Search

Assays for the molecular detection of miRNAs are typically constrained by the level of multiplexing, especially in a single tube. Here, we report a general and programmable diagnostic platform by combining mesophilic Clostridium perfringens Argonaute (CpAgo) with exponential isothermal amplification (EXPAR), which is a dual-signal amplification strategy, allowing for the rapid and sensitive detection of multiple miRNAs with single-nucleotide discrimination in one pot. The C pAgo-based O ne- P ot (COP) assay achieved a limit of detection of 1 zM miRNA within 30 min of turnaround time and a wide concentration range. This COP assay was applied to simultaneously detect four miRNAs in a single tube from clinical serum samples, showing superior analytical performance in distinguishing colorectal cancer patients from healthy individuals. This programmable, one-pot, multiplex, rapid, and specific strategy offers great promise in scientific research and clinical applications.

Background & Aims: Non-small cell lung cancer (NSCLC) accounts for about 80% of lung cancer diagnoses across the world. Despite recent appreciable improvements in treatment plans for patients with NSCLC, the prognosis for those with the cancer still remains poor. Recently, a growing number of studies have shown that N-myristoyltransferases (NMTs) may be critical in carcinogenesis, however, the functional and clinical significance of this pathway in NSCLC remains unclear and requires further research., Methods: Initially, we evaluated the expression levels of NMT1 or NMT2 in a clinical cohort comprising of 303 paired primary NSCLC tissues and matched normal mucosae by using ELISA. We subsequently performed a tissue microarray analysis (TMA) to confirm its expression pattern in an independent validation cohort (n = 78). Then, we used a publicly available KM plotter database (n = 1921) to evaluate the prognostic impact of NMT1 and NMT2 in NSCLC. Lastly, a series of in-vitro molecular/cellular and animal experiments were performed for mechanistic understanding of the role of N-myristoyltransferases in NSCLC., Results: Our ELISA data revealed that the expression level of NMT1 and NMT2 was down-regulated in tumor tissues (n = 303, P < 0.0001), which was confirmed in an independent validation cohort by TMA (n = 78, P = 0.014 for NMT1 and P < 0.0001 for NMT2). On the other hand, patients with low expression of NMT1 or NMT2 had shorter overall survival (P = 0.013, HR = 0.85 for NMT1; P = 0.00059, HR = 0.8, for NMT2). Mechanistically, we revealed that the interaction and co-localization of NMT1 and NMT2 in NSCLC, and N-terminus of NMT1 and NMT2 was observed to be crucial for their interaction as well as for their catalytic activity. Moreover, we found that NMT1 can significantly promote the expression of NMT2 by enhancing its stability. We corroborated these findings by performing functional assays in which the knockout of NMT1 and NMT2 resulted in enhanced cell proliferation, migration and invasion as well as increased tumorxenograftgrowth. In addition, we identified miR-182 as a novel regulator of both NMT1 and NMT2. More specifically, the overexpression or inhibition of miR-182 modulated globe N-myristoylation level, contributed to phenotypic alterations in NSCLS cells., Conclusions: NMT1 and NMT2 can act as potential tumor suppressors in NSCLC, and the inhibition of miR-182 expression or therapeutic NMTs replenishment may be a promising treatment option for patients with NSCLC., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The simultaneous analysis of the levels of multiple microRNAs (miRNAs) is critical to the early diagnosis of cancer. However, this analysis is challenging because of the low concentrations of miRNAs and their high sequence homology. Here, we report a general and programmable diagnostic strategy for miRNA analysis: Thermus thermophilus Argonaute (TtAgo)-assisted exponential isothermal amplification for multiplex detection (TEAM). This system combines exponential isothermal amplification (EXPAR), for target amplification, with programmable TtAgo cleavage, for the generation of the reporting signal. The TEAM assay achieved attomolar sensitivity with a rapid turnaround time (30-35 min). Because of the single-nucleotide precision of TtAgo, the system demonstrated robust multiplex capability in the simultaneous detection of four miRNA targets and the classification of let-7 family members. The TEAM assay was superior in differentiating colorectal cancer patients from healthy individuals relative to the conventional EXPAR and reverse transcription polymerase chain reaction (RT-PCR) methods. This tunable and scalable approach is a powerful nucleic acid analysis tool that holds promise in scientific and clinical applications.

Long noncoding RNAs (lncRNAs) are abnormally expressed in many malignant tumors and involved in regulating the malignant phenotypes of cancer cells. However, the role of LINC00665 in colorectal cancer (CRC) and its regulatory mechanism remain unclear. In this study, real-time polymerase chain reaction (RT-PCR) was used to detect the expressions of LINC00665, miR-9-5p and activating transcription factor 1 (ATF1) mRNA in CRC tissues. The expression of ATF1 in CRC tissues was also detected by immunohistochemistry and Western blot. CCK-8 and colony formation assays were employed to detect cell proliferation. Cell cycle and apoptosis were detected by flow cytometry analysis. Scratch healing assay and Transwell test were exploited to detect cell migration and invasion. The targeting relationships between LINC00665 and miR-9-5p, and miR-9-5p and ATF1 were validated by dual luciferase reporter assay. We found that LINC00665 was significantly overexpressed in CRC tissues, and it was also negatively correlated with the expression of miR-9-5p and positively associated with the expression of ATF1. Besides, LINC00665 promoted the proliferation, migration and invasion of CRC cells, and inhibited cell apoptosis by sponging miR-9-5p. ATF1 was proved to be the downstream target of miR-9-5p and was indirectly regulated by LINC00665. Collectively, it is concluded that LINC00665 contributes to the progression of CRC by regulating miR-9-5p/ATF1 axis.

Purpose: Colorectal cancer is one of the most common malignancies worldwide. Recently, a novel circular RNA, ciRS-7, was proposed to be a potential miR-7 sponge. As miR-7, a putative tumor-suppressor, regulates the expression of several important drivers of colorectal cancer, we analyzed the clinical significance of ciRS-7 in colorectal cancer patients. Experimental Design: Initially, we evaluated the expression levels of ciRS-7 in a training cohort comprising of 153 primary colorectal cancer tissues and 44 matched normal mucosae. We subsequently confirmed its clinical relevance in an independent validation cohort ( n = 165), and evaluated the effect of ciRS-7 on miR-7, and its target genes EGFR and RAF1. Functional analyses were performed in cell lines and an animal model to support clinical findings. Results: Our data revealed that ciRS-7 was significantly upregulated in colorectal cancer tissues compared with matched normal mucosae ( P = 0.0018), and its overexpression was associated with poor patient survival ( P = 0.0224 and 0.0061 in the training and validation cohorts, respectively). Multivariate survival analysis revealed that ciRS-7 emerged as an independent risk factor for overall survival ( P = 0.0656 and 0.0324 in the training and validation cohorts, respectively). Overexpression of ciRS-7 in HCT116 and HT29 cells led to the blocking of miR-7 and resulted in a more aggressive oncogenic phenotype, and ciRS-7 overexpression permitted the inhibition of miR-7 and subsequent activation of EGFR and RAF1 oncogenes. Conclusions: CiRS-7 is a promising prognostic biomarker in colorectal cancer patients and may serve as a therapeutic target for reducing EGFR-RAF1 activity in colorectal cancer patients. Clin Cancer Res; 23(14); 3918-28. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Background & Aims: Nearly 20% of the global cancer burden can be linked to infectious agents. Fusobacterium nucleatum promotes tumor formation by epithelial cells via unclear mechanisms. We aimed to identify microRNAs (miRNAs) induced by F nucleatum and evaluate their ability to promote colorectal carcinogenesis in mice., Methods: Colorectal cancer (CRC) cell lines were incubated with F nucleatum or control reagents and analyzed in proliferation and would healing assays. HCT116, HT29, LoVo, and SW480 CRC cell lines were incubated with F nucleatum or phosphate-buffered saline (PBS [control]) and analyzed for miRNA expression patterns and in chromatin immunoprecipitation assays. Cells were incubated with miRNAs mimics, control sequences, or small interfering RNAs; expression of reporter constructs was measured in luciferase assays. CRC cells were incubated with F nucleatum or PBS and injected into BALB/C nude mice; growth of xenograft tumors was measured. C57BL adenomatous polyposis coli min/+ , C57BL miR21a -/- , and C57BL mice with full-length miR21a (controls) were given F nucleatum by gavage; some mice were given azoxymethane and dextran sodium sulfate to induce colitis and colon tumors. Intestinal tissues were collected and tumors were counted. Serum samples from mice were analyzed for cytokine levels by enzyme-linked immunosorbent assay. We performed in situ hybridization analyses to detect enrichment of F nucleatum in CRC cells. Fusobacterium nucleatum DNA in 90 tumor and matched nontumor tissues from patients in China were explored for the expression correlation analysis; levels in 125 tumor tissues from patients in Japan were compared with their survival times., Results: Fusobacterium nucleatum increased proliferation and invasive activities of CRC cell lines compared with control cells. CRC cell lines infected with F nucleatum formed larger tumors, more rapidly, in nude mice than uninfected cells. Adenomatous polyposis coli min/+ mice gavaged with F nucleatum developed significantly more colorectal tumors than mice given PBS and had shorter survival times. We found several inflammatory factors to be significantly increased in serum from mice given F nucleatum (interleukin 17F, interleukin 21, and interleukin 22, and MIP3A). We found 50 miRNAs to be significantly up-regulated and 52 miRNAs to be significantly down-regulated in CRCs incubated with F nucleatum vs PBS; levels of miR21 increased by the greatest amount (>4-fold). Inhibitors of miR21 prevented F nucleatum from inducing cell proliferation and invasion in culture. miR21a -/- mice had a later appearance of fecal blood and diarrhea after administration of azoxymethane and dextran sodium sulfate, and had longer survival times compared with control mice. The colorectum of miR21a -/- mice had fewer tumors, of smaller size, and the miR21a -/- mice survived longer than control mice. We found RASA1, which encodes an RAS GTPase, to be one of the target genes consistently down-regulated in cells that overexpressed miR21 and up-regulated in cells exposed to miR21 inhibitors. Infection of cells with F nucleatum increased expression of miR21 by activating Toll-like receptor 4 signaling to MYD88, leading to activation of the nuclear factor-κB. Levels of F nucleatum DNA and miR21 were increased in tumor tissues (and even more so in advanced tumor tissues) compared with non-tumor colon tissues from patients. Patients whose tumors had high amounts of F nucleatum DNA and miR21 had shorter survival times than patients whose tumors had lower amounts., Conclusions: We found infection of CRC cells with F nucleatum to increase their proliferation, invasive activity, and ability to form xenograft tumors in mice. Fusobacterium nucleatum activates Toll-like receptor 4 signaling to MYD88, leading to activation of the nuclear factor-κB and increased expression of miR21; this miRNA reduces levels of the RAS GTPase RASA1. Patients with both high amount of tissue F nucleatum DNA and miR21 demonstrated a higher risk for poor outcomes., (Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Purpose: Colorectal cancer ranks as the third most frequent cancer type, and its incidence continues to rise gradually worldwide, highlighting the need to identify previously unrecognized molecular events that propel development of this malignancy. Recent evidence suggests that dysregulated expression of FOX family of transcription factors may be critical in various genetic disorders as well as cancer; however, the functional and clinical significance of this pathway in colorectal cancer remains unclear., Experimental Design and Results: Herein, we performed a systematic and comprehensive discovery step by evaluating the expression of FOX family members, and identified that FOXM1 and FOXQ1 are frequently overexpressed in colorectal cancer. We subsequently confirmed these findings in two large testing cohorts (n = 550) and an independent clinical validation cohort (n = 134), in which high expression of FOXM1 and FOXQ1 emerged as an independent prognostic factor in colorectal cancer patients. We corroborated these findings by performing functional assays in which knockdown of FOXM1 and FOXQ1 resulted in inhibited cell proliferation and suppressed migration and invasion in colorectal cancer cells. Furthermore, using bioinformatic approaches, we identified miR-342 as a novel regulator of both FOXM1 and FOXQ1. Overexpression or inhibition of miR-342 modulated the expression of both genes and contributed to phenotypic alterations in colorectal cancer cells, which was subsequently validated in a xenograft animal model., Conclusions: Collectively, we have firstly identified FOXM1 and FOXQ1 as promising prognostic biomarkers in colorectal cancer patients, and provided novel evidence that therapeutic targeting of these genes or miR-342 may be a potential treatment approach in colorectal cancer patients. Clin Cancer Res; 22(19); 4947-57. ©2016 AACR., (©2016 American Association for Cancer Research.)

Colorectal carcinogenesis represents a sequential progression of normal colonic mucosa from adenoma to carcinoma. It has become apparent that miRNA deregulation contributes to the initiation and progression of colorectal cancer (CRC). These oncogenic or tumor-suppressive miRNAs interact with intracellular signaling networks and lead to alteration of cell proliferation, apoptosis, metastasis and even response to chemotherapeutic treatments. This article aims to review the cutting edge progress in the discovery of the role of novel mechanisms for miRNAs in the development of CRC. We will also discuss the potential use of miRNAs as biomarkers for early diagnosis and prognosis of CRC. Furthermore, with advancements in RNA delivery technology, it is anticipated that manipulation of miRNAs may offer an alternative therapy for CRC treatment.

Renal cell carcinoma (RCC) is a common urological malignancy. It remains unclear, however, whether Yin Yang 1 (YY1) plays a functional role in the development of human RCC. In the present study, we demonstrated that levels of YY1 were significantly increased in primary RCC tissues when compared to these levels in the matched healthy tissues. YY1 knockdown inhibited cell growth, migration and invasion of RCC cells. Additionally, we highlighted a positive feedback-loop pathway resulting in YY1 upregulation. We observed that overexpression of YY1 caused repression of C/EBPα and the inhibition of C/EBPα led to the suppression of miR-34a. Since YY1 is a direct target of miR-34a, the low level of miR-34a increased the expression of YY1, promoting the aggressiveness of RCC cells. Furthermore, this feedforward mechanism was found in RCC tissues. We observed that miR-34a was downregulated in the pools of cancer tissues when compared to that of the normal tissues. The expression of miR-34a displayed an inverse correlation with YY1, but a positive correlation with C/EBPα. In conclusion, our study highlights the importance of a novel regulatory circuitry for YY1 activation in maintaining the aggressive phenotypes of RCC.

Aim: Organometallic arene Ru(II) complexes have long been considered as most promising substitutes for cisplatinum as an anti-tumor drug, with low toxicity towards human normal cells and high selectivity to tumor cells. In this study, we synthesized a novel arene Ru(II) drug named Rawq01. We evaluated its activity in an in vitro model of esophageal cancer (ESCC) and further explored the cellular signaling pathways altered by Rawq01., Materials and Methods: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphe nyltetrazolium bromide (MTT) assay, colony forming assay and apoptosis assays were used to evaluate the antitumor activity of Rawq01. We treated ESCC cells with Rawq01 alone or combined with microRNA-21(miR-21) and LY294002 to explore whether Rawq01 altered the effect of miR-21 on PTEN/AKT signaling pathway in ESCC cell., Results: ESCC cells were sensitive to RAWQ01. We also found that Rawq01 up-regulated the expression of PTEN through mir-21 inhibition and therefore inhibited the PI3K-AKT pathway. Furthermore, when we combined Rawq01 treatment with miR-21 inhibitor or LY294002, the inhibitory effect of Rawq01 was significantly enhanced., Conclusion: The effect of miR-21 on PTEN/AKT signaling pathway is abrogated by the novel arene Ru(II) drug Rawq01. Our data may be useful for the future development of a chemosensitizing strategies through manipulating microRNA expression for tumor treatment.

Unlabelled: Ovarian cancer is one of the most common gynecological malignancies. Limited efficacy of cytotoxic chemotherapy is a key obstacle in the treatment of advanced ovarian cancer. This study aimed to investigate whether Mir-375 enhances Rawq01 (a ruthenium derived compound) induced cell death in ovarian cancer., Methods: Three human ovarian cancer cell lines were selected, and independently treated with Rawq01+mir-375 and Rawq01+control. MTT assay and flow cytometry were performed to detect the growth of ovarian cancer cells. Western blot was carried out to determine the expression of apoptotic associated proteins. In addition, ovarian cancer xenografts were established to explore whether mir-375 increased the in vivo chemosensitivity of ovarian cancer cells to RAWQ01., Results: Over-expression of mir-375 sensitized the ovarian cancer cells to RAWQ01. Mir-375 enhanced the in vitro sensitivity of ovarian cancer cells to RAWQ01 by inducing apoptosis. Mir-375 also increased the in vivo chemosensitivity of ovarian cancer cells to RAWQ01., Conclusion: Mir-375 can enhance Rawq01 induced cell death in human ovarian cancer both in vitro and in vivo.