The Study Of HSF4 On Protein Expression Of HLECs And Relation With Senile Cataract

Heat shock transcription factor 4(HSF4) was reported to be related with congenital cataract for the first time in 2002.HSF4 has two isoforms,HSF4a and HSF4b.In eye lens,HSF4b but not HSF4a is existed.Congenital cataract is the frequent cause of children blindness.The lens is the highest protein content of any tissue of the body.Proteomics,especially differential proteomics is applied widely in disease studies,iTRAQ is one of the methods appeared recently in differential proteomics.Which proteins are changed by the mutation of HSF4 in a Chinese family which was reported for the first time?We try to find the effect of the mutation on Human lens cells(HLECs) using iTRAQ.Senile cataract is the most frequent cataract.Genetic factors account partly for senile cataract.It is quite reasonable to evaluate the effect of a gene to cataract on the whole.Besides congenital cataract,HSF4 may play a role in senile cataract.So we collected sporadic senile cataract patients who were free from other diseases and natural population to study.PartⅠ:The proteome study of HLECs transfected with mutated HSF4bPurpose To study the proteome changes of HLECs,SRA01/04,which were transfected with mutated HSF4b(T to C transition at nucleotide 348).Methods Human lens cells(HLECs),SRA01/04,were cultured in vitro. The plasmid,pcDNA3.1-HSF4b was mutated at certain site.Plasmid was amplified on a large scale.HLECs were transfected with plasmids using lipofection(pcDNA3.1-HSF4b,mutant pcDNA3.1-HSF4b,empty plasmid vector pcDNA3.1).The whole cell proteins of four groups(three groups were transfected with different plasmid and one group without transfection) were extracted and were labeled with iTRAQ after digestion.The proteome changes were studied using liquid chromatography and mass spectrometry.Results One hundred and four proteins were identified on the whole.Comparing with other three groups,the cells transfected with mutant pcDNA3.1-HSF4b had 20 proteins up-regulated and 14 proteins down-regulated.Twelve proteins in the group transfected with mutant pcDNA3.1-HSF4b and 14 proteins in the group transfected with pcDNA3.1-HSF4b were up-or down-regulated compared with transfected with empty vector pcDNA3.1,among which Vinculin was up-regulated and Protein S100-A13 was down-regulated.Conclusions Some proteins were up-or down-regulated after HLECs were transfected with mutant pcDNA3.1-HSF4b.These changed proteins were related with heat shock response,cell differentiation,protein biosynthesis and DNA binding.PartⅡ:The study of HSF4 and age related cataractPurpose To explore the role of HSF4 in the development of senile cataract.Methods We screened sequence of all exons of the HSF4 gene in 150 senile cataract patients and natural population of 100 from Shanghai.Results In individuals of natural population,we detected no single-nucleotide polymorphism with frequency higher than 5%in a complete coding region and their exon-intron boundaries.In senile cataract patients,we found 7 sequence variances,among which 5 were present only in 150 senile cataract patients(c.1020-25G＞A,c.1078A＞G,c.1223C＞T,c.1286C＞T,c.1256+25C＞T) and 2 were present both in 150 patients and 100 control subjects(c.1019+9C＞T,c.1243G＞A).Conclusions We identified 5 new HSF4 gene mutations in 150 senile cataract patients,which indicated that HSF4 mutation account partly for senile cataracts.