LncRNA OTUD6B-AS1 inhibits many cellular processes in colorectal cancer by sponging miR-21-5p and regulating PNRC2

2021 ◽  
pp. 096032712199797 ◽  
Author(s):  
Y Cai ◽  
Y Li ◽  
C Shi ◽  
Z Zhang ◽  
J Xu ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Bioinformatic Analysis ◽  
Cellular Process ◽  
Luciferase Reporter ◽  
Nuclear Receptor Coactivator ◽  
Rna Molecules ◽  
Cellular Processes ◽  
Inhibitory Function ◽  
Reporter Assays ◽  
Competitive Endogenous Rna

Accumulating evidence has revealed that long noncoding RNAs (lncRNAs) play essential roles in regulating cellular process of various cancers. There have been many studies on the biological functions of lncRNAs in colorectal cancer (CRC). In this research, we explored the role and mechanism of lncRNA ovarian tumor domain containing 6B antisense RNA1 (OTUD6B-AS1) in CRC. Here, we detected OTUD6B-AS1 expression in CRC tissues and cells by RT-qPCR. Functional experiments were performed to test alterations in different cellular processes. Moreover, to verify the binding ability among the indicated RNA molecules, we carried out RIP, RNA pull-down and luciferase reporter assays. According to our data, OTUD6B-AS1 expression was low in CRC tissues and cells. Functionally, overexpression of OTUD6B-AS1 inhibited cell proliferation, migration, invasion and EMT, and promoted cell apoptosis. Bioinformatic analysis and mechanistical experiments confirmed that OTUD6B-AS1 could act as a competitive endogenous RNA (ceRNA) to upregulate Proline-Rich Nuclear Receptor Coactivator 2 (PNRC2) expression by sequestering miR-21-5p. Further rescue experiments validated the inhibitory function of the OTUD6B-AS1/miR-21-5p/PNRC2 axis in cellular process of CRC. Overall, OTUD6B-AS1 inhibits cellular development in CRC by sponging miR-21-5p and upregulating PNRC2, providing a novel insight into the exploration on CRC treatment.

scholarly journals Hsa_circ_0026628 promotes the development of colorectal cancer by targeting SP1 to activate the Wnt/β-catenin pathway

2021 ◽  
Vol 12 (9) ◽  
Author(s):  
Xuexiu Zhang ◽  
Jianning Yao ◽  
Haoling Shi ◽  
Bing Gao ◽  
Haining Zhou ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Transcription Factor ◽  
Circular Rna ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
Transcriptional Level ◽  
Sp1 Transcription Factor ◽  
Reporter Assays ◽  
Sphere Formation

AbstractCircular RNAs (circRNAs) have been reported to play crucial roles in the progression of various cancers, including colorectal cancer (CRC). SP1 (Sp1 transcription factor) is a well-recognized oncogene in CRC and is deemed to trigger the Wnt/β-catenin pathway. The present study was designed to investigate the role of circRNAs which shared the same pre-mRNA with SP1 in CRC cells. We identified that hsa_circ_0026628 (circ_0026628), a circular RNA that originated from SP1 pre-mRNA, was upregulated in CRC cells. Sanger sequencing and agarose gel electrophoresis verified the circular characteristic of circ_0026628. Functional assays including CCK-8, colony formation, transwell, immunofluorescence staining, and sphere formation assay revealed the function of circ_0026628. RNA pull-down and mass spectrometry disclosed the proteins interacting with circ_0026628. Mechanistic assays including RIP, RNA pull-down, CoIP, ChIP, and luciferase reporter assays demonstrated the interplays between molecules. The results depicted that circ_0026628 functioned as a contributor to CRC cell proliferation, migration, EMT, and stemness. Mechanistically, circ_0026628 served as the endogenous sponge of miR-346 and FUS to elevate SP1 expression at the post-transcriptional level, thus strengthening the interaction between SP1 and β-catenin to activate the Wnt/β-catenin pathway. In turn, the downstream gene of Wnt/β-catenin signaling, SOX2 (SRY-box transcription factor 2), transcriptionally activated SP1 and therefore boosted circ_0026628 level. On the whole, SOX2-induced circ_0026628 sponged miR-346 and recruited FUS protein to augment SP1, triggering the downstream Wnt/β-catenin pathway to facilitate CRC progression.

scholarly journals miR-128 Targets the SIRT1/ROS/DR5 Pathway to Sensitize Colorectal Cancer to TRAIL-Induced Apoptosis

2018 ◽  
Vol 49 (6) ◽  
pp. 2151-2162 ◽  
Author(s):  
Bo Lian ◽  
Dongxiang Yang ◽  
Yanlong Liu ◽  
Gang Shi ◽  
Jibin Li ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Flow Cytometry ◽  
Western Blot ◽  
Cancer Cells ◽  
Cell Lines ◽  
Sirtuin 1 ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Reporter Assays ◽  
Induced Apoptosis

Background/Aims: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an ideal anti-tumor drug because it exhibits selective cytotoxicity against cancer cells. However, certain cancer cells are resistant to TRAIL, and the potential mechanisms are still unclear. The aim of this study was to reduce the resistance of colorectal cancer (CRC) cells to TRAIL. Methods: Quantitative real-time PCR analysis was performed to detect the expression of microRNA-128 (miR-128) in tissues from patients with CRC and CRC cell lines. MTT assays were used to evaluate the effect of miR-128 on TRAIL-induced cytotoxicity against CRC cell lines. The distribution of death receptor 5 (DR5) and the production of reactive oxygen species (ROS) were detected by flow cytometry analysis. Western blot, flow cytometry, and luciferase reporter assays were performed to evaluate the potential mechanism and pathway of miR-128-promoted apoptosis in TRAIL-treated CRC cells. Results: MiR-128 expression was downregulated in tumor tissues from patients with CRC as well as in CRC cell lines in vitro. The enforced expression of miR-128 sensitized CRC cells to TRAIL-induced cytotoxicity by inducing apoptosis. Mechanistically, bioinformatics, western blot analysis, and luciferase reporter assays showed that miR-128 directly targeted sirtuin 1 (SIRT1) in CRC cells. miR-128 overexpression suppressed SIRT1 expression, which promoted the production of ROS in TRAIL-treated CRC cells. This increase of ROS subsequently induced DR5 expression, and thus increased TRAIL-induced apoptosis in CRC cells. Conclusion: The combination of miR-128 with TRAIL may represent a novel approach for the treatment of CRC.

scholarly journals Polo-like kinase 3 inhibits glucose metabolism in colorectal cancer by targeting HSP90/STAT3/HK2 signaling

2019 ◽  
Vol 38 (1) ◽  
Author(s):  
Baochi Ou ◽  
Hongze Sun ◽  
Jingkun Zhao ◽  
Zhuoqing Xu ◽  
Yuan Liu ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Glucose Metabolism ◽  
Transcriptional Activation ◽  
Lactate Production ◽  
Luciferase Reporter ◽  
Transcriptional Level ◽  
Atp Production ◽  
Reporter Assays

Abstract Background Polo-like kinase 3 (PLK3) has been documented as a tumor suppressor in several types of malignancies. However, the role of PLK3 in colorectal cancer (CRC) progression and glucose metabolism remains to be known. Methods The expression of PLK3 in CRC tissues was determined by immunohistochemistry. Cells proliferation was examined by EdU, CCK-8 and in vivo analyses. Glucose metabolism was assessed by detecting lactate production, glucose uptake, mitochondrial respiration, extracellular acidification rate, oxygen consumption rate and ATP production. Chromatin immunoprecipitation, luciferase reporter assays and co-immunoprecipitation were performed to explore the signaling pathway. Specific targeting by miRNAs was determined by luciferase reporter assays and correlation with target protein expression. Results PLK3 was significantly downregulated in CRC tissues and its low expression was correlated with worse prognosis of patients. In vitro and in vivo experiments revealed that PLK3 contributed to growth inhibition of CRC cells. Furthermore, we demonstrated that PLK3 impeded glucose metabolism via targeting Hexokinase 2 (HK2) expression. Mechanically, PLK3 bound to Heat shock protein 90 (HSP90) and facilitated its degradation, which led to a significant decrease of phosphorylated STAT3. The downregulation of p-STAT3 further suppressed the transcriptional activation of HK2. Moreover, our investigations showed that PLK3 was directly targeted by miR-106b at post-transcriptional level in CRC cells. Conclusion This study suggests that PLK3 inhibits glucose metabolism by targeting HSP90/STAT3/HK2 signaling and PLK3 may serve as a potential therapeutic target in colorectal cancer.

scholarly journals Circular RNA hsa_circ_0007142 Is Upregulated and Targets miR-103a-2-5p in Colorectal Cancer

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Chang-li Zhu ◽  
Xiaofeng Sha ◽  
Yuan Wang ◽  
Jin Li ◽  
Men-yan Zhang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Bioinformatic Analysis ◽  
Circular Rna ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Clinical Samples ◽  
Circular Rnas ◽  
Aberrant Expression ◽  
Poor Differentiation ◽  
Hct 116 Cells

Circular RNAs (circRNAs) are a large class of endogenous noncoding RNAs that regulate gene expression and mainly function as microRNA sponges. This study aimed to explore the aberrant expression of circRNAs in colorectal cancer (CRC). Using a circRNA microarray, we identified 892 differentially expressed circRNAs between six pairs of CRC and adjacent paracancerous tissues. Among them, hsa_circ_0007142 was significantly upregulated. Further analysis in 50 CRC clinical samples revealed that hsa_circ_0007142 upregulation was associated with poor differentiation and lymphatic metastasis of CRC. Bioinformatic analysis and luciferase reporter assay showed that hsa_circ_0007142 targeted miR-103a-2-5p in CRC cells. Moreover, the silencing of hsa_circ_0007142 by siRNAs decreased the proliferation, migration, and invasion of HT-29 and HCT-116 cells. Taken together, these findings suggest that hsa_circ_0007142 is upregulated in CRC and targets miR-103a-2-5p to promote CRC.

scholarly journals The Mechanisms of Colorectal Cancer Cell mesenchymal-epithelial Transition Induced by Hepatocyte Exosome-derived miR-203a-3p

2021 ◽  
Author(s):  
Heyang Xu ◽  
Qiusheng Lan ◽  
Yongliang Huang ◽  
Yang Zhang ◽  
Yujie Zeng ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Liver Metastasis ◽  
Epithelial To Mesenchymal Transition ◽  
Luciferase Reporter ◽  
Mesenchymal Transition ◽  
Real Time Quantitative Pcr ◽  
Mesenchymal To Epithelial Transition ◽  
Reporter Assays ◽  
Phosphatase Of Regenerating Liver

Abstract Background: Liver metastasis is the most common cause of death in patients with colorectal cancer (CRC). Phosphatase of regenerating liver-3 induces CRC metastasis by epithelial-to-mesenchymal transition, which promotes CRC cell liver metastasis. Mesenchymal-to-epithelial transition (MET), the opposite of epithelial-to-mesenchymal transition, has been proposed as a mechanism for the establishment of metastatic neoplasms. However, the molecular mechanism of MET remains unclear. Methods: Using Immunohistochemistry, western blotting，invasion assays, real-time quantitative PCR, chromatin immunoprecipitation, luciferase reporter assays, human miRNA arrays, and xenograft mouse model, we determined the role of hepatocyte exosome-derived miR-203a-3p in CRC MET.Results: In our study, we found that miR-203a-3p derived from hepatocyte exosomes increased colorectal cancer cells E-cadherin expression, inhibited Src expression, and reduced activity. In this way miR-203a-3p induced the decreased invasion rate of CRC cells.Coclusion: MiR-203a-3p derived from hepatocyte exosomes plays an important role of CRC cells to colonize in liver.

scholarly journals Circ_0026344 overexpression inhibits the migration, invasion, and enhances apoptosis of colorectal cancer cells by sponging miR-31

2020 ◽  
Author(s):  
Ye Jin ◽  
Lingli Yu ◽  
Bin Zhang ◽  
Yun Chen ◽  
Changfeng Liu ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Flow Cytometry ◽  
Wound Healing ◽  
Cell Lines ◽  
Opposite Effect ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Colorectal Cancer Cells ◽  
Reporter Assays ◽  
E Cadherin

Abstract Background: Circ_0026344 was reported to be associated with the metastasis of colorectal cancer (CRC). This study aimed to investigate the expression of circ_0026344 in CRC and the effect mechanisms of circ_0026344 on CRC.Methods: The expressions of circ_0026344 and miR-31 in clinical CRC tissues or CRC cell lines were analyzed by qPCR. The target of circ_0026344 was predicted and verified by CircInteractome and dual-luciferase reporter assays. The correlation between circ_0026344 and miR-31 expression was analyzed using Pearson analysis. After the CRC cells were overexpressed circ_0026344 or miR-31 or silenced circ_0026344, the viability, apoptosis, migration, and invasion of CRC cells were evaluated by CCK-8, flow cytometry, wound healing, and transwell. Also, the expressions of miR-31, Bcl-2, Bax, E-cadherin, and N-cadherin in the cells were detected by qPCR or Western blot. Results: Circ_0026344 was low-expressed in CRC tissues and cell lines. Circ_0026344 sponged miR-31 which was high-expressed in CRC tissues. The expression of circ_0026344 was negatively correlated to the expression of miR-31. The miR-31 expression could be down-regulated by circ_0026344 overexpression. Circ_0026344 overexpression inhibited the cell viability, migration, and invasion; and enhanced the apoptosis of CRC cells. Circ_0026344 overexpression decreased the expressions of Bcl-2 and N-cadherin and increased the expressions of Bax and E-cadherin in CRC cells. Circ_0026344 silencing and miR-31 overexpression had an opposite effect on CRC cells as circ_0026344 overexpression. Furthermore, miR-31 overexpression counteracted the effect of circ_0026344 overexpression.Conclusion: Circ_0026344 overexpression inhibited the migration, invasion, and enhanced apoptosis of CRC cells by sponging miR-31.

scholarly journals Circular RNA CCDC66 Improves Murine Double Minute 4 (MDM4) Expression through Targeting miR-370 in Colorectal Cancer

2022 ◽  
Vol 2022 ◽  
pp. 1-9
Author(s):  
Yang Mo ◽  
Qin Lu ◽  
Qi Zhang ◽  
Jie Chen ◽  
Youming Deng ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Circular Rna ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Colon And Rectal Cancer ◽  
Double Minute ◽  
Reporter Assays ◽  
Hct 116 ◽  
Murine Double Minute

Introduction. Colorectal cancer (CRC), a common digestive tract tumor that contains colon and rectal cancer, is one of the three most common cancers globally. circRNAs are involved in the occurrence and development of CRC, but the mechanism of how they participate in this process remains unclear. Methods. We adopted PCR for expression measure, CCK-8 for cell proliferation detection, Transwell for cell migration and invasion detection, and dual-luciferase reporter assays to detect the potential downstream targets of CCDC66 in CRC. Results. This study showed that circRNA CCDC66 was overexpressed in CRC tissues, and after knockdown, it inhibited the proliferation, migration, and invasion of CRC cells (RKO and HCT-116) in vitro. In addition, the dual-luciferase reporter assay showed that there was a binding site between circCCDC66 and miR-370, as well as between miR-370 and murine double minute 4 (MDM4). That is, circCCDC66 upregulated the expression of MDM4 through competitively binding to miR-370. The expression of circCCDC66 in CRC tissues was positively correlated with MDM4 and negatively correlated with miR-370. Conclusion. In summary, our results indicate that circCCDC66 is a key upregulation of CRC. circCCDC66 upregulates MDM4 through competitive binding to miR-370, thereby enhancing the metastatic ability of CRC cells and promoting the development of CRC.

scholarly journals Long Noncoding RNA LINC00958 Suppresses Apoptosis and Radiosensitivity of Colorectal Cancer Through Targeting miR-422a

2021 ◽  
Author(s):  
Hong Liang ◽  
Qiuyan Zhao ◽  
Zhonglin Zhu ◽  
Chao Zhang ◽  
Hui Zhang
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Noncoding Rna ◽  
Luciferase Reporter ◽  
Flow Cytometric ◽  
Potential Biomarker ◽  
Reporter Assays ◽  
Cancer Tissues

Abstract Background: Long non-coding RNAs (lncRNAs) have been elucidated to participate in the development and progression of various cancers. In this study, we aim to explore the underlying functions and mechanisms of LINC00958 in colorectal cancer. Methods: LINC00958 expression in colorectal cancer tissues was examined by qRT-PCR. The associations between LINC00958 expression with clinical characteristics and prognosis were evaluated. The biological functions of LINC00958 were detected by CCK-8, MTT, colony formation and Flow cytometric analyses. RNA-pull down, RIP and luciferase reporter assays were used to confirm the regulation of LINC00958 on miR-422a. Rescue experiments were performed to detect the effects of miR-422a on the roles of LINC00958. Results: LINC00958 was upregulated in colorectal cancer tissues and cell lines; high LINC00958 level was significantly associated with tumor differentiation, T stage and TNM stage, and also predicted poor prognosis. Cell experiments showed that LINC00958 promoted cell proliferation and suppressed apoptosis and the sensitivity of radiotherapy in vitro, and promoted cell growth in vivo. Bioinformatics analysis predicted the binding site of miR-422a on LINC00958. Mechanistically, RNA-pull down, RIP and luciferase reporter assays demonstrated that LINC00958 specially targeted miR-422a. In addition, we provided evidence that miR-422a suppressed MAPK1 expression through directly binding to the 3’-UTR of MAPK1, thereby inhibiting cell proliferation and enhancing apoptosis and the radiosensitivity. Furthermore, miR-422a rescued the roles of LINC00958 on promoting MAPK1 expression and cell proliferation and decreasing apoptosis and the radiosensitivity. Conclusions: LINC00958 promoted MAPK1 expression and cell proliferation and suppressed apoptosis and the radiosensitivity through targeting miR-422a, highlighting a potential biomarker for the prognosis and treatment of colorectal cancer.

scholarly journals The tsRNAs (tRFdb-3013a And tRFdb-3013b) Serve As Novel Biomarkers For Colon Adenocarcinomas

2021 ◽  
Author(s):  
Xiaoling Wu ◽  
Lihong Tan ◽  
Zhurong Tang ◽  
Chunjie Wen ◽  
Huan Chen ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Colon Adenocarcinoma ◽  
Bioinformatic Analysis ◽  
Molecular Signature ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Diagnosis And Prognosis ◽  
Adenocarcinoma Cells ◽  
Reporter Assays ◽  
Novel Biomarkers

Abstract Background: The tsRNAs (tRNA-derived small RNAs) are novel class of small non-coding RNAs derived from transfer-RNAs. Colon adenocarcinoma (COAD) are well known malignant intestinal tumors. This study focused on the identification and characterization of tsRNA biomarkers in colon adenocarcinomas. Methods: Data processing, bioinformatic analysis and visualization were performed with R or Python software. The cell proliferation, migration and invasion ability were described by CCK-8 and transwell assays. Luciferase reporter assays were performed to test the binding of tsRNA with its target.Results: With computational approaches, we identified the tsRNA fragments profiles within COAD datasets, and discriminated forty-two differentially expressed tsRNAs between colon adenocarcinomas and non-tumor controls. Among the fragments derived from the 3′ end of mature tRNA-His-GUG (a histidyl-transfer-RNA), tRFdb-3013a and tRFdb-3013b (tRFdb-3013a/b) were significantly decreased in colon adenocarcinomas, especially, tRFdb-3013a/b may tend to down-regulated in the patients with lymphatic or vascular invasion present. The clinical survival of colon adenocarcinomas patients with low tRFdb-3013a/b expression was significantly worse than that of high expression patients. In colon adenocarcinoma cells, tRFdb-3013a could suppressed cell proliferation, and reduced cell migration and invasion ability. The enrichment analyses showed that most of tRFdb-3013a-related genes were enriched in the extracellular matrix associated GO terms, phagosome pathway, and a GSEA molecular signature. Mechanically, the 3′UTR of ST3GAL1 mRNA was predicted to contain the putative binding sites of tRFdb-3013a/b, tRFdb-3013a/b might directly target ST3GAL1 and regulate ST3GAL1 expression in colon adenocarcinomas. Conclusions: These results suggested that tRFdb-3013a and tRFdb-3013b might serve as novel biomarkers for diagnosis and prognosis of colon adenocarcinomas, and play an important role in tumor progression of colon adenocarcinomas.

scholarly journals HOX Transcript Antisense RNA HOTAIR Abrogates Vasculogenic Mimicry by Targeting the AngiomiR-204/FAK Axis in Triple Negative Breast Cancer Cells

2020 ◽  
Vol 6 (2) ◽  
pp. 19
Author(s):  
Allan Lozano-Romero ◽  
Horacio Astudillo-de la Vega ◽  
María Cruz del Rocío Terrones-Gurrola ◽  
Laurence A. Marchat ◽  
Daniel Hernández-Sotelo ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Migration ◽  
Cancer Cells ◽  
Triple Negative Breast Cancer ◽  
Breast Cancer Cells ◽  
Triple Negative ◽  
Vasculogenic Mimicry ◽  
Bioinformatic Analysis ◽  
Luciferase Reporter ◽  
Reporter Assays

HOX transcript antisense RNA (HOTAIR) is an oncogenic long non-coding RNA frequently overexpressed in cancer. HOTAIR can enhance the malignant behavior of tumors by sponging microRNAs with tumor suppressor functions. Vasculogenic mimicry is a hypoxia-activated process in which tumor cells form three-dimensional (3D) channel-like networks, resembling endothelial blood vessels, to obtain nutrients. However, the role of HOTAIR in vasculogenic mimicry and the underlying mechanisms are unknown in human cancers. In the current study, we investigated the relevance of HOTAIR in hypoxia-induced vasculogenic mimicry in metastatic MDA-MB-231 and invasive Hs-578t triple negative breast cancer cells. Analysis of The Cancer Genome Atlas (TCGA) database using cBioPortal confirmed that HOTAIR was upregulated in clinical breast tumors relative to normal mammary tissues. Our quantitative RT-PCR assays showed a significant increase in HOTAIR levels after 48 h hypoxia relative to normoxia in breast cancer cell lines. Remarkably, knockdown of HOTAIR significantly abolished the hypoxia-induced vasculogenic mimicry which was accompanied by a reduction in the number of 3D channel-like networks and branch points. Likewise, HOTAIR silencing leads to reduced cell migration abilities of cancer cells. Bioinformatic analysis predicted that HOTAIR has a potential binding site for tumor suppressor miR-204. Luciferase reporter assays confirmed that HOTAIR is a competitive endogenous sponge of miR-204. Congruently, forced inhibition of HOTAIR in cells resulted in augmented miR-204 levels in breast cancer cells. Further bioinformatic analysis suggested that miR-204 can bind to the 3′ untranslated region of focal adhesion kinase 1 (FAK) transcript involved in cell migration. Western blot and luciferase reporter assays confirmed that FAK is a novel target of miR-204. Finally, silencing of HOTAIR resulted in low levels of cytoplasmic FAK protein and alterations in the organization of cellular cytoskeleton and focal adhesions. In summary, our results showed, for the first time, that HOTAIR mitigates cell migration and vasculogenic mimicry by targeting the miR-204/FAK axis in triple negative breast cancer cells.

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