Down-regulation of BTG1 by miR-454-3p enhances cellular radiosensitivity in renal carcinoma cells.
ABSTRACT: BACKGROUND: B cell translocation gene 1 (BTG1) has long been recognized as a tumor suppressor gene. Recent reports demonstrated that BTG1 plays an important role in progression of cell cycle and is involved in cellular response to stressors. However, the microRNAs mediated regulatory mechanism of BTG1 expression has not been reported so far. MicroRNAs can effectively influence tumor radiosensitivity by preventing cell cycle progression, resulting in enhancement of the cytotoxicity of radiotherapy efficacy. This study aimed to demonstrating the effects of microRNAs on the BTG1 expression and cellular radiosensitivity. METHODS: The human renal carcinoma 786-O cells were treated with 5 Gy of X-rays. Expressions of BTG1 gene and miR-454-3p, which was predicted to target BTG1 by software algorithm, were analyzed by quantitative polymerase chain reaction. Protein expressions were assessed by Western blot. Luciferase assays were used to quantify the interaction between BTG1 3'-untranslated region (3'-UTR) and miR-454-3p. The radiosensitivity was quantified by the assay of cell viability, colony formation and caspase-3 activity. RESULTS: The expression of the BTG1 gene in 786-O cells was significantly elevated after treatments with X-ray irradiation, DMSO, or serum starvation. The up-regulation of BTG1 after irradiation reduced cellular radiosensitivity as demonstrated by the enhanced cell viability and colony formation, as well as the repressed caspase-3 activity. In comparison, knock down of BTG1 by siRNA led to significantly enhanced cellular radiosensitivity. It was found that miR-454-3p can regulate the expression of BTG1 through a direct interaction with the 3'-UTR of BTG1 mRNA. Decreasing of its expression level correlates well with BTG1 up-regulation during X-ray irradiation. Particularly, we observed that over-expression of miR-454-3p by transfection inhibited the BTG1 expression and enhanced the radiosensitivity. In addition, cell cycle analysis showed that over-expression of miR-454-3p shifted the cell cycle arrest from G2/M phase to S phase. CONCLUSIONS: Our results indicate that BTG1 is a direct target of miR-454-3p. Down-regulation of BTG1 by miR-454-3p renders tumor cells sensitive to radiation. These results may shed light on the potential application in tumor radiotherapy.
Project description:Accumulating evidence has demonstrated that circular RNAs (circRNAs) play important roles in regulating gene expression involved in tumor development. However, the role of circRNAs in modulating the radiosensitivity of oral squamous cell carcinoma (OSCC) and its potential mechanisms have not been documented. We performed high-throughput RNA sequencing (RNA-seq) to investigate the circRNA expression profile in OSCC patients and discovered that the circATRNL1 expression was significantly downregulated and closely related to tumor progression. The circATRNL1 was structurally validated via Sanger sequencing, RNase R treatment, and specific convergent and divergent primer amplification. Importantly, the expression levels of circATRNL1 decreased after irradiation treatment, and upregulation of circATRNL1 enhanced the radiosensitivity of OSCC through suppressing proliferation and the colony survival fraction, inducing apoptosis and cell-cycle arrest. Moreover, we observed that circATRNL1 could directly bind to microRNA-23a-3p (miR-23a-3p) and relieve inhibition for the target gene PTEN. In addition, the tumor radiosensitivity-promoting effect of circATRNL1 overexpression was blocked by miR-23a-3p in OSCC. Further experiments also showed that PTEN can reverse the inhibitory effect of OSCC radiosensitivity triggered by miR-23a-3p. We concluded that circANTRL1 may function as the sponge of miR-23a-3p to promote PTEN expression and eventually contributes to OSCC radiosensitivity enhancement. This study indicates that circANTRL1 may be a novel therapeutic target to improve the efficiency of radiotherapy in OSCC.
Project description:BACKGROUND:microRNAs (miRNAs) are non-coding RNAs that alter the stability and translation efficiency of messenger RNAs. Ionizing radiation (IR) induces rapid and selective changes in miRNA expression. Depletion of the miRNA processing enzymes Dicer or Ago2 reduces the capacity of cells to survive radiation exposure. Elucidation of critical radiation-regulated miRNAs and their target proteins offers a promising approach to identify new targets to increase the therapeutic effectiveness of the radiation treatment of cancer. PRINCIPAL FINDINGS:Expression of miR-525-3p is rapidly up-regulated in response to radiation. Manipulation of miR-525-3p expression in irradiated cells confirmed that this miRNA mediates the radiosensitivity of a variety of non-transformed (RPE, HUVEC) and tumor-derived cell lines (HeLa, U2-Os, EA.hy926) cell lines. Thus, anti-miR-525-3p mediated inhibition of the increase in miR-525-3p elevated radiosensitivity, while overexpression of precursor miR-525-3p conferred radioresistance. Using a proteomic approach we identified 21 radiation-regulated proteins, of which 14 were found to be candidate targets for miR-525-3p-mediated repression. Luciferase reporter assays confirmed that nine of these were indeed direct targets of miR-525-3p repression. Individual analysis of these direct targets by RNAi-mediated knockdown established that ARRB1, TXN1 and HSPA9 are essential miR-525-3p-dependent regulators of radiation sensitivity. CONCLUSION:The transient up-regulation of miR-525-3p, and the resultant repression of its direct targets ARRB1, TXN1 and HSPA9, is required for cell survival following irradiation. The conserved function of miR-525-3p across several cell types makes this microRNA pathway a promising target for modifying the efficacy of radiotherapy.
Project description:Radiotherapy is frequently applied for clinically localized prostate cancer while its efficacy could be significantly hindered by radioresistance. MicroRNAs (miRNAs) are important regulators in mediating cellular responses to ionizing radiation (IR), and strongly associate with radiosensitivity in many cancers. In this study, enhancement of radiosensitivity by miR-29b-3p was demonstrated in prostate cancer cell line LNCaP in vitro. Results showed that miR-29b-3p expression was significantly upregulated in response to IR from both X-rays and carbon ion irradiations. Knockdown of miR-29b-3p resulted in radioresistance while overexpression of miR-29b-3p led to increased radiosensitivity (showing reduced cell viability, suppressed cell proliferation and decreased colony formation). In addition, miR-29b-3p was found to directly target Wnt1-inducible-signaling protein 1 (WISP1). Inhibition of WISP1 facilitated the mitochondrial apoptosis pathway through suppressing Bcl-XL expression while activating caspase-3 and poly (ADP-ribose) polymerase (PARP). The results indicated that miR-29b-3p was a radiosensitizing miRNAs and could enhance radiosensitivity of LNCaP cells by targeting WISP1. These findings suggested a novel treatment to overcome radioresistance in prostate cancer patients, especially those with higher levels of the WISP1 expression.
Project description:Esophageal cancer (ESCA) is the eighth most common cause of cancer-associated mortality in humans. An increasing number of studies have demonstrated that microRNAs (miRs) serve important roles in mediating tumor initiation and progression. miR-454-3p has been found to be involved in the development of various human malignancies; however, little is known about the role of miR-454-3p in esophageal cancer. In the present study, the protein and gene expression levels of miR-454-3p in ESCA tissues and cells were downregulated compared with adjacent normal tissues and normal human esophageal epithelial cells. Additionally, miR-454-3p downregulation resulted in improved survival rates in patients with ESCA, and miR-454-3p overexpression significantly suppressed cell proliferation, migration and invasion and promoted apoptosis in four ESCA cell lines (EC9706, ECA109, TE-1 and TE-8). It was found that miR-454-3p overexpression inhibited the expression of insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) at the protein and mRNA expression levels. Furthermore, it was demonstrated that miR-454-3p inhibited ESCA cell proliferation, migration and apoptosis by targeting IGF2BP1 via the ERK and AKT signaling pathways in a subcutaneous xenograft tumor mouse model. These results showed that miR-454-3p functioned as an important tumor suppressor in ESCA by targeting IGFBP1. Therefore, miR-454-3p may be a novel prognostic biomarker and therapeutic target for patients with ESCA.
Project description:Lung cancer is the leading cause of cancer-related mortality worldwide. Radiotherapy is often applied for treating lung cancer, but it often fails because of the relative non-susceptibility of lung cancer cells to radiation. MicroRNAs (miRNAs) have been reported to modulate the radiosensitivity of lung cancer cells and have the potential to improve the efficacy of radiotherapy. The purpose of this study was to identify a miRNA that can adjust radiosensitivity in lung adenocarcinoma cells. Two lung adenocarcinoma cell lines (CL1-0 and CL1-5) with different metastatic ability and radiosensitivity were used. In order to understand the regulatory mechanisms of differential radiosensitivity in these isogenic tumor cells, both CL1-0 and CL1-5 were treated with 10 Gy radiation, and were harvested respectively at 0, 1, 4, and 24 h after radiation exposure. The changes in expression of miRNA upon irradiation were examined using Illumina Human microRNA BeadChips. Twenty-six miRNAs were identified as having differential expression post-irradiation in CL1-0 or CL1-5 cells. Among these miRNAs, miR-449a, which was down-regulated in CL1-0 cells at 24 h after irradiation, was chosen for further investigation. Overexpression of miR-449a in CL1-0 cells effectively increased irradiation-induced DNA damage and apoptosis, altered the cell cycle distribution and eventually led to sensitization of CL1-0 to irradiation.
Project description: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.
Project description:Oral squamous cell carcinoma (OSCC) is the most common malignancy of head and neck. Although radiotherapy is used for OSCC treatment, the occurrence of radioresistant cancer cells limits its efficiency. MicroRNAs (miRNAs) are non-coding RNAs with lengths of 18-25 base pairs and known to be involved in carcinogenesis. We previously demonstrated that by targeting B lymphoma Mo-MLV insertion region 1 homolog (Bmi1), miR-494-3p functions as a putative tumor suppressor miRNA in OSCC. In this study, we further discovered that miR-494-3p could enhance the radiosensitivity of SAS OSCC cells and induce cellular senescence. The overexpression of miR-494-3p in SAS cells increased the population of senescence-associated ?-galactosidase positive cells, the expression of p16(INK4a) and retinoblastoma 1 (RB1), as well as downregulated Bmi1. The knockdown of Bmi1 by lentiviral-mediated delivery of specific short hairpin RNAs (shRNAs) also enhanced the radiosensitivity of SAS cells and the activation of the senescence pathway. Furthermore, the inverse correlation between Bmi1 and miR-494-3p expression was observed among OSCC tissues. Results suggest that miR-494-3p could increase the radiosensitivity of OSCC cells through the induction of cellular senescence caused by the downregulation of Bmi1.
Project description:Serum microRNAs (miRNAs) have been reported as novel biomarkers for various diseases. But circulating biomarkers for predicting the radiosensitivity of nasopharyngeal carcinoma (NPC) have not been used in clinical practice. To screen out of differently expressed serum miRNAs from NPC patients with different radiosensitivity may be helpful for its individual therapy. NPC patients with different radiosensitivity were enrolled according to the inclusion and exclusion criteria. RNA was isolated from serum of these NPC patients before treatment. We investigated the differential miRNA expression profiles using microarray test (GSE139164), and the candidate miRNAs were validated by reverse transcription-quantitative real time polymerase chain reaction (RT-qPCR) experiments. Receiver operating characteristic (ROC) analysis has been applied to estimate the diagnostic value. In this study, 37 serum-specific miRNAs were screened out from 12 NPC patients with different radiosensitivity by microarray test. Furthermore, RT-qPCR analysis confirmed that hsa-miR-1281 and hsa-miR-6732-3p were significantly downregulated in the serum of radioresistant NPC patients (P?<?0.05), which was consistent with the results of microarray test. ROC curves demonstrated that the AUC for hsa-miR-1281 was 0.750 (95% CI: 0.574-0.926, SE 87.5%, SP 57.1%). While the AUC for hsa-miR-6732-3p was 0.696 (95% CI: 0.507-0.886, SE 56.3%, SP 78.6%). These results suggested that hsa-miR-1281 and hsa-miR-6732-3p in serum might serve as potential biomarkers for predicting the radiosensitivity of NPC.
Project description:The Wnt/?-catenin pathway is constitutively active and promotes multiple tumor processes, including breast cancer metastasis. However, the underlying mechanism by which the Wnt/?-catenin pathway is constitutively activated in breast cancer metastasis remains unclear. Inhibition of Wnt antagonists is important for Wnt/?-catenin signaling activation, and post-transcriptional regulation of these antagonists by microRNAs (miRNAs) might be a possible mechanism underlying signaling activation. Regulation of nuclear pre-mRNA domain-containing 1A (RPRD1A) is a known inhibitor of cell growth and Wnt/?-catenin signaling activity, but the function and regulatory mechanism of RPRD1A in breast cancer have not been clarified. The aim of this study was to understand how regulators of the Wnt/?-catenin pathway may play a role in the metastasis of this cancer. <b>Methods:</b> RPRD1A expression and its association with multiple clinicopathological characteristics was analyzed immunohistochemically in human breast cancer specimens. miR-454-3p expression was analyzed using real-time PCR. RPRD1A or miR-454-3p knockdown and overexpression were used to determine the underlying mechanism of their functions in breast cancer cells. Xenografted tumor model, 3D invasive culture, cell migration and invasion assays and sphere formation assay were used to determine the biofunction of RPRD1A and miR-454-3p in breast cancer. Electrophoretic mobility shift assay (EMSA), luciferase reporter assay, and RNA immunoprecipitation (RIP) were performed to study the regulation and underlying mechanisms of RPRD1A and miR-454-3p expression and their correlation with the Wnt/?-catenin pathway in breast cancer. <b>Results:</b> The Wnt/?-catenin signaling antagonist RPRD1A was downregulated and its upstream regulator miR-454-3p was amplified and overexpressed in metastatic breast cancer, and both were correlated with overall and relapse-free survival in breast cancer patients. The suppression by miR-454-3p on RPRD1A was found to activate Wnt/?-catenin signaling, thereby promoting metastasis. Simultaneously, three other negative regulators of the Wnt/?-catenin pathway, namely, AXIN2, dickkopf WNT signaling pathway inhibitor (DKK) 3 and secreted frizzled related protein (SFRP) 1, were also found to be targets of miR-454-3p and were involved in the signaling activation. miR-454-3p was found to be involved in early metastatic processes and to promote the stemness of breast cancer cells and early relapse under both <i>in vitro</i> and <i>in vivo</i> conditions. <b>Conclusions:</b> The findings indicate that miR-454-3p-mediated suppression of Wnt/?-catenin antagonist RPRD1A, as well as AXIN2, DKK3 and SFRP1, sustains the constitutive activation of Wnt/?-catenin signaling; thus, miR-454-3p and RPRD1A might be potential diagnostic and therapeutic targets for breast cancer metastasis.
Project description:BACKGROUND:PTPRG antisense RNA 1 has been well-documented to exert an oncogenic role in diverse neoplasms. However, the precise role of PTPRG antisense RNA 1 in regulating radiosensitivity of nonsmall cell lung cancer cells remains largely elusive. METHODS:Expression levels of PTPRG antisense RNA 1 and miR-200c-3p in nonsmall cell lung cancer tissues and cells were detected by quantitative real-time polymerase chain reaction, while transcription factor 4 expression was examined by immunohistochemistry and Western blot. After nonsmall cell lung cancer cells were exposed to X-ray with different doses in vitro, Cell Counting Kit-8 assay and colony formation assay were conducted to determine the influence of PTPRG antisense RNA 1 on cell viability. Interaction between miR-200c-3p and PTPRG antisense RNA 1 as well as transcription factor 4 was investigated by dual luciferase reporter assay. RESULT:In nonsmall cell lung cancer tissues, the expressions of PTPRG antisense RNA 1 and transcription factor 4 were significantly upregulated, whereas the expression of miR-200c-3p was downregulated. It was also proved that PTPRG antisense RNA 1 and 3'-untranslated region of transcription factor 4 can bind to miR-200c-3p. Under X-ray irradiation, overexpressed PTPRG antisense RNA 1 could promote the viability and enhance the radioresistance of nonsmall cell lung cancer cells, and this effect was partially weakened by miR-200c-3p mimics. Transcription factor 4 was identified as a target gene of miR-200c-3p, which could be positively regulated by PTPRG antisense RNA 1. CONCLUSION:PTPRG antisense RNA 1 reduces the radiosensitivity of nonsmall cell lung cancer cells via modulating miR-200c-3p/TCF4 axis.