Expression Levels Research Articles

Overcoming Low mRNA Expression in White Matter: A Protocol for RNA Extraction From the Optic Nerve in Large Animals for Transcriptomic Analysis.

White matter (WM) abnormalities are associated with various central nervous system (CNS) disorders, and the optic nerve provides a unique opportunity to study WM pathology. Large animal models offer a more suitable platform for preclinical testing of novel therapeutic strategies for optic neuropathy due to their similarities to humans in size and relevant anatomy. Transcriptomic analyses of optic nerve tissue are essential for understanding the underlying pathological mechanisms. However, extracting high-quality RNA from the optic nerve in large animals remains challenging. We utilized in situ hybridization and single-nucleus RNA sequencing (snRNA-seq) to examine mRNA expression in WM cells and gray matter (GM) cells. We discovered that mRNA expression levels in WM cells were only 15% to 66% of those in GM neurons. To overcome the low mRNA yield, we developed a specialized RNA extraction protocol for the intra-canalicular optic nerve in large animal models, achieving an RNA integrity number (RIN) of 6.8 ± 0.06. For single-cell transcriptomics (scRNA-seq), we obtained a cell density of 1.0 × 105 cells/µL, cell viability of 93% ± 1.84%, and an agglomeration rate of 5.37% ± 0.75%. This approach is also applicable for postmortem human optic nerve with a RIN of 8.3 ± 0.3 using snRNA-seq. We first discovered that the mRNA expression in the WM was significantly lower than that in the GM. Our RNA extraction protocol from large animal models enhances transparency and reproducibility in transcriptomic studies of optic nerve and other WM tissues.

Asiatic Acid Alleviates LPS-Induced Pyroptosis and Endoplasmic Reticulum Stress-Mediated Apoptosis via Inhibiting the HMGB1/TLR4/NF-κB Pathway in Broiler Hepatocytes.

Lipopolysaccharides (LPS) is the major compoent of Gram-negative bacteria and an important factor in inducing inflammation, which usually leads to multiple organ failure in broilers, seriously affecting the growth performance of broilers and hindering the development of poultry farming. Under the policy of prohibiting antibiotics in feed, it has become more urgent to find natural drugs to prevent liver damage caused by LPS in broilers. Asiatic acid (AA) is a pentacyclic triterpene that has been proven to have anti-inflammatory and antioxidant effects. However, the protective effects of AA in LPS-induced liver damage in broilers still need to be clarified. This study aims to explore the complete mechanism of AA against LPS-induced acute liver injury (ALI) in broilers. A total of 60 broilers (1 day old) were randomly divided into the control group, LPS group, LPS+AA (15, 30 and 60 mg/kg) group and control+AA (60 mg/kg) group. At 16, 18 and 20 days of age, the broilers were attacked with LPS (0.5 mg/kg) to construct liver injury model. H&E staining assessed liver pathological changes. The mRNA and protein expression levels related tothe HMGB1/TLR4/NF-κB pathway, pyroptosis, and ERS-mediated apoptosis in the liver tissue were detected. Our results founded that intraperitoneal injection of LPS in broilers increased the activities of AST and ALT, as well as raising the related gene and protein expression of the HMGB1/TLR4/NF-κB pathway, pyroptosis, and ERS-mediated apoptosis. Interestingly, AA improved LPS-induced liver damage and decreased the activities of AST and ALT in broilers. Additionally, AA mitigated LPS-induced ALI by reducing the mRNA levels and protein expressions of the HMGB1/TLR4/NF-κB pathway, pyroptosis and ERS-mediated apoptosis. In conclusion, the present study investigated that AA mitigated LPS-induced ALI in broilers by reducing pyroptosis and ERS-mediated apoptosis via inhibition of the HMGB1/TLR4/NF-κB pathway. Therefore, AA may serve as a potential feed additive for the prevention of LPS-induced ALI in broilers.

MDM4 inhibits ferroptosis in p53 mutant colon cancer via regulating TRIM21/GPX4 expression.

MDM4 is one of the major regulators of p53. The biological effect of MDM4 on tumor is controversial, its role and molecular mechanism in colon cancer progression and prognosis are still unclear. In this study, we identify that MDM4 is significantly overexpressed in human colon cancer and high MDM4 expression was associated with poor prognosis of colon cancer with mutant p53. MDM4 inhibits the ubiquitination of the ferroptosis marker protein GPX4 at K167 and K191 by upregulating the protein expression level of the E3 ubiquitin ligase TRIM21, which promotes the polyubiquitination of GPX4 transfer from K48- to K63- linked ubiquitination. Thereby, MDM4 enhances the stability of GPX4 protein, inhibiting ferroptosis, increasing the resistance of colon cancer patients to chemotherapy, and promoting colon cancer progression. These findings elucidate the ferroptosis inhibition effect of MDM4 via regulating TRIM21/GPX4 on p53-mutated colon cancer and provide a potential therapeutic strategy for colon cancer therapy.

Fine construction of gene coexpression network analysis using GTOM and RECODE detected a critical module of neuroblastoma stages 4 and 4S.

Stage 4 neuroblastoma (NBL), a solid tumor of childhood, has a poor prognosis. Despite intensive molecular genetic studies, no targetable gene abnormalities have been identified. Stage 4S NBL has a characteristic of spontaneous regression, and elucidation of the mechanistic differences between stages 4 and 4S may improve treatment. Conventional NBL studies have mainly focused on the detection of abnormalities in individual genes and have rarely examined abnormalities in gene networks. While the gene coexpression network is expected to contribute to the detection of network abnormalities, the fragility of the network due to data noise and the extraction of arbitrary topological structures for the high-dimensional network are issues. The present paper concerns the classification method of stages 4 and 4S NBL patients using highly accurate gene coexpression network analysis based on RNA-sequencing data of transcription factors (TFs). In particular, after applying a noise reduction method RECODE, generalized topological overlapping measure (GTOM), which weighs the connections of nodes in the network structure, succeeded in extracting a cluster of TFs that showed high classification performance for stages 4 and 4S. In addition, we investigated how these clusters correspond to clinical information and to TFs which control the normal adrenal tissue and NBL characters. A clustering method is presented for finding intermediate-scale clusters of TFs that give considerable separation performance for distinguishing between stages 4 and 4S. It is suggested that this method is useful as a way to extract factors that contribute to the separation of groups from multiple pieces of information such as gene expression levels.

Abstract A003: Snord67 promotes breast cancer metastasis through U6-mediated alternative splicing

Abstract Small nucleolar RNAs (snoRNAs) a class of ncRNAs that canonically guide post-transcriptional modifications, including 2'-O-methylation, on ribosomal RNA (rRNA) and small nuclear RNA (snRNA). While traditionally considered housekeeping genes, snoRNAs have increasingly been found to function in diverse physiologic and pathologic processes, including cancer. In an immune-competent murine model of triple negative breast cancer (TNBC) lymphatic metastasis, we identified the snoRNA Snord67 as one of the most upregulated noncoding RNAs in axillary LN (AxLN) tumors relative to mammary fat pad (MFP) tumors and lung metastases. Loss of Snord67 resulted in decreased colony formation and spheroid size in murine and human TNBC cell lines, and led to decreased lymph node tumor growth and decreased distant metastases in two immune-competent murine models of TNBC lymphatic dissemination. To determine the mechanism by which Snord67 promotes tumor growth and metastasis, we examined the impact of Snord67 on 2'-O-methylation, gene expression, and alternative splicing. Loss of Snord67 in TNBC cell lines led to decreased 2'-O-methylation at the C60 nucleotide (Cm60) in the core spliceosome component U6 snRNA. Re-introduction of wild-type Snord67 rescued Cm60 in U6 snRNA and rescued the colony formation and spheroid phenotypes of the Snord67 knockout cell lines. However, a mutant Snord67 incapable of guiding U6 Cm60 only partially rescued these in vitro phenotypes, suggesting that Snord67 promotes in vitro tumor cell growth at least in part by guiding Cm60 in U6 snRNA. We then performed RNA sequencing of Snord67 knockout and wild-type murine and human TNBC cell lines. We found that loss of Snord67 led to widespread changes in alternative splicing, consistent with its role in guiding 2'-O-methylation of the core spliceosome component U6. We further demonstrated that the inclusion of alternatively spliced cassette exons in MYO18A and NFYA was not only downregulated upon Snord67 knockout in a human TNBC cell line, but also positively correlated with Snord67 expression levels in primary breast tumors and lymph node metastases from breast cancer patients. Based on these results, we propose a model in which Snord67 guides U6 Cm60, which leads to a pro-metastatic alternative splicing program and thereby promotes lymph node tumor growth and distant metastasis. Citation Format: Katherine I Zhou, Yvonne Chao, Kwame K Forbes, Alessandro Porrello, Gabrielle M Gentile, Aaron C Chack, Dixcy J.S. John Mary, Haizhou Liu, Yinzhou Zhu, Eric Cockman, Lincy Edatt, Grant A Goda, Justin Zhao, Hala Abou Assi, Hannah J Wiedner, Yi-Hsuan Tsai, Lily Wilkinson, Amanda E Van Swearingen, Lisa A Carey, Jimena Giudice, Daniel Dominguez, Christopher L Holley, Chad V Pecot. Snord67 promotes breast cancer metastasis through U6-mediated alternative splicing [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A003.

Abstract B013: Investigation of a novel lncRNA, TROLL-9, in breast cancer formation and progression

Abstract Breast cancer (BC) is the most diagnosed cancer and the second leading cancer-related cause of mortality among women in the US. Despite advances in treatment, BC remains a challenge due to its metastatic potential. The most aggressive BC subtype, triple negative (TN), often harbors mutations in the tumor suppressor gene, p53. One of the effects of mutant p53 involves the inhibition of the tumor and metastatic suppressive functions of TAp63, a p53 family member. Loss of TAp63 leads to the formation of metastatic mammary adenocarcinomas in mice, in part through the regulation of a group of nine long non-coding RNAs (lncRNAs), that we identified and named TAp63 regulated oncogenic lncRNAs (TROLLs). We previously characterized two TROLLs, TROLL-2 and TROLL-3, as markers of tumor progression in multiple cancer types via the activation of the AKT pathway. Currently we are investigating the role of another member, TROLL-9. To investigate the biological role of TROLL-9, we performed in vivo experiments using preclinical mouse models. Specifically, MCF-DCIS, MCF-CA1D, and MDA-MB-231 triple negative breast cancer (TNBC) cells constitutively expressing TROLL-9 were generated and utilized for two mouse models: orthotopic xenografts for primary tumor growth and spontaneous metastasis. The former was designed to identify the potential role of TROLL-9 in tumor formation, whereas the latter involved the resection of a primary tumor and monitoring for metastases via bioluminescence imaging. Notably, TROLL-9 overexpression led to increased formation of lung and livers metastases in all these models, suggesting a role for TROLL-9 in the metastatic cascade. In order to elucidate the mechanism of function of TROLL-9, two complementary approaches were used. Firstly, pulldown using MS2-TRAP followed by mass spectrometry (MS) was performed and led to the identification of putative protein interactors. In parallel, Laser Capture Microscopy (LCM) of the lung metastases followed by RNA-sequencing was used to assess for any gene expression changes due to TROLL-9 overexpression. Integration of the two datasets is currently being performed to unveil the network of genes and proteins perturbed by TROLL-9. Furthermore, to understand the relevance in BC patients, TROLL-9 expression levels were analyzed in the TCGA BC dataset. Interestingly, differences in TROLL-9 levels across BC subtypes were observed, thus supporting its putative role in TNBC. Overall, our data suggest a role for TROLL-9 in BC progression. Our findings provide evidence for further characterizing its mechanism of function as a potential therapeutic target for the treatment of invasive BC, particularly TNBC harboring TP53 mutations. Citation Format: Avani A Deshpande, Marco Napoli, John H Lockhart, Christina L Carr, Jaden R Baldwin, Elsa R Flores. Investigation of a novel lncRNA, TROLL-9, in breast cancer formation and progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B013.

Experimental study of early evaluation of radiosensitivity in mouse models of lung cancers using 89Zr-anti-γH2AX-TAT PET imaging.

Early evaluation of radiation sensitivity in lung cancer patients can facilitate the transition to personalized treatment strategies. To this end, we assessed the capability of 89Zr-anti-γH2AX-TAT microPET imaging in determining the radiosensitivity of lung cancer xenograft models. We prepared and conducted quality control on 89Zr-anti-γH2AX-TAT. The radiosensitivity of human non-small cell lung cancer cells (H460) and adenocarcinoma cells (A549) was analyzed through clonogenic survival experiments. Additionally, the role of γH2AX as a biomarker for radiosensitivity was validated by quantifying γH2AX foci via fluorescence staining. Subsequently, the H460 and A549 xenograft mouse models were subjected to irradiation, followed by 89Zr-anti-γH2AX-TAT microPET imaging. Concurrently, we performed immunofluorescence staining for γH2AX in tumor tissues to establish a correlation between the uptake of 89Zr-anti-γH2AX-TAT and γH2AX expression. The surviving fraction 2Gy (SF2) values of H460 and A549 indicating that A549 adenocarcinoma has higher radiosensitivity. The cell immunofluorescence experiment showed that the repair of γH2AX foci in H460 cells after irradiation was significantly higher than that in A549 cells, which also confirmed that A549 has higher radiosensitivity. The microPET imaging results showed the uptake of 89Zr-anti-γH2AX-TAT in the tumor of the A549 models after radiotherapy was higher than H460 models. The immunofluorescence staining of tumor tissue confirmed that the expression level of γH2AX was higher and the correlation with microPET imaging uptake was good. 89Zr-anti-γH2AX-TAT allows PET imaging of radiosensitivity in lung cancer xenograft models, and is expected to become an early evaluation method for lung cancer radiosensitivity.

The impact of sex on memory during aging and Alzheimer's disease progression: Epigenetic mechanisms.

Alzheimer's disease (AD) is a leading cause of dementia, disability, and death in the elderly. While the etiology of AD is unknown, there are several established risk factors for the disease including, aging, female sex, and genetics. However, specific genetic mutations only account for a small percentage (1-5%) of AD cases and the much more common sporadic form of the disease has no causative genetic basis, although certain risk factor genes have been identified. While the genetic code remains static throughout the lifetime, the activation and expression levels of genes change dynamically over time via epigenetics. Recent evidence has emerged linking changes in epigenetics to the pathogenesis of AD, and epigenetic alterations also modulate cognitive changes during physiological aging. Aging is the greatest risk factor for the development of AD and two-thirds of all AD patients are women, who experience an increased rate of symptom progression compared to men of the same age. In humans and other mammalian species, males and females experience aging differently, raising the important question of whether sex differences in epigenetic regulation during aging could provide an explanation for sex differences in neurodegenerative diseases such as AD. This review explores distinct epigenetic changes that impact memory function during aging and AD, with a specific focus on sexually divergent epigenetic alterations (in particular, histone modifications) as a potential mechanistic explanation for sex differences in AD.

Live-Cell Imaging of miRNAs with the Combination of CHA and HCR Techniques.

The abnormal expression of miRNA-21 is closely related to many cancers, and its sensitive detection plays an important role in the diagnosis and treatment of cancers. In the report, we designed a non-enzymatic amplification sensing system based on the catalytic hairpin assembly (CHA) and palindrome-based hybridization chain reaction (PHCR) technique for the detection and imaging of miRNA in living cells. Based on PHCR technology, two ingeniously designed palindrome-contained fluorescently labeled hairpin probes are sequentially opened upon the stimulation of short oligonucleotide triggers, promoting the autonomous assembly of cross-linked network-like structures (CNSs) and generating fluorescence resonance energy transfer (FRET) signal. Moreover, the PHCR technique can be combined with CHA technology to sufficiently improve amplification efficiency and signal output. By utilizing the CHA-PHCR sensing system, one miRNA-21 activates many triggers through CHA process and in turn initiates the assembly of CNSs by PHCR reaction, efficiently amplifying the FRET signal output. As such, the sensing system can detect target miRNAs down to 10pm with high detection specificity. Moreover, the CNS exhibits the enhanced intracellular stability, enabling the living cell imaging of miRNA-21. The CHA-PHCR sensing system can also screen the expression level of miRNA-21 in different living cells and differentiate cancer cells from healthy cells, which is consistent with the gold-standard PCR method.

Neural network-based predictions of antimicrobial resistance phenotypes in multidrug-resistant Acinetobacter baumannii from whole genome sequencing and gene expression.

Whole genome sequencing (WGS) potentially represents a rapid approach for antimicrobial resistance genotype-to-phenotype prediction. However, the challenge still exists to predict fully minimum inhibitory concentrations (MICs) and antimicrobial susceptibility phenotypes based on WGS data. This study aimed to establish an artificial intelligence-based computational approach in predicting antimicrobial susceptibilities of multidrug-resistant Acinetobacter baumannii from WGS and gene expression data. Antimicrobial susceptibility testing (AST) was performed using the broth microdilution method for 10 antimicrobial agents. In silico multilocus sequence typing (MLST), antimicrobial resistance genes, and phylogeny based on cgSNP and cgMLST strategies were analyzed. High-throughput qPCR was performed to measure the expression level of antimicrobial resistance (AMR) genes. Most isolates exhibited a high level of resistance to most of the tested antimicrobial agents, with the majority belonging to the IC2/CC92 lineage. Phylogenetic analysis revealed undetected transmission events or local outbreaks. The percentage agreements between AMR phenotype and genotype ranged from 70.08% to 89.96%, with the coefficient of agreement (κ) extending from 0.025 and 0.881. The prediction of AST employed by deep neural network models achieved an accuracy of up to 98.64% on the testing data set. Additionally, several linear regression models demonstrated high prediction accuracy, reaching up to 86.15% within an error range of one gradient, indicating a linear relationship between certain gene expressions and the corresponding antimicrobial MICs. In conclusion, neural network-based predictions could be used as a tool for the surveillance of antimicrobial resistance in multidrug-resistant A. baumannii.

FXR deficiency induced ferroptosis via modulation of the CBP-dependent p53 acetylation to suppress breast cancer growth and metastasis.

Farnesoid X receptor (NR1H4/FXR) functions as a scavenger of lipid peroxide products and drives the proliferation and metastasis of various cancers. However, the underlying molecular mechanisms remain poorly understood. In our study, we found that the expression levels of FXR, vimentin and SLC7A11 were significantly higher in breast cancer tissues, particularly in metastatic cancer tissues compared to non-metastatic ones. Furthermore, the increased FXR expression was positively correlated with vimentin and SLC7A11 in clinical tumor specimens. In addition, a high level of FXR correlated with poor prognosis in patients with breast cancer. Both Z-Guggulsterone (Z-GS), as a pharmacological inhibitor of FXR, and silencing FXR curbed proliferation and migration of breast cancer cells by promoting ferroptosis. Notably, our results showed that FXR competitively bound to CREB-binding protein (CBP) to suppress the interaction between p53 and CBP in the nucleus, and thus prevented p53 acetylation at lys382, which was essential for upregulating the expression of SLC7A11. Conversely, FXR knockdown increased the interaction between p53 and CBP and promoted p53 acetylation, which ultimately led to facilitating ferroptosis in breast cancer cells. More importantly, we also found that Z-GS inhibited TGF-β1-induced tumor growth and metastasis of breast cancer primarily through ferroptosis via regulating CBP-dependent p53 acetylation in nude mice. In conclusion, the FXR was first reported as a tumor promoter that enhanced the proliferation and metastasis of breast cancer cells through regulating CBP-dependent p53 K382 acetylation. It proposes that FXR may serve as a potential therapeutic target for the treatment of breast cancer.

Wuzi Yanzong Decoction Ameliorates Oligoasthenozoospermia by Up-Regulating Methyltransferase and Increasing Spata, Bcl, and Pik3 Series Genes Methylation Level.

Wuzi Yanzong decoction (WZYZD) belongs to the traditional formula for treating male infertility caused by oligoasthenozoospermia (OLI). This research aims to elucidate the therapeutic substance basis and potential pharmacological mechanisms of WZYZD in treating OLI. A total of 52 chemical ingredients were identified from WZYZD. HE and TUNEL staining demonstrated that WZYZD can markedly alleviate OLI. Immunofluorescence analysis showed that WZYZD can significantly increase the expression levels of DNMT3 A, PIWIL1, SETDB1, and PRMT5. Methyl capture sequencing proved that WZYZD can markedly upregulate the methylated level of Spata, Bcl, and Pik3 series genes. Network pharmacology analysis proved that WZYZD can ameliorate OLI through BCL-2 and PI3 K-AKT signaling pathways. The immunofluorescence assay of BCL-2 and SPATA18 proved the aforementioned results. The potential mechanism of WZYZD in treating OLI mainly involved recruiting methyltransferase DNMT3 A, PIWIL1, PRMT5, and SETDB1 and increasing the methylation degree of Spata, Bcl, and Pik3 series genes.

LncRNA UCA1 enhances NRF2 expression through the m6A pathway to mitigate oxidative stress and ferroptosis in aging cardiomyocytes.

To explore the regulatory mechanism of lncRNA UCA1 and NRF2 in cardiomyocyte aging. In this study, we explored how lncRNA UCA1 regulates NRF2 and its effect on cardiomyocyte aging. H9c2 cardiomyocytes were cultured and treated with H2O2 to simulate cardiomyocyte aging in vitro. The expression levels of lncRNA UCA1 and NRF2 in cells were detected using qRT-PCR. Cell viability was assessed using the CCK8 assay, and cell aging was detected via Sa-β-gal staining. The levels of oxidative stress markers (SOD, MDA, ROS) and the expressions of ferroptosis-related proteins (ACSL4, TFR1, FTH1, GPX4) were measured. The regulatory mechanism between UCA1 and NRF2 was investigated using RIP-qPCR. Additionally, changes in m6A modification levels and the expression of m6A modification-related proteins in cells after UCA1 overexpression were analyzed by western blot. Our results indicate that H2O2 treatment significantly downregulated the expression of lncRNA UCA1 and NRF2. UCA1 overexpression promoted H9c2 cell proliferation, inhibited cell aging, increased SOD activity and the expression of FTH1 and GPX4 proteins, and decreased MDA and ROS content as well as ACSL4 and TFR1 protein expression. RIP-qPCR verified that UCA1 can promote the expression of NRF2 in cells. Overexpression of UCA1 significantly increased the expression of the demethylase FTO, leading to a reduction in m6A modification levels. Furthermore, there was significant enrichment between FTO and NRF2, and overexpression of FTO improved the expression of NRF2 protein in cells. Taken together, lncRNA UCA1 inhibits oxidative stress and ferroptosis, thereby preventing cardiomyocyte aging. This protective effect is likely mediated by increasing the expression of demethylase FTO and reducing m6A modification, which promotes the expression of NRF2.

Abstract B005: A robust NSCLC biomarker: miR-7-5p: in-silico validation and potential SPR- based probe for detection

Abstract The abundance of micro RNAs as a specific biomarker for accurately detecting cancer metastasis has arisen recently. The expression levels of miRNA are analyzed to get insights into cancer tissue detection and subtypes. Similar to other cancer types, the miRNA shows high levels of target mRNA dysregulation in association with non-small cell lung carcinoma (NSCLC). Among many promising cancer biomarkers for NSCLC, miR-7-5p has shown significant downregulation in the NSCLC tissues and targets proto-oncogenes like PAK2 and NOVA2. The expression levels of different proto-oncogenes targeting the miR-7-5p in NSCLC show that the EGFR- mutated NSCLC has an experimental validation. And, the target validation of the miR-7-5p could be analyzed using SPR (Surface plasmon resonance) based sensors at a single nanoparticle level such as Au nanocube due to its high specificity and accountability. Despite being an accountable tool for cancer diagnosis, miRNA-based biomarkers sometimes cause poor diagnostic specificity and reproducibility due to their heterogenicity and immunogenicity in cancer detection. To overcome these shortcomings, the biomarkers need to be validated according to recent clinical studies. Citation Format: Chandrajeet Dhara, Anindita Dhara, Saumyatika Gantayat. A robust NSCLC biomarker: miR-7-5p: in-silico validation and potential SPR- based probe for detection [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B005.

LncRNA CASC11 regulates the progress of delayed fracture healing via sponging miR-150-3p.

Long non-coding RNA (lncRNA) plays a pivotal role in bone regeneration by interaction with microRNAs (miRNAs) and constructing a lncRNA-miRNA regulatory network. This research aimed to elucidate the role of lncRNA CASC11 in the delayed healing process of tibial fractures and to explore its potential regulatory mechanisms. The expression levels of CASC11 and miR-150-3p in serum samples were detected and the predictive capability of CASC11 regarding delayed healing in fracture patients. Furthermore, the study confirmed the accuracy of the binding sites between CASC11 and miR-150-3p. Subsequently, overexpression/interference plasmids of CASC11, along with overexpression plasmids co-transfected with both CASC11 and miR-150-3p, were systematically introduced into MC3T3-E1 cells to investigate their effects on the expression of osteogenic marker genes, as well as their influence on cellular proliferation and apoptosis. The expression levels of CASC11 were significantly elevated, while miR-150-3p levels were markedly decreased in individuals exhibiting delayed fracture healing (P < 0.001). CASC11 was observed to suppress the expression of osteogenic marker genes, inhibit the proliferation of MC3T3-E1 cells, and promote cell apoptosis (P < 0.05). Furthermore, the overexpression of miR-150-3p effectively countered the inhibitory impact of CASC11 on osteogenic differentiation and the promoting effect on cell apoptosis (P < 0.05). The sponging effect of CASC11 on miR-150-3p led to delayed fracture healing. CASC11 emerges as a potential target for treating delayed fracture healing.

The LINC01094/miR-545-3p/SLC7A11 Signaling Axis Promotes the Development of Gastric Cancer by Regulating Cell Growth and Ferroptosis.

This study aimed to investigate the role and mechanism of action of LINC01094 in the development of gastric cancer (GC). The expression levels of LINC01094 in GC patients and healthy individuals were analyzed online using the Cancer Genome Atlas database. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analyses were performed to determine the expression of LINC01094/miR-545-3p/SLC7A11 in GC tissues and cells. Functional experiments (MTT assay, colony formation assay, and flow cytometry) were conducted to assess the effect of LINC01094 and miR-545-3p on cell proliferation, viability, apoptosis, cell cycle, and reactive oxygen species. Correlations between LINC01094 and miR-545-3p, as well as SLC7A11, were analyzed and validated using the dual-luciferase reporter assay and RNA immunoprecipitation. The levels of Fe2+, malondialdehyde, and glutathione in the cells were measured biochemically, and the protein expression levels of Bcl-2, cleaved caspase3, Cyclin D1, and p21 were detected by Western blotting. LINC01094 was significantly upregulated in the GC tissues and cells with a targeting relationship with miR-545-3p; the expression levels of LINC01094 and miR-545-3p were negatively correlated. Knockdown of LINC01094 notably inhibited the proliferation and viability of GC cells and promoted cell ferroptosis, which, however, was abrogated by the silencing of miR-545-3p. These findings indicate that miR-545-3p could target and positively correlate with SLC7A11 expression. Additionally, LINC01094 could promote GC cell progression and affect cellular ferroptosis by regulating the miR-545-3p/SLC7A11 signaling axis.

Predictive Value of Neutrophil Extracellular Traps in Neoadjuvant Chemotherapy for Muscle-Invasive Bladder Cancer.

Cisplatin-based chemotherapy is the recommended therapy for muscle-invasive bladder cancer (MIBC). However, the efficacy of MIBC for chemotherapy is only about 40%. Therefore, predictors of therapy response are urgently needed. Neutrophils form neutrophil extracellular traps (NETs), a network structure, and growing evidence indicated that it could be a prognostic and predictive marker in cancer. In MIBC, the predictive role of NETs in chemotherapy resistance is unclear. We used the Least Absolute Shrinkage and Selection Operator (LASSO) logistic regression analyses to develop a NETs-associated signature score (NETs-score) for therapeutic response prediction in the discovery cohort (GSE169455). Then the NETs score-based risk stratification was verified in two validation cohorts (Taber et al.'s cohort, our institutional cohort). In the training cohort, high NETs-score was associated with poor chemotherapy response (AUC = 0.781) and reduced recurrence-free survival (RFS) (hazard ratio [HR] = 2.07, 95% confidence interval [CI]: [1.26-3.40], p = 0.003) in MIBC patients. The NETs-score was also demonstrated to be a predictive factor for the efficacy of neoadjuvant chemotherapy in the validation cohort (AUC = 0.731). The accuracy of the NETs-score was superior to other chemotherapy response predictors such as Ba/Sq expression subtype (AUC = 0.711), BRCA2 mutation (AUC = 0.692) and ERCC2 mutation (AUC = 0.548). Furthermore, in our center cohort, the expression level of H3Cit showed a significant difference between the response and no-response group (p = 0.01). Through immunohistochemical validation, NETs was an independent predictor of MIBC neoadjuvant chemotherapy efficacy as determined by the multivariate logistic regression analysis (OR = 5.94, 95% CI: 1.20-45.50, p = 0.045). Patients with high levels of NETs predicted poor response to neoadjuvant chemotherapy. This study was the first to reveal the correlation between the level of NETs in MIBC and the efficacy of chemotherapy, which may provide a theoretical basis regarding NETs inhibitors.

Assessment of mRNA Decay and Calculation of Codon Occurrence to mRNA Stability Correlation Coefficients after 5-EU Metabolic Labeling.

mRNA translation and decay are tightly connected. This chapter describes a method to assess the influence of each codon identity on mRNA stability in cultured cells. The technique involves metabolic labeling of the nascent mRNAs by addition of the nucleoside analog 5-ethynyluridine (5-EU), purification of the RNA at different time-points after chase of the 5-EU, then biotinylation with Click chemistry, pull-down, and sequencing. The transcripts' half-lives are calculated from the expression level of each mRNA at the different time-points. Finally, the method describes the calculation of the Codon occurrence to mRNA Stability correlation Coefficient, or CSC, as a correlation between the codon occurrence in a transcript and the transcript half-life, for each codon.

Pullulan nanoparticles inhibit the pathogenicity of Candida albicans by regulating hypha-related gene expression.

The pathogenic process of Candida albicans, the primary causative species of candidiasis, involves hyphal growth, biofilm formation, and secretion of virulence factors. Of these factors, the biofilm, created by the secretion of extracellular matrix from adherent cells, shields cells from external threats, enabling them to withstand high concentrations of antifungal agents. Therefore, suppressing biofilm formation is a crucial aspect of combating candidiasis. This study developed phthalic pullulan nanoparticles (PPNPs) as a novel material for inhibiting C. albicans' pathogenicity. PPNPs were internalized within Candida cells and reduced pathogenicity at the gene expression level, resulting in reduced in vitro biofilm formation, adhesion to human cells, and mortality of infected Caenorhabditis elegans. Moreover, PPNPs exhibited these effects without toxicity to human cells and host animals. These findings not only indicate that PPNPs can be employed to hinder in vitro biofilm formation but also suggest their potential as a novel treatment for candidiasis.

Astragalus polysaccharide ameliorates diabetic retinopathy by inhibiting the SHH-Gli1-AQP1 signaling pathway in streptozotocin-induced type 2 diabetic rats.

This study aims to investigate the effects of astragalus polysaccharide (APS) on diabetic retinopathy through the SHH-Gli1-AQP1 pathway. The anti-type 2 diabetes mellitus (T2DM) targets of APS were identified through comprehensive searches of drug and disease-related databases. A protein-protein interaction network was then constructed, followed by GO and KEGG enrichment analyses. Molecular docking simulations were performed to evaluate the interactions of APS and metformin with Gli1 and AQP1. An in vivo T2DM rat model was established via streptozotocin (STZ) injection and treated with metformin and varying doses of APS for 12 weeks. Histological changes in retinal cells were assessed using H&E and PAS staining. The expression levels of AQP1, Gli1, and SHH in the retina were measured using immunohistochemistry, Western blotting, immunofluorescence, and ELISA. Additionally, mRNA expression of AQP1, Gli1, and SHH was quantified by RT-qPCR. Bioinformatic analyses indicated that Gli1 and AQP1, key components of the SHH-Gli1- AQP1 signaling pathway, may be associated with T2DM. Subsequent experiments demonstrated that the STZ-induced T2DM rats exhibited significant retinal damage, which was notably mitigated by both APS and metformin treatments. Furthermore, the SHH-Gli1-AQP1 signaling pathway was found to be overactivated in STZ-induced T2DM rats. Treatment with APS and metformin significantly reduced the elevated expression levels of SHH, Gli1, and AQP1. APS effectively inhibits retinal damage of STZ-induced T2DM rats by restraining the SHH-Gli1-AQP1 signaling pathway.