Telomease-mediated Telomere Elongation In Human Embryonic Stem Cells (HESCs)&Pathological Changes In HER2Transgenic Mice

Abstract1:Telomerase-mediated telomere elongation in human embryonic stem cells (HESCs)Purposes:Human embryonic stem cells (hESCs) are derived from the inner cell mass of human embryos; they have self-renew and multidirectional differentiation capability. In the differentiation process, hESCs lose telomerase activity and telomere length gradually, implied the association of telomere maintenance and self-renewal. However, the details of telomere maintenance in hESCs are still unclear. Here, we investigated telomere maintenance in the bank of Chinese hESCs to investigate the feature and mechanism of telomere maintenance in hESCs.Materials:For this study,96hESC lines including the initial passages, the later passages and clinical unused blastocysts samples were used. These samples were obtained with the consent of the parents and of the Ethics Committee of Reproductive and Genetic Hospital of CITIC-Xiangya.Methods::1, Telomere length were compared by Quantitative telomere in situ hybridization (Q-FISH) and terminal restriction fragment assay (TRF).2, Telomerase repeat amplification protocol and real-time PCR analysis telomerase core unit (TERT) expression were performed to detect telomerase expression.3, Immunofluorescence combined DNA-FISH were used to detect APBs; telomere sister chromatin exchange were performed to investigate the recombination based telomere maintenance path way.4, Lentivirus mediated telomerase interfere RNA (shTERT) was performed to evaluate the telomerase function on telomere elongation.5, Telomeric chromatin methylation was detected by double-IF, H3K9me3and H4K20me3colocalizes to TRF1were counted.6, Pluripotency and multidirectional differentiation detections of HESCs were carry out by routine staining, in vitro embryonic body formation and in vivo teratoma formation.Results:1, Here, we found telomere length undergo elongation during hESCs early culture until around passage15th.2, We further demonstrated that the telomere elongation relies on telomerase which maintains highly expression in human blastocysts and in long-term cultured hESCs, and hTERT knockdown partially inhibits telomere elongation. In addition, recombination based alternative telomere lengthening (ATL) pathway is inhibited in hESCs.3, Telomerase core unit hTERT is regulated by Wnt-β-catenin signaling.4, Further more, we found decreased chromatin modification of trimethylation of H3K9and H4K20occurred at telomeric regions during early culture, when telomere elongation occurs.5, Telomere elongation may not associated with hESCs pluripotency.6, Telomere length remains relatively stable in hESCs at around12.02±1.01kb up to passage95th, the telomere length range between2.5th and97.5th percentile is9.8kb to14.4kb. Unexpected variations of telomere length were found in hESCs with genomic instability and hESCs derived teratoma.Conclusions:Our research demonstrated that the telomerase dependent, Wnt-β-catenin regulated, telomere length restoring is a natural process in hESCs. Telomeres are important for the acquisition and maintenance of self-renew capability during the in vitro derivation of hESC lines. Our data also suggest a proper and stable telomere length as a new potential biomarker for stable hESCs. Abstract2:Pathological changes in HER2transgenic micePurposes:ERBB2/HER2/NEU, a member of the epidermal growth factor receptor family, is overexpressed in more than25%of non-small cell lung cancer and is considered to be a significant and independent prognostic factor in lung cancer. Here we take the advantage on HER2overexpression in lung tissue in transgenic mouse model to demonstrate the mechanism of HER2in lung cancer development.Materials:In this model, HER2was driven by the human surfactant protein-C promoter to investigate its role in pulmonary carcinogenesis and progression. Positive generations of F1were used in this study, negative litter mates were set as control.Methods:Mice of different ages were sacrificed and their lung tissues were collected for phenotype analysis. Immunohistology were performed to detect the HER2and inflammation associated factors expression. Real-time PCR were used to detect gene expression in lung tissue.Results:Notably, significant pathological changes, including lymphocyte infiltration and mesenchymal cell hyperplasia, were found in the lung tissue of transgenic mice aged from4to12months. The occurrence and severity of those lesions increased as the mice aged. Some inflammatory factors, such as tumor necrosis factor (TNF-alpha), interleukin1(IL-1) and interleukin6(IL-6), were upregulated in lung tissue of transgenic mice, implying that long-term HER2overexpression could induce serious lung inflammation and some precancerous lesions.Conclusions:This model would be useful for studying the mechanism of HER2involvement in lung carcinogenesis and for understanding the relationship between carcinogenesis and inflammation.