Dissertation > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)

Preliminary Study of Human HSPC117Gene on Cell Migration and Its Apparent Regulatory

Author ZhangXu
Tutor LiuZuo
School Northeast Agricultural University
Course Animal Genetic Breeding and Reproduction
Keywords Human HSPC117gene Cell migration Subcellular localization TSA 5-aza-CdR
Type Master's thesis
Year 2013
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HSPC117(Hematopoietic stem/proenitor cells117) is a cell adhesion associated protein that wasfound recently. It exsits in many protein complexes, and expresses abroadly in cells, tissues and organs ofmammals and rodent. HSPC117is able to inhibit cell adhesion and spreadability, it can also regulatedevelopments of certain tissues and organs, and also parent-child intergenerational phnotypic changes.Indeed it is closely related to the normal occur and development of cloned embryos. Above all we carried outa research on HSPC117gene, on the one hand it would provide more necessary informations about thestructure and functions of the gene itself, on the other hand it might make a foundation for other scientificstudies that was associated with it.Human HSPC117gene was picked as the research object in the study, and the pEGFP-C1-HSPC117eukaryotic recombinant vector was builded through cloning and sequencing, carrier restructing and othermethods. Then the recombinant vector was transfected into human choriocarcinoma cell lines JEG-3withliposome-mediated technology, and the expression position of HSPC117gene was observed on cell level.Real-time PCR method was used to detect the relative expression levels of MMP-2gene, MMP-14gene andTIMP-2gene in JEG-3cells as while as the HSPC117gene was over-expressed. Meanwhile the cell scratchwas utilized to build the cultured cell wound model, in order to assess relative JEG-3cells migration rate inthe condition of HSPC117was over-expressed. Then trichostain A (TSA) which was a kind of histonedeacetylase inhibitors and5-aza-2’-deoxycytidine (5-aza-CdR) which was one kind of DNAmethyltransferases inhibitors were used to treat JEG-3cells in different ways to detect the expression levelchanges of HSPC117gene. The results were as follows:(1) The completely coding sequence of human HSPC117gene was successfully cloned, it contained1518bp, encoded505amino acids.(2) pEGFP-C1-HSPC117eukaryotic recombinant vector was successfully constructed, and it wastransiently transfected into JEG-3cells. The HSPC117protein was mainly expressed in the cytoplasm, andthere was no obvious membrane localization phenomenon through the observing in fluorescence invertedmicroscope.(3) Real-time PCR method was applied to detect the relative expression level changes of MMP-2gene,MMP-14gene, TIMP-2gene in JEG-3cells after pEGFP-C1-HSPC117eukaryotic recombinant vector was transiently transfected for48hours. The results showed that as HSPC117gene was over-expressed in JEG-3cells, the relative expression levels of MMP-2gene and MMP-14gene was reduced notablely (P<0.05),while TIMP-2gene was increased notablely (P<0.05).(4) JEG-3cells that transiently transfected with pEGFP-C1-HSPC117eukaryotic recombinant vector,pEGFP-C1vector and untreated group were replated to24well plates for scratches experiments at the sametime, the relative migration rate of the scratched cells were observed after0,24,48,72hours. The resultsshowed that relative cell migration rate was inhibited in JEG-3cells that over-expressed of HSPC117genecompared with the control group, and the inhibition was the most significant at24hours(P<0.05).(5) Methyl thiazolyl tetrazolium bromide (MTT) method was used to assay the JEG-3cells’ growthwith different concentrations of TSA and5-aza-CdR, the results showed that both of the two drugs couldinhibite cells’ growth, and the inhibition effects were in a concentration-dependent manner.75nmol/L TSAand50nmol/L5-aza-CdR were the strongest concentration for inhibiting the growth of JEG-3cellsrespectively.(6) JEG-3cells was treated with75nmol/L TSA and50nmol/L5-aza-CdR in respectively and combinedtreatment ways, all the mRNA relative expressions of HSPC117gene had elevated except the DMSO group,the two drugs combined treatment group was the most significantly group(P<0.05).

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