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Visually Intracellular Detection of Telomerase Activity Based on DNA Strand Displacement Reaction and Gold Nanoparticle Labeling

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Chinese Journal of Chemistry 2022年第6期40卷 693-698页

作者： Yajun Zhang Shen Yan Zhaoxin Chen Xin Jiang Shuang Rao Zhuoran Jiang Shanshan Qin Xiang Zhou Yuhao Du College of Chemistry and Molecular Sciences Key Laboratory of Biomedical Polymers of Ministry of EducationWuhan UniversityWuhanHubei 430072China

Telomerase,which is regarded as a common biomarker for early cancer diagnostics and a potential target for clinical therapies,has attracted considerable interests concerning its detection and monitoring.Herein,we propose a sensitive method by designing a gold nanoparticle(AuNP)probe for visually intracellular detection of Telomerase activity.The AuNPs were functionalized with a telomer-ase substrate primer(SH-prime).A 6-carboxy-fluorescein(FAM)modified strand(FAM-probe)was attached to the surface of AuNP through its complementary stand(SH-attach).In the absence of Telomerase,the fluorescence resonance energy transfer(FRET)from FAM to AuNPs results in efficient fluorescence quenching.In the presence of Telomerase,SH-primers on AuNPs were extended with the repeat units(TTAGGGG)n.The extension sequence triggered the strand displacement of FAM-probe to restore the fluorescence signals.It is worth mentioning that the proposed strategy does not need to design complex hairpin structure and allows the meas-urement of Telomerase in crude cell extracts down to 0.5 HeLa cells/μL in 2 h.In addition,the present sensing platform can be ap-plied to the visually intracellular detection of Telomerase activity in living cells.

关键词： Telomerase Nanoparticles DNA strand displacement Intracellular FRET

Telomerase reactivation is associated with hepatobiliary and pancreatic cancers

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Hepatobiliary & Pancreatic Diseases International 2020年第5期19卷 420-428页

作者： Vito Sansone Marco Le Grazie Jenny Roselli Simone Polvani Andrea Galli Francesco Tovoli Mirko Tarocchi Department of Medical and Surgical Sciences University of BolognaBolognaItaly Department of Experimental and Clinical Biomedical Sciences"Mario Serio" University of Florence50139 FirenzeItaly

Background:Human Telomerase reverse transcriptase(hTERT)and its components play a significant role in cancer progression,but recent data demonstrated that telomeres and Telomerase alterations could be found in other diseases;increasing evidence suggests a key role of this enzyme in the fields of hepatobiliary and pancreatic diseases.Data sources:We performed a PubMed search with the following keywords:Telomerase,hepatocellular carcinoma,cholangiocarcinoma,pancreatic adenocarcinoma by December 2019.We reviewed the relevant publications that analyzed the correlation between Telomerase activity and hepatobiliary and pancreatic diseases.Results:Telomerase reactivation plays a significant role in the development and progression of hepatobiliary and pancreatic tumors and could be used as a diagnostic biomarker for hepatobiliary and pancreatic cancers,as a predictor for prognosis and a promising therapeutic target.Conclusions:Our review summarized the evidence about the critical role of hTERT in cancerous and precancerous lesions of the alteration and its activity in hepatobiliary and pancreatic diseases.

关键词： Telomerase Telomere Cancer Hepatobiliary and pancreatic disease

Mitochondrion-processed TERC regulates senescence without affecting Telomerase activities

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Protein & Cell 2019年第9期10卷 631-648页

作者： Qian Zheng Peipei Liu Ge Gao Jiapei Yuan Pengfeng Wang Jinliang Huang Leiming Xie Xinping Lu Fan Di Tanjun Tong Jun Chen Zhi Lu Jisong Guan Geng Wang MOE Key laboratory of Bioinformatics Cell Biology and Development CenterSchool of Life SciencesTsinghua University Beijing 100084China Peking University Research Center on Aging Beijing 100191China Department of Biochemistry and Molecular Biology Peking University Health Science CenterBeijing 100191China

Mitochondrial dysfunctions play major roles in ageing.How mitochondrial stresses invoke downstream responses and how specificity of the signaling is achieved, however, remains unclear. We have previously discovered that the RNA component of Telomerase TERC is imported into mitochondria, processed to a shorter form TERC-53, and then exported back to the cytosol. Cytosolic TERC-53 levels respond to mitochondrial functions, but have no direct effect on these functions, suggesting that cytosolic TERC-53 functions downstream of mitochondria as a signal of mitochondrial functions. Here, we show that cytosolic TERC-53 plays a regulatory role on cellular senescence and is involved in cognition decline in 10 months old mice, independent of its Telomerase function. Manipulation of cytosolic TERC-53 levels affects cellular senescence and cognition decline in 10 months old mouse hippocampi without affecting Telomerase activity, and most importantly, affects cellular senescence in terc^-/- cells. These findings uncover a senescence-related regulatory pathway with a non-coding RNA as the signal in mammals.

关键词： mitochondria retrograde signal nucleus transcription regulation non-coding RNA Telomerase

Cytotoxic effect of Rosa canina extract on human colon cancer cells through repression of Telomerase expression

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Journal of Pharmaceutical Analysis 2018年第6期8卷 394-399页

作者： Ibrahim Turan Selim Demir Kagan Kilinc Serap Ozer Yaman Sema Misir Hanife Kara Berna Genc Ahmet Mentese Yuksel Aliyazicioglu Orhan Deger Department of Genetic and Bioengineering Faculty of Engineering and Natural Sciences Gumushane University Medicinal Plants Traditional Medicine Practice and Research Center Gumushane University Department of Nutrition and Dietetics Faculty of Health Sciences Karadeniz Technical University Department of Medical Biochemistry Faculty of Medicine Karadeniz Technical University Department of Biochemistry Faculty of Pharmacy Cumhuriyet University Program of Medical Laboratory Techniques Vocational School of Health Sciences Karadeniz Technical University

Rosa canina is a member of the genus Rosa that has long been used for medical objectives. Several studies have reported cytotoxic effects of different Rosa species, but there has been only limited investigation of the cytotoxic effect of R. canina. The purpose of the current study was to examine the potential effect of R.canina extract on cell viability, the cell cycle, apoptosis, and the expression of Telomerase in human colon cancer(WiDr) cells. The cytotoxic effect of the extract was determined using MTT assay. The mechanism involved in the cytotoxic effect of the extract was then evaluated in terms of apoptosis and the cell cycle using flow cytometry. Mitochondrial membrane potential(MMP) was investigated using the fluorometric method, and expression levels of Telomerase were studied using RT-PCR. R. canina extract exhibited a selective cytotoxic effect on WiDr cells compared with normal colon cells. The extract induced cell cycle arrest at the S phase and apoptosis via reduced MMP in WiDr cells. R. canina extract significantly repressed Telomerase expressions at treatment times of 48 and 72 h in WiDr cells. Our results suggest that R. canina may have considerable potential for development as a novel natural product-based anticancer agent.

关键词： Apoptosis Colon cancer Cytotoxicity Rosa canina Telomerase

Telomerase and mTOR in the brain:the mitochondria connection

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Neural Regeneration Research 2017年第3期12卷 358-361页

作者： Satomi Miwa Gabriele Saretzki Ageing Biology Centre and Institute for Cell and Molecular Biosciences Campus for Ageing and VitalityNewcastle UniversityNewcastle upon TyneUK

Telomerase is an enzyme that maintains telomeres in dividing cells using a template on its inherent RNA component.Additionally,the protein part TERT（Telomerase Reverse Transcriptase） has various non-canonical functions.For example,it can localize to mitochondria under increased stress and protect cells in vitro from oxidative stress,DNA damage and apoptosis.Recently it has been demonstrated that TERT protein persists in adult neurons in the brain and data emerge suggesting that it might have a protective function in these post-mitotic cells as well.We have recently published that TERT protein accumulated in mitochondria from brain tissue of mice that have undergone short-term dietary restriction（DR） and rapamycin treatment.This localization correlated to lower levels of oxidative stress in these brain mitochondria.Since rapamycin treatment decreases mTOR signaling which is also thought to play an important role for the beneficial effects of DR,we conclude that the mTOR pathway might be involved in the TERT localization and its effects in brain mitochondria in vivo.These data are in line with previous findings from our group about increased mitochondrial localization of TERT in Alzheimer＇s disease（AD） brains and a protective function of TERT protein in neurons in vitro against pathological tau.

关键词： Telomerase brain neuron mitochondria roTOR ROS dietary restriction rapamycin

Transplantation of human Telomerase reverse transcriptase gene-transfected Schwann cells for repairing spinal cord injury

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Neural Regeneration Research 2015年第12期10卷 2040-2047页

作者： Shu-quan Zhang Min-fei Wu Jia-bei Liu Ye Li Qing-san Zhu Rui Gu Department of Orthopedics Tianjin Nankai Hospital Department of Spine Surgery Orthopedic HospitalSecond HospitalClinical HospitalJilin University Department of Orthopedics China-Japan Union HospitalJilin University

Transfection of the human Telomerase reverse transcriptase（h TERT）gene has been shown to increase cell proliferation and enhance tissue repair.In the present study,h TERT was transfected into rat Schwann cells.A rat model of acute spinal cord injury was established by the modified free-falling method.Retrovirus PLXSN was injected at the site of spinal cord injury as a vector to mediate h TERT gene-transfected Schwann cells（1×10^（10）/L;10μL）or Schwann cells（1×10^（10）/L;10μL）without h TERT gene transfection.Between 1 and 4 weeks after model establishment,motor function of the lower limb improved in the h TERT-transfected group compared with the group with non-transfected Schwann cells.Terminal deoxynucleotidyl transferase-mediated d UTP nick-end labeling and reverse transcription-polymerase chain reaction results revealed that the number of apoptotic cells,and gene expression of aquaporin 4/9 and matrix metalloproteinase 9/2decreased at the site of injury in both groups;however,the effect improved in the h TERT-transfected group compared with the Schwann cells without h TERT transfection group.Hematoxylin and eosin staining,PKH26 fluorescent labeling,and electrophysiological testing demonstrated that compared with the non-transfected group,spinal cord cavity and motor and sensory evoked potential latencies were reduced,while the number of PKH26-positive cells and the motor and sensory evoked potential amplitude increased at the site of injury in the h TERT-transfected group.These findings suggest that transplantation of h TERT gene-transfected Schwann cells repairs the structure and function of the injured spinal cord.

关键词： nerve regeneration spinal cord injury Schwann cells transplantation motor function Telomerase reverse transcriptase proliferation modification cells neural regeneration

A homeopathic nosode, Hepatitis C 30 demonstrates anticancer effect against liver cancer cells in vitro by modulating Telomerase and topoisomerase Ⅱ activities as also by promoting apoptosis via intrinsic mitochondrial pathway

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Journal of Integrative Medicine 2016年第3期14卷 209-218页

作者： Jesmin Mondal Jayeeta Das Rajesh Shah Anisur Rahman Khuda-Bukhsh Cytogenetics and Molecular Biology Laboratory Department of Zoology University of Kalyani Life Force 411 Krushal Commercial Complex

OBJECTIVE： Homeopathic nosodes have seldom been scientifically validated for their anticancer effects. This study was conducted to examine if a recently developed hepatitis C nosode has demonstrable anticancer potential in cancer cells in vitro.METHODS： Anticancer effects of Hepatitis C 30C（Hep C 30）, if any, were initially tested on three cancer cell lines, HepG2（liver cancer）, MCF-7（breast cancer） and A549（lung cancer） and one normal liver cell line WRL-68 cells and subsequently a more thorough study using further scientific protocols was undertaken on HepG2 cells（against WRL-68 cells as the normal control） as HepG2 cells showed better anticancer response than the other two. Three doses, one at 50% lethal dose（LD50） and the other two below LD50, were used on HepG2 cells subsequently. Protocols like apoptosis induction and its possible signaling mechanism were deployed using immunoblots of relevant signal proteins and confocal microscopy, with particular reference to Telomerase and topoisomerase Ⅱ（Top Ⅱ） activities, two strong cancer biomarkers for their direct relationship with divisional activities of cells and DNAs. RESULTS： Hep C 30 induced apoptosis, caused distorted cell morphology typical of apoptotic cells, increased reactive oxygen species generation and produced increased DNA nicks. Further it enhanced pro-apototic signal proteins like Bax, cytochrome c and inhibited anti-apoptotic signal proteins, Bcl-2, cytochrome c and caspase-3, changed mitochondrial membrane potential and caused externalization of phosphatidylserine. The drug also decreased expression of two cancer biomarkers, Top Ⅱ and Telomerase, consistent with its anticancer effect. CONCLUSION： Hep C 30 has demonstrable anticancer effects against liver cancer cells in vitro.

关键词： formularies,homeopathic antineoplastic agents Telomerase DNA topoisomerases,type Ⅱ membrane potential,mitochondrial

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World Journal of Gastroenterology 2024年第9期30卷 1224-1236页

作者： Hai-Yang Li Lin-Lin Zheng Nan Hu Zhi-Hao Wang Chang-Cheng Tao Ya-Ru Wang Yue Liu Zulihumaer Aizimuaji Hong-Wei Wang Rui-Qi Zheng Ting Xiao Wei-Qi Rong Department of Hepatobiliary Surgery National Cancer Center/National Clinical Research Center for Cancer/Cancer HospitalChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing 100021China Department of Hepatobiliary Hernia Surgery Liaocheng Dongcangfu People’s HospitalLiaocheng 252000Shandong ProvinceChina State Key Laboratory of Molecular Oncology Department of Etiology and CarcinogenesisNational Cancer Center/National Clinical Research Center for Cancer/Cancer HospitalChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing 100021China

BACKGROUND As a critical early event in hepatocellular carcinogenesis,Telomerase activation might be a promising and critical biomarker for hepatocellular carcinoma(HCC)patients,and its function in the genesis and treatment of HCC has gained much attention over the past two decades.AIM To perform a bibliometric analysis to systematically assess the current state of research on HCC-related Telomerase.METHODS The Web of Science Core Collection and PubMed were systematically searched to retrieve publications pertaining to HCC/Telomerase limited to“articles”and“reviews”published in English.A total of 873 relevant publications related to HCC and Telomerase were identified.We employed the Bibliometrix package in R to extract and analyze the fundamental information of the publications,such as the trends in the publications,citation counts,most prolific or influential writers,and most popular journals;to screen for keywords occurring at high frequency;and to draw collaboration and cluster analysis charts on the basis of coauthorship and co-occurrences.VOSviewer was utilized to compile and visualize the bibliometric data.RESULTS A surge of 51 publications on HCC/Telomerase research occurred in 2016,the most productive year from 1996 to 2023,accompanied by the peak citation count recorded in 2016.Up to December 2023,35226 citations were made to all publications,an average of 46.6 citations to each paper.The United States received the most citations(n=13531),followed by China(n=7427)and Japan(n=5754).In terms of national cooperation,China presented the highest centrality,its strongest bonds being to the United States and Japan.Among the 20 academic institutions with the most publications,ten came from China and the rest of Asia,though the University of Paris Cité,Public Assistance-Hospitals of Paris,and the National Institute of Health and Medical Research(INSERM)were the most prolific.As for individual contributions,Hisatomi H,Kaneko S,and Ide T were the three most prolific authors.Kaneko S ranked first by H-index,G-index,

关键词： Telomerase Bibliometric analysis Telomerase reverse transcriptase Prognosis Treatment Hepatocellular carcinoma

Simultaneous and Spatial Quantification of Telomerase Activity and DNA Methylation in Living Cells by a Deformable Satellite Nanocapsule

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CCS Chemistry 2021年第4期3卷 1231-1244页

作者： Shan Huang Cheng Ma Jiahao Lin Wenjing Wang Yizhu Xu Xingxin Wu Jian-Rong Zhang Jun-Jie Zhu School of Chemistry and Chemical Engineering State Key Laboratory of Analytical Chemistry for Life ScienceNanjing UniversityNanjingJiangsu 210023 College of Science State Key Laboratory of Agricultural MicrobiologyHuazhong Agricultural UniversityWuhanHubei 430070 School of Life Sciences State Key Laboratory of Pharmaceutical BiotechnologyNanjing UniversityNanjingJiangsu 210023 School of Chemistry and Life Science Jinling CollegeNanjing UniversityNanjingJiangsu 210089

Telomerase plays an essential role in many biological processes.DNA methylation regulates the expression of many genes,including Telomerase.Here,we propose a deformable satellite nanocapsule fluorescein isothiocyanate(FITC)-hollowbowl mesoporous organicsilica@gold nanoparticles-methyl-CpG-binding protein 2(MECP 2)-silver nanoclusters(FHBMO@AMA),for simultaneous quantitative detection of both cytoplasmic Telomerase activity and the degree of DNA methylation.This strategy enabled spatial-based detection in cells.The total cytoplasmic Telomerase activity was detected by fluorescence energy resonance transfer(FRET)between FHBMO and gold nanoparticles(Au NPs),while the DNA methylation in the nucleus was detected by enhanced fluorescence of silver nanoclusters(Ag NCs).Furthermore,FHBMO@AMA could intuitively distinguish between the differences in Telomerase expression in cells during the DNA synthesis period at the mitotic phase(S/M)of the cell cycle.Interestingly,the ratio of the two detections(Telomerase activity/DNA methylation)significantly correlated with the efficacy of anticancer drugs.At the same time,there was no apparent linear relationship between any single detection target and the efficacy of the anticancer drugs.Therefore,based on the relationship between Telomerase activity and DNA methylation,our newly developed approach serves as new and feasible method for evaluating the efficacy of anticancer drugs,thereby,extending the technology toolbox for precision in medical and pharmaceutical analysis of drug potency.

关键词： Telomerase DNA methylation simultaneous detection single-cell level the efficacy of anticancer drugs