Molecular and Cellular Probes最新文献

The clinical treatment of hepatocellular carcinoma (HCC) is still a heavy burden worldwide. Intracellular microRNAs (miRNAs) commonly express abnormally in cancers, thus they are potential therapeutic targets for cancer treatment. miR-21 is upregulated in HCC whereas miR-122 is enriched in normal hepatocyte but downregulated in HCC. In our study, we first generated a reporter genetic switch compromising of miR-21 and miR-122 sponges as sensor, green fluorescent protein (GFP) as reporter gene and L7Ae:K-turn as regulatory element. The reporter expression was turned up in miR-21 enriched environment while turned down in miR-122 enriched environment, indicating that the reporter switch is able to respond distinctly to different miRNA environment. Furthermore, an AAT promoter, which is hepatocyte-specific, is applied to increase the specificity to hepatocyte. A killing switch with AAT promoter and an apoptosis-inducing element, Bax, in addition to miR-21 and miR-122 significantly inhibited cell viability in Huh-7 by 70 % and in HepG2 by 60 %. By contrast, cell viability was not affected in five non-HCC cells. Thus, we provide a novel feasible strategy to improve the safety of miRNA-based therapeutic agent to cancer.

Backgroud

Cellular senescence is closely related to human aging and multiple aging-related diseases, and impaired mitochondrial energy metabolism is an important mechanism of cellular senescence. Notably, microRNA-125b-1-3p (miR-125b-1-3p) is a microRNA (miR, miRNA) that may be associated with mitochondrial energy metabolism. Ubiquinol-cytochrome c reductase binding protein (UQCRB) gene, predicted by bioinformatics tools to be targeted by miR-125b-1-3p, could serve as a novel diagnostic indicator and therapeutic target for cellular senescence-associated diseases, as well as a new idea for delaying aging.

Methods

First, the dual-luciferase reporter gene assay was used to identify UQCRB as a target gene of miR-125b-1-3p. Next, miRNA interference technology was conducted to verify that miR-125b-1-3p could negatively regulate the expression of UQCRB. Subsequently, the influence of miR-125b-1-3p on mitochondrial energy metabolism function was explored by observing the internal substances and ultrastructure of mitochondria. Further, an in vitro model of cellular senescence was established in rat renal tubular epithelial cells, which was characterized by detecting senescence-related proteins p16 and p21 and beta-galactosidase (β-gal) activity. Finally, the mitochondrial energy metabolism function of hydrogen peroxide (H₂O₂)-incubated cells was explored.

Results

The experimental results revealed that miR-125b-1-3p affected the mitochondrial energy metabolism function by inhibiting the target gene UQCRB. Meanwhile, the level of mitochondrial energy metabolism function in H₂O₂-incubated senescent cells was lower than that in normal cells.

Conclusion

In this study, we identified the target gene, UQCRB, of miR-125b-1-3p, and demonstrated its role in the pathway of mitochondrial energy metabolism, as well as its possible effect on cellular senescence through this pathway. The ameliorative effects on cellular senescence can be further explored in subsequent studies to provide additional options for delaying aging or treating aging-related diseases.

Recently, it has been discovered surprisingly that tRNA can be cleaved into specific small fragments under certain conditions. Most importantly, these tRNA-derived fragments (tRFs) participate in the regulation of gene expression, playing pivotal roles in various physiological and pathological processes and thus attracting widespread attention. Detecting tRF expression in tissues and cells often involves using tRF-specific stem-loop primers for reverse transcription. However, the high specificity offered by this method limits it to transcribing only one specific tRF sequence per reaction, necessitating separate reverse transcription and qPCR steps for multiple tRFs, leading to substantially increased time and resource consumption. This becomes especially challenging in precious samples with limited RNA availability. To address these issues, there is an urgent need for a universal and cost-effective tRF identification method. This study introduces a versatile tRF detection approach based on the uniform polyadenylation of all tRFs, allowing reverse transcription with a universal oligo(dT) primer. This method enables simultaneous reverse transcription of all target tRFs in one reaction, greatly facilitating subsequent qPCR analysis. Furthermore, it demonstrates exceptional sensitivity and specificity, offering significant value in tRF-related research.

Verrucous carcinoma (VC) is a rare subtype of squamous cell carcinoma (SCC) characterized by its histological presentation as a low-grade tumor with no potential for metastasis, setting it apart from invasive SCC. However, distinguishing VC from its benign counterpart, verrucous hyperplasia (VH), is challenging due to their clinical and morphological similarities. Despite the importance of accurate diagnosis for determining treatment strategies, diagnosis for of VH and VC relied only on lesion recurrence after resection. To address this challenge, we generated RNA profiling data from tissue samples of VH and VC patients to identify novel diagnostic markers. We analyzed differentially expressed (DE) mRNA and long non-coding RNA (lncRNA) in tissue samples from VH and VC patients. Additionally, ChIP-X Enrichment Analysis 3 (ChEA3) was conducted to identify the top five transcription factors potentially regulating the expression of DE mRNAs in VH and VC. Our analysis of mRNA and lncRNA expression profiles in VH and VC provides insights into the underlying molecular characteristics of these diseases and offers potential new diagnostic markers. The identification of specific DE genes and lncRNAs may enable clinicians to more accurately differentiate between VH and VC, leading to better treatment choices.

Objective

Utilizing transcriptome analysis to investigate the mechanisms and therapeutic approaches for cisplatin resistance in non-small cell lung cancer (NSCLC).

Methods

Firstly, the biological characters of A549 cells and A549/DDP cells were detected by RNA sequencing, CCK-8 and hippocampal energy analyzer. Then, the differential Genes were functionally enriched by GO and KEGG and the competitive endogenous RNA network map was constructed. Finally, the effects of the predicted biogenesis pathway on the biological functions of A549/DDP cells were verified by in vitro and in vivo experiments.

Result

The differentially transcribed genes of A549 and A549/DDP cells were analyzed by enrichment analysis and cell biological characteristics detection. The results showed that A549/DDP cells showed significantly increased resistance to cisplatin, glucose metabolism signaling pathway and glycolysis levels compared with A549 cells. Among glycolysis-related transcription genes, PKM had the most significant difference Fold Change is 8. LncRNA PCIF1 is a new marker of A549/DDP cells and can be used as a molecular sponge to regulate the expression of PKM. LncRNA PCIF1 targets miR-326 to induce PKM expression, promote glycolysis level, and enhance the resistance of A549/DDP cells to cisplatin.

Conclusion

LncRNA PCIF1 as biomarkers of A549/DDP cells, higher expression can induce the PKM, promote cell glycolysis, lead to the occurrence of cisplatin resistance. LncRNA PCIF1 can be considered as a potential target for treating cisplatin-resistant NSCLC.

Ovarian cancer (OC) is the fifth most common cause of death in women worldwide. Chemoresistance is a key reason for treatment failure, causing high mortality. As a member of the tripartite motif-containing (TRIM) protein family, tripartite motif 47 (TRIM47) plays a vital role in the carcinogenesis and drug resistance of various cancers. This study investigated the impact and mechanisms of TRIM47 on cisplatin (DDP) chemosensitivity and apoptosis in OC. OC cell viability was assessed with a cell counting kit-8 assay and OC cell apoptosis was assessed using flow cytometry, caspase-3 and caspase-9 activity, and Bax and Bcl-2 expression assays while gene and protein expression were assessed using qRT–PCR and Western blot assays. The expression of TRIM47 was significantly increased in both DDP-resistant tissues from patients with OC tissues and in cancer cell lines compared with that in normal tissue or parental cell lines. The increased level of TRIM47 correlated with poor prognosis in patients with OC. Functional assays demonstrated that TRIM47 promoted DDP resistance both in vitro and in vivo. The increased viability and reduced apoptosis of OC cells induced by TRIM47 can be rescued by the endoplasmic reticulum (ER) stress–inducer tunicamycin, suggesting that TRIM47 inhibits OC cell apoptosis by suppressing ER stress. Therefore, TRIM47 may be targeted as a therapeutic strategy for DDP resistance in OC.

Context

DNA mismatch repair (MMR) deficiency (dMMR) testing is now recommended in endometrial cancer. Defect identification in the molecules participating in this pathway, or the presence of microsatellite instability, are commonly employed for this purpose. Novel methods are continuously evolving to report dMMR/microsatellite instability and to easily perform routine diagnoses.

Objective

The main aim of this study was to compare the concordance of the Idylla microsatellite instability test for the identification of dMMR endometrial cancer samples defined by immunohistochemistry and MMR genomic status.

Design

We applied the Idylla MSI test to 126 early-stage endometrial cancer cases with MMR testing by immunohistochemistry and genomic characterization (methylation in MLH1 and sequence alterations in MLH1, PMS2, MSH2 and MSH6). Individual markers and overall specific performance indicators were explored.

Results

The Idylla platform achieved a higher global concordance rate with MMR genomic status than with immunohistochemistry (75 % and 66 %, respectively). Sensitivity and specificity are also higher (75 % vs 66 % and 96 % vs 90 %, respectively). Clustering analysis split the patients into 2 well-differentiated clusters, the pMMR and the dMMR group, represented by MLH1/PMS2 loss and the MLH1 methylated promoter. Overall, immunohistochemistry and MMR genomic status identified more dMMR cases than did the Idylla test, although correlations were improved with a modified Idylla test cut-off.

Conclusions

Performance of the Idylla test was better correlated with MMR genomic status than MMR immunohistochemistry status, which improved with a modified test cut-off. Further studies are needed to confirm the cut-off accuracy.

The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed millions of people and continues to wreak havoc across the globe. This sudden and deadly pandemic emphasizes the necessity for anti-viral drug development that can be rapidly administered to reduce morbidity, mortality, and virus propagation. Thus, lacking efficient anti-COVID-19 treatment, and especially given the lengthy drug development process as well as the critical death tool that has been associated with SARS-CoV-2 since its outbreak, drug repurposing (or repositioning) constitutes so far, the ideal and ready-to-go best approach in mitigating viral spread, containing the infection, and reducing the COVID-19-associated death rate. Indeed, based on the molecular similarity approach of SARS-CoV-2 with previous coronaviruses (CoVs), repurposed drugs have been reported to hamper SARS-CoV-2 replication. Therefore, understanding the inhibition mechanisms of viral replication by repurposed anti-viral drugs and chemicals known to block CoV and SARS-CoV-2 multiplication is crucial, and it opens the way for particular treatment options and COVID-19 therapeutics. In this review, we highlighted molecular basics underlying drug-repurposing strategies against SARS-CoV-2. Notably, we discussed inhibition mechanisms of viral replication, involving and including inhibition of SARS-CoV-2 proteases (3C-like protease, 3CL^pro or Papain-like protease, PL^pro) by protease inhibitors such as Carmofur, Ebselen, and GRL017, polymerases (RNA-dependent RNA-polymerase, RdRp) by drugs like Suramin, Remdesivir, or Favipiravir, and proteins/peptides inhibiting virus-cell fusion and host cell replication pathways, such as Disulfiram, GC376, and Molnupiravir. When applicable, comparisons with SARS-CoV inhibitors approved for clinical use were made to provide further insights to understand molecular basics in inhibiting SARS-CoV-2 replication and draw conclusions for future drug discovery research.

Background

Exosome (EXOs) are rapidly being identified as key mediators of cell-to-cell communication. They convey biologically active molecules to target cells, serve important roles in a range of physiological and pathological processes, and have enormous potential as novel therapeutic strategies.

Methods

Preclinical research published between 2019 and 2023 provided the study's data searched on different medline search engine, and clinicaltrials.gov was searched for clinical data. These papers were chosen because they are relevant to the research of mesenchymal stem cell-derived exosomes (MSC-EXOs). Thematic synthesis and meta-analysis were used to perform the meta-analysis of diabetic wound healing.

Results

For data extraction, a total of 18 preclinical and 4 clinical trials were selected. Preclinical investigations involving EXOs across various animal wound healing models showed promising potential for treatment. Specifically, following EXO treatment, there was a notable correlation with wound closure rates, with a pooled proportion of 46 % (95 % CI: 0.34; 0.59) and τ² of 0.0593 after 3 ± 2 days, 54 % (95 % CI: 0.43; 0.65) and τ² of 0.0465 after 7 ± 2 days, and 69 % (95 % CI: 0.62; 0.76) and τ² of 0.0221 after 14 ± 2 days, with an egger's test p-value of <0.01. Further investigation into heterogeneity was conducted through subgroup analysis based on the source of EXO and the animal model utilized in the study.

Conclusions

EXOs are proving to be viable platforms for the treatment of a wide range of disorders in clinical trials. MSC-EXOs exhibited significant diabetic wound healing capabilities across diverse outcomes including wound closure, increase angiogenesis, immunomodulatory ability and skin regeneration with its typical structure and functions.

Objective

This study aimed to conclude the effect and mechanism of ZIC2 on immune infiltration in lung adenocarcinoma (LUAD).

Methods

Expression of ZIC2 in several kinds of normal tissues of TCGA data was analyzed and its correlation with the baseline characteristic of LUAD patients were analyzed. The immune infiltration analysis of LUAD patients was performed by CIBERSORT algorithm. The correlation analysis between ZIC2 and immune cell composition was performed. Additionally, the potential upstream regulatory mechanisms of ZIC2 were predicted to identify the possible miRNAs and lncRNAs that regulated ZIC2 in LUAD. In vitro and in vivo experiments were also conducted to confirm the potential effect of ZIC2 on cell proliferation and invasion ability of LUAD cells.

Results

ZIC2 expression was decreased in various normal tissues, but increased in multiple tumors, including LUAD, and correlated with the prognosis of LUAD patients. Enrichment by GO and KEGG suggested the possible association of ZIC2 with cell cycle and p53 signal pathway. ZIC2 expression was significantly correlated with T cells CD4 memory resting, Macrophages M1, and plasma cells, indicating that dysregulated ZIC2 expression in LUAD may directly influence immune infiltration. ZIC2 might be regulated by several different lncRNA-mediated ceRNA mechanisms. In vitro experiments validated the promotive effect of ZIC2 on cell viability and invasion ability of LUAD cells. In vivo experiments validated ZIC2 can accelerate tumor growth in nude mouse.

Conclusion

ZIC2 regulated by different lncRNA-mediated ceRNA mechanisms may play a critical regulatory role in LUAD through mediating the composition of immune cells in tumor microenvironment.