| BackgroundThe development of the lens determines the function of the eye.The lens protein changes and becomes cloudy,which will lead to the occurrence of cataract.The structure of the lens is composed of two parts,a layer of lens epithelium in the anterior capsule of the lens and semi-encapsulated fibers by the epithelium.During the development of the lens,the epithelial cells of the anterior capsule of the lens will denucleate in the equatorial region of the lens and gradually differentiate into lens fiber cells toward the center of the lens.Primary fiber cells are arranged in the center of the lens to form the core-free zone(OFZ),which ensures the transparency of the lens.Heat shock transcription factor(HSF)encodes the expression of heat shock protein and activates heat shock response under external environment or intracellular stress.HSF regulates the expression of heat shock proteins by identifying heat shock elements on heat shock protein promoters.Heat shock transcription factor 4(Hsf4)includes two splices,Hsf4 a and Hsf4 b.Hsf4b is specifically expressed in the lens,and Hsf4 mutation can lead to the occurrence of familial congenital cataract.Hsf4 is a key transcription factor in the regulation of lens development,the expression of structural proteins in the lens,and the differentiation of lens epithelial cells.Studies have confirmed that the removal of Hsf4 in mice and zebrafish leads to the occurrence of cataracts.However,the specific molecular mechanism by which Hsf4 regulates lens development remains unclear.Cytoskeleton is involved in the development of lens and is important for the differentiation of lens.There are three known cytoskeletons that are associated with fibroblast differentiation and morphogenesis,including microtubules(/α-tubulin),filaments(F-actin)and intermediate filament(IF).Microtubules are mainly composed of α-Tubulin and β-Tubulin,which are essential for elongation and vesicle trafficking of fibroblasts,and play an important role in n-cadherin regulation of cell-cell connectivity.Microfilaments are filaments formed by the helical polymerization of actin molecules,also known as actin filaments.They are necessary for the remodeling of fibrous cell membranes and mechanical stiffness,and are generally used as actin networks to regulate the development of the lens.E-cadherin is an essential adhesion system in epithelial cells and is involved in adhesion between epithelial cells and fibroblasts,and f-actin filaments are also involved.Intermediate fibers play an important role in maintaining the mechanical integrity of the lens and maintaining its transparency.Vimentin is a type of intermediate fiber,and studies have confirmed that Hsf4 regulates the expression of Vimentin by binding to the promoter DNA of Vimentin to regulate the development of lens.In the preliminary experiment,we found that Aim-1 is a new downstream target gene of Hsf4,and it has been found that Aim-1 co-locates with f-actin.Through analysis of the cellular function of Aim-1 and the study of the molecular function during the development of the lens,we explored the molecular mechanism of Hsf4 regulating lens development.PurposeIn this study,human lens epithelial cell line HLE B3,mouse lens epithelial cell line mlec,mouse knockout and overexpression of Hsf4 b lens epithelial cell line mlec-hsf4b-/-and mlec-HA-hsf4 b,wild-type C57 and Aim-1-/-mice were used as the research objects,and the molecular function and mechanism of Hsf4’s new downstream target gene Aim-1 in the development of lens were studied in depth.Methods1.Polyclonal antibodies no.1 and no.2 were customized to recognize mouse Aim-1 protein,and the full-length Aim-1 protein was expressed to verify the specificity of the antibody.The Aim-1 protein with flag tag was overexpressed in human lens epithelial cells(HLE B3);small interfering RNA that targeting Aim-1(si-Aim-1)and control small interfering RNA(si-normal control,si-NC)was transfected into mouse fibroblasts(MEF),Western blot was used to detect the expression of Aim-1,the interference effect of si-Aim-1 and the specificity of custom antibody No.1 and No.2 for Aim-1 protein recognition.2.Western blot was used to detect the expression of Aim-1 in knockout and overexpressed mouse lens epithelial cell lines mlec-hsf4-/-and mlec-HA-hsf4 b.At the same time,si-Aim1 and si-NC were transfected into mouse lens epithelial cell line mlec to detect the expression of alphaB-crystallin(cryab)by western blot.3.Transfected si-Aim-1 and si-NC in mouse lens epithelial cell lines mlec-Hsf4b-/-and mlec-HAHsf4 b and the mRNA levels of Aim-1,cryab,heat shock protein 25(Hsp25)and SRC kinase-associated phosphoprotein2(skap2)were detected by real-time fluorescence quantitative PCR.Overexpression PEBG-αB-FL and Aim-1 truncations in HLE B3 cell lines and the interaction of cryab with each truncation of Aim-1 was examined by GST-pull down assay.4.Immunofluorescence assay was performed to observe the localization mode of each fragment and full length of Aim-1 in cells after overexpression of GFP-Aim-1-n-2k,GFP-Aim-1-n-3k,GFP-Aim-1-c-2k,and full-length plasmid 3×flag-Aim-1-fl in HLE B3 cells.The Aim-1 N telomere GFP-N-2k-NLS with fusion GFP and nuclear localization sequence(NLS)was overexpressed in HLE B3 cells,and the localization of Aim-1 truncated bodies with nuclear localization sequence was observed by immunofluorescence assay.5.3×flag-Aim-1-FL plasmids were overexpressed in HLE B3 cells,and immunofluorescence experiments were performed to stain F-actin with phalloidin to observe the positioning of Aim-1 and F-actin.3×flag-Aim-1-FL plasmids were overexpressed in HLE cells,and immunofluorescence experiments were performed to stain the endogenous alpha-Tubulin(α-Tubulin)to observe the localization of Aim-1 and α-Tubulin.6.3×flag-cmv7.1 plasmids and 3×flag-Aim-1-FL plasmids were overexpressed in human renal epithelial cell line(293t),flag antibody for IP assay to detect the interaction between α-Tubulin and Aim-1.7.The wild type(WT)mice eye of 5 months were taken to fixed by 4% paraformaldehyde(PFA)and dehydrate,embed and slice them for immunohistochemical experiments,and observe the localization of Aim-1 in the lens.8.Screening and authenticating the mouse model of Aim-1 knockout constructed by crispr-cas9 technology,total RNA was extracted from the kidney tissue of mice and reversely transcribed into cDNA,and the Aim-1 cDNA sequence near the gRNA target amplified by PCR was sent for sequencing to verify the genotype of the knockout mice.9.Aim-1 knockout mice and WT mice eye of 6 months and 8 months were taken to fixed by 4%paraformaldehyde(PFA)and dehydrate,embed and slice them for HE staining.The lens phenotypes of Aim-1 knockout mice and WT mice were observed.Results1.The full-length protein of Aim-1 was overexpressed in lens epithelial cells,and siRNA was used to knock down Aim-1 in MEF cells to verify the specificity of the customized Aim-1 polyclonal antibody.The results showed that both Aim-1 polyclonal antibodies could recognize Aim-1 protein.2.Hsf4 up-regulates Aim-1,knocking down Aim-1 leads to a decrease in the expression of the downstream protein cryab of HSF4.And there is an interaction between Aim-1 and cryab.3.The whole length of Aim-1 was located in the cytoplasm of cells,and its peptide chain contained NLS sequence,and the Aim-1 truncated body with NLS sequence was located in the nucleus.4.Aim-1 co-localizes with α-Tubulin and F-actin in lens epithelial cells,and interactes with alphatubulin.5.Aim-1 was localized in the cytoplasm of lens epithelial cells of WT adult mice.6.The deletion of exon 2 of Aim-1 in Aim-1-/-mice resulted in the termination of Aim-1 from the leucine shift at position 214 to 16 unrelated amino acids,resulting in the change of Aim-1 from 1,691 amino acids to 230 truncated amino acids,and the loss of 12 / domains and rich lectin B domains.7.Knockout of Aim-1 in mice may not affect the early development and differentiation of the lens.Conclusions1.The whole length of Aim-1 was located in the cytoplasm of cells and co-located with f-actin.Aim-1 also interacts with the cytoskeleton protein alpha-tubulin,which may be a cytoskeleton protein.2.Aim-1 knockout mice were successfully constructed,but the knockout of Aim-1 in mice may not affect the early development and differentiation of the lens. |
PDF Full Text Request