The Function Of A New Secretory Protein Plac9

Secretory proteins are a kind of important protein in human proteome which account for one tenth of total proteins.Some important enzymes,hormones and cytokines in the human body are secreted proteins.It also participates in cell proliferation,differentiation,apoptosis,signal transduction between cells,individual development,blood coagulation,immune defense and other processes.During the study of embryonic development,we found a new secretory protein Plac9.The results of previous embryonic development research suggested that Plac9 may involve in organ development during embryogenesis.However,the research of Plac9 is still limited.Before performing the functional experiments of Plac9,we firstly explore the biological characteristics of Plac9.First,by using the method of bioinformatics,we found that Plac9 was located in human chromosome 10 long arm 22.3 area.The mounts of nucleotides were 691 bp.The cDNA of Plac9 contains four exons,and could encode an around 10 KD protein.Then Plac9's secondary structure and tertiary structure were predicted by the bioinformatics analysis,and the tertiary structure was similar to a small spring.In addition,the hydrophobicity and signal peptide of Plac9 were analyzed,and the results predicted that Plac9 scould be secreted into extracellular space.It was well known that there are two secreted pathways,classical pathway and non-classical pathway.A series of experiments of subcellular co-location about Plac9 and endoplasmic reticulum and golgi were performed to prove that Plac9 protein could secret into extracellular space by classical secretory pathway.After analyzing the structure and secretion pathway of Plac9 protein,we further analyzed its function.First,the expression of Plac9 gene was examined in various bone cell lines,lung cell lines and liver cell lines.The expressions of Plac9 in different cell lines were various.Recent research indicated that Plac9 might paticipate in liver development.L02 cell line was chosen to do the following studies.Firstly the L02 cell lines that overexpressed Plac9 gene were constructed.Subsequently,we analyzed whether Plac9 protein could effect the proliferation of L02 cells.A series of experiments such as MTT,and colony formation verified that Plac9 could inhibit cell proliferation.Further,we wanted to know whether Plac9 protein affects cell cycle.By flow cytometry we found that Plac9 protein could arrest cells in G2/M phase.The expression of related cyclic protein gene in L02 cell lines of overexpresed Plac9 was detected.From the results it could be seen that the expressions of genes related to G2/M phase were regulated.Furthermore,we wonder whether Plac9 perform proliferation-inhibited function by secreting outside the cell.Through MTT experiments,we found that Plac9 could inhibit proliferation of cells via secreting extracellularly.In the first part,we overexpressed Plac9 in L02 cells.Then,in the second part,we tend to edit Plac9 genes to find effects of decreased Plac9 on cell proliferation.CRISPR/Cas9 is an adaptive immune defense system in bacteria and archaea which was formed during the long-term evolution,mainly against the virus,and the invasion of exogenous DNA.CRISPR/Cas9 is an effective gene editing technique,which was widely used in recent years.In the CRISPR/Cas9 system,the combination of its compounds(crRNA(CRISPR-derived RNA)and tracrRNA(trans-activating RNA))could guide nuclease Cas9 to shear double-stranded DNA.As a result,cells start the homologous recombination repair function to repair double chain rupture.The process of repairin could cause gene mutation,insertions or deletions,which could be applied in gene editing.We wondered if we could use this technique to build the knockout cell lines of the Plac9 gene to perform further experiments on this gene.Using CRISPR/Cas9 technology we designed 4 sgRNAs linked with PX458 carrier,and transfected into 293 T cells.Then the cells was separated into individual cells by using flow cytometer.The transfected cells were cultured for 15 days then identified.The sequencing analysis and a series of detecting results verified Plac9 gene was knocked.It turns out that we successfully constructed the Plac9 gene knockout cell line for further study.Taken together,our research results show that Plac9 is a classcical secretory protein,which could inhibit cell proliferation and induce G2/M phase arrest.Furthermore,we successfully constructed Plac9 knockout 293 T cell lines by CRISPR/Cas9 technology for further study.