The Biological Effect In Prostate Cancer Cell LNCap By Stable Silence Of HOXB13 With Lentiviral-mediated SiRNA

Objective:Prostate cancer(PCa) is the most common cancers of men.It accounts for about 25%(186,320) of incident cases in men.In our country,the incident of PCa has been increased.In the early stage of this disease,PCa is treated by surgery or local radition theraphy.The first choice to treat advanced PCa is surgery or durg to achieve castration.However,sensitivity to hormonal therapy in PCa patients had become steroid-dependent,even metastases.It's very import to do researches on mechanisms.HOXB13 gene is one of homeogene,and it plays an important role in the course of embryonic and tumor development.The role and functions of HOXB13 gene in PCa development and carcinogenesis need to be further studies.Nowadays,the technique of RNAi becomes a new heated point of gene therapy.It is a credible method for inhibition of gene expression.In this study,we constructed a cell model with HOXB13 knockout lentivirusmediated RNA interferent.And we investigated the influence in proliferation ability and invasion character of LNCap cells.This research will improve the pathogenesy of PCa.Materials and methods:1.According to the Tuschl's criteria and RNAi vector rule,the siRNA was designed and converted into shRNA specific to human HOXB13 gene.The cDNA was synthesized and inserted into plasmid tele-sh003 which was linearized by restriction endonucleases.The recombinant plasmid was transformed into competent E.coli cells.The positive recombinant colony was selected by ampicillin medium agar and identified by DNA sequencing.The positive recombinant was contransfected along with pRsv-REV,pMDLg/pRRE and pMD2.G into 293FT to package lentivirus particles.Then the lentiviral particles in the 293FT media supernatant were collected and concentrated by centrifuging.2.The experimental objects were divided into three groups,the HOXB13 RNAi infected group,the HOXB13-neg infected group and the uninfected group. Transwell assay were used to study the migration of LNCap;MTT and cell counted methods were used to study the proliferation of LNCap cells.3.TRUS-guided biopsies were performed in the region of cancer and non-cancer,respectively.With real-time quantitative PCR,the amplification curve and melting curve were obtained.The relationships between the expression of HOXB13 and the level of tPSA,fPSA and fPSA/tPSA,PSAD,Gleason Score were analyzed,respective.Results:1.Lentivirus vectors of targeting HOXB13 gene for RNAi were contructed. The results suggested that HOXB13 RNAi could silence the HOXB13 expression. Lentivirus vectors were packaged.2.MTT test indicated proliferation velocity of LNCap-HOXB13 was higher than orther two groups.Down-regulation of HOXB13 in LNCap cells increases cell invasion and the cell's migration ability.3.The SYBR Gree I Real-time fluorescent quantitative PCR was a highly accurate,sensitive,and fesible DNA Methylation quantitative method.The Ct was diefferent in PCa tissues and para-PCa tissues.Conclusion:The RNAi of viralvector target HOXB13 can efficiently silence HOXB13 expression.Blocking the HOXB13 expression can increase the proliferation, invasion and migration abilities.