Biological Character Of PC Cell Expressing BRP44 Stably And High Efficiently

BackgroundProstate cancer has become one of the most deadly human malignant tumors and the first leading cause of cancer death in male of western countries.Recently,the number of prostate cancer patients is increasing in China because of the increasing age,changing life style ,advanced medical examination.Using Serial analysis of gene expression(SAGE) ,We can contrast dates from normal tissues and tumor tissues that are absorbed in National Cancer for Biotechnology(NCBI).By the free and on-line analysis. so few are provided by the Cancer Genome Anatomy Project(CGAP),screening and searching the genes differentially expressed between malignant and benign prostate tissues。It might provide us a clue to find importantly new prostate related genes,We may successfully get the basic function of the novel gene that we are interested in by the verification of biological experimental methods.It will benefit not only fundamental research about intresting functional genomics but also clinical research of prostate cancer about new diagnostic and therapeutic method by using genetic ways.In the early studying,we have obtained the differential-expression gene between prostate cancer and normal tissues by means of SAGE databases,determining that the totally unknown-function gene BRP44 is the object next to research.Northern blot experiment has verified that the gene BRP44 expression abundance in malignant prostate tissues is higher than that of normal prostate tissues.At the same time,gene BRP44 was expressed in LNCaP cell but not in PC-3 cell.According to the deducetion of initial bioinformatics process ,BRP44 is a up-regulated expression gene related with prostate cancer. In order to research more function of gene BRP44 ,We may establish PC-3 cell line expressing BRP44 Stably and efficiently firstly ,then We may test Biological character of the cell lines,For example the cellular capability of proliferation。invasion and tumorigenesis.After getting the message of the role of BRP44 in carcino genesis of prostate cancer,We maybe evaluate the gene BRP44 if it play an important role in the development of prostate cancer.MethodsFirstly,we should cultivate LNCaP cell and PC-3 cell to a stable phase,then reverse transcription PCR of total DNA extracted from LNCaP cell line was performed. The products of which were cloned into a highly efficient eukaryotic expression vercto pcDNA3.1/myc-His A.The recombinants were sequenced and identified by restrictive endonuclease digestion and then transfected into the PC-3 cell line by lipofectamine 2000. The highly expressing clones were identified by Northern bloting and RT- PCR.After PC-3 cell line expressing BRP44 stably and efficiently was established,we test the biological character of the cell :cel lgrowth and proliferation of the PC-3 cells was detected by Alamar Blue,cell cycle was detected by flow cytometry ,The mobility of the cells was examined by trapswell cell layer infiltration assay .ResultsRT- PCR of gene BRP44 from LNCaP cell produce a strap about 500 bp, which is identical to the expecting clip. After eukaryotic expression vercto pcDNA3.1/myc-His A-BRP44 was digested by restrictive endonuclease , The recombinant was sent to Sangon company in ShangHai to sequence and the results indicated that the sequence was equal to the nucleotide sequence of gene BRP44 publicated,meanwhile,the inserting clip pocess correct location and direction,which means the eukaryotic expression recombinant vercto pcDNA3.1/myc-His A-BRP44 was constructed.After the stable transfectants were screened by G418 repeatedly, RT-PCR of gene BRP44 from transfected PC-3 cell produce a specific strap about 500 bp,identical to the expecting clip.Northern bloting verified that subclones cells expressed objective gene in distinct degree.Selecting the strongest cell subclone expressing gene BRP44 from them , we decide to evaluate the function of gene BRP44 through testing the biological character of the cell.then cultivating cells in good quantity to further study.Compared to the PC3-PcDNA and normal PC3 cells,The cells with high expression of BRP44 had faster proliferation and strong invasive ability。PC3-BRP44 cells had grown than t he PC3-PcDNA and normal PC3 cell.(p<0.05).ConclusionPC-3 Cell subclones stably and efficiently expressing gene BRP44 was established and could be used for testing biological character .Frome thebiological character,we may drawt the conclusion:The high expression of BRP44 is closely associated with malignant phenotypes of PC3 cell line among prostate cancer ,BRP44 maybe play a very important role in development of prostate cancer.