play an important role in the functional modification of cancer cells and recent studies have reported miRNA-mediated radiotherapy resistance. However, further research is necessary to reveal the regulation mechanisms, and treatment strategies using miRNA are yet to be established for ESCC. We compared the miRNA expression profiles of ESCC parental (TE-4) and acquired radioresistance (TE-4R) cell lines using a miRNA microarray and qRT-PCR. Our data showed that miR-338-5p, one of the target miRNA biomarkers, was significantly downregulated in TE-4R. Ectopic overexpression of miR-338-5p induced apoptosis and sensitivity to radiation treatment by interfering with survivin, which is a known inhibitor of apoptosis. Overexpression of survivin reversed miR-338-5p-induced apoptosis. Tumor xenograft experiments indicated that therapeutic delivery of the miR-338-5p mimics via direct injection into tumor mass increased sensitivity to radiation therapy. In conclusion, our findings suggest that miR-338-5p is a potential radiosensitizer and may be a therapeutic biomarker for radioresistant in ESCC.

Several miRNAs have been correlated with patient survival and may be useful in the prediction and modification of anticancer treatments, including radiotherapy [19][20][21] . However, miRNA expression profiling data of radioresistant ESCC cell lines are limited and potential miRNA functions in therapeutic strategies remain unclear.
In this study, we predicted that miR-338-5p is a regulator of radioresistance in ESCC based on miRNA expression profiling of ESCC parental (TE-4) and acquired radioresistance (TE-4R) cell lines. We checked for binding sites for miR-338-5p in the 3′ UTR of survivin, one of the key regulators of apoptosis inhibition. Based on our findings, we report that miR-338-5p increases radiation-induced apoptosis and enhances radiosensitivity by downregulating survivin expression.

Establishment of a radioresistant ESCC cell line. To establish a radioresistant ESCC cell line, we used
γ-rays to select radioresistant populations from parental TE-4 ESCC cells. TE-4 cells were exposed to fractionated radiation and surviving cells formed colonies. The colonies were pooled, and the radiation treatment was repeated (Fig. 1A). Cells derived from this selection were named TE-4R. To determine the radiosensitivity of TE-4R cells as compared with parental TE-4 cells, a clonogenic survival assay was performed. The surviving fraction of TE-4R cells was significantly larger than that of the TE-4 cells at various doses (Fig. 1B). Cell viability analysis by MTS assay at different time points (1, 3, 5, and 7 days) after irradiation with 4 Gy showed that TE-4R viability was significantly higher at day 5, and even more markedly so at day 7 after irradiation ( Fig. 1C) (p < 0.05). To evaluate the cellular response to radiation, the levels of apoptosis-related proteins were analyzed. Cleavaged Poly(ADPribose) polymerase (PARP) and caspase3, which is considered to be a hallmark of apoptosis, was elevated in TE-4 cells than TE-4R cells as indicated by increased levels of cleaved PARP and caspase3 in the former (Fig. 1D). These data indicated that TE-4R cells are more resistant to cell death than the parental cells after irradiation.

MiR-338-5p is downregulated in radioresistant ESCC cells.
To identify miRNAs that regulate radiosensitivity in ESCC cell lines, we used the NanoString nCounter miRNA expression assay. The miRNA expression array indicated that 20 miRNA were increased (fold change > 1.5) and 49 miRNA were decreased (fold change < 1.5) in TE-4R cells as compared to their parental cell line ( Fig. 2A). We selected miR-338-5p for further analysis because its function and mechanism in radiosensitization have not been characterized. MiR-338-5p was downregulated in the TE-4R cell line (fold change = −5.1). Real-time PCR analysis confirmed the microarray data (Fig. 2B). To investigate the effect of miR-338-5p as a radiosensitizer, TE-4 cells were transiently transfected with control (miR-con) or miR-338-5p mimic (miR-338), and their survival after irradiation was determined. Western blot results confirmed that transfection was successful (Fig. S1). A clonogenic survival assay showed that miR-338-5p significantly decreased the surviving cell fraction as compared to miR-con (Fig. 2C). The effect of miR-338-5p expression on cell growth after irradiation was also examined by MTS assay; miR-338-5p-treated cells showed reduced numbers as compared to miR-con-treated cells at 3, 5, and 7 days (p < 0.05) (Fig. 2D). Taken together, these results demonstrated that miR-338-5p expression in ESCC cells significantly decreases radioresistance in vitro; thus, miR-338-5p can sensitize ESCCs cells to irradiation.
MiR-338-5p regulates the expression of survivin. To understand how miR-338-5p modulates radiation-induced apoptosis, we analyzed target genes of miR-338-5p predicted by TargetScan (www.targetscan. org) and miRanda (www.microrna.org). Most notably, one of the predicted targets is survivin, which is known to inhibit apoptosis. Previous studies have suggested that survivin is important in the development of radioresistance through the regulation of apoptosis [22][23][24][25] . When compared with TE-4 cells, TE-4R cells exhibited much higher survivin expression (Fig. 4A). Thus, we assumed that miR-338-5p targets survivin. To analyze the targeted regulation of survivin by miR-338-5p in ESCC cells further, the miR-338-5p level was manipulated by transfecting control or miR-338-5p mimic were exposed to 4 Gy radiation and cell viability was analyzed using the MTS cell proliferation assay at 1, 3, 5, and 7 days after irradiation. Data in the bar chart are the mean ± SD of three independent experiments (p < 0.05).
SCIenTIfIC RepoRts | 7: 10932 | DOI:10.1038/s41598-017-10977-9 the cells with miR-338-5p mimic and inhibitor. MiR-338-5p mimic transfection significantly reduced survivin expression, whereas its expression increased in anti-338-transfected cells as compared with their controls, in both the TE-4 and TE-14 cell lines (Fig. 4B,C). To confirm survivin as a direct target of miR-338-5p, we constructed a luciferase reporter plasmid containing the binding site of the survivin 3′ UTR (3′ UTR WT) and one harboring a deletion of the binding site (3′ UTR muta). We co-transfected these plasmids with miR-con or miR-338 into TE-4 cells. The results revealed significantly reduced luciferase activity in cells transfected with 3′ UTR WT-and miR-338, whereas the mutant form did not affect luciferase activity (Fig. 4D). These findings suggested that survivin is a direct target of miR-338-5p.

Survivin overexpression rescues cells from apoptosis induced by miR-338-5p.
To confirm the interaction between miR-338-5p and survivin, we silenced the latter using siRNA and examined the effect on apoptosis. Western blot assay and flow cytometry analysis of Annexin V-stained cells showed that the knockdown of endogenous survivin expression could imitate the effects associated with miR-338-5p overexpression, including increased cleaved PARP and caspase3 expression levels and promotion of apoptosis ( Fig. 5A and B). To clarify whether apoptosis induction by miR-338-5p is mediated through suppressing survivin expression, a pCMV survivin expression plasmid was constructed. A rescue experiment was conducted to explore whether survivin could functionally reverse radiation-induced apoptosis by miR-338-5p. The pCMV-survivin vector and pCMV control vector were transfected into TE-4 cells with miR-con or miR-338-5p, and the cells were examined by flow cytometry after irradiation. The level of apoptotic cells was decreased in cells overexpressing survivin as compared to control cells transfected with miR-338-5p (Fig. 5C). This rescue experiment revealed that survivin can reverse the apoptosis induced by miR-338-5p.
MiR-338-5p enhances radiosensitivity of ESCC in xenograft models. To explore whether miR-338-5p enhances radiosensitivity in vivo, we generated subcutaneous tumors in nude mice using TE-4 cells. As illustrated in Fig. 6A, control or miR-338-5p mimic were injected into the tumors 4 times at 1-week intervals before and after ionizing radiation of 6 Gy, and tumor size was measured every 3 days. After irradiation, control tumors continued to increase in volume; however, miR-338-5p-injected TE-4 tumors grew evidently slower (Fig. 6B). Additionally, survivin expression was downregulated in miR-338-transfected xenografts (Fig. 6C). Taken together, these results demonstrated that miR-338-5p expression obviously decreases radioresistance in vivo, suggesting that in vivo administration of miR-338-5p mimic has considerable potential for radiosensitization.

Discussion
Radiotherapy plays an important role in the treatment of esophageal cancers as part of combined-modality therapy together with chemotherapy and surgery. This treatment approach has improved tumor control and survival rates. However, tumor recurrence because of radioresistance occurs in a high proportion of patients; therefore, therapeutic strategies to improve the response to radiation in ESCC need to be developed. Here, we identified miR-338-5p as a radiosensitizer. Additionally, we found the miR-338-5p target survivin to be an important inhibitor of radiation-induced apoptosis (Fig. 6D).
Abundant miRNAs regulate a wide range of biological processes in cancers. Using a miRNA expression profiling approach, we identified miR-338-5p as a downregulated miRNA in radioresistant ESCC. In a previous study, miR-338-5p was considered to function as an oncomiR by inhibiting PIK3C3 expression and autophagy in colorectal cancer 26 . However, miR-338-5p might have different functions in different cancers. Our results demonstrated that miR-338-5p acts as a radiosensitizer through inhibition of apoptosis in ESCC. Results from several other groups have confirmed miRNAs to be involved in the radioresistance of ESCC. Huang et al. identified miR-21 as an activator of radioresistance through inhibition of PTEN 27 , and Su et al. recently found a set of miRNAs aberrantly expressed in acquired radioresistant ESCC cells that conferred resistance through the Wnt/B-catenin signal pathway 21 . Although these studies reported on miRNAs associated with radioresistance in ESCC, our study is the first to show that miR-338-5p as radiosensitizer inhibits radiation-induced apoptosis in vitro and in vivo. We showed that survivin expression was markedly enhanced in the radioresistant cell line as compared to the parental cells. We and others previously showed that survivin is associated with radioresistance in pancreatic cancer, rectal cancer, head and neck squamous carcinoma, as well as lung cancer 22,23,28,29 . Survivin is tightly associated with stemness-promoting pathways such as Notch 30 , Oct4, and STAT3 31 . Moreover, survivin was shown to be a major target of different pro-survival signaling pathways, such as the PI3K/AKT pathway 32 . These pathways are known to control radiosensitivity; thus, survivin might induce radioresistance in ESCC through the regulation of these signaling pathways. Additionally, accumulation of survivin and interaction with components of the DNA-double-strand break (DSB) repair machinery in glioblastoma cells indicated that survivin regulates DSB repair, significantly improving survival in these cells 25 . It will be interesting to study the different roles of survivin, including not only the inhibitory effect on caspase activity during apoptosis but also functions in DNA damage repair in ESCC radioresistant cells. Future studies are required to test this hypothesis.
To the best of our knowledge, this is the first report of the roles of miR-338-5p and survivin in radioresistance of ESCC. We reported a novel role for miR-338-5p as a key regulator of apoptosis and showed that miR-338-5p sensitizes ESCC to irradiation by inhibiting survivin expression suggesting miR-338-5p as a potential therapeutic strategies for improving the response of ESCC patients to radiation therapy. Irradiation and clonogenic assay. TE-4 and TE-4R cells were seeded at 2 × 10 3 cells per well in 6-well culture plates, cultured for 24 h, and irradiated with different radiation doses (0, 2, 4, 6, and 8 Gy) using a 137 Cs γ-ray source (Atomic Energy of Canada Ltd, Chalk River, Canada) at a dose rate of 3.81 Gy/min. After 14 days of culture, the cells were fixed, stained with 2% crystal violet, and cell colonies were counted using the ImageJ software. Transfection. One day before transfection, cells were seeded in 6-well plates and cultured to 60-80% confluence. Then, the cells were treated with 50 nM control microRNA, miR-338-5p mimic; 20 nM siNC or siSurvivin (Bioneer, Daejeon, Korea); or 1 μg survivin expression plasmid or vehicle (Origene, Rockville, MD, USA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). After 6 h, the cells were transferred to complete growth medium. On the third day after transfection, the cells were harvested and used in further experiments.

Methods
MiRNA expression profiling using a NanoString array. MiRNAs were prepared using a miRNeasy Mini kit (Qiagen, Hilden, Germany) and hybridized to the array in the nCounter miRNA Expression Assay (NanoString Technologies, Seattle, WA, USA) following the manufacturer's instructions. Western blotting. For western blot analysis, cells were washed with cold PBS and lysed in RIPA buffer (Thermo Fisher Scientific, Grand Island, NY, USA). Proteins were quantified using the Bradford method and equal amounts of protein were resolved by SDS-PAGE and analyzed by western blotting as previously described 33 . The membranes were incubated with primary antibodies against cleaved PARP, caspase3, survivin (Cell Signaling Technology, Beverly, MA, USA) and β-actin (Sigma-Aldrich, St. Louis, MO, USA) overnight at 4 °C and with a secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. Proteins were visualized using enhanced chemiluminescence (Thermo Fisher Scientific). Western blot images were analyzed with a LAS-4000 mini (GE Healthcare Life Sciences, Uppsala, Sweden) and Bio-Rad ChemiDoc (Bio-Rad, Richmond, CA, USA). Cell proliferation assay. Cells were seeded in 96-well plates and allowed to attach for 24 h. Then, the cells were irradiated with 4 Gy. At various time points, 10 μl of (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyp henyl)-2-(4-sulfophenyl)-2H-tetrazolium; MTS) of the CellTiter 96 AQueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI, USA) was added to 100 μl of culture media for 1 h. The absorbance at 490 nm was read using a plate reader.
Dual luciferase reporter assay. TE-4 cells were seeded into 6-well plates one day before transfection. The cells were transfected with 1 μg of pmiRGLO vector (Promega) containing predicted miR-338-5p-binding sites of the survivin 3′ UTR and 50 nM microRNA mimic using Lipofectamine 2000. pRL-TK, which encodes Renilla luciferase, was included in all transfections to normalize for transfection efficiency. At 48 h after transfection, assays were performed using a dual luciferase reporter system (Promega) according to manufacturer's instructions. Firefly luciferase activity was normalized to that of Renilla, and then compared with those of the respective controls.
Animal experiments. Female nude mice at 6 weeks of age were subcutaneously injected with 1 × 10 7 TE-4 cells into the right flank of the back. At 7 days post-injection, 1.6 μl JetPEI and 10 µg miR-con or miR-338-5p were directly injected into the tumor, and injections were repeated every week for 4 weeks. Then, the mice were irradiated with a single dose of 6 Gy using an X-RAD 320 X-ray irradiator (Softex, Goyang, Korea) at a dose rate of 2 Gy/min. This study was carried out in accordance with relevant guidelines and regulations. The animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of Korea Institute of Radiological and Medical Sciences (protocol number: KIRAMS 2016-0032). The animal care facility is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care.

Statistical analysis.
Data obtained from at three experiments are expressed as the mean ± standard deviation. Statistical significance of differences was analyzed by Student's t-test, and p < 0.05 was considered significant.