scholarly journals N1, N12-Diacetylspermine Is Elevated in Colorectal Cancer and Promotes Proliferation through the miR-559/CBS Axis in Cancer Cell Lines

2021 ◽  
Vol 2021 ◽  
pp. 1-15
Author(s):  
Teng Mu ◽  
Tingguang Chu ◽  
Wenxin Li ◽  
Qianze Dong ◽  
Yong Liu
Keyword(s):  
Colorectal Cancer ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
Polyclonal Antibodies ◽  
Biological Effects ◽  
Clinical Stage ◽  
Luciferase Reporter ◽  
E Proteins ◽  
Atp Production ◽  
Underlying Mechanisms

N1, N12-Diacetylspermine (DiAcSpm) has been reported to be upregulated in the urine of cancer patients. Mass spectrometry has shown elevated DiAcSpm expressions in colorectal cancer (CRC) tissues. However, the diagnostic application of DiAcSpm is not available due to a lack of diagnostic grade antibodies. Also, its biological roles in CRC cells remain unexplored. In the present study, we developed an antibody that directly detected DiAcSpm expression in paraffin-embedded tissues. We also characterized its biological characteristics and underlying mechanisms. Polyclonal antibodies were generated by immunizing animals with a synthetic product of DiAcSpm. Antibody DAS AB016 showed strong sensitivity against DiAcSpm in CRC tissues. Immunohistochemistry results showed that DiAcSpm expression was significantly elevated in CRC tissues. High levels of DiAcSpm correlated with the clinical stage and Ki67 index. DiAcSpm treatment increased levels of proliferation, cell cycle progression, and cyclin D1 and cyclin E proteins in CRC cell lines, SW480 and Caco-2. DiAcSpm also upregulated ATP production in these two cell lines. RNA-sequencing showed that DiAcSpm downregulated miR-559, which was confirmed using RT-qPCR. The luciferase reporter assay, western blotting, and RT-qPCR showed that cystathionine β-synthase (CBS) was the target of miR-559. miR-559 inhibited, while CBS accelerated, CRC proliferation. In addition, CBS siRNA knockdown blocked the biological effects of DiAcSpm on CRC cells. In conclusion, DiAcSpm was found to be increased in CRC tissues using a newly developed antibody. DiAcSpm accelerated CRC proliferation by regulating the miR-559/CBS axis.

scholarly journals Circ_0003266 sponges miR-503-5p to suppress colorectal cancer progression via regulating PDCD4 expression

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Caihong Wen ◽  
Xiaoqing Feng ◽  
Honggang Yuan ◽  
Yong Gong ◽  
Guangsheng Wang
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Cell Lines ◽  
Cancer Progression ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Circular Rnas ◽  
Pdcd4 Expression ◽  
Novel Target

Abstract Background Circular RNAs (circRNAs) feature prominently in tumor progression. However, the biological function and molecular mechanism of circ_0003266 in colorectal cancer (CRC) require further investigation. Methods Circ_0003266 expression in 46 pairs CRC tissues / adjacent tissues, and CRC cell lines was detected by quantitative real-time polymerase chain reaction (qRT-PCR); after circ_0003266 was overexpressed or knocked down in CRC cells, cell proliferation, apoptosis, migration, and invasion were evaluated by the cell counting kit-8 (CCK-8), flow cytometry, and Transwell assays, respectively; the interaction among circ_0003266, miR-503-5p, and programmed cell death 4 (PDCD4) was confirmed using bioinformatics analysis and dual-luciferase reporter assay; PDCD4 protein expression in CRC cells was quantified using Western blot. Results Circ_0003266 was significantly lowly expressed in CRC tissues and cell lines. Circ_0003266 overexpression markedly repressed CRC cell proliferation, migration, and invasion, and accelerated the cell apoptosis, but its overexpression promoted the malignant phenotypes of CRC cells. PDCD4 was a direct target of miR-503-5p and circ_0003266 promoted PDCD4 expression by competitively sponging miR-503-5p. Conclusion Circ_0003266 suppresses the CRC progression via sponging miR-503-5p and regulating PDCD4 expressions, which suggests that circ_0003266 may serve as a novel target for the treatment of CRC.

Long Non-Coding RNA-Colon Cancer Associated Transcript 1 Down-Regulation Inhibits Cell Proliferation and Promotes Apoptosis in Colorectal Cancer Through Enhancing miR-152 Expression

2020 ◽  
Vol 10 (12) ◽  
pp. 1766-1772
Author(s):  
Jindong Li ◽  
Xi Wang ◽  
Xin Huang ◽  
Na Li ◽  
Ya Ling ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Viability ◽  
Cancer Cell ◽  
Cancer Progression ◽  
Biological Effects ◽  
Colorectal Cancer Cell ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Cancer Tissue ◽  
Colorectal Cancer Progression

Colorectal cancer is a common malignant cancer that is characterized by high mortality rate. CCAT1 is a type of newly discovered lncRNA. This research was conducted to study the role of CCAT1 in colorectal cancer. The findings showed that there was significant up-regulation of CCAT1 expression in colorectal cancer. Then, online bioinformatic database and dual-luciferase reporter assay to prove CCAT1 and miR-152 have direct binding sites. Many researches demonstrated that miR-152 played a crucial role in development of colorectal cancer. Therefore, we then explored the relationship between CCAT1 and miR-152 in colorectal cancer. qRT-PCR analysis showed that miR-152 was lowly expressed in cancer tissue and cells. We then explored the effect of CCAT1 and miR-152 on the biological effects of colorectal cancer cells. MiR-152 up-regulation significantly reduced colorectal cancer cell viability and enhanced apoptosis. Furthermore, CCAT1-shRNA inhibited colorectal cancer cell viability and enhanced cell apoptosis were significantly eliminated by miR-152 inhibitor. Combined with all results, CCAT1/miR-152 axis was related to colorectal cancer progression regulation, which might be used as new therapeutic targets for colorectal cancer treatment.

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 MicroRNA-374a Governs Aggressive Cell Behaviors of Glioma by Targeting Prokineticin 2

2019 ◽  
Vol 18 ◽  
pp. 153303381882140 ◽  
Author(s):  
Ye Zhang ◽  
Rui Zhang ◽  
Rui Sui ◽  
Yi Chen ◽  
Haiyang Liang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Glioma Cell ◽  
Cell Cycle Progression ◽  
Luciferase Reporter ◽  
Cell Behaviors ◽  
Prokineticin 2 ◽  
Glioma Cell Lines ◽  
Glioma Cell Proliferation ◽  
Cancer Types

MicroRNA-374a has been abnormally expressed in several cancer types; however, its role in glioma remains unclear. Therefore, we aimed to investigate whether microR-374a participated in the progression of glioma. Expression of microR-374a in glioma cell lines and normal cell line was measured by quantitative real-time polymerase chain reaction. Luciferase reporter assay and Western blot were used to detect the targets of microR-374a. In vitro functional experiments were conducted to investigate the biological role of microR-374a. Low expression of microR-374a was found in glioma cell lines. Prokineticin 2 was identified as a direct target of microR-374a in glioma. Investigations on the mechanisms related to glioma progression showed that microR-374a inhibited glioma cell proliferation, cell cycle progression, and cell invasion through targeting Prokineticin 2. Taken together, these results revealed that microR-374a functions as tumor suppressor by targeting Prokineticin 2, suggesting it might be a novel therapeutic target for glioma.

Regulation of the Cyclin D1 Proto-Oncogene by Cell Cycle Regulatory MicroRNAs.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1801-1801
Author(s):  
Anagha Borwankar ◽  
Alessandro Pastore ◽  
Aniruddha Deshpande ◽  
Yvonne Zimmermann ◽  
Christian Buske ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Cell Lines ◽  
Binding Sites ◽  
Cell Cycle Progression ◽  
Luciferase Activity ◽  
Full Length ◽  
Luciferase Reporter ◽  
Microrna Target ◽  
The Impact

Abstract Mutations, activation or overexpression of cyclin D1 are common features of several human cancers including mantle cell lymphoma (MCL) which bears the characteristic t(11;14) translocation juxtaposing the cyclin D1 gene downstream of the immunoglobulin heavy chain enhancer. The loss of the 3’UTR of this gene has been reported in a majority of MCL patients as well as in cell lines. In order to assess the impact of the 3’UTR on cyclin D1 expression levels, we used YFP tagged cyclin D1 reporter plasmids to quantify cyclin D1 expression in cell lines with different mutations of the 3’UTR. Interestingly, protein expression was significantly higher upon deletion of the cyclin D1 3’UTR compared to the full-length cyclin D1 gene as assessed by flow cytometry (2.1 fold; n=3, P < 0.05). Applying a more sensitive dual-luciferase reporter assay where a constitutively expressed luciferase gene was fused to the cyclin D1 3’UTR, the normalized firefly luciferase activity was reduced significantly to 23% as compared to luciferase only (the empty vactor). We then introduced 3’UTR mutations observed in MCL patients (insertion of adenosine between nucleotides 2308 and 2309 and a deletion of the tri-nucleotide sequence TCA from 2309–2311 of the full length cyclin D1-YFP reporter cDNA), which resulted in a significant increase of cyclin D1 expression (1.3 fold both in Ins308 and Δ309-311) compared to full length cyclin D1, (P< 0.05) showing that these mutations contribute to cyclinD1 overexpression in these patients. Subsequently, the 3’UTR was scanned for elements potentially regulating cyclin D1 expression, and putative microRNA binding sites were identified using the TargetScan and PicTar microRNA target prediction software. The most interesting candidate microRNAs include the miR-15/16 family and the miR-17–92 cluster, both of which have been shown to be involved in lymphoid malignancies and regulate cell cycle progression. In order to confirm whether the cyclin D1 3’UTR is a direct target of these microRNAs, we cloned the cyclin D1 3’UTR target region containing putative miR-15/16 or miR-17/20a binding sites and transfected these reporter constructs into HeLa cells. Upon introduction of oligonucleotide mimics of the miR15/16 microRNAs or a plasmid expressing microRNAs of the miR-17 cluster, the normalized luciferase activity of the respective luciferase reporters was reduced significantly to 41% (miR-15), 33% (miR-16) and 79% (miR-17/20a), respectively. Moreover, introduction of mutations in the seed sequences of the putative microRNA recognition sites rendered these constructs insensitive to inhibition by these microRNAs, confirming the specificity of the microRNA::target interaction. These data confirm that the binding of these microRNAs play an important role in the repression of cyclin D1 mediated by the 3’UTR and mutation or deletion result in cyclin D1 overexpression in MCL as well as other human tumors.

scholarly journals Inhibitory Effects of Bangladeshi Medicinal Plant Extracts on Interactions between Transcription Factors and Target DNA Sequences

2008 ◽  
Vol 5 (3) ◽  
pp. 303-312 ◽  
Author(s):  
Ilaria Lampronti ◽  
Mahmud T. H. Khan ◽  
Monica Borgatti ◽  
Nicoletta Bianchi ◽  
Roberto Gambari
Keyword(s):  
Transcription Factors ◽  
Medicinal Plants ◽  
Cell Lines ◽  
Plant Extracts ◽  
Dna Sequences ◽  
Cell Cycle Progression ◽  
Biological Effects ◽  
Mobility Shift ◽  
Medicinal Plant Extracts ◽  
Target Dna

Several transcription factors (TFs) play crucial roles in governing the expression of different genes involved in the immune response, embryo or cell lineage development, cell apoptosis, cell cycle progression, oncogenesis, repair and fibrosis processes and inflammation. As far as inflammation, TFs playing pivotal roles are nuclear factor kappa B (NF-kB), activator protein (AP-1), signal transducer and activator of transcription (STATs), cAMP response element binding protein (CREB) and GATA-1 factors. All these TFs regulate the expression of pro-inflammatory cytokines and are involved in the pathogenesis of a number of human disorders, particularly those with an inflammatory component. Since several medicinal plants can be employed to produce extracts exhibiting biological effects and because alteration of gene transcription represents a very interesting approach to control the expression of selected genes, this study sought to verify the ability of several extracts derived from Bangladeshi medicinal plants in interfering with molecular interactions between different TFs and specific DNA sequences. We first analyzed the antiproliferative activity of 19 medicinal plants on different human cell lines, including erythroleukemia K562, B lymphoid Raji and T lymphoid Jurkat cell lines. Secondly, we employed the electrophoretic mobility shift assay as a suitable technique for a fast screening of plant extracts altering the binding between NF-kB, AP-1, GATA-1, STAT-3, CREB and the relative target DNA elements.

scholarly journals Hsa_circRNA_000166 facilitated cell growth and limited apoptosis through targeting miR-326 / LASP1 axis in colorectal cancer

2020 ◽  
Author(s):  
Qin Hao ◽  
Zhongtao Zhang
Keyword(s):  
Colorectal Cancer ◽  
Cell Growth ◽  
Cell Lines ◽  
Direct Interaction ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
Western Blot Assay ◽  
Apoptosis Assay ◽  
Geo Database

Abstract Background: Circular RNAs(circRNAs) belong to non-coding RNAs and widely expressed in a variety of cell species, including cancers. However, the function and mechanism of circRNAs in colorectal cancer (CRC) has not been well investigated. Methods: Microarray data of CRC from Gene Expression Omnibus (GEO) database was used to obtain DEGs. QRT-PCR and western blot assay were performed to determine the mRNA and protein levels of multiple genes, respectively. Cell growth and apoptosis assay were conducted to measure CRC cell proliferation and apoptosis, respectively. Luciferase assay was utilized to confirm the direct interaction between hsa_circRNA_000166 and miR-326. Results: We downloaded and analyzed the circRNA expression profile of CRC from the GEO database and identified 181 differentially expressed circRNAs between 10 pairs of CRC and adjacent normal tissues. Interestingly, we observed that the expression of hsa_circRNA_000166 was the top increased among these circRNAs. Then, we confirmed an upregulation of hsa_circRNA_000166 in CRC tissues and cell lines and observed that higher expression of hsa_circRNA_000166 was associated with poor 5-year survival rate of patients with CRC. Cell growth and apoptosis assay revealed that hsa_circRNA_000166 regulated the cell growth and apoptosis in CRC cell lines. Furthermore, we identified that hsa_circRNA_000166 targeted miR-326/LASP1 pathway using bioinformatic analysis and luciferase reporter assay. Finally, overexpression of miR-326 could sufficiently rescued the aberrant cell growth and apoptosis in CRC cell lines. Conclusion: Taken together, our results indicated that downregulation of hsa_circRNA_000166 inhibited the cell growth and facilitated apoptosis during CRC development by sponging miR-326 / LASP1 pathway.

scholarly journals SKP1 promotes YAP-mediated colorectal cancer stemness via suppressing RASSF1

2020 ◽  
Author(s):  
Cong Tian ◽  
Tingyuan Lang ◽  
Jiangfeng Qiu ◽  
Kun Han ◽  
Lei Zhou ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Stem Cells ◽  
Protein Level ◽  
Drug Target ◽  
Target Genes ◽  
Xenograft Model ◽  
Luciferase Reporter ◽  
Hippo Signaling ◽  
Self Renewal ◽  
Underlying Mechanisms

Abstract Background: Cancer stem cells (CSCs) have been recognized as an important drug target, however, the underlying mechanisms have not been fully understood. SKP1 is a traditional drug target for cancer therapy, while, whether SKP1 promotes colorectal cancer (CRC) stem cells (CRC-SCs) and the underlying mechanisms have remained elusive. Methods: Human CRC cell lines and primary human CRC cells were used in this study. Gene manipulation was performed by lentivirus system. The mRNA and protein levels of target genes were examined by qRT-PCR and western blot. The sphere-forming and in vitro migration capacities were determined by sphere formation and transwell assay. The self-renewal was determined by limiting dilution assay. The tumorigenicity and metastasis of cancer cells were examined by xenograft model. The promoter activity was examined by luciferase reporter assay. Nuclear run-on and Chromatin immunoprecipitation-PCR (ChIP-PCR) assay were employed to examine the transcription and protein-DNA interaction. Co-immunoprecipitation assay was used to test protein-protein interaction. The relationship between gene expression and survival was analyzed by Kaplan-meier analysis. The correlation between two genes was analyzed by Spearman analysis. Data are represented as mean ± s.d. and the significance was determined by Student’s t-test.Results: SKP1 was upregulated in CRC-SCs and predicted poor prognosis of colon cancer patients. Overexpression of SKP1 promoted the stemness of CRC cells reflected by increased sphere-forming, migration and self-renewal capacities as well as the expression of CSCs markers. In contrast, SKP1 depletion produced the opposite effects. SKP1 strengthened YAP activity and knockdown of YAP abolished the effect of SKP1 on the stemness of CRC cells. SKP1 suppressed RASSF1 at both mRNA and protein level. Overexpression of RASSF1 abolished the effect of SKP1 on YAP activity and CRC stemness. Conclusion: Our results demonstrated that SKP1 suppresses RASSF1 at both mRNA and protein level, attenuates Hippo signaling, activates YAP, and thereby promoting the stemness of CRC cells.

scholarly journals LncRNA-URHC Functions as a ceRNA to Regulate Danjb9 Expression by Competitively Binding to miR-5007-3p in Hepatocellular Carcinoma

2021 ◽  
Author(s):  
kunwei niu ◽  
Shibin Qu ◽  
Xuan Zhang ◽  
Jimin Dai ◽  
Jianlin Wang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Biological Effects ◽  
Luciferase Reporter ◽  
Underlying Mechanisms ◽  
Proliferation In Vitro ◽  
Hcc Cells

Abstract Background: Hepatocellular carcinoma (HCC) is often diagnosed at a late stage, when the prognosis is poor. The regulation of long non-coding RNAs (lncRNAs) plays a crucial role in HCC. However, the precise regulatory mechanisms of lncRNA signaling in HCC remain largely unknown. We study aim to investigate the underlying mechanisms of lncRNA (upregulated in hepatocellular carcinoma) URHC in HCC. Methods: RT-qPCR, fluorescence in situ hybridization (FISH) staining, EdU, colony formation, and tumor xenografts experiments were used to identify localized and biological effects of URHC on HCC cells in vitro and in vivo. The bioinformatics analysis, Dual-luciferase reporter assay, and rescue experiments revealed the potential mechanism of URHC.Results: URHC silencing may inhibit the HCC cells proliferation in vitro and in vivo. We found that URHC was mainly localized in the cytoplasm. The expression of miR-5007-3p was negatively regulated by URHC. And miR-5007-3p could reverse the effect of URHC in HCC cells. The expression of DNAJB9 was negatively regulated by miR-5007-3p but positively regulated by URHC. These suggesting of lncRNA-URHC positively regulated the level of DNAJB9 by sponging miR-5007-3p.Conclusion: Together, our study elucidated the role of URHC as a miRNA sponge in HCC, and shed new light on lncRNA-directed diagnostics and therapeutics in HCC.

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