Supermolecular Confined Silicon Phosphorescence Nanoprobes for Time-Resolved Hypoxic Imaging Analysis.

Room temperature phosphorescence (RTP) nanoprobes play crucial roles in hypoxia imaging due to their high signal-to-background ratio (SBR) in the time domain. However, synthesizing RTP probes in aqueous media with a small size and high quantum yield remains challenging for intracellular hypoxic imaging up to present. Herein, aqueous RTP nanoprobes consisting of naphthalene anhydride derivatives, cucurbit[7]uril (CB[7]), and organosilicon are reported via supermolecular confined methods. Benefiting from the noncovalent confinement of CB[7] and hydrolysis reactions of organosilicon, such small-sized RTP nanoprobes (5-10 nm) exhibit inherent tunable phosphorescence (from 400 to 680 nm) with microsecond second lifetimes (up to ∼158.7 µs) and high quantum yield (up to ∼30%). The as-prepared RTP nanoprobes illustrate excellent intracellular hypoxia responsibility in a broad range from ∼0.1 to 21% oxygen concentrations. Compared to traditional fluorescence mode, the SBR value (∼108.69) of microsecond-range time-resolved in vitro imaging is up to 2.26 times greater in severe hypoxia (<0.1% O2), offering opportunities for precision imaging analysis in a hypoxic environment.

NIR-Triggered On-Demand Nitric Oxide Release for Enhanced Synergistic Phototherapy of Hypoxic Tumor.

Hypoxia of tumor microenvironments is a major factor restricting tumor treatment, which causes progression and metastasis of tumor. The hypoxic tumor microenvironment not only makes the traditional treatment method, such as chemotherapy, ineffective but also hinders the O2-dependent treatments, such as photodynamic therapy (PDT). Recently, stimuli-responsive nitric oxide (NO) donors have attracted extensive research interest in hypoxic tumor treatment because the NO release process is O2-independent. Besides, NO can distribute more uniformly than drug molecules and more widely than the PDT-generated active species due to its strong diffusion ability (200 µm in cells) and long lifetime (2 s in cells). Encouraged by these advantages, a near infrared light-triggered NO release polymeric nanoplatform (P1-CapNO NPs) was constructed by a thermally sensitive NO release unit, a photothermal unit, and a hydrophilic polyethylene glycol unit. P1-CapNO NPs possess strong absorption in the NIR region (the wavelength of maximal absorption peak was 790 nm with a molar absorption coefficient of 2.4 × 105 M-1 cm-1), great photothermal conversion efficiency (23.8%), and NO release ability (the released NO concentration can reach 1.3 µM) under 808 nm laser irradiation. Owing to these advantages, the great synergistic antitumor effect can be achieved in vitro and in vivo even under the hypoxic environment. The synergistic therapeutic strategy in this work could bypass the obstacles caused by hypoxia in tumor treatment and provide a reference for building a NO-involved therapeutic platform.

Dual-emissive Iridium(III) Complexes as Phosphorescent Probes with Orthogonal Responses to Analyte Binding and Oxygen Quenching.

Phosphorescent probes often show sensitive response toward analytes at a specific wavelength. However, oxygen quenching usually occurs at the same wavelength and thus hinders the accurate detection of analytes. In this study, we have developed dual-emissive iridium(III) complexes that exhibit phosphorescence responses to copper(II) ions at a wavelength distinct from that where oxygen quenching occurs. The complexes displayed colorimetric phosphorescence response in aqueous solutions under different copper(II) and oxygen conditions. In cellular imaging, variation in oxygen concentration over a large range from 5 % to 80 % can modulate the intensity and lifetime of green phosphorescence without affecting the response of red phosphorescence toward intracellular copper(II) ions.

circEXOC6B interacting with RRAGB, an mTORC1 activator, inhibits the progression of colorectal cancer by antagonizing the HIF1A-RRAGB-mTORC1 positive feedback loop.

BACKGROUND: In recent years, an increasing number of studies have indicated that circular RNA plays crucial roles in regulating tumor development and chemoresistance. Using two high-throughput RNA sequence datasets, we previously found that circEXOC6B was downregulated in colon cancer. However, its role and mechanism in colorectal cancer (CRC) remained unknown. METHODS: Real-time quantitative PCR was used to examine the expression of circEXOC6B in CRC tissues. In vivo and in vitro functional experiments were performed to determine the suppressor role of circEXOC6B in CRC progression. RNA pull-down, mass spectrometry, RNA-binding protein immunoprecipitation, co-immunoprecipitation, fluorescence in situ hybridization, and immunofluorescence were applied to investigate the possible mechanisms connecting circEXOC6B to CRC growth and 5-fluorouracil-induced apoptosis. Chromatin immunoprecipitation, dual-luciferase assay, western blot, and immunohistochemistry were used to explore the mechanisms underlying the HIF1A regulation of RRAGB transcription. RESULTS: circEXOC6B was downregulated in CRC tissues, and its lower expression was associated with poor prognosis of patients. Functional experiments showed that circEXOC6B inhibited growth and increased the 5-fluorouracil-induced apoptosis of CRC cells in vitro and in vivo. Mechanistically, circEXOC6B inhibited the heterodimer formation of RRAGB by binding to it, thereby suppressing the mTORC1 pathway and HIF1A level. In addition, HIF1A upregulated the transcription of RRAGB by binding to its promoter region. Altogether, the results demonstrated that a HIF1A-RRAGB-mTORC1 positive feedback loop drives tumor progression in CRC, which could be interrupted by circEXOC6B. CONCLUSIONS: circEXOC6B inhibits the progression of CRC and enhances the chemosensitivity of CRC cells to 5-fluorouracil by antagonizing the HIF1A-RRAGB-mTORC1 positive feedback loop. circEXOC6B is a possible therapeutic target for CRC treatment.

Millisecond-Range Time-Resolved Bioimaging Enabled through Ultralong Aqueous Phosphorescence Probes.

Probes featuring room-temperature phosphorescence (RTP) are promising tools for time-resolved imaging. It is worth noting that the time scale of time-resolved bioimaging generally ranges around the microsecond level, because of the short-lived emission. Herein, the first example of millisecond-range time-resolved bioimaging is illustrated, which is enabled through a kind of ultralong aqueous phosphorescence probes (i.e., cyclo-(Arg-Gly-AspD-Tyr-Cys)-conjugated zinc-doped silica nanospheres), with a RTP emission lasting for ≈5 s and a lifetime as long as 743.7 ms. We demonstrate that live cells and deep tumor tissue in mice can be specifically targeted through immune-phosphorescence imaging, with a high signal-to-background ratio (SBR) value of ≈69 for in vitro imaging, and ≈627 for in vivo imaging, respectively. We further show that, compared to that of fluorescence imaging, the SBR enhancement of millisecond-range time-resolved in vivo bioimaging is up to 105 times.

Overcoming Tumor Hypoxia through Multiple Pathways Using an All-in-One Polymeric Therapeutic Agent to Enhance Synergistic Cancer Photo/Chemotherapy Effects.

Hypoxia is a significant characteristic of tumors, which causes aggressive tumor growth and strong therapy resistance. Inspired by the improved therapeutic efficacy of synergistic treatment, herein, an all-in-one polymeric therapeutic agent was developed, which could overcome tumor hypoxia through multiple pathways. Multiple therapeutic agents were incorporated into the polymer, including the singlet oxygen (1O2) carrier unit to store cytotoxic reactive oxygen species, the photosensitized and photothermal unit to trigger the capture and release of 1O2, and the hypoxia-responsive prodrug unit to maintain a long-term tumor inhibition. In addition, the hydrophilic polyethylene glycol unit was also introduced to improve water-solubility and biocompatibility. Importantly, this study achieved the capture and controllable release of 1O2 just by regulating the power of an 808 nm laser for the first time, which is more convenient and flexible than previous works. As expected, the as-prepared copolymer displayed reduced oxygen dependence, accompanied with promising synergistic anti-tumor and anti-recurrence efficacies under hypoxic in vitro and in vivo environments. Consequently, this synergistic anti-hypoxia strategy may open up new avenues in the design of all-in-one therapeutic platforms for promoting the development of accurate, efficient, and long-acting treatment in clinical studies.

Bioorthogonal "Labeling after Recognition" Affording an FRET-Based Luminescent Probe for Detecting and Imaging Caspase-3 via Photoluminescence Lifetime Imaging.

Bis-labeling with a luminescent energy donor/acceptor pair onto biological substrates affords probes which give FRET readouts for the detection of interaction partners. However, the covalently bound luminophores bring about steric hindrance and nonspecific interaction, which probably perturb the biological recognition. Herein, we designed a highly sensitive and specific "labeling after recognition" sensing approach, where luminophore labeling occurred after the biological recognition. Taking the cutting enzyme caspase-3 as an example, we demonstrated the detection of its catalytic activity in solution and apoptotic cells using the tetrapeptide motif Asp-Glu-Val-Asp (DEVD) as the cleavable substrate, and an iridium(III) complex and a rhodamine derivative as the energy donor/acceptor pair. The DEVD tetrapeptide was modified with an azide and a GK-norbornylene groups at the amino and carboxyl terminuses, respectively, which allowed donor/acceptor bis-labeling via two independent catalysis-free bioorthogonal reactions. The phosphorescence lifetime of the iridium(III) complex was quenched upon bis-labeling owing to the intracellular FRET to the rhodamine derivative, and significantly elongated upon the peptide being catalytically cleaved by caspase-3. Interestingly, the sensitivity and efficiency of the lifetime responses were much higher in the "labeling after recognition" sensing approach. Molecular docking analysis showed that the steric hindrance and nonspecific interactions partially inhibited the biological recognition of the DEVD substrate by caspase-3. The imaging of the catalytic activity of caspase-3 in apoptotic cells was demonstrated via photoluminescence lifetime imaging microscopy. Lifetime analysis not only confirmed the occurrence of intracellular bioorthogonal bis-labeling and catalytic cleavage, but also showed the extent to which the two dynamic processes occurred.

Mineral acid-triggered multicolor room-temperature phosphorescence nanoprobes for time-resolved bioimaging.

We present a facile strategy to achieve color-tunability room-temperature phosphorescence (RTP) nanoprobes by doping mineral acids (i.e., boric acid and phosphoric acid) in an organic silicon scaffold through a cross-linking process. Such RTP nanoprobes exhibit inherent tunable phosphorescence (from 420-650 nm) with long lifetime (emission lasting for ∼5-15 s, RTP lifetime: ∼0.53-2.11 s) and high quantum yields (∼13.1-43.0%). Therefore, the as-prepared nanoprobes enable multiple imaging in live cells with a high signal-to-background ratio value of ∼52.

Phosphorescence Enzyme-Mimics for Time-Resolved Sensitive Diagnostics and Environment-Adaptive Specific Catalytic Therapeutics.

Enzyme mimics (EMs) with intrinsic catalysis activity have attracted enormous interest in biomedicine. However, there is a lack of environmentally adaptive EMs for sensitive diagnosis and specific catalytic therapeutics in simultaneous manners. Herein, the coordination modulation strategy is designed to synthesize silicon-based phosphorescence enzyme-mimics (SiPEMs). Specifically, the atomic-level engineered Co-N4 structure in SiPEMs enables the environment-adaptive peroxidase, oxidase, and catalase-like activities. More intriguingly, the internal Si-O networks are able to stabilize the triplet state, exhibiting long-lived phosphorescence with lifetime of 124.5 ms, suitable for millisecond-range time-resolved imaging of tumor cells and tissue in mice (with high signal-to-background ratio values of ∼60.2 for in vitro and ∼611 for in vivo). Meanwhile, the SiPEMs act as an oxidative stress amplifier, allowing the production of ·OH via cascade reactions triggered by the tumor microenvironment (∼136-fold enhancement in peroxidase catalytic efficiency); while the enzyme-mimics can scavenge the accumulation of reactive oxygen species to alleviate the oxidative damage in normal cells, they are therefore suitable for environment-adaptive catalytic treatment of cancer in specific manners. We innovate a systematic strategy to develop high-performance enzymemics, constructing a promising breakthrough for replacing traditional enzymes in cancer treatment applications.

Full-color persistent room temperature phosphorescent elastomers with robust optical properties.

Persistent room temperature phosphorescent materials with unique mechanical properties and robust optical properties have great potential in flexible electronics and photonics. However, developing such materials remains a formidable challenge. Here, we present highly stretchable, lightweight, and multicolored persistent luminescence elastomers, produced by incorporating ionic room temperature phosphorescent polymers and polyvinyl alcohol into a polydimethylsiloxane matrix. These prepared elastomers exhibit high optical transparency in daylight and emit bright persistent luminescence after the removal of 365 nm excitation. The homogeneous distribution of polymers within the matrix has been confirmed by confocal fluorescence microscopy, scanning electron microscopy, and atomic force microscopy. Mechanical property investigations revealed that the prepared persistent luminescence elastomers possess satisfactory stretchability. Impressively, these elastomers maintain robust optical properties even under extensive and repeated mechanical deformations, a characteristic previously unprecedented. These fantastic features make these persistent luminescence elastomers ideal candidates for potential applications in wearable devices, flexible displays, and anti-counterfeiting.

The prognosis analysis of organ metastatic patterns in lung large cell neuroendocrine carcinoma: A population-based study.

Lung large cell neuroendocrine carcinoma (LCNEC) is a rare and highly aggressive malignancy with a dismal prognosis. This study was designed to depict patterns of distant organ metastatic and to analyze prognosis of LCNEC patients. We gathered data from the Surveillance, Epidemiology, and End Results (SEER) database between 2010 and 2015. We conducted the Kaplan-Meier method to calculate overall survival (OS) and compare different variables. Cox proportional hazards regression models in univariate and multivariate analyses were employed to further explore prognostic factors. A total of 1335 LCNEC patients were eventually selected from the SEER database, of which 348 patients (26.0%) had single organ metastasis and 197 patients (14.8%) had multiple metastases. Our study indicates that patients with single organ metastasis generally have a poor prognosis, with a median OS of 8 months for both lung and brain metastasis with 1-year survival rates of 33% and 29% respectively. Patients with multiple metastases exhibited the worst prognosis, with a median OS of only 4 months and a 1-year OS of 8%. Multivariate analysis revealed that age, T stage, N stage, chemotherapy and radiation in metastatic patients were independently associated with OS. In conclusion, LCNEC exhibits a high metastatic rate when diagnosed. The most common metastatic organ is the brain in single-site metastatic patients. Patients with single or multiple metastases exhibit a significantly worse prognosis than those with non-organ metastases. In the group of single organ metastases, patients with brain and lung metastases had a better prognosis than those with bone and liver metastases.

HRK inhibits colorectal cancer cells proliferation by suppressing the PI3K/AKT/mTOR pathway.

Background: As one of the most common malignant tumor, colorectal cancer (CRC) continues to have a high incidence and mortality rate. HRK belongs to the BCL-2 protein family, which has been shown to have antitumor effects in prostate cancer. However, its role in colorectal cancer is not yet known. Methods: In this study, we verified the expression levels of HRK in colorectal cancer tissues by public database search as well as immunohistochemistry. Next, we analyzed HRK expression levels in CRC tissues,adjacent non-cancerous tissues, cell lines and normal intestinal epithelial cells by qPCR and Western blotting. CCK-8 proliferation assays, transwell assays, wound healing assays, colony assays and flow cytometry were performed to clarified the effect of HRK on CRC cells. Western blotting and rescue experiments were used to determine the role of HRK in regulating PI3K/AKT/mTOR signaling pathway. Results: HRK expression was lower in CRC tissues and cell lines. Gain and loss of function experiments showed that HRK decreased proliferation, invasion and migration of CRC cells. Low expression of HRK inhibited CRC cell apoptosis as well as activated the PI3K/AKT/mTOR signaling pathway. In addition, rapamycin inhibits the activation of PI3K/AKT/mTOR signaling pathway and reverses HRK-induced alterations in cell biological functions. Conclusion: Our study demonstrates that HRK is lowly expressed in colorectal cancer tissues. And for the first time, HRK was shown to promote apoptosis and inhibit proliferation of colorectal cancer cells by inhibiting PI3K/AKT/mTOR signaling pathway. HRK represents a potential target for the treatment of CRC.

Cellular imaging properties of phosphorescent iridium(III) complexes substituted with ester or amide groups.

Phosphorescent iridium(III) complexes have been extensively investigated as cellular imaging reagents and sensors. The intracellular localization of the complexes is known to be closely related to their formal charge, molecular size, lipophilicity, and bioactive pendants. Herein, we reported four phosphorescent iridium(III) complexes with the diimine ligands being modified with ester or amide groups as imaging reagents for living cells. The complexes have the same positive charge and very similar molecular size and weight. The lipophilicity of the complexes is similar ranging from 1.45 to 2.14. Upon internalization into living HeLa cells, while complexes 2-4, like most other iridium(III) complexes, were localized in the cytoplasm, complex 1 unexpectedly stained the whole cells including nuclei. The nuclear uptake of complex 1 was not observed when the cells were pretreated with chlorpromazine or nocodazole, suggesting that clathrin and microtubules mediated the nuclear uptake of complex 1. Additionally, the nuclear uptake efficiency is related to the cell division cycle. The complex was mainly concentrated in the nucleus when the cells were in mitosis, and distributed in whole cells when the cells were in the interphases. Furthermore, complex 1 exhibited a longer luminescence lifetime in the nucleus than in the cytoplasm as revealed by photoluminescence lifetime imaging microscopy (PLIM). Incubation of the cells in the hypoxia environment elongated the lifetime of the cytoplasmic complex, but hardly affected the luminescence properties of the intranuclear complex.

Dual-lifetime luminescent probe for time-resolved ratiometric oxygen sensing and imaging.

Fluorescent/phosphorescent dual-emissive polymers or hybrids consisting of both fluorophore and phosphor have been used as self-calibrating probes and imaging reagents for cellular molecular oxygen. Oxygen selectively quenches the phosphorescence and the fluorescence serves as an internal reference. The phosphorescence/fluorescence ratio is used as a quantitative indicator of oxygen content. In wavelength-ratiometric probes, the fluorophore and phosphor are designed to emit at different wavelengths. It is easy to achieve spectral separation, but the phosphorescence/fluorescence ratio fluctuates due to the difference in the absorption and scattering of light at different wavelengths by biological samples. Herein we reported a lifetime-ratiometric luminescent polymeric probe where the fluorophore and phosphor emitted at the same wavelength. Spectral separation was achieved based on the difference in their excited-state lifetimes via time-resolved luminescence analysis and imaging. The probe exhibited a phosphorescence lifetime of about 931 ns with a phosphorescence/fluorescence ratio of 4.49 in deaerated aqueous buffer. The lifetime was shortened to 251 ns and the ratio decreased to 1.08 in oxygen saturated solution because of phosphorescence quenching. The utilization of the probe for quantitative oxygen sensing and mapping in living HeLa cells was demonstrated using calibration curves obtained from fixed cells.

Manipulating Electroluminochromism Behavior of Viologen-Substituted Iridium(III) Complexes through Ligand Engineering for Information Display and Encryption.

Electrically controlling photoluminescence has attracted great research interest and offers many opportunities for technological developments. Electroluminochromic materials undergo redox reactions under low-voltage stimuli to achieve reversible luminescence switching. Till now, photoluminescence switching of a single molecule caused by electrical stimuli is restricted to intensity response because the redox-active moieties are good electron donors or acceptors and electrical stimuli can regulate the photoinduced electron-transfer and affect the luminescence intensity. In this work, the manipulation of the electroluminochromism behavior of a series of viologen-substituted iridium(III) complexes through the regulation of ligand orbital energy levels and electronic communication between the viologen pendants and the iridium(III) complex core is reported. Electrochemical redox reactions reversibly modulate either the luminescence quenching effect or the push-pull electronic effect of the viologen substituents, achieving multicolor "on-off" luminescence response toward electrical stimuli and luminescence manipulation between two emissive states with different wavelengths and lifetimes. To illustrate the promising applications of these electroluminochromic materials, recording and displaying luminescence information under electrical stimuli are demonstrated. Information encryption is realized by letting the electroluminochromism occur in the near-infrared region or in the time domain. Near-infrared camera or time-resolved luminescence analysis can be used to help read the invisible information.

Time-resolved analysis of photoluminescence at a single wavelength for ratiometric and multiplex biosensing and bioimaging.

Simultaneous analysis of luminescence signals of multiple probes can improve the accuracy and efficiency of biosensing and bioimaging. Analysis of multiple signals at different wavelengths usually suffers from spectral overlap, possible energy transfer, and difference in detection efficiency. Herein, we reported a polymeric luminescent probe, which was composed of a phenothiazine-based fluorescent compound and a phosphorescent iridium(iii) complex. Both luminophores emitted at around 600 nm but their luminescence lifetimes are 160 times different, allowing time-resolved independent analysis. As the fluorescence was enhanced in response to oxidation by hypochlorite and the phosphorescence was sensitive toward oxygen quenching, a four-dimensional relationship between luminescence intensity, fluorescence/phosphorescence ratio, hypochlorite concentration, and oxygen content was established. In cellular imaging, time-resolved photoluminescence imaging microscopy clearly showed the independent fluorescence response toward hypochlorite and phosphorescence response toward oxygen in separated time intervals. This work opens up a new idea for the development of multiplex biosensing and bioimaging.

Novel PI3K/Akt/mTOR pathway inhibitors plus radiotherapy: Strategy for non-small cell lung cancer with mutant RAS gene.

Non-small cell lung cancer (NSCLC) with RAS -mutant gene has been the most difficult obstacle to overcome. Over 25% of muted lung adenocarcinomas have RAS mutation. The prognosis of NSCLC patients with RAS-mutant genes is always poor because there is no effective drug to suppress RAS-mutant genes. NSCLC patients with RAS-mutant usually develop resistance to radiotherapy and chemotherapy, which in some cases leads to a 5-10% survival rate for non-small cell lung cancer (NSCLC). As little clinical symptom of NSCLC was presented at its early stages, thus it always brings in disappointing treatment outcome. Currently, NSCLC presents the highest morbidity and mortality all over the world. The combination of PI3K/AKT/mTOR pathway inhibitors with radiotherapy is a novel strategy to improve radiosensitivity and therapeutic outcome of NSCLC with a RAS-mutant gene. There have been many preclinical studies and clinical trials on the effect of PI3K/AKT/mTOR pathway inhibitors combined with radiotherapy in NSCLC with a RAS-mutant gene have been reported in the past years. This review provides current knowledge of the combination of PI3K/Akt/mTOR pathway inhibitors with radiotherapy, which prove to be a significant improvement for the treatment of NSCLC patients with RAS mutations and will benefit NSCLC patients with RAS mutations.

Cyclometalated iridium(III) complexes containing an anthracene unit for sensing and imaging singlet oxygen in cellular mitochondria.

Singlet oxygen (1O2), as a highly reactive oxygen species, plays an important role in the physical, chemical and biomedical fields, especially during photodynamic therapy (PDT) process. In this work, two iridium(III) complexes containing an anthracene unit in their diimine ligand were designed and synthesized to monitor 1O2 in living cells. The complexes were weakly emissive owing to the photoinduced electron transfer process, but exhibited intense luminescence upon capturing 1O2, resulting from the formation of the corresponding endoperoxide analogues. The remarkable turn-on luminescence response was specific toward 1O2 and in preference to other reactive oxygen species. The utilization of one of the complexes for imaging 1O2 in living cells has also been demonstrated using three different cells lines. Cells incubated with the complexes were hardly emissive. Further light irradiation at 475 nm triggered intracellular emission turn on, indicative of the production of 1O2 photochemically. The emissive pattern was well colocalized with commercially available MitoTracker, suggesting the potential applications of the complexes for imaging mitochondria 1O2. The 1O2 capturing properties rendered the complexes low photocytotoxicity since 1O2-caused oxidative damage toward cellular molecules and structures was inhibited.

Genome-wide profiling of chemoradiation­induced changes in alternative splicing in colon cancer cells.

Alternative splicing is a key mechanism that regulates protein diversity and has been found to be associated with colon cancer progression and metastasis. However, the function of alternative splicing in chemoradiation­resistant colon cancer remains elusive. In this study, we constructed a chemoradiation­resistant colon cancer cell line. Through RNA-sequencing of normal and chemoradiation­resistant colon cancer cells (HCT116), we found 818 genes that were highly expressed in the normal HCT116 cells, whereas 285 genes were highly expressed in the chemoradiation-resistant HCT116 (RCR-HCT116) cells. Gene ontology (GO) analysis showed that genes that were highly expressed in the HCT116 cells were enriched in GO categories related to cell cycle and cell division, whereas genes that were highly expressed in the RCR-HCT116 cells were associated with regulation of system processes and response to wounding. Analysis of alternative splicing events revealed that exon skipping was significantly increased in the chemoradiation­resistant colon cancer cells. Moreover, we identified 323 alternative splicing events in 293 genes that were significantly different between the two different HCT116 cell types. These alternative splicing­related genes were clustered functionally into several groups related with DNA replication, such as deoxyribonucleotide metabolic/catabolic processes, response to DNA damage stimulus and helicase activity. These findings enriched our knowledge by elucidating the function of alternative splicing in chemoradiation-resistant colon cancer.