Establishment Of PC-3 Cell Line Expressing BRP44 Stably And Efficiently

BackgroundProstate cancer is one of the most common human malignant tumors and the second leading cause of cancer death in male of western countries.Nowadays,the number of prostate cancer patients is increasing in China because of the increasing age,changing life style and improving medical examination.Serial analysis of gene expression(SAGE) datsbase form normal tissues and tumor tissues are absorbed in National Cancer for Biotechnology(NCBI).By the free and on-line analysis sofeware provided by the Cancer Genome Anatomy Project(CGAP),screening and searching genes differentially expressed between malignant and benign prostate tissues might provide us a clue to find novel prostate related genes,though the results require the verification of biological experimental methods.It will benefit not only fundamental research about functional genomics but also clinical research of prostate cancer about novel diagnostic and therapeutic method.In the past experiment,we have obtained the differential-expression genes between prostate cancer and normal tissues by means of SAGE databases,determining that the wholly 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 expressed in LNCaP cell but not in PC-3 cell.According of the deducetion of initial bioinformatics process ,BRP44 is a up-regulated expression gene related with prostate cancer. The protein of BRP44 expressed in both nuclei and cytoplasm of LNCaP cell but the expression in cytoplasm is stronger than in nuclei.BRP44 is possibly a resolved protein regulated by oct-1 and revolved in regulating the gene expressing in cellular mitochondrion.BRP44 negatively regulated proliferation and growth of LNCaP cell line,but had no key effect on PSA secreted pathway.On the other hand,the effect of gene BRP44 on PC-3 cell is just the core of this experiment topic.MethodsAbove all cultivate LNCaP cell and PC-3 cell to a stable character phase,then reverse transcription PCR of total DNA extracted from LNCaP cell line was performed. The products of which were cloned into a highly efficitnt 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 stable transfectants werw screened by G418.Cell subclones were isolated by gradient dilution.The highly expressing clones were identified by Northern bloting and reverse transcription PCR.At last, PC-3 cell line expressing BRP44 stably and efficiently was established.ResultsReverse transcription PCR of gene BRP44 from LNCaP cell produce a specific strap about 500 bp, identical to the expecting clip. After eukaryotic expression vercto pcDNA3.1/myc-His A-BRP44 was digested by restrictive endonuclease ,a specific strap about 500 bp was revealed the same as PCR amplification of gene BRP44.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 werw screened by G418 repeatedly,15 Cell subclones were obtained in all. Reverse transcription 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 5 subclones screened from G418 expressed objective gene in distinct degree.Selecting the strongest cell subclone expressing gene BRP44 from them and then cultivating and freezing in quantity in order to further study.ConclusionAfter eukaryotic expression vercto pcDNA3.1/myc-His A-BRP44 was transfected into PC-3 cell,cell subclones stably and efficiently expressing gene BRP44 was gained.Cell subclones stably and efficiently expressing gene BRP44 can be used for further study such as changes of PC-3 cell subclones bio-character in order to acquire the function hints of gene BRP44 and the contributions to development and growth of prostate cancer.