Telomerase Reverse Transcriptase mRNA Research Articles

An association between elevated telomerase reverse transcriptase expression and the immune tolerance disruption of dendritic cells

BackgroundTo elucidate the mechanism of dysfunction of tolerogenic dendritic cells (DCs) is of significance. Telomerase involves the regulation of the cell fate and activities. The objective of this study is to investigate the role of telomerase reverse transcriptase (TERT) in regulating the tolerogenic feature of DCs.MethodsThe telomerase was assessed in DCs, which were collected from patients with allergic rhinitis (AR), healthy control (HC) subjects, and mice. RNAs were extracted from DCs, and analyzed by RNA sequencing (RNAseq), real-time quantitative RT-PCR, and Western blotting.ResultsThe results showed that expression of TERT was higher in peripheral DCs of AR patients. The expression of IL10 in DCs was negatively correlated with the levels of TERT expression. Importantly, the levels of TERT mRNA in DCs were associated with the AR response in patients with AR. Endoplasmic reticulum (ER) stress promoted the expression of Tert in DCs. Sensitization with the ovalbumin-aluminum hydroxide protocol increased the expression of Tert in DCs by exacerbating ER stress. TERT interacting with c-Maf (the transcription factor of IL-10) inducing protein (CMIP) in DCs resulted in CMIP ubiquitination and degradation, and thus, suppressed the production of IL-10. Inhibition of Tert in DCs mitigated experimental AR.ConclusionsElevated amounts of TERT were detected in DCs of patients with AR. The tolerogenic feature of DCs was impacted by TERT. Inhibited TERT attenuated experimental AR.

Increased Prolylcarboxypeptidase Expression Can Serve as a Biomarker of Senescence in Culture.

Prolylcarboxypeptidase (PRCP, PCP, Lysosomal Pro-X-carboxypeptidase, Angiotensinase C) controls angiotensin- and kinin-induced cell signaling. Elevation of PRCP appears to be activated in chronic inflammatory diseases [cardiovascular disease (CVD), diabetes] in proportion to severity. Vascular endothelial cell senescence and mitochondrial dysfunction have consistently been shown in models of CVD in aging. Cellular senescence, a driver of age-related dysfunction, can differentially alter the expression of lysosomal enzymes due to lysosomal membrane permeability. There is a lack of data demonstrating the effect of age-related dysfunction on the expression and function of PRCP. To explore the changes in PRCP, the PRCP-dependent prekallikrein (PK) pathway was characterized in early- and late-passage human pulmonary artery endothelial cells (HPAECs). Detailed kinetic analysis of cells treated with high molecular weight kininogen (HK), a precursor of bradykinin (BK), and PK revealed a mechanism by which senescent HPAECs activate the generation of kallikrein upon the assembly of the HK-PK complex on HPAECs in parallel with an upregulation of PRCP and endothelial nitric oxide (NO) synthase (eNOS) and NO formation. The NO production and expression of both PRCP and eNOS increased in early-passage HPAECs and decreased in late-passage HPAECs. Low activity of PRCP in late-passage HPAECs was associated with rapid decreased telomerase reverse transcriptase mRNA levels. We also found that, with an increase in the passage number of HPAECs, reduced PRCP altered the respiration rate. These results indicated that aging dysregulates PRCP protein expression, and further studies will shed light into the complexity of the PRCP-dependent signaling pathway in aging.

Engineered Exosomes with Growth Differentiation Factor-15 Overexpression Enhance Cardiac Repair After Myocardial Injury.

Cardiac repair remains a thorny issue for survivors of acute myocardial infarction (AMI), due to the regenerative inertia of myocardial cells. Cell-free therapies, such as exosome transplantation, have become a potential strategy for myocardial injury. The aim of this study was to investigate the role of engineered exosomes in overexpressing Growth Differentiation Factor-15 (GDF-15) (GDF15-EVs) after myocardial injury, and their molecular mechanisms in cardiac repair. H9C2 cells were transfected with GDF-15 lentivirus or negative control. The exosomes secreted from H9C2 cells were collected and identified. The cellular apoptosis and autophagy of H2O2-injured H9C2 cells were assessed by Western blotting, TUNEL assay, electron microscopy, CCK-8 and caspase 3/7 assay. A rat model of AMI was constructed by ligating the left anterior descending artery. The anti-apoptotic, pro-angiogenic effects of GDF15-EVs treatment, as well as ensuing functional and histological recovery were evaluated. Then, mRNA sequencing was performed to identify the differentially expressed mRNAs after GDF15-EVs treatment. GDF15-EVs inhibited apoptosis and promoted autophagy in H2O2 injured H9C2 cells. GDF15-EVs effectively decreased the infarct area and enhanced the cardiac function in rats with AMI. Moreover, GDF15-EVs hindered inflammatory cell infiltration, inhibited cell apoptosis, and promoted cardiac angiogenesis in rats with AMI. RNA sequence showed that telomerase reverse transcriptase (TERT) mRNA was upregulated in GDF15-EVs-treated H9C2 cells. AMPK signaling was activated after GDF15-EVs. Silencing TERT impaired the protective effects of GDF15-EVs on H2O2-injured H9C2 cells. GDF15-EVs could fulfil their protective effects against myocardial injury by upregulating the expression of TERT and activating the AMPK signaling pathway. GDF15-EVs might be exploited to design new therapies for AMI.

Electroacupuncture pretreatment preserves telomerase reverse transcriptase function and alleviates postoperative cognitive dysfunction by suppressing oxidative stress and neuroinflammation in aged mice.

Elderly patients often exhibit postoperative cognitive dysfunction (POCD), a postsurgical decline in memory and executive function. Oxidative stress and neuroinflammation, both pathological characteristics of the aged brain, contribute to this decline. This study posits that electroacupuncture (EA) stimulation, an effective antioxidant and anti-inflammatory modality, may enhance telomerase reverse transcriptase (TERT) function, the catalytic subunit of telomerase known for its protective properties against cellular senescence and oxidative damage, to alleviate POCD in aged mice. The animal POCD model was created by subjecting aged mice to abdominal surgery, followed by EA pretreatment at the Baihui acupoint (GV20). Postoperative cognitive function was gauged using the Morris water maze (MWM) test. Hippocampal TERT mRNA levels and telomerase activity were determined through qPCR and a Telomerase PCR ELISA kit, respectively. Oxidative stress was assessed through superoxide dismutase (SOD), reactive oxygen species (ROS), and malondialdehyde (MDA) levels. Iba-1 immunostaining determined the quantity of hippocampal microglia. Additionally, western blotting assessed TERT, autophagy markers, and proinflammatory cytokines at the protein level. Abdominal surgery in aged mice significantly decreased telomerase activity and TERT mRNA and protein levels, but increased oxidative stress and neuroinflammation and decreased autophagy in the hippocampus. EA-pretreated mice demonstrated improved postoperative cognitive performance, enhanced telomerase activity, increased TERT protein expression, improved TERT mitochondrial localization, and reduced oxidative damage, autophagy dysfunction, and neuroinflammation. The neuroprotective benefits of EA pretreatment were diminished following TERT knockdown. Our findings underscore the significance of TERT function preservation in alleviating surgery-induced oxidative stress and neuroinflammation in aged mice. Anovel neuroprotective mechanism of EA stimulation is highlighted, whereby modulation of TERT and telomerase activity reduces oxidative damage and neuroinflammation. Consequently, maintaining TERT function via EA treatment could serve as an effective strategy for managing POCD in elderly patients.

The Landscape of Telomere Length and Telomerase in Human Embryos at Blastocyst Stage.

The telomere length of human blastocysts exceeds that of oocytes and telomerase activity increases after zygotic activation, peaking at the blastocyst stage. Yet, it is unknown whether aneuploid human embryos at the blastocyst stage exhibit a different profile of telomere length, telomerase gene expression, and telomerase activity compared to euploid embryos. In present study, 154 cryopreserved human blastocysts, donated by consenting patients, were thawed and assayed for telomere length, telomerase gene expression, and telomerase activity using real-time PCR (qPCR) and immunofluorescence (IF) staining. Aneuploid blastocysts showed longer telomeres, higher telomerase reverse transcriptase (TERT) mRNA expression, and lower telomerase activity compared to euploid blastocysts. The TERT protein was found in all tested embryos via IF staining with anti-hTERT antibody, regardless of ploidy status. Moreover, telomere length or telomerase gene expression did not differ in aneuploid blastocysts between chromosomal gain or loss. Our data demonstrate that telomerase is activated and telomeres are maintained in all human blastocyst stage embryos. The robust telomerase gene expression and telomere maintenance, even in aneuploid human blastocysts, may explain why extended in vitro culture alone is insufficient to cull out aneuploid embryos during in vitro fertilization.

Inhibition of Posterior Capsule Opacification by Adenovirus-Mediated Delivery of Short Hairpin RNAs Targeting TERT in a Rabbit Model

Purpose Posterior capsule opacification (PCO) is the most common postoperative complication after cataract surgery and cannot yet be eliminated. Here, we investigated the inhibitory effects of telomerase reverse transcriptase (TERT) gene silencing on PCO in a rabbit model. Methods After rabbit lens epithelial cells (LECs) were treated with adenovirus containing short hairpin RNAs (shRNA) targeting TERT (shTERT group), adenovirus containing scramble nonsense control shRNA (shNC group) or PBS (control group), quantitative real-time polymerase chain reaction and Western blotting were used to measure the expression levels of TERT, and a scratch assay was performed to assess the LEC migration. New Zealand white rabbits underwent sham cataract surgery followed by an injection of adenovirus carrying shTERT into their capsule bag. The intraocular pressure and anterior segment inflammation were evaluated on certain days, and EMT markers (α-SMA and E-cadherin) were evaluated by Western blotting and immunofluorescence. The telomerase activity of the capsule bag was detected by ELISA. At 28 d postoperatively, hematoxylin and eosin staining of the cornea and iris and electron microscopy of the posterior capsule were performed. Results Application of shTERT to LECs downregulated the expression levels of TERT mRNA and protein. The scratch assay results showed a decrease in the migration of LECs in the shTERT group. In vivo, shTERT decreased PCO formation after cataract surgery in rabbits and downregulated the expression of EMT markers, as determined by Western blotting and immunofluorescence. In addition, telomerase activity was suppressed in the capsule bag. Despite slight inflammation in the iris, histologic results revealed no toxic effects in the cornea and iris. Conclusion TERT silencing effectively reduces the migration and proliferation of LECs and the formation of PCO. Our findings suggest that TERT silencing may be a potential preventive strategy for PCO.

Abstract 2743: Synergistic efficacy of telomerase-specific oncolytic adenoviral therapy and histone deacetylase inhibition in human hepatocellular carcinoma

Abstract Background: The telomerase-specific oncolytic adenovirus Telomelysin along with the histone deacetylase inhibitor AR42 have both demonstrated anticancer effects in preclinical models of human hepatocellular carcinoma (HCC). However, the clinical development of Telomelysin may be hindered by human antiviral immunity and tumor resistance. Combining oncolytic and epigenetic therapies has been reported to be a viable approach for treating various cancers. We investigated the potential synergism of Telomelysin and AR42 and the relevant underlying mechanisms. Method: HCC cell lines (PLC5 and Hep3B) were tested and median effect analysis was performed to estimate potential synergistic antiproliferative effects. Apoptosis was assessed by flow cytometry and Western blotting. The expression of coxsackievirus and adenovirus receptor (CAR) was measured through flow cytometry and immunofluorescence staining. Human telomerase reverse transcriptase (hTERT) mRNA expression was assessed by reverse transcription polymerase chain reaction. Western blotting was conducted to examine the activation of pivotal signaling pathways and the interferon (IFN) response. HCC cells constitutively overexpressing Akt were cloned for molecular mechanism verification. MK-2206 was used to inhibit the activation of Akt signaling. In vivo efficacy was determined through xenograft models of human HCC. Results: Telomelysin and AR42 exhibited synergistic antiproliferative effects in human HCC models in vitro and in vivo. Apoptosis induced by Telomelysin was significantly enhanced by AR42 in both PLC5 and Hep3B HCC cells. AR42 treatment unexpectedly attenuated the expression of the CAR and the mRNA levels of hTERT, which may be positively associated with the cytotoxicity of Telomelysin. Meanwhile, the cellular antiviral interferon response was not altered by AR42 treatment. Further, we found that Telomelysin enhanced Akt phosphorylation in HCC cells. AR42 reduced Telomelysin-induced phospho-Akt activation and enhanced Telomelysin-induced apoptosis. The correlation of Akt phosphorylation with drug-induced apoptosis was validated in HCC cells with upregulated or downregulated Akt signaling. Conclusions: Combination therapy with Telomelysin and AR42 demonstrates synergistic anti-HCC efficacy. Clinical trials investigating this new combination regimen are warranted. Keywords: Hepatocellular carcinoma, oncolytic adenovirus, Telomelysin, HDAC inhibitor, Akt Citation Format: Zhong-Zhe Lin, Mickey C-T. Hu, Chiun Hsu, Yao-Ming Wu, Yen-Shen Lu, Ja-An Annie Ho, Shiou-Hwei Yeh, Pei-Jer Chen, Ann-Lii Cheng. Synergistic efficacy of telomerase-specific oncolytic adenoviral therapy and histone deacetylase inhibition in human hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2743.

TERT promoter mutations and methylation for telomerase activation in urothelial carcinomas: New mechanistic insights and clinical significance.

Telomerase, an RNA-dependent DNA polymerase synthesizing telomeric TTAGGG sequences, is primarily silent in normal human urothelial cells (NHUCs), but widely activated in urothelial cell-derived carcinomas or urothelial carcinomas (UCs) including UC of the bladder (UCB) and upper track UC (UTUC). Telomerase activation for telomere maintenance is required for the UC development and progression, and the key underlying mechanism is the transcriptional de-repression of the telomerase reverse transcriptase (TERT), a gene encoding the rate-limiting, telomerase catalytic component. Recent mechanistic explorations have revealed important roles for TERT promoter mutations and aberrant methylation in activation of TERT transcription and telomerase in UCs. Moreover, these TERT-featured genomic and epigenetic alterations have been evaluated for their usefulness in non-invasive UC diagnostics, recurrence monitoring, outcome prediction and response to treatments such as immunotherapy. Importantly, the detection of the mutated TERT promoter and TERT mRNA as urinary biomarkers holds great promise for urine-based UC liquid biopsy. In the present article, we review recent mechanistic insights into altered TERT promoter-mediated telomerase activation in UCs and discuss potential clinical implications. Specifically, we compare differences in senescence and transformation between NHUCs and other types of epithelial cells, address the interaction between TERT promoter mutations and other factors to affect UC progression and outcomes, evaluate the impact of TERT promoter mutations and TERT-mediated activation of human endogenous retrovirus genes on UC immunotherapy including Bacillus Calmette-Guérin therapy and immune checkpoint inhibitors. Finally, we suggest the standardization of a TERT assay and evaluation system for UC clinical practice.

In Vitro Cytotoxic Effects and Mechanisms of Action of Eleutherine Isolated from Eleutherine plicata Bulb in Rat Glioma C6 Cells.

Gliomas are the most common primary malignant brain tumors in adults, and have a poor prognosis, despite the different types of treatment available. There is growing demand for new therapies to treat this life-threatening tumor. Quinone derivatives from plants have received increased interest as potential anti-glioma drugs, due to their diverse pharmacologic activities, such as inhibiting cell growth, inflammation, tumor invasion, and promoting tumor regression. Previous studies have demonstrated the anti-glioma activity of Eleutherine plicata, which is related to three main naphthoquinone compounds-eleutherine, isoeleutherine, and eleutherol-but their mechanism of action remains elusive. Thus, the aim of this study was to investigate the mechanism of action of eleutherine on rat C6 glioma. In vitro cytotoxicity was evaluated by MTT assay; morphological changes were evaluated by phase-contrast microscopy. Apoptosis was determined by annexin V-FITC-propidium iodide staining, and antiproliferative effects were assessed by wound migration and colony formation assays. Protein kinase B (AKT/pAKT) expression was measured by western blot, and telomerase reverse transcriptase mRNA was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Eleutherine reduced C6 cell proliferation in a dose-dependent manner, suppressed migration and invasion, induced apoptosis, and reduced AKT phosphorylation and telomerase expression. In summary, our results suggest that eleutherine has potential clinical use in treating glioma.

Lab-on-chip assay of tumour markers and human papilloma virus for cervical cancer detection at the point-of-care

Cervical cancer affects over half a million people worldwide each year, the majority of whom are in resource-limited settings where cytology screening is not available. As persistent human papilloma virus (HPV) infections are a key causative factor, detection of HPV strains now complements cytology where screening services exist. This work demonstrates the efficacy of a handheld Lab-on-Chip (LoC) device, with an external sample extraction process, in detecting cervical cancer from biopsy samples. The device is based on Ion-Sensitive Field-Effect Transistor (ISFET) sensors used in combination with loop-mediated isothermal amplification (LAMP) assays, to amplify HPV DNA and human telomerase reverse transcriptase (hTERT) mRNA. These markers were selected because of their high levels of expression in cervical cancer cells, but low to nil expression in normal cervical tissue. The achieved analytical sensitivity for the molecular targets resolved down to a single copy per reaction for the mRNA markers, achieving a limit of detection of 102 for hTERT. In the tissue samples, HPV-16 DNA was present in 4/5 malignant and 2/5 benign tissues, with HPV-18 DNA being present in 1/5 malignant and 1/5 benign tissues. hTERT mRNA was detected in all malignant and no benign tissues, with the demonstrated pilot data to indicate the potential for using the LoC in cervical cancer screening in resource-limited settings on a large scale.

Synergistic effects of telomerase reverse transcriptase and regulator of telomere elongation helicase 1 on aggressiveness and outcomes in adrenocortical carcinoma

Adrenocortical carcinoma (ACC) is one of the deadliest endocrine malignancies and telomere maintenance by activated telomerase is critically required for ACC development and progression. Because telomerase reverse transcriptase (TERT) and regulator of telomere elongation helicase 1 (RTEL1) play key roles in telomere homeostasis, we determined their effect on ACC pathogenesis and outcomes. Analyses of TCGA and GEO datasets showed significantly higher expression of RTEL1 but not TERT in ACC tumors, compared to their benign or normal counterparts. Furthermore, gains/amplifications of both TERT and RTEL1 genes were widespread in ACC tumors and their expression correlated with their gene copy numbers. Higher expression of either TERT or RTEL1 was associated with shorter overall and progression-free survival (OS and PFS) in the TCGA ACC patient cohort, and higher levels of both TERT and RTEL1 mRNA predicted the shortest patient OS and PFS. However, multivariate analyses showed that only RTEL1 independently predicted patient OS and PFS. Gene set enrichment analysis further showed enrichments of wnt/β-catenin, MYC, glycolysis, MTOR, and DNA repair signaling pathways in ACC tumors expressing high TERT and RTEL1 mRNA levels. Taken together, TERT and RTEL1 promote ACC aggressiveness synergistically and may serve as prognostic factors and therapeutic targets for ACC.

Establishment and characterization of a sheep endometrial epithelial cell line

Endometrial epithelial cells play a significant role in the "dialogue" between the embryo and the mother, but in vitro studies to clarify this are hampered by the limited lifespan of primary cells. As such, it is necessary to develop an in vitro model to study endometrial function. Morphological analysis showed that the pEECs were homogeneous, formed characteristic cobblestone monolayers, and expressed the epithelial cell-specific marker, cytokeratin 18. The isolated and purified cells were transfected with a plasmid encoding human telomerase reverse transcriptase (TERT) gene, pCI-neo-TERT, to establish an immortal endometrial epithelial cell line (iEECs). The transfected cells were cultured with G418 and monoclonal cells were selected for expanded culture. Expression of TERT mRNA was detected by RT-qPCR and protein was quantitated by Western blot. TERT expression was stable and continued to be active with no signs of aging. Assays for cell proliferation and apoptosis indicated higher proliferation and cellular activity in iEECs than pEECs. After stimulated by interferon tau (IFN-τ), both iEECs and pEECs showed similar upregulation levels in all the underlying genes. Taken together, these findings demonstrate that iEECs retained the basic morphology and function of pEECs, providing a robust in vitro model for study of the function of ovine endometrial epithelial cells.

High prevalence of TERT aberrations in myxoid liposarcoma: TERT reactivation may play a crucial role in tumorigenesis.

Myxoid liposarcoma (MLPS) is genetically characterized by FUS‐DDIT3 or EWSR1‐DDIT3 gene fusion and the high frequency of hotspot mutations (C228T or C250T) in the promoter region of telomerase reverse transcriptase (TERT) that encodes the TERT protein. The latter leads to telomerase reactivation, a mechanism of telomere maintenance. Although the TERT promoter hotspot mutation is a poor prognostic factor in various tumors, its effect on MLPS has not been reported in detail. In the present study, we examined the clinicopathological characteristics, prognosis, and telomere maintenance mechanisms in 83 primary tumor samples of MLPS, which were resected surgically at the Cancer Institute Hospital, Japanese Foundation for Cancer Research, Tokyo, Japan, from 2008 to 2020. TERT promoter hotspot mutations were observed in 77% (63/82) cases, and alternative lengthening of telomeres (ALT) was absent in all cases. Among the cases without TERT promoter hotspot mutations, TERT rearrangements, and minor point mutations in the TERT promoter region were found in 3 and 2 cases, respectively. TERT mRNA expression was observed consistently even in patients for whom no genomic TERT aberrations were detected, and the presence of TERT promoter hotspot mutation did not correlate significantly with either overall and metastasis‐free survival (P = .56, P = .83, respectively) or clinicopathological features. Therefore, patients with MLPS characteristically shows TERT expression and a high prevalence of TERT aberrations. Our findings suggest that TERT aberration is not prognostic factor, but might occur at an early stage and play a key role in tumorigenesis.

Cycloastragenol and Astragaloside IV activate telomerase and protect nucleus pulposus cells against high glucose-induced senescence and apoptosis.

In diabetes-induced intervertebral disc degeneration (Db-IVDD), senescence and apoptosis of nucleus pulposus cells (NPCs) are major contributing factors. Telomere attrition and telomerase downregulation are some of the main reasons for senescence and eventual apoptosis. The derivatives of the Chinese herb Astragalus membranaceus, Cycloastragenol (CAG) and Astragaloside IV (AG-IV), are reportedly effective telomerase activators against telomere shortening; however, their effect in Db-IVDD have not been explored. The present study simultaneously investigated the regulation of these derivatives on senescence, apoptosis, telomeres and telomerase a model of high-glucose (HG)-induced stress using rat primary NPCs. The NPCs were stimulated with HG (50 mM) to evoke HG-induced stress, and the effects of CAG and AG-IV were observed on: i) The expression level of senescence marker p16; ii) β-Gal staining; iii) the expression levels of apoptosis markers cleaved-caspase 3 (c-C3), BAX and Bcl-2; iv) telomerase activation with telomerase reverse transcriptase (TERT) mRNA and protein expression, while telomere length was measured with reverse transcription-quantitative PCR. Cell proliferation was determined using the Cell Counting Kit-8 assay. Results demonstrated an upregulation in the expression levels of p16, c-C3 and BAX, and increased β-Gal staining; while the expression level of Bcl-2 was downregulated in a concentration-dependent manner. Pre-treatment of the NPCs with CAG and AG-IV downregulated the protein expression levels of p16, c-C3 and BAX, and decreased the percentage of β-Gal and FITC staining; while upregulating the Bcl-2 expression. These effects protected the cells from HG stress-induced senescence and apoptosis. HG also downregulated the expression profile of TERT and shortened the telomere length in a glucose concentration-dependent manner. While pretreatment with CAG and AG-IV upregulated TERT expression and ameliorated the telomere attrition. CAG and AG-IV also increased cell proliferation and improved cell morphology in HG conditions. Overall, these findings indicated that CAG and AG-IV suppressed HG stress-induced senescence and apoptosis, in addition to enhancing telomerase activation and lengthening of the Telomere. Therefore, CAG and AG-IV prolonged the replicative capability and longevity of the NPCs and they have the potential to be therapeutic agents in Db-IVDD.

Nuclear compartmentalization of TERT mRNA and TUG1 lncRNA is driven by intron retention

The spatial partitioning of the transcriptome in the cell is an important form of gene-expression regulation. Here, we address how intron retention influences the spatio-temporal dynamics of transcripts from two clinically relevant genes: TERT (Telomerase Reverse Transcriptase) pre-mRNA and TUG1 (Taurine-Upregulated Gene 1) lncRNA. Single molecule RNA FISH reveals that nuclear TERT transcripts uniformly and robustly retain specific introns. Our data suggest that the splicing of TERT retained introns occurs during mitosis. In contrast, TUG1 has a bimodal distribution of fully spliced cytoplasmic and intron-retained nuclear transcripts. We further test the functionality of intron-retention events using RNA-targeting thiomorpholino antisense oligonucleotides to block intron excision. We show that intron retention is the driving force for the nuclear compartmentalization of these RNAs. For both RNAs, altering this splicing-driven subcellular distribution has significant effects on cell viability. Together, these findings show that stable retention of specific introns can orchestrate spatial compartmentalization of these RNAs within the cell. This process reveals that modulating RNA localization via targeted intron retention can be utilized for RNA-based therapies.

MESENCHYMAL STEM CELLS RIF1, TCL1A, AND TERT MARKERS AS INDICATORS OF AGE ESTIMATION IN RATS

Background: Research on stem cells has become one of the most promising and advanced scientific topics. Nevertheless, there is a lack of this type of research in the field of forensic diagnostics. Age estimation is one of the main worked-on issues in forensic practice and research. Still, there is a need to explore new complementary reliable methods. This study aimed to detect the possibility of age estimation via age-associated alteration of RIF-1 (Replication timing regulatory factor 1), TCL1A (T-cell leukemia/lymphoma-1A), and TERT (Telomerase reverse transcriptase) gene expression in rats bone marrow stem cells. Methods: Isolation of bone marrow mesenchymal stem cells from rats were undertaken at different ages; 2, 4, 6, 8, 10, and 12 weeks old. RNA extraction was performed then total RNA was reverse transcribed to cDNA to detect the quantitative expression of RIF-1, TLC1A, and TERT by using the qRT-PCR then immunoblotting. Results: There were statistically significant increases in RIF1 and TCL1A mRNA and protein expression and a decrease in TERT mRNA and protein expression with age. Conclusion: Our results showed that the mRNA and protein expression of RIF-1, TCL1A, and TERT in bone marrow cells of rats could be useful indicators for age estimation in forensic practice.

Nuclear-specific accumulation of telomerase reverse transcriptase (TERT) mRNA in TERT promoter mutated follicular thyroid tumours visualised by in situ hybridisation: a possible clinical screening tool?

AimsUpregulation of the telomerase reverse transcriptase (TERT) gene is a frequent finding in follicular thyroid carcinomas (FTCs) with metastatic features. The augmented expression is usually caused by TERT promoter mutations....

Berberine Inhibits Telomerase Activity and Induces Cell Cycle Arrest and Telomere Erosion in Colorectal Cancer Cell Line, HCT 116.

Colorectal cancer (CRC) is the most common cancer among males and females, which is associated with the increment of telomerase level and activity. Some plant-derived compounds are telomerase inhibitors that have the potential to decrease telomerase activity and/or level in various cancer cell lines. Unfortunately, a deeper understanding of the effects of telomerase inhibitor compound(s) on CRC cells is still lacking. Therefore, in this study, the aspects of telomerase inhibitors on a CRC cell line (HCT 116) were investigated. Screening on HCT 116 at 48 h showed that berberine (10.30 ± 0.89 µg/mL) is the most effective (lowest IC50 value) telomerase inhibitor compared to boldine (37.87 ± 3.12 µg/mL) and silymarin (>200 µg/mL). Further analyses exhibited that berberine treatment caused G0/G1 phase arrest at 48 h due to high cyclin D1 (CCND1) and low cyclin-dependent kinase 4 (CDK4) protein and mRNA levels, simultaneous downregulation of human telomerase reverse transcriptase (TERT) mRNA and human telomerase RNA component (TERC) levels, as well as a decrease in the TERT protein level and telomerase activity. The effect of berberine treatment on the cell cycle was time dependent as it resulted in a delayed cell cycle and doubling time by 2.18-fold. Telomerase activity and level was significantly decreased, and telomere erosion followed suit. In summary, our findings suggested that berberine could decrease telomerase activity and level of HCT 116, which in turn inhibits the proliferative ability of the cells.

GDF15 as a biomarker of ageing.

The ageing process is accompanied by the gradual development of chronic systemic inflammation (inflamm-ageing). Growth differentiation factor 15 (GDF15) is associated with inflammation and known to be a stress-induced factor. The present study aimed to explore the association of GDF15 with ageing. In this cross-sectional study, serum GDF15, hematological parameters, and biomedical parameters were determined in 120 healthy individuals (23-83years old, males). Three telomere related parameters, including telomere length, telomerase activity, and the expression of human telomerase reverse transcriptase (hTERT) mRNA were also quantified. Our results showed that the older group has a higher levels of GDF15 and lower expression of hTERT mRNA, and PBMC telomerase activity (p<0.001). In individuals with high GDF15 levels, they were older, and presented with the lower level of hTERT mRNA and T/S ratio (p<0.01). Spearman correlation analysis shows that GDF15 positively correlated with age (r=0.664, p<0.001), and negatively correlated with telomere length (r=-0.434, p<0.001), telomerase activity (r=-0.231, p=0.012), and hTERT mRNA (r=-0.206, p=0.024). Furthermore, in multivariate regression analysis, GDF15 levels showed a statistically significant linear and negative relationship with PBMC telomerase activity (β-coefficient=-0.583, 95% CI -1.044 to -0.122, p=0.014), telomere length (β-coefficient=-0.200, 95% CI -0.305 to -0.094, p<0.001), and hTERT mRNA (β-coefficient=-0.207, 95% CI -0.312 to -0.102, p<0.001) after adjusting for confounders. These results support that circulating GDF15 is the potential biomarker of ageing that may influence the risk and progression of multiple ageing conditions.

Hepatitis B Virus-Telomerase Reverse Transcriptase Promoter Integration Harnesses Host ELF4, Resulting in Telomerase Reverse Transcriptase Gene Transcription in Hepatocellular Carcinoma.

Background and AimsHepatitis B virus (HBV) integrations are common in hepatocellular carcinoma (HCC). In particular, alterations of the telomerase reverse transcriptase (TERT) gene by HBV integrations are frequent; however, the molecular mechanism and functional consequence underlying TERT HBV integration are unclear.Approach and ResultsWe adopted a targeted sequencing strategy to survey HBV integrations in human HBV‐associated HCCs (n = 95). HBV integration at the TERT promoter was frequent (35.8%, n = 34/95) in HCC tumors and was associated with increased TERT mRNA expression and more aggressive tumor behavior. To investigate the functional importance of various integrated HBV components, we employed different luciferase reporter constructs and found that HBV enhancer I (EnhI) was the key viral component leading to TERT activation on integration at the TERT promoter. In addition, the orientation of the HBV integration at the TERT promoter further modulated the degree of TERT transcription activation in HCC cell lines and patients’ HCCs. Furthermore, we performed array‐based small interfering RNA library functional screening to interrogate the potential major transcription factors that physically interacted with HBV and investigated the cis‐activation of host TERT gene transcription on viral integration. We identified a molecular mechanism of TERT activation through the E74 like ETS transcription factor 4 (ELF4), which normally could drive HBV gene transcription. ELF4 bound to the chimeric HBV EnhI at the TERT promoter, resulting in telomerase activation. Stable knockdown of ELF4 significantly reduced the TERT expression and sphere‐forming ability in HCC cells.ConclusionsOur results reveal a cis‐activating mechanism harnessing host ELF4 and HBV integrated at the TERT promoter and uncover how TERT HBV‐integrated HCCs may achieve TERT activation in hepatocarcinogenesis.