Studying The Mechanism Of Senescence In Lens Cell Caused By Knocking Out Hsf4 By CRISPR/Cas9

BackgroundHeat shock response(HSR)is an adaptive protective response characterized by expressing a number of heat shock protein with external stimulus.Heat shock proteins help the denatured protein structure correctly folded or promote the degradation of denatured protein through its molecular chaperone activity to stabilize the protein homeostasis in cells.Heat shock factors(HSFs)are the main transcription factors to regulate the heat shock response.Heat shock transcription factors regulate heat shock proteins expression by binding the heat shock element(HSE)at the target gene promoter.There are 4 HSF family members in mammals,Hsf1,Hsf2,Hsf3 and Hsf4.Hsf4,with tissue-specific expression pattern,is the primary transcription factor for the heat shock response in the lens.The mutations of Hsf4 lead to congenital and age-related cataracts,while knockout of Hsf4 in mice leads to lens development abnormalities and cataracts.However,the molecular mechanism of lens differentiation and the occurrence of cataracts regulated by Hsf4 is not clear.CRISPR-Cas9 is a natural immune mechanism formed by bacteria and archaea during long-term evolution,and is mainly used for against viruses and exogenous DNA.The DNA fragments from infected virus or plasmids are integrated CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats,clustered,regularly spaced short palindromic repeat)trough the CRISPR / Cas9 system.The bacterium utilizes transcription to produce the corresponding CRISPR RNAs(crRNAs)to guide the degradation of the homologous sequence of the Cas9 protein,thereby protecting the bacteria.The CRISPR / Cas9 system was able to perform precise digestion for the target sequence and quickly applied to gene editing.On the basis of the principle,the gRNA(guide RNA)targeting Hsf4 was designed to direct the cleavage of the Cas9 protein at the desired site in the genome.To screen conveniently the Hsf4 fusion fragment was used as a template to complete the expected gene recombination in the genome gap to obtain cell lines knocking out Hsf4 with green fluorescence.Lens epithelial cells play an important role in maintaining the homeostasis and the function of lens.Studies result that age-related effects of lens epithelial cell life-long growth and the occurrence of cataract is relating to lens epithelial cell aging.In aging lens epithelial cells,upregulation of NDRG2 associated with cell proliferation and differentiation leads to human age-related cataracts.The inactivation of Hsf4 induces tumor cell senescence,while inhibiting tumorigenesis in vivo.Given the function of Hsf4 in lens differentiation,it is not clear whether Hsf4 participates in the regulation of lens development and function by regulating cell senescence.In this study,we knock out Hsf4 by CRISPR / Cas9 system in the mouse lens epithelial cell to explore the molecular mechanism of Hsf4 deficiency leading to cell senescence.ObjectivesTo study the molecular mechanism of cell senescence in the mouse lens epithelial cells which Hsf4 gene was knocked out by CRISPR / Cas9 system.Methods and results1.Firstly,we construct the mouse Hsf4 CRISPR / Cas9 gRNA plasmids and mouse Hsf4 fusion fragment plasmid.And we designed 2 gRNA loci downstream of the Hsf4 coding region ATG to increase the cutting efficiency.2.Second,The mouse Hsf4 CRISPR / Cas9 gRNA plasmid and the mouse Hsf4-GFP fusion fragment plasmid are co-transfected into mouse lens epithelial cells(mLEC).3.Clone genome sequence at the cleavage site by PCR.Detect the expression level of Hsf4 downstream genes by western blot.Observe green fluorescent cells.These prove the Hsf4 gene recombination at the designed site.4.Screen the green fluorescent monoclonal cells knocking out Hsf4 by finite dilution and monoclonal culture.Monoclonal cells with GFP signals were identified with finite dilution and monoclonal culture methods.Cells with GFP signals indicated that Hsf4 has been knocked out.5.The expression of GFP and Hsf4 downstream genes Hsp25 and αB-crystallin were detected by western blot.Our results suggested that the expression levels of GFP increased while Hsf4 downstream genes decreased.These data indicateed that Hsf4 gene has been knocking out in monoclonal cell.6.We aim at proving whether Hsf4 is involved in cell aging process through β galactosidase staining experiments,senescence associated heterochromatin foci(SAHF)detection and MTS cell proliferation assay.Staining enhances in β galactosidase staining experiments.SAHF increases with immunofluorescence.The rate of cell proliferation slows down with MTS cell proliferation assay.The results indicate that Hsf4 knockout leads to lens epithelial cell aging.7.The moleculars p21 and p16 are the main regulators of the cell aging process.So as to explore the molecular mechanism of Hsf4 regulation of cell senescence,we detecte the expression of two moleculars in Hsf4 knockout cells.We detecte the same expression of senescence related gene p16 by western blot,and the increased expression of p21 significantly,while detecte the mRNA level of p21 increased significantly by q-PCR.8.In order to confirm the function of Hsf4 in lens cell senescence in vivo,we gather the young and aged mouse lens tissue.We find that the heat shock response ability of senile mouse lens significantly decreases and the expression of Hsf4 decreases,while the expression of p21 significantly increases by realtime PCR.ConclusionsThe deletion of the Hsf4 gene leads to cell senescence and increase the expression of p21 in the lens epithelial cell line knocked out the Hsf4 gene by the CRISPR / Cas9 system.