Radiosensitivity Of Colorectal Cancer Research Articles

MiR-378a-5p exerts a radiosensitizing effect on CRC through LRP8/β-catenin axis

ABSTRACT Background MiRNAs are closely related to tumor radiosensitivity. MiR-378a-5p level is down-regulated in colorectal cancer (CRC). Therefore, this study intends to explore the role of miR-378a-5p in CRC, especially radiosensitivity. Methods The expression of miR-378a-5p was analyzed in CRC samples. CRC cell lines were treated with different doses of X-rays. Bioinformatics analysis, dual-luciferase reporter assay and RT-qPCR were used to detect the expressions and binding relationship of miR-378a-5p and low-density lipoprotein receptor-related protein 8 (LRP8). MiR-378a-5p inhibitor or/and siLRP8 were transfected into CRC cells with or without irradiation. Subsequently, clonogenic assay, flow cytometry and in vivo experiments including tumorigenesis assay, immunohistochemistry, RT-qPCR and Western blot were performed to clarify the role of miR-378a-5p/LRP8 axis in the radiosensitivity of CRC. Results The down-regulated expression of miR-378a-5p in CRC is related to histological differentiation and tumor-node-metastasis (TNM) stage. After irradiation, the survival fraction of CRC cells was decreased, while the apoptotic rate and the level of miR-378a-5p were increased. Restrained miR-378a-5p repressed apoptosis and apoptosis-related protein expressions, yet promoted the proliferation and the radioresistance of cells by regulating β-catenin in CRC cells. LRP8 was highly expressed in CRC, and targeted by miR-378a-5p. SiLRP8 improved radiosensitivity and reversed the effect of miR-378a-5p down-regulation on CRC cells. Overexpressed miR-378a-5p and irradiation enhanced the level of miR-378a-5p, yet suppressed the expressions of Ki67 and LRP8 as well as tumorigenesis. Conclusion MiR-378a-5p may exert a radiosensitizing effect on CRC through the LRP8/β-catenin axis, which may be a new therapeutic target for CRC radioresistance.

Protective effect of total flavonoids of Engelhardia roxburghiana Wall. leaves against radiation-induced intestinal injury in mice and its mechanism.

Irradiation-induced intestinal injury (RIII) often occurs during radiotherapy in patients, which would result in abdominal pain, diarrhea, nausea, vomiting, and even death. Engelhardia roxburghiana Wall. leaves, a traditional Chinese herb, has unique anti-inflammatory, anti-tumor, antioxidant, and analgesic effects, is used to treat damp-heat diarrhea, hernia, and abdominal pain, and has the potential to protect against RIII. To explore the protective effects of the total flavonoids of Engelhardia roxburghiana Wall. leaves (TFERL) on RIII and provide some reference for the application of Engelhardia roxburghiana Wall. leaves in the field of radiation protection. The effect of TFERL on the survival rate of mice was observed after a lethal radiation dose (7.2 Gy) by ionizing radiation (IR). To better observe the protective effects of the TFERL on RIII, a mice model of RIII induced by IR (13 Gy) was established. Small intestinal crypts, villi, intestinal stem cells (ISC) and the proliferation of ISC were observed by haematoxylin and eosin (H&E) and immunohistochemistry (IHC). Quantitative real-time PCR (qRT-PCR) was used to detect the expression of genes related to intestinal integrity. Superoxide dismutase (SOD), reduced glutathione (GSH), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the serum of mice were assessed. In vitro, cell models of RIII induced by IR (2, 4, 6, 8 Gy) were established. Normal human intestinal epithelial cells HIEC-6 cells were treated with TFERL/Vehicle, and the radiation protective effect of TFERL on HIEC-6 cells was detected by clone formation assay. DNA damage was detected by comet assay and immunofluorescence assay. Reactive oxygen species (ROS), cell cycle and apoptosis rate were detected by flow cytometry. Oxidative stress, apoptosis and ferroptosis-related proteins were detected by western blot. Finally, the colony formation assay was used to detect the effect of TFERL on the radiosensitivity of colorectal cancer cells. TFERL treatment can increase the survival rate and time of the mice after a lethal radiation dose. In the mice model of RIII induced by IR, TFERL alleviated RIII by reducing intestinal crypt/villi structural damage, increasing the number and proliferation of ISC, and maintaining the integrity of the intestinal epithelium after total abdominal irradiation. Moreover, TFERL promoted the proliferation of irradiated HIEC-6 cells, and reduced radiation-induced apoptosis and DNA damage. Mechanism studies have found that TFERL promotes the expression of NRF2 and its downstream antioxidant proteins, and silencing NRF2 resulted in the loss of radioprotection by TFERL, suggesting that TFERL exerts radiation protection by activating the NRF2 pathway. Surprisingly, TFERL reduced the number of clones of colon cancer cells after irradiation, suggesting that TFERL can increase the radiosensitivity of colon cancer cells. Our data showed that TFERL inhibited oxidative stress, reduced DNA damage, reduced apoptosis and ferroptosis, and improved IR-induced RIII. This study may offer a fresh approach to using Chinese herbs for radioprotection.

TRAF4-mediated ubiquitination-dependent activation of JNK/Bcl-xL drives radioresistance

The E3 ligase TNF receptor-associated factor 4 (TRAF4) is upregulated and closely associated with tumorigenesis and the progression of multiple human malignancies. However, its effect on radiosensitivity in colorectal cancer (CRC) has not been elucidated. The present study found that TRAF4 was significantly increased in CRC clinical tumor samples. Depletion of TRAF4 impaired the malignant phenotype of CRC cells and sensitized irradiation-induced cell death. Irradiation activated the c-Jun N-terminal kinases (JNKs)/c-Jun signaling via increasing JNKs K63-linked ubiquitination and phosphorylation. Furthermore, c-Jun activation triggered the transcription of the antiapoptotic protein Bcl-xL, thus contributing to the radioresistance of CRC cells. TRAF4 was positively correlated with c-Jun and Bcl-xL, and blocking TRAF4 or inhibiting Bcl-xL with inhibitor markedly promoted ionizing radiation (IR)-induced intrinsic apoptosis and sensitized CRC cells to radiotherapy in vitro and in vivo. Our findings illustrate a potential mechanism of radioresistance, emphasizing the clinical value of targeting the TRAF4/Bcl-xL axis in CRC therapy.

MiR-7-5p/KLF4 signaling inhibits stemness and radioresistance in colorectal cancer

Resistance to radiotherapy remains a major unmet clinical obstacle in the treatment of locally advanced rectal cancer. Cancer stem cells (CSCs) are considered to mediate tumor development and radioresistance. However, the role of CSCs in regulating resistance to radiotherapy in colorectal cancer (CRC) remains largely unknown. We established two radioresistant CRC cell lines, HCT116-R and RKO-R, using fractionated irradiation. Analysis using miRNA sequencing and quantitative real-time PCR confirmed lower levels of miR-7-5p in both of the radioresistant cells compared to their parental cells. Subsequently, we validated that miR-7-5p expression was decreased in cancerous tissues from radiotherapy-resistant rectal cancer patients. The Cancer Genome Atlas (TCGA) database analyses revealed that low miR-7-5p expression was significantly correlated with poor prognosis in CRC patients. Overexpression of miR-7-5p led to a rescue of radioresistance and an increase in radiation-induced apoptosis, and attenuated the stem cell-like properties in HCT116-R and RKO-R cells. Conversely, knocking down miR-7-5p in parental HCT116 and RKO cells suppressed the sensitivity to radiation treatment and enhance cancer cell stemness. Stemness-associated transcription factor KLF4 was demonstrated as a target of miR-7-5p. Rescue experiments revealed that miR-7-5p/KLF4 axis could induce radiosensitivity by regulating CSCs in colorectal cancer cells. Furthermore, we used CRC tumor tissues which exhibited resistance to neoadjuvant radiotherapy to establish a patient-derived xenograft (PDX) mouse model. Tail vein injection of magnetic nanoparticles carrying miR-7-5p mimics into the PDX mice significantly inhibited tumor growth with or without irradiation treatment in vivo. Our current studies not only demonstrate an anti-cancer function of miR-7-5p in regulating CSC properties and radiosensitivity in colorectal cancer, but also provide a novel potential strategy for delaying or reverse radiation resistance in preoperative radiotherapy of CRC patients.

CircRNA ITGA7 restrains growth and enhances radiosensitivity by up-regulating SMAD4 in colorectal carcinoma.

Circular RNAs have been reported to be widely involved in cancer cell tumorigenesis and drug resistance; here, the aim of this study was to investigate whether circRNA Integrin Subunit Alpha 7 (ITGA7) (circ_ITGA7) was associated with the tumor growth and radiosensitivity of colorectal cancer (CRC). We found that circ_ITGA7 expression was lower in CRC tissues and cells than those in the normal tissues and cell lines according to quantitative real-time polymerase chain reaction. As shown by cell counting kit-8 assay, flow cytometry, colony formation assay, and xenograft experiment, ectopic overexpression of circ_ITGA7 remarkably restrained CRC tumor growth and enhanced radiosensitivity in vitro and in vivo. Mechanistically, circ_ITGA7 could target microRNA (miR)-766 to prevent the degradation of its target gene mothers against decapentaplegic homolog 4 (SMAD4), the binding between miR-766 and circ_ITGA7 or SMAD4 was first verified by dual-luciferase activity assay. Additionally, miR-766 up-regulation reversed the inhibitory effects of circ_ITGA7 on CRC growth and radiosensitivity. Besides that, inhibition of miR-766 reduced CRC cell growth and sensitized cells to radiotherapy, and these effects mediated by miR-766 inhibitor were rescued by the silencing of SMAD4. In all, circ_ITGA7 suppressed CRC growth and enhanced radiosensitivity by up-regulating SMAD4 through sequestering miR-766, providing an insight for the further development of CRC treatment.

Targeting the DNA Damage Response and DNA Repair Pathways to Enhance Radiosensitivity in Colorectal Cancer.

Simple SummaryThe DNA damage response pathway plays a critical role in maintaining genomic integrity. Therefore, inhibition of activation of cell-cycle checkpoints involved in this pathway may increase the sensitivity of tumor cells to DNA damage induced by ionizing radiation. In this review, we provide an overview of mechanisms, preclinical studies and advances in clinical trials of DNA-PKcs, ATM/ATR, CHK1/CHK2, WEE1 and PARP1 kinase inhibitors combined with radiotherapy for colorectal cancer treatment. We evaluate the potential of developing high-efficiency and low-toxicity radiosensitizers targeting the DNA damage response and DNA repair pathways to enhance the response to radiotherapy in colorectal cancer.Radiotherapy is an important component of current treatment options for colorectal cancer (CRC). It is either applied as neoadjuvant radiotherapy to improve local disease control in rectal cancers or for the treatment of localized metastatic lesions of CRC. DNA double-strand breaks (DSBs) are the major critical lesions contributing to ionizing radiation (IR)-induced cell death. However, CRC stem cells promote radioresistance and tumor cell survival through activating cell-cycle checkpoints to trigger the DNA damage response (DDR) and DNA repair after exposure to IR. A promising strategy to overcome radioresistance is to target the DDR and DNA repair pathways with drugs that inhibit activated cell-cycle checkpoint proteins, thereby improving the sensitivity of CRC cells to radiotherapy. In this review, we focus on the preclinical studies and advances in clinical trials of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related kinase (ATR), checkpoint kinase 1 (CHK1), checkpoint kinase 2 (CHK2), WEE1 and poly (ADP-ribose) polymerase 1 (PARP1) kinase inhibitors in CRC. Importantly, we also discuss the selective radiosensitization of CRC cells provided by synthetic lethality of these inhibitors and the potential for widening the therapeutic window by targeting the DDR and DNA repair pathways in combination with radiotherapy and immunotherapy.

Role of non-coding RNAs in radiosensitivity of colorectal cancer: A narrative review

Colorectal cancer (CRC) is a global public health concern because of its high prevalence and mortality. Although radiotherapy is a key method for treating CRC, radioresistance is an obstacle to radiotherapy use. The molecular mechanisms underlying the radioresistance of CRC remain unclear. Increasing evidence has revealed the multiple regulatory functions of non-coding RNAs (ncRNAs) in numerous malignancies, including CRC. Several ncRNAs have been reported to be involved in the determination of radiosensitivity of CRC cells, and some have excellent potential to be prognostic biomarkers or therapeutic targets in CRC treatment. The present review discusses the biological functions and underlying mechanisms of ncRNAs (primarily lncRNA, miRNA, and circRNA) in the regulation of the radiosensitivity of CRC. We also evaluate studies that examined ncRNAs as biomarkers of response to radiation and as therapeutic targets for enhancing radiosensitivity.

Abstract 5240: S-phase cyclinA associated protein in the ER(SCAPER)rs3812908 T>C promotes the nuclear translocation of CyclinA2 and enhances DNA damage repair in radioresistance of colorectal cancer

Abstract Radiotherapy resistance is still an obstacle of improving the survival of patients with locally advanced colorectal cancer. In this study, whole-exome sequencing and targeted sequencing were preformed on colorectal cancer tumor tissue samples which were colloected from multiple centers, and the pathologic complete response predictive model PGS-LARC(Prediction Genotype Signature for Locally Advanced Rectal Cancer) was established. A candidate SNV-SCAPER rs3812908 T>C was screened using this model. To investigate the relationship between SCAPER rs3812908 T>C and radiosensitivity of colorectal cancer, targeted sequencing was performed and the results revealed that proportion of CC genotype was significantly higher in resistant sequencing cohort than in sensitive ones (30% vs. 22%). And Immunohistochemistry (IHC) results revealed that SCAPER protein expressed less in CC genotype colorectal cancer tissues than in TT genotype. Here, the cell proliferation results showed that SCAPER-KD cells and SCAPER rs3812908 CC cells were more radioresistant than SCAPER-NC and TT cells. Furthermore, the immunofluorescence assay showed SCAPER rs3812908 T>C promoted translocation of S phase associated protein-Cyclin A2 into nucleus and western blotting results showed that the phosphorylation of ERK and JNK(p-ERK and p-JNK) increased significantly in SCAPER CC cells, and the protein p-ATR, p-ATM, p-CDC25C and CDC25A related to homologous recombination repair were also increased in SCAPER-KD and SCAPER CC cells.Taken together, the SCAPER gene rs3812908 T>C plays an important role in radioresistance of colorectal cancer. The SNV of SCAPER rs3812908 T>C significantly promotes Cyclin A2 nuclear translocation, activates the MAPKs pathway, and increases homologous recombination repair, and finally leads to radioresistance. Citation Format: Hai-na Yu, Ting Jiang, Shuang Liu, Yan Yuan, Shao-yan Xi, Zhi-wei Guo, Yuan-hong Yuan, Yan Li, Wei-wei Xiao. S-phase cyclinA associated protein in the ER(SCAPER)rs3812908 T>C promotes the nuclear translocation of CyclinA2 and enhances DNA damage repair in radioresistance of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5240.

Radiosensitivity of colorectal cancer and radiation-induced gut damages are regulated by gasdermin E.

Although radiotherapy is an important clinical option available for colorectal cancer (CRC), its use is restricted due to low radiosensitivity of CRC and high toxicity to surrounding normal tissues. The purpose of this study is to investigate the molecular mechanism by which CRC is not sensitive to radiation and radiation causes toxicity to surrounding normal tissues. Here we found that GSDME was silenced in CRC but markedly expressed in their surrounding normal tissues. GSDME determines radiation-induced pyroptosis in CRC cells and normal epithelial cells through the caspase-3-dependent pathway. GSDME expression sensitizes radioresistant CRC cells to radiation. In the homograft model, after radiation treatment, the tumor volume and weight were significantly decreased in GSDME-expressed homograft tumors compared to GSDME-knockout homograft tumors. On the mechanism, radiation induced GSDME-mediated pyroptosis in CRC cells, which recruited and activated NK cells to enhance antitumor immunity. In addition, GSDME-knockout mice were protected from radiation-induced weight loss and tissue damages in the intestine, stomach, liver and pancreas compared to wild-type control littermates. In summary, we show that GSDME determines CRC radiosensitivity and radiation-related toxicity to surrounding normal tissues through caspase-3-dependent pyroptosis. Our finding reveals a previously unrecognized link between radiation and pyroptosis.

Exosomal circ_PTPRA inhibits tumorigenesis and promotes radiosensitivity in colorectal cancer by enriching the level of SMAD4 via competitively binding to miR-671-5p.

Accumulating evidence supports that exosomal RNAs are crucial in tumor microenvironment and may be used as diagnostic biomarkers for cancers. This study aimed to determine the role of exosomal circular RNA_protein tyrosine phosphatase receptor type A (circ_PTPRA) in colorectal cancer (CRC). The morphology of exosomes was identified by transmission electron microscopy (TEM), and several exosome-specific proteins were quantified by western blot. The expression of circ_PTPRA, miR-671-5p and SMAD family member 4 (SMAD4) was detected using quantitative polymerase chain reaction (qPCR). Cell cycle was assessed using flow cytometry assay. Cell proliferation was assessed by MTT assay. Radiosensitivity was observed according to colony growth and cell apoptosis rate by colony formation assay and flow cytometry assay. The protein levels of proliferation- and apoptosis-related markers and SMAD4 were measured by western blot. The predicted relationship between miR-671-5p and circ_PTPRA or SMAD4 was verified by dual-luciferase reporter assay. Animal study was performed to investigate the role of exosomal circ_PTPRA in vivo. Circ_PTPRA expression was declined in serumal exosomes from CRC patients and CRC cell lines. Exosomal circ_PTPRA induced CRC cell cycle arrest and inhibited cell proliferation. Besides, exosomal circ_PTPRA promoted radiosensitivity of CRC cells, leading to inhibitory colony formation and increased apoptotic rate. In mechanism, circ_PTPRA functioned as a competing endogenous RNA (ceRNA) to increasing SMAD4 level by binding to miR-671-5p. Rescue experiments concluded that circ_PTPRA inhibited CRC growth and radioresistance by decreasing miR-671-5p expression, and miR-671-5p inhibition also inhibited CRC growth and radioresistance by enriching SMAD4 expression. Moreover, exosomal circ_PTPRA blocked tumor growth in vivo. Exosomal circ_PTPRA enhanced CRC cell radiosensitivity and inhibited CRC malignant development partially by regulating the miR-671-5p/SMAD4 pathway, hinting that exosomal circ_PTPRA might be used as a potential predicted and therapeutic target for CRC.

IGF-1R depletion sensitizes colon cancer cell lines to radiotherapy.

Insulin like growth factor receptor 1 (IGF-1R) has been documented to play a key role in radiation response, thereby offering an attractive drug target to enhance tumor sensitivity to radiotherapy. Here, we investigated wether knockdown of IGF-1R can sensitize colorectal cancer (CRC) cell lines to radiation. Human colon carcinoma SW480 and HT-29 cells were transfected with specific small interference RNA (siRNA) to mediate IGF-1R depletion. The expression of IGF-1R mRNA and protein among transfected and untransfected cells was detected by Western blot analysis. Changes in cell proliferation and radiosensitivity were evaluated by the clonogenic survival assay. NVP-ADW742, an IGF-1R inhibitor, in combination with radiation was studied. RAD51, a measure for homologous recombination repair, and 53BP1, a maker for non-homologous end-joining (NHEJ), were determined by immunofluorescence for double-strand breaks (DSB) repair pathways. Cell cycle was also examined in the IGF-1R knockdown and IGF-1R-inhibited cells. CRC cell lines were selectively sensitized to radiation after siRNA-mediated IGF-1R depletion. NVP-ADW742 efficiently increases cancer cell response to radiation. Furthermore, initial formation of RAD51 foci after IR, and 53BP1 foci were significantly reduced in IGF-1R-depleted or with IGF-1R Inhibitor CRC cell lines. Lastly, IGF-1R-depleted or with IGF-1R Inhibitor caused more G2 phase cell arrest. Our findings demonstrate that depletion of IGF-1R lead to an increase in radiosensitivity in CRC.

Long noncoding RNA LINC00958 suppresses apoptosis and radiosensitivity of colorectal cancer through targeting miR-422a

BackgroundLong noncoding RNAs (lncRNAs) have been elucidated to participate in the development and progression of various cancers. In this study, we aimed to explore the underlying functions and mechanisms of LINC00958 in colorectal cancer.MethodsLINC00958 expression in colorectal cancer tissues was examined by qRT-PCR. The correlations between LINC00958 expression and clinical characteristics and prognosis were evaluated. The biological functions of LINC00958 were detected by CCK-8, MTT, colony formation and flow cytometric analyses. RNA pulldown, RIP and luciferase reporter assays were used to confirm the regulatory effects of LINC00958 on miR-422a. Rescue experiments were performed to detect the effects of miR-422a on the roles of LINC00958.ResultsLINC00958 was upregulated in colorectal cancer tissues and cell lines. High LINC00958 levels were positively associated with T stage and predicted poor prognosis. Cell experiments showed that LINC00958 promoted cell proliferation and suppressed apoptosis and sensitivity to radiotherapy in vitro and promoted tumor growth in vivo. Bioinformatics analysis predicted the binding site of miR-422a on LINC00958. Mechanistically, RNA pulldown, RIP and luciferase reporter assays demonstrated that LINC00958 specifically targeted miR-422a. In addition, we found that miR-422a suppressed MAPK1 expression by directly binding to the 3’-UTR of MAPK1, thereby inhibiting cell proliferation and enhancing cell apoptosis and radiosensitivity. Furthermore, miR-422a rescued the roles of LINC00958 in promoting MAPK1 expression and cell proliferation and decreasing cell apoptosis and radiosensitivity.ConclusionsLINC00958 promoted MAPK1 expression and cell proliferation and suppressed cell apoptosis and radiosensitivity by targeting miR-422a, which suggests that it is a potential biomarker for the prognosis and treatment of colorectal cancer.

Exosomal circ_IFT80 Enhances Tumorigenesis and Suppresses Radiosensitivity in Colorectal Cancer by Regulating miR-296-5p/MSI1 Axis.

BackgroundExosomal circular RNAs (circRNAs) can act as biomarkers and play crucial roles in colorectal cancer (CRC) and radiosensitivity. The aim of this study was to explore the functions and regulatory mechanism of exosomal circRNA intraflagellar transport 80 (circ_IFT80) in tumorigenesis and radiosensitivity of CRC.MethodsExosomes were detected using transmission electron microscopy (TEM). Protein levels were determined by Western blot assay. The expression of circ_IFT80, microRNA-296-5p (miR-296-5p) and musashi1 (MSI1) was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell cycle distribution, cell apoptosis, and cell proliferation were detected by flow cytometry and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, respectively. Colony formation assay was used to determine the radiosensitivity of cells. The interaction between miR-296-5p and circ_IFT80 or MSI1 was verified by dual-luciferase reporter assay. A xenograft tumor model was established to explore the role of exosomal circ_IFT80 in vivo.ResultsCirc_IFT80 was upregulated in exosomes derived from CRC patient serum and CRC cells. Exosomal circ_IFT80 or circ_IFT80 overexpression facilitated tumorigenesis by increasing cell proliferation and reducing apoptosis, and inhibited radiosensitivity via promoting colony formation and inhibiting apoptosis. Additionally, circ_IFT80 acted as a sponge of miR-296-5p, and miR-296-5p reversed the effects of circ_IFT80 on tumorigenesis and radiosensitivity. Moreover, MSI1 was a direct target of miR-296-5p. Furthermore, miR-296-5p overexpression inhibited tumorigenesis and promoted radiosensitivity by downregulating MSI1. Exosomal circ_IFT80 also accelerated tumor growth in vivo.ConclusionExosomal circ_IFT80 promoted tumorigenesis and reduced radiosensitivity by regulating miR-296-5p/MSI1 axis, which might provide a novel avenue for treatment of CRC.

Downregulation of miR-423-5p Contributes to the Radioresistance in Colorectal Cancer Cells.

Resistance to radiotherapy is the main reason causing treatment failure in locally advanced rectal cancer. MicroRNAs (miRNAs) have been well demonstrated to regulate cancer development and progression. However, how miRNAs regulate radiotherapy resistance in colorectal cancer remains unknown. Herein, we established two human colorectal cancer cell lines resistant to radiotherapy, named HCT116-R and RKO-R, using the strategy of fractionated irradiation. The radioresistant phenotypical changes of the two cell lines were validated by cell viability assay, colony formation assay and apoptosis assay. The miRNA expression profilings of HCT116-R and RKO-R were determined using RNA-seq analyses, and further confirmed by quantitative real-time PCR. Multiple miRNAs, including miR-423-5p, miR-7-5p, miR-522-3p, miR-3184-3p, and miR-3529-3p, were identified with altered expression in both of the radiotherapy-resistant cells, compared to the parental cells. The downregulation of miR-423-5p was further validated in the rectal cancer tissues from radiotherapy-resistant patients. Silencing of miR-423-5p in parental HCT116 and RKO cells decreased the sensitivity to radiation treatment, and inhibited the radiation-induced apoptosis. In consistence, overexpression of miR-423-5p in HCT116-R and RKO-R cells partially rescued their sensitivity to radiotherapy, and promoted the radiation-induced apoptosis. Bcl-xL (Bcl-2-like protein 1) was predicted to be a potential target gene for miR-423-5p, and miR-423-5p/Bcl-xL axis could be a critical mediator of radiosensitivity in colorectal cancer cells. The current finding not only revealed a novel role of miR-423-5p in regulating the radiosensitivity in colorectal cancer, but also suggested miR-423-5p as a molecular candidate for combination therapy with radiation to treat colorectal cancer.

Depletion of insulin-like growth factor 1 receptor increases radiosensitivity in colorectal cancer.

Although radiation therapy for advanced colorectal cancer (CRC) is very effective in some patients, treatment resistance limits its efficacy. Insulin-like growth factor 1 receptor (IGF1R) can affect tumor responsiveness and sensitivity to radiation in several cancer types. Herein, we studied the underlying function of IGF1R in the resistance of advanced CRC to radiation therapy and the possible use of drugs targeting IGF1R to overcome this resistance in patients with CRC. Differences in the expression levels of the IGF1R were assessed in CRC samples from patients who were radiosensitive or radioresistant. Two radio-resistant colorectal cancer cell lines, SW480 and HT29, were selected for in vitro studies, and the involvement of the IGF1R in their radiation resistance was elucidated by suppressing its expression through a targeted siRNA and through the use of a specific IGF1R inhibitor, BMS-754807. We assessed radiosensitivity in these human CRC cells lines by examining their proliferation and colony formation, as well as cell cycle analysis. Activation of the Akt pathway was assessed using western blotting. Compared with tissues from radiosensitive patients, higher IGF1R expression levels were found in patients with radiation-resistant colorectal cancer, while BMS-754807 had improved radiosensitivity and reversed radiation tolerance in both colorectal cancer cell lines. Pre-treatment with BMS-754807 prior to irradiation inhibited Akt phosphorylation, induced cell cycle arrest, and increased DNA damage. Therefore, the IGF1R contributes to radiation resistance of CRC cells in vitro. This study supports the notion that the radiosensitivity of radiation-resistant colorectal cancer cells can be enhanced by directly targeting IGF1R expression or activity. Ultimately, the combination of radiotherapy with IGF1R targeted inhibitors could potentially increase its effectiveness in the treatment of advanced colorectal cancer.

γ-H2AX as a potential indicator of radiosensitivity in colorectal cancer cells.

Preoperative radiotherapy improves local disease control and disease-free survival in patients with advanced rectal cancer; however, a reliable predictive biomarker for the effectiveness of irradiation has yet to be elucidated. Phosphorylation of H2A histone family member X (H2AX) to γ-H2AX is induced by DNA double-strand breaks and is associated with the development of colorectal cancer (CRC). The current study aimed to clarify the relationship between γ-H2AX expression and CRC radiosensitivity in vitro and in vivo. H2AX levels were analyzed in datasets obtained from cohort studies and γ-H2AX expression was investigated by performing immunohistochemistry and western blotting using clinical CRC samples from patients without any preoperative therapy. In addition, the CRC cell lines WiDr and DLD-1 were subjected to irradiation and/or small interfering RNA-H2AX, after which the protein levels of γ-H2AX were examined in samples obtained from patients undergoing preoperative chemoradiotherapy. To quantify the observable effect of treatment on cancer cells, outcomes were graded as follows: 1, mild; 2, moderate; and 3, marked, with defined signatures of cellular response. Datasets obtained from cohort studies demonstrated that H2AX mRNA levels were significantly upregulated and associated with distal metastasis and microsatellite instability in CRC tissues, in contrast to that of normal tissues. In addition, γ-H2AX was overexpressed in clinical samples. In vitro, following irradiation, γ-H2AX expression levels increased and cell viability decreased in a time-dependent manner. Combined irradiation and γ-H2AX knockdown reduced the viability of each cell line when compared with irradiation or γ-H2AX knockdown alone. Furthermore, among clinical CRC samples from patients undergoing preoperative chemoradiotherapy, levels of γ-H2AX in the grade 1 group were significantly higher than those in grade 2 or grade 3. In conclusion, γ-H2AX may serve as a novel predictive marker and target for preoperative radiotherapy effectiveness in patients with CRC.

MiR-1587 Regulates DNA Damage Repair and the Radiosensitivity of CRC Cells via Targeting LIG4.

DNA is subject to a range of endogenous and exogenous insults that can impair DNA replication and lead to DNA double-strand breaks (DSBs). The repair capacity of cancer cells mediates their radiosensitivity, but the roles of miR-1587 during radiation resistance are poorly characterized. In this study, we explored whether miR-1587 regulates the growth and radiosensitivity of colorectal cancer (CRC) cells through its ability to regulate DNA Ligase4 (LIG4). We found that CRC cells in which miR-1587 was overexpressed inhibited cell growth and promoted apoptosis through increasing DSBs and promoting cell cycle arrest. We found that overexpression of miR-1587 significantly inhibited LIG4 messenger RNA and protein expression and further revealed the ability of miR-1587 to directly bind to the LIG4-3′-untranslated region through dual-luciferase reporter assays. More notably, miR-1587 mimics increased the radiosensitivity of CRC cells. Taken together, we show that miR-1587 overexpression enhances the formation of DSBs, arrests CRC cell growth, and enhances the radiosensivity of CRC cells through the direct repression of LIG4 expression. These results reveal novel roles for miR-1587 during DNA damage repair and the radiosensivity of CRC cells. This highlights miR-1587 as a novel therapeutic target for CRC.

Long non-coding RNA HOTAIR knockdown enhances radiosensitivity through regulating microRNA-93/ATG12 axis in colorectal cancer

Colorectal cancer (CRC) is a global healthcare problem. Radioresistance is a huge setback for CRC radiotherapy. In this text, the roles and molecular mechanisms of long non-coding RNA HOTAIR in CRC tumorigenesis and radioresistance were further investigated. ATG12 mRNA, HOTAIR, and microRNA-93 (miR-93) levels were measured by quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay. Protein levels of LC3 I, LC3 II, p62, ATG12, cleaved caspase 3, Bax, and Bcl-2 were detected by western blotting assay in cells and were examined by immunohistochemistry (IHC) assay in tissues. Cell survival fractions, viability, and apoptotic rates were determined by clonogenic survival assay, CCK-8 assay, and flow cytometry analysis, respectively. The relationships of HOTAIR, miR-93, and ATG12 were tested by bioinformatics analysis and luciferase reporter assay. Mouse xenograft tumor models were established to investigate the influence of HOTAIR knockdown on CRC radioresistance in vivo. We found that HOTAIR expression was markedly upregulated in plasma from CRC patients after radiotherapy and CRC cells after irradiation. HOTAIR knockdown, miR-93 overexpression, or ATG12 silencing weakened cell viability, induced cell apoptosis, inhibited cell autophagy, and enhanced cell radiosensitivity in CRC. HOTAIR exerted its functions by downregulating miR-93. Moreover, HOTAIR functioned as a molecular sponge of miR-93 to regulate ATG12 expression. ATG12 protein expression was markedly upregulated and associated with miR-93 and HOTAIR expression in CRC tissues. Furthermore, HOTAIR knockdown enhanced radiosensitivity of CRC xenograft tumors by regulating miR-93/ATG12 axis. In conclusion, HOTAIR knockdown potentiated radiosensitivity through regulating miR-93/ATG12 axis in CRC, further elucidating the roles and molecular basis of HOTAIR in CRC radioresistance.

The combination of temsirolimus and chloroquine increases radiosensitivity in colorectal cancer cells.

The PI3K/AKT/mTOR pathway and autophagy are known to play important roles in cancer radioresistance. The aim of the present study was to investigate whether the combination of temsirolimus (TEM), an mTOR inhibitor, and chloroquine(CQ), an autophagy inhibitor, can increase radiosensitivity in colorectal cancer(CRC) cells. The efficacies of TEM and/or CQ as radiosensitizers were examined using clonogenic assays in CRC cell lines SW480 and HT‑29. The expression levels of the phosphorylated isoforms of S6 and 4E‑BP1, downstream proteins of mTOR, as well as the expression levels of p62 and LC3, autophagy‑related proteins, were assessed by western blot analysis. The formation of acidic organelles was detected in acridine orange‑stained cells. Apoptosis and caspase activity were assessed using flow cytometry. The results revealed that ionizing radiation(IR) activated the downstream proteins of mTOR and induced autophagy. In the clonogenic assays, neither TEM nor CQ influenced the efficacy of IR, whereas their combination significantly increased the dose‑dependent efficacy of IR. TEM inhibited phosphorylation of the downstream proteins of mTOR and induced autophagy. CQ inhibited autophagy in the late phase and did not influence the downstream proteins of mTOR. TEM and CQ inhibited both the phosphorylation of downstream proteins of mTOR and autophagy. Cell death analysis revealed that the combination of TEM and CQ strongly induced apoptosis in cells exposed to IR. In conclusion, the combination of TEM and CQ increased radiosensitivity in CRC cells through co‑inhibition of mTOR and autophagy.

MiR-185 enhances radiosensitivity of colorectal cancer cells by targeting IGF1R and IGF2

ObjectiveRadioresistance is a significant obstacle for effective treatment of colorectal cancer (CRC). Recent studies have indicated that miR-185 inhibits proliferation, survival, and invasion of CRC; however, the role of this miRNA in radioresistance of CRC has not been identified yet. The aim of this study is to investigate the role of miR-185 in radiosensitivity of CRC. MethodsAfter transfecting the cells with mimic miR-185, expressions of IGF1R and IGF2 were evaluated by real-time PCR and western blot. The radiation response of transfected cells was also examined by colony forming assay. Sub-G1 fraction analysis through flow cytometry and caspase 3 activity was used to evaluate apoptosis. ResultsThe results of real-time PCR and western blot indicated that IGF1R and IGF2 are downregulated in the transfected cells. Colony forming assay revealed that transfected cells were more radiosensitive than other cells. On the other hand,following irradiation the rate of apoptosis was significantly higher in the transfected cells than in the other cells. ConclusionIn summary, our study is the first to show that upregulation of miR-185 enhances the sensitivity of CRC cells to ionizing radiation. miR-185 may act as a novel biomarker of radioresistance and may clinically enhance the radiation response of CRC.