대장암의 방사선 저항성과 관련된 유전적·분자적 기전 연구 = Gene Expression-Based Analysis of Radiation Resistance in Colorectal Cancer

다국어 초록 (Multilingual Abstract)

Gene Expression-Based Analysis of Radiation Resistance in Colorectal Cancer Beamjun Park Department of Medical Life Science of Soonchunhyang University Asan, Korea (Supervised by Professor Taesung Ann) Radiation therapy is a fundamental component of colorectal cancer (CRC) treatment, yet its therapeutic efficacy varies considerably among patients due to intrinsic and acquired resistance of CRC cells. To address this challenge, the present study investigated changes in gene expression profiles in colorectal cancer tissues before and after radiation therapy to identify potential biomarker genes associated with radiosensitivity or radioresistance. This study employed small interfering RNA (siRNA) technology to suppress the expression of four genes(BAMBI, GADD34, NFKBIA, and NFKBID) in colorectal cancer cell lines SW480 and HCT116, and investigated their functional roles in colorectal cancer. The effects of gene suppression were evaluated through molecular and cellular assays, including cell proliferation, migration, invasion, TUNEL staining, quantitative real-time PCR, and radiation response experiments. The siRNA induced knockdown of the four target genes resulted in a notable decrease in colorectal cancer cell proliferation, migration, and invasion. Furthermore, gene silencing increased sensitivity to radiation, as reflected by enhanced apoptotic activity and reduced cell viability compared with untreated controls. Collectively, these results suggest that BAMBI, GADD34, NFKBIA, and NFKBID are critically involved in colorectal cancer progression and resistance to radiation therapy. This study provides an important foundation for future research on the mechanisms of radioresistance and underscores the potential for personalized radiotherapy strategies that predict patient outcomes and prevent unnecessary radiation treatment. Key word Colorectal cancer cell, Radiation resistance, Key genes in radiation resistance Ⅰ. INTRODUCTION The incidence of colorectal cancer (CRC) has steadily declined over the past decade; however, it remains the second leading cause of cancer-related mortality worldwide, following lung cancer [1–4]. Recent studies have explored strategies to overcome radiation resistance from an epigenetic perspective, identifying gene targets and regulatory pathways that contribute to this resistance and compromise the efficacy of radiotherapy [5, 6]. Across various cancer types, molecular targets within key signaling cascades—such as ERK/MAPK, Wnt/β-catenin, TGF- β/SMAD, and PI3K–AKThave been explored as potential modulators of radioresistance [5]. In rectal cancer, radioresistance has been linked to elevated carcinoembryonic antigen (CEA) levels that promote M2 macrophage polarization and to dysregulation of DNA repair genes regulated by miR-31. In the present study, we analyzed colorectal cancer patient tissues to identify genes that were differentially expressed between normal and tumor samples, as well as between pre- and post-radiation therapy specimens. Candidate genes were subsequently selected and validated through a systematic screening process. This analysis led to the identification of four genes (BAMBI; BMP and Activin Membrane-Bound Inhibitor , GADD34; Growth Arrest and DNA Damage-Inducible Protein 34 , NFKBIA; NF-κB Inhibitor Alpha , and NFKBID; NF-κB Inhibitor Delta) for further characterization. Their functional roles were investigated using siRNA-mediated knockdown to assess how their regulation influences colorectal cancer cell behavior and radiosensitivity. Ⅱ. MATERIALS AND METHODS 1. Gene Selection Tissue samples were obtained from six patients newly diagnosed with colorectal cancer. Paired normal and tumor tissues were collected via sigmoidoscopic biopsy prior to radiotherapy and again four weeks after the completion of radiotherapy. Next-generation sequencing (NGS) analysis was subsequently conducted to identify differentially expressed genes associated with the radiotherapy response. Genes with a fold change ≥ 1.5 and a raw p- value < 0.05 were considered statistically significant. 2. Cell Lines and Culture Among various colorectal cancer cell lines, HCT116 and SW480 were selected for this study based on their consistent proliferation rates, stable culture conditions, and high siRNA transfection efficiency, all of which contribute to reliable experimental reproducibility. HCT116 is characterized by its rapid growth rate, while SW480 exhibits low metastatic potential and relatively low radiosensitivity. Both cell lines were obtained from the Korea Cell Line Bank (Korean cell line bank, Seoul, Republic of Korea). Cells were grown in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS)(YounginFrontier, Korea) and 1% penicillin streptomycin solution (ABS)(Corning, USA) at 37 °C in a humid atmosphere containing 5% CO2. 3. Small Interfering RNA(siRNA) and Transient Transfection Human CRC cell lines were seeded with 2 × 106 cells in 10cm cell culture dish. After 24 h, the medium was changed to siRNA, Opti MEM (Gibco, Massachusetts, USA, 31985-062) and Hiperfect® (QIAGEN, Germany, 301707) were mixed and replaced the existing media. siRNA was used to silence genes. Preliminary experiments were performed using three siRNA concentrations: half, normal, and double the recommended dose, in order to determine optimal transfection conditions. Based on these trials, the optimal knockdown efficiency was achieved at a concentration of 20 nmol/µL with a 48-h incubation period. Cell function tests and radiosensitivity assays were subsequently performed using cells transfected under these optimized conditions. To establish appropriate control conditions, an additional set of preliminary experiments was carried out by varying the cell seeding density (3 × 105, 5 × 105, and 7 × 105 cells per well) and incubation times (24, 48, and 72 h). From these experiments, the optimal experimental parameters were determined. All main experiments were performed in triplicate, and average values were calculated. 4. Cell Proliferation The cells were cultured in T-75 flasks and subcultured when reaching approximately 80–90% confluency. For subculturing, cells were seeded at a density of 1 × 106 cells per flask. To perform the experiment, cells were seeded into 96-well plates containing membranes. At 24, 48, 72 h after incubation, EZ-Cytox solution was added to each well, followed by a 2 h incubation in a humidified incubator at 37 °C with 5% CO2. Absorbance was subsequently measured at 450 nm using a microplate reader (Thermo Fisher, Waltham, MA,USA,51119200). 5. Transwell Migration Assay A migration assay was performed to evaluate the migratory capacity of the cells, using a 24- well plate equipped with a 6.5 mm polycarbonate membrane insert with a pore size of 8.0 µm (Corning, Somerville, MA, USA). A total of 250 µL of RPMI 1640 medium containing 3 × 105 cells was added to the upper chamber. In the bottom chamber, 750 µL of RPMI 1640 medium, containing 10% FBS and 1% ABS, was added. This was incubated for 48 h in a humidified incubator containing CO2 and at 37 °C. Then, the medium in the bottom chamber and the Transwell chamber was removed and the chamber was washed three times using phosphate buffered saline (PBS)(YounginFrontier, Korea). To fix the cells in the membrane of the washed chamber, 4% formaldehyde was applied to the Transwell and bottom chambers and reacted for 2 m. After washing 3 times with PBS, 100% methyl alcohol was applied and reacted for 10 m. In addition, cells were stained by immersing the Transwell chamber in 0.5% crystal violet solution for 2 m. Using an optical microscope, 5 sites of the membrane were randomly photographed (900 × 1200 µm2). 6. Transwell Invasion Assay Invasion assays were performed using 24-well inserts with 6.5 mm polycarbonate membranes and a pore size of 8.0 µm (Corning, Somerville, MA, USA). The inside of the Transwell was covered with a non-growth factor 1:4 ratio Matrigel dilution in RPMI 1640 and incubated for 1 h at 37 ◦C. A concentration of 5 × 105 cells was then plated in the 750 µL of RPMI 1640 medium with 10% FBS and 1%ABS was added in the bottom chamber. After 72 h of incubation, cells were fixed with 4% formaldehyde and stained with 0.05‰ crystal violet. 7. Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Assay Cells were seeded in the 12-well plate with an added cover slide. The TUNEL Assay kit detects the DNA fragmentation of apoptotic cells by marking DNA breaks using standard immunohistochemical techniques. Thus, after treatment with the nanoparticles, the cells were fixed by incubation in 4% formaldehyde for 30 m. Cells were permeabilized using 0.2% Triton X-100 (in PBS) for 45 m at 37 ◦C to facilitate staining. All wells received a DNA marking solution (10 µL of reaction buffer, 0.75 µL of TdT enzyme, 8.0 µL of BrdUTP and 31.25 µL of dH2O) and were incubated for 60 m at 37 °C. The nuclei were counterstained with DAPI. 8. Radiation Irradiation HCT116 colorectal cancer (CRC) cells were subjected to varying doses of radiation to evaluate their response to irradiation. Cells were irradiated with doses ranging from 0 to 900 Gray (Gy) in 100 Gy increments using a calibrated linear accelerator. Cell viability was measured at 0, 24, 48, 72, and 96h post-irradiation to determine the impact of radiation dose and exposure time. Based on the survival data, 500 Gy was selected as the optimal dose for further experiments. Subsequent irradiation of HCT116 cells was conducted at 500 Gy to assess the effect of radiation on cell proliferation and viability under various experimental conditions. Cell survival was evaluated using the EZ-Cytox assay, and apoptotic cell populations were analyzed using TUNEL staining as described in the earlier sections. 9. Real-Time Polymerase Chain Reaction (PCR) Colon cancer cells were seeded at a density of 1 × 104 cells per well in 6-well cell culture plates. These were cultured after 24 h, and 1ml RiboEx (GeneAll, Seoul, Republic of Korea, 301-001) was added to each well according to the manufacturer’s protocol. Total RNA was isolated from all cell lines using the Hybrid-R™ kit (GeneAll, Seoul, Republic of Korea, 305- 101). Reverse transcription was performed using ReverTra Ace™ kit (TOYOBO, Osaka, Japan, FSQ-101). Quantitative qRT-PCR was performed using the SYBR® Green (TOYOBO, Osaka, Japan, QPK-201). The PCR cycle included one cycle at 95°C for 1 m, followed by 39 cycles at 95 °C for 15 s, 60 °C at 15 s, and 72 °C for 25 s. The primers used for real time PCR were based on data from the ensemble (Table 1). 10. Statistical Analysis In order to improve the reproducibility and statistical robustness of our experiments, each experiment was conducted at least three times. The graphs were constructed based on the mean values and standard deviations derived from these repeated measurements. Statistical analyses were performed using SPSS version 26.0 for Windows 11 (SPSS Inc, USA). Student’s t-tests were used to compare knockdown validation, cell proliferation, migration, and invasion assays in CRC cell lines. A p-value of < 0.01 was indicated by a single asterisk (*), and p < 0.05 was indicated by a double asterisk (**). Ⅲ. RESULTS 1. Gene Expression Profiling of Colorectal Cancer Patient Samples. In colorectal cancer patient samples, genes showing a fold change of ≥1.5 with a raw p-value below 0.05 were initially identified (Figure 1). Differentially expressed genes between tumor and matched normal tissues were further presented using a volcano plot (Figure 2). Among the 39 genes that exhibited significant expression alterations before and after radiotherapy, four(BAMBI, GADD34, NFKBIA, and NFKBID)were prioritized. The final selection was based on the magnitude of expression change, supporting evidence from previous studies, and their relevance to colorectal cancer as documented in the Human Protein Atlas Figure 1. A heatmap generated through hierarchical clustering (Euclidean distance, complete linkage) showing gene expression similarities across samples. Significant genes were grouped according to their expression patterns, allowing visualization of how samples and genes cluster based on overall expression profiles. Figure 2. Volcano plot visualizing the differential gene expression between tumor and matched normal tissues. Genes meeting the significance threshold (fold change ≥ 1.5 and p < 0.05) are marked for emphasis. The x-axis represents the log2 fold change, while the y-axis denotes the −log10 p-value. 2. Functional Assays Following Gene Silencing in CRC Cell Lines Figure 3. Validation of gene knockdown in CRC cell lines using quantitative real-time PCR. mRNA expression levels of BAMBI (A, E), GADD34 (B, F), NFKBIA (C, G), and NFKBID (D, H) were measured in SW480 and HCT116 cells following siRNA-mediated silencing. Significance: p < 0.01. The mRNA expression levels of BAMBI, GADD34, NFKBIA, and NFKBID were first evaluated in colorectal cancer cell lines SW480 and HCT116. To explore the effects of these genes on cellular behavior, siRNA-mediated knockdown models for each gene were generated in both colorectal cancer cell lines. Expression of all four genes decreased by more than 60% following siRNA transfection (Figure 3). Figure 4. Cell proliferation analysis following gene knockdown in CRC cell lines. Proliferative activity was assessed in cells transfected with siBAMBI (A), siGADD34 (B), siNFKBIA (C), and siNFKBID (D). Statistical significance: *p < 0.01, **p < 0.05. To assess functional changes associated with gene loss, siRNA-untreated cells were compared to individual knockdown groups. In cell proliferation assays, no significant differences were observed in cells lacking BAMBI, GADD34, or NFKBIA during the initial 48 hours. Conversely, NFKBID knockdown cells exhibited reduced survival at this early stage. By 72 hours, all gene silencing groups exhibited a statistically significant reduction in proliferation of at least 20% compared to controls, suggesting that suppression of these four genes inhibits CRC cell growth (Figure 4). Figure 5. Migration analysis of SW480 cells following gene-specific knockdown. Migrated cells were stained with crystal violet, examined under a light microscope, and quantified by manually counting cells in at least three randomly selected fields for each experimental condition. Statistical significance: p < 0.01. Panels correspond to siBAMBI (A), siGADD34 (B), siNFKBIA (C), and siNFKBID (D). Next, migration and invasion capabilities were examined following knockdown of each gene. Migration was markedly reduced in CRC cell lines where all four genes were silenced. Specifically, BAMBI knockdown resulted in nearly a 9-fold decrease in migration compared to control cells, while NFKBIA and NFKBID knockdown groups showed reductions exceeding 80% (Figure 5). GADD34 loss also reduced migration activity to a statistically significant level, though the degree of change was less pronounced compared to the other three genes. Figure 6. Invasion assay of SW480 cells following individual gene knockdown. Invaded cells were stained with crystal violet, examined under a light microscope, and quantified by manually counting cells in at least three randomly chosen fields for each condition. Statistical significance: p < 0.01. Panels represent siBAMBI (A), siGADD34 (B), siNFKBIA (C), and siNFKBID (D). Reduction of BAMBI, GADD34, NFKBIA, and NFKBID expression led to a marked decrease in the invasive capacity of CRC cells. All four knockdown groups exhibited more than a 50% reduction in invasion, demonstrating that these genes contribute to the invasive phenotype of colon cancer cells (Figure 6). Collectively, these findings show that BAMBI, GADD34, NFKBIA, and NFKBID promote key malignant behaviors in CRC, including enhanced proliferation, migration, and invasion. 3. Analysis of Gene Expression Related to Radiation Sensitivity in CRC Cells Figure 7. Evaluation of radiation response in CRC cell lines using cell viability analysis. (A) Temporal changes in cell viability measured at multiple time points after irradiation. (B) Viability profiles assessed 72 hours post-irradiation across increasing radiation doses. To establish suitable irradiation conditions for evaluating radiation sensitivity in CRC cell lines, cell viability was monitored at multiple time points following exposure to various radiation doses (0, 24, 48, 72, and 96 hours; Figure 7A). A marked decline in viability exceeding 50% was observed at 72 and 96 hours post-irradiation. For determination of the half-maximal inhibitory concentration (IC₅₀), the incubation time was set at 72 hours, after which cell viability was reassessed across the same range of radiation doses. As shown in Figure 7B, approximately 50% viability was achieved at a dose of 500 Gy. Based on these findings, all subsequent experiments investigating the effects of gene knockdown on radiosensitivity were performed using 500 Gy irradiation followed by a 72hour incubation period. Figure 8. Evaluation of radiosensitivity in CRC cell lines following gene-specific knockdown based on cell viability measurements. Statistical significance: p < 0.01. The HCT116 and SW480 colorectal cancer cell lines were exposed to 500 Gy of ionizing radiation, and cell viability was assessed 72 hours afterward. Viability outcomes were compared between control cells and cells in which each of the four target genes had been individually silenced. Absorbance values obtained using the Ez-Cytox assay were used to quantify cell survival, with the viability of siRNA untreated controls normalized to 100%. All knockdown groups exhibited a significant decrease in radiation survival relative to the controls. Among the four genes, NFKBIA knockdown produced the mildest reduction, whereas NFKBID knockdown showed variable effects depending on the cell line. In contrast, silencing BAMBI or GADD34 led to a substantial decline in survival, with viability dropping below 80% in both CRC cell lines (Figure 8). 4. Confocal Imaging of Radiation Response Modulated by Gene Expression Figure 9. Radiation-induced cell death in CRC cells. representative confocal images showing DAPI- stained nuclei and TUNEL-positive apoptotic cells, together with quantitative measurements of the percentage of TUNEL-positive cells in control versus gene-silenced groups. (Statistical significance: p < 0.01) To visualize cell survival after irradiation, control and gene-silenced groups were prepared following the same procedure used in the EZ-Cytox viability assay and subsequently examined by confocal microscopy. Cells were plated at identical densities onto coverslips, exposed to 500 Gy of radiation, and incubated for 72 hours. Nuclear staining with DAPI (blue) was used to assess total cell numbers. A noticeably higher number of DAPI-positive nuclei was observed in the control group compared with the four knockdown groups, indicating reduced cell survival following gene silencing. Apoptotic cells were identified using TUNEL staining (red fluorescence). For each condition, both total cells and TUNEL-positive cells were manually counted in four randomly selected microscopic fields. The percentage of apoptotic cells was calculated and expressed as mean ± standard deviation. All four gene knockdown groups displayed at least a threefold increase in TUNEL-positive cells relative to the control, demonstrating that suppression of these genes enhances radiation-induced cell death (Figure 9). Ⅳ. DISCUSSION In this study, we aimed to clarify mechanisms underlying colorectal cancer (CRC) radiation resistance by investigating four genes (BAMBI, GADD34, NFKBIA, and NFKBID) identified as potential contributors to the cellular response to radiotherapy. By suppressing these genes, we observed broad inhibition of tumor-associated behaviors and enhanced radiation-induced cell death, suggesting their relevance as modulators of CRC radiosensitivity. Across all gene-silenced groups, proliferation, migration, and invasion were consistently reduced, although the magnitude of change differed among genes. In particular, migration was reduced by >80% following knockdown of BAMBI, NFKBIA, or NFKBID, whereas GADD34 knockdown showed a less pronounced effect. Previous studies indicate that GADD34 modulates intestinal epithelial motility through myosin IIA upregulation, which may account for the more modest decline in migration [8, 9]. Conversely, decreases in proliferation and invasion following GADD34 silencing appear to involve STAT3 signaling, as GADD34 enhances the production of inflammatory mediators such as TNF-α and IL-6, thereby activating IL-6/JAK/STAT3 signaling and promoting epithelial proliferation [10]. Since STAT3 contributes to DNA damage repair through ATM–Chk2 and ATR– Chk1 pathways [11], reduced STAT3 signaling may partially explain the increased radiosensitivity seen in GADD34-deficient CRC cells. The two NF-κB–related genes examined NFKBIA and NFKBID regulate canonical and non- canonical NF-κB pathways, respectively. NFKBIA encodes IκB-α, a negative regulator that prevents NF-κB nuclear translocation, whereas NFKBID participates in Th17/Treg-associated NF-κB signaling but does not directly modulate apoptosis. NF-κB activation is rapidly induced by irradiation and promotes cell survival through crosstalk with major pathways such as PI3K–AKT– mTOR and STING. Prior research demonstrates that inhibiting radiation-induced NF-κB activation increases apoptosis and reduces tumor cell proliferation and clonogenic survival [12]. However, the functional outcome depends on the specific NF-κB arm: canonical inhibition may suppress irradiation-induced antitumor immunity, while non-canonical inhibition enhances type I interferon responses and strengthens dendritic cell and CD8+ T-cell activity [13, 14]. Because canonical NF- κB activation is transient, it is possible that our post-irradiation validation primarily captured early- phase events. These complexities highlight the need for time-resolved studies to fully characterize NF-κB’s dual roles in radiosensitivity. BAMBI, a pseudoreceptor that antagonizes TGF-β/SMAD signaling, is typically discussed in the context of metastatic CRC; however, our results indicate that BAMBI influences proliferation, migration, and invasion even in non-metastatic CRC models. Prior work has reported that BAMBI knockdown decreases Wnt10B, p53, and Bcl-2 expression, resulting in reduced proliferation [16]. TGF-β signaling itself plays divergent roles, suppressing metastasis in CRC but promoting EMT and metastatic progression in other tumor types [15]. Importantly, TGF-β dysregulation is strongly associated with radioresistance across several cancers [4]. Radiation has been shown to downregulate BAMBI in myeloid-derived suppressor cells (MDSCs), thereby enhancing TGF-β activity and contributing to extrinsic radiation resistance in murine MC38 and B16 models [17]. However, those findings do not address tumor-cell–intrinsic BAMBI activity, and their translational relevance is limited by reliance on mouse cell lines. In our system, BAMBI knockdown before irradiation reduced CRC cell viability, implying that BAMBI-mediated inhibition of TGF-β signaling was diminished, thereby enhancing TGF-β–driven apoptotic responses. Supporting this interpretation, earlier studies have shown that BAMBI overexpression in ovarian cancer increases proliferation and motility while markedly reducing TGF-β–induced apoptosis [18, 19]. Together, these findings suggest that BAMBI influences radiosensitivity through modulation of TGF-β/SMAD signaling, though the temporal dynamics of this regulation following irradiation require further investigation. Collectively, our results identify BAMBI, GADD34, NFKBIA, and NFKBID as critical contributors to CRC progression and radiotherapy response. These genes represent promising therapeutic targets that may enable more personalized interventions to overcome radiation resistance. Nonetheless, several limitations must be acknowledged. This work was conducted exclusively in vitro, and additional validation using animal models and clinical specimens is necessary to establish clinical relevance. Moreover, mechanistic connections between these genes and immune-related pathways remain insufficiently resolved. Future studies using CRISPR-Cas9 gene editing, rescue experiments, and detailed pathway interrogation will help clarify gene-specific functions and further define their roles in CRC radiosensitivity. Ⅴ. CONCLUSIONS Colorectal cancer (CRC) exhibits substantial biological heterogeneity, a characteristic that contributes to considerable variation in patient responses to radiotherapy. The cellular reaction to radiation involves a complex series of early and late regulatory mechanisms, and these multilayered processes often limit the predictability of treatment outcomes. In this study, suppression of BAMBI, GADD34, NFKBIA, and NFKBID reduced key malignant properties such as proliferation, migration, and invasion, while also increasing the susceptibility of CRC cells to apoptosis triggered by radiation exposure. The results of this work indicate that these genes are involved in shaping the intrinsic radiosensitivity of CRC and that alterations in their expression can influence the cellular decision between survival and death following irradiation. By defining the functional relevance of these targets, this study provides a framework for understanding how specific molecular regulators contribute to radiation response in CRC. The findings also suggest that therapeutic approaches directed toward these pathways may help overcome radioresistance and strengthen the effectiveness of conventional radiotherapy. Furthermore, this study offers foundational evidence for the development of future translational strategies, such as integrating gene modulation with standard treatment protocols or identifying biomarkers that predict radiation response. 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국문 초록 (Abstract)

대장암의 방사선 저항성과 관련된 유전적·분자적 기전 연구 주제어: 대장암, 방사선 저항성, 방사선 저항성 유전자 방사선치료는 직장암 및 대장암 치료의 핵심 치료법 중 하나이나, 그 효율성은 종종 암세포에 존재하는 내재적 또는 후천적 방사선 저항성으로 인해 제한된다. 본 연구에서는 대장암 세포주 SW480 및 HCT116을 이용해 siRNA 매개 유전자 발현 저하 기법을 적용함으로써, 네 가지 후보 유전자(BAMBI, GADD34, NFKBIA, NFKBID)가 대장암 세포의 증식, 이동 및 침윤 기능과 방사선 감수성에 미치는 역할을 규명하였다. 유전자 각각을 침묵시켰을 때, 세포 증식·이동·침윤 모두 유 의하게 억제되었으며, 특히 방사선 조사 후 세포 생존율이 대조군 대비 현저히 감소하고 세포사멸(아폽토시스) 비율이 증가함을 확인하였다. 이러한 결과는 BAMBI, GADD34, NFKBIA, NFKBID가 대장암의 진행 및 방사선 저항성 조절에 중요한 기능을 수행함을 시사한다. 본 연구는 대장암에서 방사선 저항성의 기전 을 분자 수준에서 제시함으로써, 향후 분자표지자 기반 맞춤형 방사선 치료 전 략 개발의 토대를 제공한다.

목차 (Table of Contents)

• INDEX
• Index of Tables and Figures
• ABSTRACT
• Ⅰ. INTRODUCTION 1
• Ⅱ. MATERIALS AND METHODS 2
• 1. Gene Selection 2
• 2. Cell Lines and Culture 2
• 3. Small Interfering RNA(siRNA) and Transient Transfection 2
• 4. Cell Proliferation· 3
• 5. Transwell Migration Assay 3
• 6. Transwell Invasion Assay 4
• 7. Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Assay 4
• 8. Radiation Irradiation 5
• 9. Real-Time Polymerase Chain Reaction (PCR) 5
• 10. Statistical Analysis 6
• Ⅲ. RESULT 7
• 1. Gene Expression Profiling of Colorectal Cancer Patient Samples 7
• 2. Functional Assays Following Gene Silencing in CRC Cell Lines 10
• 3. Analysis of Gene Expression Related to Radiation Sensitivity in CRC Cells 14
• 4. Confocal Imaging of Radiation Response Modulated by Gene Expression 17
• Ⅳ. DISCUSSION 19
• Ⅴ. CONCLUSIONS 22
• References 23
• 국문초록 36
• INDEX OF TABLE AND FIGURES
• i. A heatmap generated through h i erarchical clustering 8
• ii. Volcano plot visualizing the differential gene expression b e t w e e n t u m o r a n d m a t c h e d n o r m a l t i s s u e s 9
• iii. Validation of gene knockdown in CRC cell lines using quantitative real-time PCR 10
• iv. Cell proliferation analysis following gene knockdown in CRC cell lines 11
• v. Migration analysis of SW480 cells following gene-specific k n o c k d o w n 12
• vi. Invasion assay of SW480 cells following individual gene knockdown 13
• vii. Evaluation of radiation response in CRC cell lines using cell viability analysis 15
• viii. Evaluat ion of radiosens i t iv i ty in CRC cell lines 17
• ix. < F i g u r e 9 > R a d i a t i o n - i n d u c e d c e l l d e a t h i n C R C cells 19