Construction Of Eukaryotic Cell Expression Vector Containing Human Wild Type PTEN Gene And Green Fluorescence Protein Gene And Its Inhibition On Human Carcinoma Of Breast

Background:Breast cancer is one of the most lethal cancers among women. At present, therapies for breast cancer includes surgical therapy, chemotherapy, radiotherapy and endocrine therapy.Following with the application and study of medical molecular biology, Gene therapy is becoming an important component of biotherapy for tumors and possessesing a good applied perspective of breast cancer therapeutics. After p53 and Rb gene,PTEN gene is hitherto the first tumor suppressor gene encoding a dual specificity protein–lipid phosphatase that plays an important role in cell apoptosis, blockage of cell cycle ,cell migration and tumorigenesis. In recent years,the studies on PTEN gene mostly have been focusing on its various kinds of functional mechanisms in tumorigenesis and tumor growth ,which seldom involved in its effects on tumor cells,solid tumors and related pharmacal research and development. Therefore,we constructed eukaryotic cell expression vector containing human wild type PTEN gene and green fluorescence protein gene,observed its inhibition on human carcinoma of breast in vitro and in vivo,made a primary approach to mechanisms in its inhibition on tumors,which may provide theoretical and experimental basis of feasibility of PTEN gene treatment for breast cancer, lay a foundation for gene therapy in clinical application.Methods:1. Construction of eukaryotic cell expression vector containing human wild type PTEN gene and green fluorescence protein geneThe recombinant plasmid was constructed by using nested polymerase chain reaction. The total RNA that extracted from normal adult female placental tissue was taken as template to synthesize the first chain,The product of nested PCR fragments including PTEN gene were amplified and directly cloned into the pMD18-T Vector, the recombinant plasmid was digested by double-endonuclease and the positive plasmids obtained through screening. Both pMD18-T Vector including PTEN gene and pEGFP-C1 Vector were digested by double-endonuclease again , PTEN gene was directly ligated into enhanced green fluorescent protein expression vector pEGFP-C1, the positive recombinant plasmids obtained through screening, which were identified by double-endonuclease, sequencing and sequence alignment.2.The inhibition on human breast cancer cells by PTEN geneThe recombinant pEGFP-PTEN was transfected into breast cancer cell line ZR-75-1 by lipofection. Positive cell clones ZR-75-1-vect,ZR-75-1-PTEN were obtained by G418 screening.Fluorescence microscopy,PTEN gene DNA assay, RNA assay, SDS-PAGE, Western-blot assay and immunocytochemistry assay were used to identify if the PTEN gene be integrated into breast cancer cell line ZR-75-1 or stably expressed. Through DNA agarose gel electrophoresis, FCM Analysis, MTT assay, morphological observation by transmission electron microscope, we observed the inhibition on human breast cancer cells by PTEN gene.By detecting PTEN protein and P-Akt protein using Western-blot assay, detecting PTEN protein and mutant P53 protein using immunocytochemistry assay,we made a primary approach to mechanisms in human wild type PTEN gene`s inhibition on breast cancer cells in vitro.3. The inhibition of human carcinoma of breast by PTEN gene in vivoIn experiment of tumor formation of nude mice , ZR-75-1,ZR-75-1-vect and ZR-75-1-PTEN cells were inoculated into nude mice, We measured the size of tumors every 3 days, drew tumor growth curve.Nude mice were put to death after 21days and the tumors were dissected and weighed and tumor inhibition rate was calculated. In experiment of treatment of nude mice xenografts, We established nude mice models of human carcinoma of breast,which randomly divided into four groups—liquor natrii chloridi isotonicus group(control group), adriamycin group, PTEN group and combined therapy group.We treated the nude mice models from the sixth day ,measured the size of tumors, drew tumor growth curve every 3 days, Nude mice models were put to death after 27days and the tumors were dissected and tumor inhibition ratio was calculated too. We detected PTEN protein and COX-2 protein using immunohistochemistry assay and made a primary approach to mechanisms in PTEN gene`s inhibition on tumors in vivo.Results:1. The recombinant plasmid was identified by double-endonuclease, sequencing and sequence alignment, the sequence of which were fully consistent with that announced in Gene Bank. the results showed that the recombinant plasmid was successfully constructed.2. Fluorescence microscopy showed ZR-75-1-vect and ZR-75-1-PTEN cell lines sent out green fluorescence,whereas the ZR-75-1 cell line did not . The target gene band emerged in PTEN gene DNA assay and RNA assay. Western-blot assay showed that the contoll group cells did not express PTEN gene,but the cells transfected with PTEN gene expressed it.Furthermore, the expression of Phospho-Akt (Ser473) in transfected cells decreased.The results of MTT showed proliferation velocity of ZR-75-1-PTEN is significantly slower than that of controll group(P<0.05).FCM analysis showed that, compared with the control group, the proportion of G1 phase cells and the apoptosis rate of C group (ZR-75-1-PTEN)were significantly increased(P<0.05), the proportion of G2-M phase cells and S phase cells did not changed, The proportion of G1 phase ,S phase, G2-M phase cells and the apoptosis rate between control group and treatment grooup had no apparent difference (P>0.05).Before G1 phase of C group, there appeared hypodiploid peak(apoptotic peak).These results demonstrate that PTEN gene can inhibit proliferation of carcinoma of breast cells, induce apoptosis, cause cell arrest at G1 phase.The results of detecting PTEN protein protein by immunocytochemistry assay showed that the staining of immunocytochemistry pEGFP-C1-PTEN group was only positive(60.4%),whereas other groups were negtive, which demonstrated that the PTEN gene was integrated into breast cancer cell line ZR-75-1 and stably expressed. The results of detecting mutant P53 protein protein by immunocytochemistry assay showed that the P53 protein positive rates of ZR-75-1,ZR-75-1-vect were 32.8%,35.4% respectively,there was no significant difference between them(P>0.05),but the P53 protein positive rates of ZR-75-1-PTEN group was 10.6% compared with control group(P<0.01). It is thus clear that the expresssion of mutant P53 protein decreased after transfection.The results of transmission electron microscope showed that ultrastructure of ZR-75-1-vect cells included uniform nucleus which was composed of regular dispersed chromatin and nucleoli. on the contrary, ultrastructure of ZR-75-1-PTEN cells appeared typical morphological features of apoptosis: irregular nucleus with condensed chromatin along the nuclear envelope and condensed cytoplasm (feature of apoptosis), In the cytoplasm, double-membraned giant autophagic vacuoles (ultrastructural feature of autophagy) contain a large part of the cytoplasm with some preserved organelles like endocytoplasmic reticulum, mitochondria, ribosomes,apoptotic body was unseen.These results demonstrated that the PTEN gene was sucsessfully transfected into breast cancer cell line ZR-75-1 by lipofection which was integrated into breast cancer cell line ZR-75-1 and stably expressed, PTEN gene can induce cell apoptosis , arrest cell cycle at G1 phase and and down-regulate the expression of mutant P53 and Phospholated Akt.3. In experiment of nude mice tumorigenesis, there were clear difference between ZR-75-1-PTEN and control groups on the average tumor size,the average tumor weights and tumor growth rate(P<0.01),which demonstrate that PTEN gene can inhibit the growth of xenografts of nude mice.In experiment of treatment of nude mice xenografts,we used adriamycin,PTEN gene,combination adriamycin with PTEN gene to treat nude mice xenografts,Their tumor inhibition rate were 44.23%,72.38%,89.74% respectively(compared with control group,P<0.05), There were clear difference between treatment groups and control group on the average tumor size and the average tumor weights(P<0.01), There were also notable difference in treatment groups on the average tumor size and the average tumor weights(P<0.05), These results indicate that all the three treatment--- adriamycin,PTEN gene,combination adriamycin with PTEN gene can inhibit the growth of xenografts of nude mice,that tumor inhibition rate of PTEN gene was higher than that of adriamycin, that tumor inhibition rate of combination adriamycin with PTEN gene were higher than that of either adriamycin or PTEN gene, that the tumor inhibition effect was increased by using combination PTEN gene with adriamycin.PTEN protein and COX-2 protein were detected by immunohistochemistry assay, the results showed that tumor inhibition rate was increased following with increase of positive expression of PTEN protein, but positive expression of COX-2 protein simultaneously decreased, there was a negative correlation between PTEN and COX-2. We presume that PTEN gene inhibit growth of tumor by down-regulating the expression of COX-2 protein.Conclusion:These results indicate that we successfully constructed the eukaryotic cell expression vector containing human wild type PTEN gene and green fluorescence protein gene, the PTEN gene was successfully transfected into breast cancer cell line ZR-75-1 and stably expressed, PTEN gene can inhibit growth of tumor in vitro and in vivo. These results show the prospects for PTEN gene therapy in breast cancer treatment, also provide theoretical and experimental basis for PTEN gene in clinical application, but specific mechanisms need to be further discussed.