ABSTRACT
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system is a hotspot of gene editing and gene expression research, in which CRISPR/Cas13 system provides a new direction for RNA interference and editing. In this study, we designed and synthesized the corresponding gRNAs of CRISPR/Cas13a and CRISPR/Cas13b systems in non-homologous end joining (NHEJ) pathway, such as Ku70 and Lig4, and then detected the expression of ku70 and lig4 in HEK293T cells. The CRISPR/Cas13a system could efficiently knockdown the mRNA expression of ku70 and lig4 more than 50%, and CRISPR/Cas13b system also suppressed ku70 and lig4 about 92% and 76%, respectively. Also, CRISPR/Cas13a, b systems could down-regulate Ku70 and Lig4 proteins level to 68% and 53%, respectively. The study demonstrates that the CRISPR/Cas13 system could effectively knockdown the expression of RNA and protein in HEK293T cells, providing a new strategy for gene function and regulation research.
Subject(s)
Humans , CRISPR-Cas Systems , DNA Ligase ATP , Genetics , Gene Expression Regulation , Genetics , Gene Knockdown Techniques , HEK293 Cells , Ku Autoantigen , GeneticsABSTRACT
Aberrations of the mammalian target of rapamycin (mTOR) signaling are frequently observed in many types of cancer.Activation of mTOR regulates tumor cell proliferation,survival and metastasis,and then results tumorigenesis and tumor developmet.Blocking the mTOR signaling pathway by rapamycin and its derivatives can inhibit gastric cancer cell growth and promote tumor necrosis and such effects can be synergistically improved by combined use of other chemotherapeutic agents.Thus,rapamyein and its derivatives may be effective in the prevention and treatment of gastric cancer.
ABSTRACT
Objective To observe the effects of hyperoxia on Notch 1 receptor of alveolar epithelial type Ⅱ cells (AEC Ⅱ), in a hetcrocellular culture of alveolar epithelial type Ⅱ cells and lung flbroblasts(LF), in order to explore Notch signaling in hyperoxic induced lung injury and thus make theoretical basis for prevention and treatment of a acute/chronie neonatal lung injury. Method Twelve Spragne Dawkey female rats with 200～220 g and 3 Spragne Dawkey male rats with 220～250 g were offered from experimental animal centre of Tongji Medical Colleege, Huazhong University of Science and Technology. The AEC Ⅱ/ LF co-culture system was established successfully. AEC H s from premature rats were randomly assigned to 2 groups: air control group and hyperoxia group. Air control group was kept in room air 50% ml/L CO2 enviromnent at 37°C, while hyperoxia group was exposed to 950 ml/L O2 + 250 ml/L CO2. Immuno-histochemistry was taken to detect Notch 1. Fluorescent quantitafive PCR was used to quantify the Notch 1 mRNA. MTT method was taken to assess cen proliferation viability.Flow eytometry double label method was used to detect cell percentages. Results In hyperoxia group:Notch 1 activation was inhibited, and Notch 1 mRNA decreased to 0.43,0.29,0.11,0.03 fold of control (95% confidence limit). AEC Ⅱ percentage descended predominantly[ 24 h hyperoxia group vs. control group: (68.92±6.88)%vs. (90.35±4.01)%, P =0.006;48 h hyperoxia group vs. control group: (38.03±3.27) vs. (61.47±4.81)%, P =0.000;72 h hyperoxia group vs. control group:(20.13±4.45)% vs. (52.05±3.35)%, P =0.000;96 h hyperoxia group vs. control group:(8.17±1.99)% vs. (52.59±2.93)%, P =0.0001 while that d AECI rised[24 h hyperoxia group vs. contrd group:(0.11±0.03)% vs. (0.01±0.01)%, P=0.006;48h hyperoxia group vs. control gnmp:(49.73±3.45)% vs. (16.13±2.13)%, P =0.000;72 h hyperoxia group vs. control group: (52.43±3.14) % vs. (5.98±0.95) %, P = 0.000;96h hyperorxia group vs. control group:(19.85±3.26)% vs. (29.03±3.16)%, P =0.007]. Comclusions Hyperoxia may inhibit Notch signaling pathway, which can weaken proliferation and disdifferentiation of AEC Ⅱ s. Investigations on how to control Notch signaling will provide fresh thoughts for alveolar epithelium repairing.