CRISPR/Cas13-Based Platforms for a Potential Next-Generation Diagnosis of Colorectal Cancer through Exosomes Micro-RNA Detection: A Review

Colorectal cancer (CRC) is the third most prevalent cancer with the second highest mortality rate worldwide. CRC is a heterogenous disease with multiple risk factors associated, including obesity, smoking, and use of alcohol. Of total CRC cases, 60% are diagnosed in late stages, where survival can drop to about 10%. CRC screening programs are based primarily on colonoscopy, yet this approach is invasive and has low patient adherence. Therefore, there is a strong incentive for developing molecular-based methods that are minimally invasive and have higher patient adherence. Recent reports have highlighted the importance of extracellular vesicles (EVs), specifically exosomes, as intercellular communication vehicles with a broad cargo, including micro-RNAs (miRNAs). These have been syndicated as robust candidates for diagnosis, primarily for their known activities in cancer cells, including immunoevasion, tumor progression, and angiogenesis, whereas miRNAs are dysregulated by cancer cells and delivered by cancer-derived exosomes (CEx). Quantitative polymerase chain reaction (qPCR) has shown good results detecting specific cancer-derived exosome micro-RNAs (CEx-miRNAs) associated with CRC, but qPCR also has several challenges, including portability and sensitivity/specificity issues regarding experiment design and sample quality. CRISPR/Cas-based platforms have been presented as cost-effective, ultrasensitive, specific, and robust clinical detection tools in the presence of potential inhibitors and capable of delivering quantitative and qualitative real-time data for enhanced decision-making to healthcare teams. Thereby, CRISPR/Cas13-based technologies have become a potential strategy for early CRC diagnosis detecting CEx-miRNAs. Moreover, CRISPR/Cas13-based platforms’ ease of use, scalability, and portability also showcase them as a potential point-of-care (POC) technology for CRC early diagnosis. This study presents two potential CRISPR/Cas13-based methodologies with a proposed panel consisting of four CEx-miRNAs, including miR-126, miR-1290, miR-23a, and miR-940, to streamline novel applications which may deliver a potential early diagnosis and prognosis of CRC.

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Micro RNA ‐425‐5p regulates chemoresistance in colorectal cancer cells via regulation of Programmed Cell Death 10

Journal of Cellular and Molecular Medicine ◽

10.1111/jcmm.12742 ◽

2016 ◽

Vol 20 (2) ◽

pp. 360-369 ◽

Cited By ~ 45

Author(s):

Ye Zhang ◽

Xingqian Hu ◽

Xiaofei Miao ◽

Kuiyu Zhu ◽

Songkui Cui ◽

...

Keyword(s):

Colorectal Cancer ◽

Cell Death ◽

Programmed Cell Death ◽

Cancer Cells ◽

Micro Rna ◽

Colorectal Cancer Cells

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Hsa-miR-27a-3p overexpression in men with nonobstructive azoospermia: A case-control study

International Journal of Reproductive BioMedicine ◽

10.18502/ijrm.v13i11.7963 ◽

2020 ◽

Author(s):

Hamid Norioun ◽

Majid Motovali-bashi ◽

Seyed Morteza Javadirad

Keyword(s):

Male Infertility ◽

Case Control Study ◽

Quantitative Polymerase Chain Reaction ◽

Case Control ◽

Micro Rna ◽

Expression Level ◽

Obstructive Azoospermia ◽

Aberrant Expression ◽

Micro Rnas ◽

Control Study

Background: The role of KDM3A and its downstream genes in male fertility has been approved in animal models. Additionally, the expression shrinkage of KDM3A is significantly correlated with human azoospermia phenotype. Aberrant expression of micro-RNAs could mislead spermatogenesis and mostly lead to diverse phenotypes of male infertility. Objective: The aim of this study was to evaluate the expression level of hsa-miR-27a- 3p in azoospermic men to reveal its possible association with infertility. Materials and Methods: This case-control study was conducted on 30 azoospermic men, of whom, 19 had non obstructive azoospermia (NOA) and 11 obstructive azoospermia (OA) according to the pathological examinations. Comprehensive bioinformatics investigations were performed securely and hsa-miR-27a-3p was selected afterward. Reverse Transcriptase-quantitative polymerase chain reaction (RTqPCR) method was used and statistical analysis was performed to compare the expression level of hsa-miR-27a-3p in both OA and NOA individuals. Results: In silico analysis suggested hsa-miR-27a-3p, with its potential binding ability to target KDM3A transcripts. The expression analysis of candidate hsa-miR-27a-3p indicated its significant overexpression in NOA men. Conclusion: The hsa-miR-27a-3p was overexpressed in NOA men compared to OA-control individuals. As a consequence, the overexpressed micro-RNA could downregulate directly KDM3A and indirectly TNP1 and PRM1. Therefore, spermatogenesis could be misled and male infertility could be developed. Key words: hsa-miR-27a-3p, Male infertility, KDM3A.

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TP53 and Let-7a micro-RNA Regulate K-Ras Activity in HCT116 Colorectal Cancer Cells

PLoS ONE ◽

10.1371/journal.pone.0070604 ◽

2013 ◽

Vol 8 (8) ◽

pp. e70604 ◽

Cited By ~ 14

Author(s):

Carrie Luu ◽

Eileen L. Heinrich ◽

Marjun Duldulao ◽

Amanda K. Arrington ◽

Marwan Fakih ◽

...

Keyword(s):

Colorectal Cancer ◽

Cancer Cells ◽

Micro Rna ◽

Colorectal Cancer Cells

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Characterization of micro-RNA Profile in the Blood of Patients with Marfan's Syndrome

The Thoracic and Cardiovascular Surgeon ◽

10.1055/s-0037-1604083 ◽

2017 ◽

Vol 66 (01) ◽

pp. 116-124 ◽

Cited By ~ 4

Author(s):

Nicole Ludwig ◽

Tanja Rädle-Hurst ◽

Andreas Keller ◽

Lars Motsch ◽

Ina Marsollek ◽

...

Keyword(s):

Cardiovascular Diseases ◽

Quantitative Polymerase Chain Reaction ◽

Clinical Manifestations ◽

Micro Rna ◽

Cellular Interaction ◽

Marfan’S Syndrome ◽

Marfan's Syndrome ◽

Micro Rnas ◽

Wide Range ◽

Mirna Abundance

Background Marfan's syndrome (MFS) is an autosomal dominant inheritance disorder with a 1/5,000 live-birth prevalence. It is characterized by a wide range of clinical manifestations with more than 3,000 mutations identified in the FBN1 gene. In this study, we aimed to determine if specific patterns of circulating micro-RNAs (miRNAs) are associated with MFS-associated with cardiovascular diseases. Methods Microarray-based miRNA profiling was performed on blood samples of 12 MFS patients, and 12 healthy volunteers (HVs) controls and the differences in miRNA abundance between the two groups were validated using independent cohorts of 22 MFS and of 22 HV controls by real-time quantitative polymerase chain reaction (RT-qPCR). Enrichment analyses of altered miRNA abundance were predicted using bioinformatics tools. Results Altered miRNA abundance levels were determined between MFS (n = 34) and HVs (n = 34). In a screening phase, we analyzed 12 patients with MFS and 12 HVs by miRNA microarray. We found 198 miRNAs that were significantly altered in MFS patients as compared with HVs, including 16 miRNAs with a more than 1.5-fold change. Out of these 16 miRNAs, 10 showed a decreased abundance and 6 showed an increased abundance. In the validation phase, we analyzed independent cohorts of 22 MFS and of 22 HV controls by RT-qPCR. We confirmed the direction of abundance changes and the significance of different abundances between MFS patients and HVs for four miRNAs, namely, miR-362–5p, miR-339–3p, miR-340–5p, and miR-210–3p. Only the miR-150–5p showed a significant correlation with mitral valve prolapse (p = 0.010). The predicted targets for the validated miRNAs were associated with signal transduction, tissue remodeling, and cellular interaction pathways. Conclusion The altered abundance level of different miRNAs in whole blood of MFS patients lays the ground to the development of novel diagnostic approaches with altered miRNAs levels associated with MFS with manifestations associated with cardiovascular diseases.

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New Insights in Bladder Cancer Diagnosis: Urinary miRNAs and Proteins

Medical Sciences ◽

10.3390/medsci6040113 ◽

2018 ◽

Vol 6 (4) ◽

pp. 113 ◽

Cited By ~ 2

Author(s):

Gökçe Güllü Amuran ◽

Irem Peker Eyuboglu ◽

Ilker Tinay ◽

Mustafa Akkiprik

Keyword(s):

Bladder Cancer ◽

Early Diagnosis ◽

Cancer Diagnosis ◽

Wnt Signaling Pathway ◽

Storage Conditions ◽

Micro Rna ◽

Protein Biomarkers ◽

Cholecystokinin Receptor ◽

Micro Rnas ◽

Keywords Bladder Cancer

Bladder cancer is the 10th-most common cancer worldwide. The diagnosis and follow-up of patients require costly invasive methods and due to these expenses, bladder cancer continues to be one of the expensive malignancies. Early diagnosis is crucial in bladder cancer as it is in other cancers; therefore, non-invasive biomarkers for early diagnosis are very important. In this review, we aimed to focus on the most recent investigations on potential urinary micro RNA (miRNA) and protein biomarkers for bladder cancer diagnosis and their associated pathways. Studies performed by different groups were compiled and the biomarker properties of various proteins and miRNAs in the urine of bladder cancer patients were evaluated. Key studies were obtained by searching keywords “bladder cancer, urinary miRNA, urinary protein, urinary biomarker”. Targets and the pathways of the miRNAs and proteins were analyzed according to mirBase Catalogue and Panther Database. The major pathways that are targeted by aberrantly expressed miRNAs are Cholecystokinin receptor (CCKR), p53, Wnt signaling pathway, and feedback loops. We hereby conclude that urinary micro RNAs and proteins are promising candidates for bladder cancer diagnosis. It should be noted that urine collection, storage conditions, choice of fraction, and normalization strategies should be standardized.

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Predicting Response to Neoadjuvant Therapy in Colorectal Cancer Patients the Role of Messenger-and Micro-RNA Profiling

Cancers ◽

10.3390/cancers12061652 ◽

2020 ◽

Vol 12 (6) ◽

pp. 1652 ◽

Cited By ~ 2

Author(s):

Alberto Izzotti ◽

Chiara Ceccaroli ◽

Marta Geretto ◽

Filippo Grillo Ruggieri ◽

Sara Schenone ◽

...

Keyword(s):

Colorectal Cancer ◽

Neoadjuvant Therapy ◽

Cancer Patients ◽

Micro Rna ◽

Molecular Signature ◽

Messenger Rnas ◽

Rna Profiling ◽

Micro Rnas ◽

Colorectal Cancer Patients ◽

Study Designs

Colorectal cancer patients’ responses to neoadjuvant therapy undergo broad inter-individual variations. The aim of this systematic review is to identify a molecular signature that is predictive of colon cancer downstaging and/or downgrading after neoadjuvant therapy. Among the hundreds analysed in the available studies, only 19 messenger-RNAs (mRNAs) and six micro-RNAs (miRNAs) were differentially expressed in responders versus non-responders in two or more independent studies. Therefore, a mRNA/miRNA signature can be designed accordingly, with limitations caused by the retrospective nature of these studies, the heterogeneity in study designs and the downgrading/downstaging assessment criteria. This signature can be proposed to tailor neoadjuvant therapy regimens on an individual basis.

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The Effect of Micro RNA-34a and carboplatin combination on the inducing apoptosis of Breast Cancer Cell line

10.21203/rs.3.rs-289066/v1 ◽

2021 ◽

Author(s):

Sajjad Eslamkhah ◽

Nazila Alizadeh ◽

Sahar Safaei ◽

Mohammad Amini ◽

ahad Mokhtarzadeh ◽

...

Keyword(s):

Breast Cancer ◽

Cell Viability ◽

Cancer Cells ◽

Cell Line ◽

Caspase 3 ◽

Cancer Cell Line ◽

Micro Rna ◽

Control Group ◽

Expression Levels ◽

Micro Rnas

Abstract Aim: Breast cancer (BC) has been classified among the main causes of death owing to females' cancer. Carboplatin is a platinum-based chemotherapeutic drug that is an important treatment option for BC. But high and frequent doses of carboplatin usually reducing the reaction of cancer cells to medication. There is an immediate need to establish methods for increasing the carboplatin susceptibility to BC cells. For instance, micro RNAs (miRNAs) such as MiR34a demonstrate significant potential. Considering that, this research was planned to explore the better clinical effect and underlying mechanism of miR-34a as a possible tumor inhibitor and drug resistance regulator in compound with carboplatin chemotherapy drug in the cell lines of BC in humans. Methods: MCF-7 cell line was transfected with miR-34a to perform functional analyses. Subsequently, the MTT assay was applied to assess cell viability. Cell viability and cell death associated gene expression amounts including Bax, Bcl-2, caspase-3, MDR1, P53, and mir34-a, were examined through real-time quantitative PCR. Results: Findings showed that miR-34a upregulation significantly decreased MCF7 cell viability in comparison with control group. Furthermore, separate treatment of cells with miR-34a mimics and carboplatin could significantly increase Bax, Caspase-3, P53, and decrease in Bcl-2 mRNA expression levels evaluated to the non-treated group. Moreover, by reduction in expression levels of the MDR1 gene, BC cells' reaction to carboplatin has increased via miR-34a. Conclusion: In line with the findings, it could be inferred that miR-34a may improve the responsiveness of breast cancer cells to carboplatin chemotherapy with downregulation of MDR1.

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Differential expression of micro-RNA in tumor and non-tumor tissues of patients with colorectal cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.15_suppl.e13516 ◽

2020 ◽

Vol 38 (15_suppl) ◽

pp. e13516-e13516

Author(s):

Inna A. Novikova ◽

Natalya N. Timoshkina ◽

Oleg Ivanovich Kit ◽

Sergey I. Poluektov ◽

Andrey V. Dashkov ◽

...

Keyword(s):

Gene Expression ◽

Colorectal Cancer ◽

Differential Expression ◽

Tumor Markers ◽

Target Genes ◽

Tumor Tissue ◽

Micro Rna ◽

Differentially Expressed ◽

Tumor Tissues ◽

Micro Rnas

e13516 Background: The colorectal cancer (CRC) incidence is steadily increasing. Moreover, the problem of its early diagnosis remains unresolved due to the low specificity of known tumor markers, and the problem of creating new therapeutic approaches is due to the lack of a complete understanding of the mechanisms of regulation of gene expression in this oncopathology. The study of micro-RNAs (short non-coding RNAs that regulate gene expression) can be the solution to both problems. The aim of the study was to analyze micro-RNA differential expression in the tumor and non-tumor tissues of CRC patients. Methods: 5 patients with CRC (colon adenocarcinoma, G2) were selected for the multiple parallel micro-RNA sequencing. The mirVana miRNA Isolation Kit protocol was used to isolate small RNA fractions. The miRNA library was prepared using the TruSeq Small RNASample Preparation Kit. Sequencing of the nucleotide sequences of cDNA libraries was performed using a MiSeq (Illumina, USA). The copy numbers of micro-RNA were determined by comparing the nucleotide sequence of the sequenced molecules in each sample with the known nucleotide sequences of micro-RNA presented in the databases. When analyzing the differential expression of micro-RNA, the DESeq2 method implemented in R medium was used. Results: Six differentially expressed micro-RNAs were detected (p < 0.05): 2 that decrease expression (hsa-miR-143-3p,hsa-miR-26a-5p) and 4 increase expression in the tumor relative to non-tumor (hsa-miR-25-3p, hsa-miR-92a-3p, hsa-miR-21-5p, hsa-let-7i-5p). The highest level of expression in both tumor and non-tumor tissue was observed for hsa-miR-143-3p, the lowest one for hsa-let-7i-5p. Moreover, the largest difference in micro-RNA expression in tumor tissue relative to non-tumor was shown for hsa-miR-92a-3p (4.5 times, p = 0.02), the smallest for hsa-miR-143-3p (2.4 times, p = 0.04). For miRNAs that differentially changed their expression, a search was made for target genes using the miRWalk 3.0 database. 14573 target genes were found, of which 3346 were for hypo-expressed micro-RNAs and 11228 for hyper-expressed micro-RNAs. Conclusions: Sequencing revealed 6 differentially expressed micro-RNAs (hsa-miR-143-3p, hsa-miR-26a-5p, hsa-miR-25-3p, hsa-miR-92a-3p, hsa-miR-21-5p, hsa-let-7i-5p) in the tumor tissue is relatively non-tumor tissues of the colon. The data obtained expand the understanding of the mechanisms of gene regulation in the context of this oncopathology and may possibly become the basis for highly specific tumor markers panel.

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NDAT Targets PI3K-Mediated PD-L1 Upregulation to Reduce Proliferation in Gefitinib-Resistant Colorectal Cancer

Cells ◽

10.3390/cells9081830 ◽

2020 ◽

Vol 9 (8) ◽

pp. 1830 ◽

Cited By ~ 1

Author(s):

Tung-Yung Huang ◽

Tung-Cheng Chang ◽

Yu-Tang Chin ◽

Yi-Shin Pan ◽

Wong-Jin Chang ◽

...

Keyword(s):

Colorectal Cancer ◽

Cell Proliferation ◽

Tumor Growth ◽

Cancer Cells ◽

Tumor Cells ◽

Quantitative Polymerase Chain Reaction ◽

Combined Treatment ◽

Proliferative Effect ◽

Pi3k Activation

The property of drug-resistance may attenuate clinical therapy in cancer cells, such as chemoresistance to gefitinib in colon cancer cells. In previous studies, overexpression of PD-L1 causes proliferation and metastasis in cancer cells; therefore, the PD-L1 pathway allows tumor cells to exert an adaptive resistance mechanism in vivo. Nano-diamino-tetrac (NDAT) has been shown to enhance the anti-proliferative effect induced by first-line chemotherapy in various types of cancer, including colorectal cancer (CRC). In this work, we attempted to explore whether NDAT could enhance the anti-proliferative effect of gefitinib in CRC and clarified the mechanism of their interaction. The MTT assay was utilized to detect a reduction in cell proliferation in four primary culture tumor cells treated with gefitinib or NDAT. The gene expression of PD-L1 and other tumor growth-related molecules were quantified by quantitative polymerase chain reaction (qPCR). Furthermore, the identification of PI3K and PD-L1 in treated CRC cells were detected by western blotting analysis. PD-L1 presentation in HCT116 xenograft tumors was characterized by specialized immunohistochemistry (IHC) and the hematoxylin and eosin stain (H&E stain). The correlations between the change in PD-L1 expression and tumorigenic characteristics were also analyzed. (3) The PD-L1 was highly expressed in Colo_160224 rather than in the other three primary CRC cells and HCT-116 cells. Moreover, the PD-L1 expression was decreased by gefitinib (1 µM and 10 µM) in two cells (Colo_150624 and 160426), but 10 µM gefitinib stimulated PD-L1 expression in gefitinib-resistant primary CRC Colo_160224 cells. Inactivated PI3K reduced PD-L1 expression and proliferation in CRC Colo_160224 cells. Gefitinib didn’t inhibit PD-L1 expression and PI3K activation in gefitinib-resistant Colo_160224 cells. However, NDAT inhibited PI3K activation as well as PD-L1 accumulation in gefitinib-resistant Colo_160224 cells. The combined treatment of NDAT and gefitinib inhibited pPI3K and PD-L1 expression and cell proliferation. Additionally, NDAT reduced PD-L1 accumulation and tumor growth in the HCT116 (K-RAS mutant) xenograft experiment. (4) Gefitinib might suppress PD-L1 expression but did not inhibit proliferation through PI3K in gefitinib-resistant primary CRC cells. However, NDAT not only down-regulated PD-L1 expression via blocking PI3K activation but also inhibited cell proliferation in gefitinib-resistant CRCs.

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ΔNp73, TAp73 and Δ133p53 Extracellular Vesicle Cargo as Early Diagnosis Markers in Colorectal Cancer

Cancers ◽

10.3390/cancers13092240 ◽

2021 ◽

Vol 13 (9) ◽

pp. 2240

Author(s):

Javier Rodríguez-Cobos ◽

David Viñal ◽

Carmen Poves ◽

María J. Fernández-Aceñero ◽

Héctor Peinado ◽

...

Keyword(s):

Colorectal Cancer ◽

Early Diagnosis ◽

Cancer Patients ◽

Extracellular Vesicles ◽

Premalignant Lesions ◽

Invasive Technique ◽

Mrna Levels ◽

Specific Content ◽

Screening Programs ◽

Colorectal Cancer Patients

The early diagnosis of colorectal cancer is a key factor in the overall survival of the patients. The actual screening programs include different approaches with significant limitations such as unspecificity, high invasiveness, and detection at late stages of the disease. The specific content of extracellular vesicles derived from malignant cells may represent a non-invasive technique for the early detection of colorectal cancer. Here, we studied the mRNA levels of ΔNp73, TAp73, and Δ133p53 in plasma-derived extracellular vesicles from healthy subjects (n = 29), individuals with premalignant lesions (n = 49), and colorectal cancer patients (n = 42). Extracellular vesicles’ ΔNp73 levels were already significantly high in subjects with premalignant lesions. Δ133p53 levels were statistically increased in colorectal cancer patients compared to the other two groups and were associated with patients’ survival. Remarkably, TAp73 mRNA was not detected in any of the individuals. The evaluation of ΔNp73, Δ133p53 and CEA sensitivity, specificity and AUC values supports ΔNp73 as a better early diagnosis biomarker and CEA as the best to identify advanced stages. Thus, low levels of CEA and a high content of ΔNp73 may identify in screening programs those individuals at higher risk of presenting a premalignant lesion. In addition, Δ133p53 emerges as a potential prognosis biomarker in colorectal cancer.

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