The Effect Of KLF6 Gene Recombinant Plasmid On Prostate Cancer Cell Line PC-3

SECTION 1[Objective] To extract total RNA from the normal tissue of human liver, clone cDNA of anti-canncer gene KLF6 by RT-PCR, design and achieve plasmid vector KLF6-pEGFP-C₁ which contains anti-canncer gene KLF6.[Methods] We extracted mRNA from normal human liver, designed the primer refering to sequence of KLF6 mRNA from the GeneBank : 5'-AAGGTACCATGGACGTGCTCCCTATGTGC-3';5'-CGTCTAGATCAGAGGTGCCTCTTCATGTG-3'. We cloned cDNA of anti-cancer gene KLF6 by RT-PCR. The cDNA of KLF6 was 852bp. The cDNA of KLF6 and pEGFP-Ci were cut by endonuclease BamH I and EcoR I, then they were jointed together. We gained recombined plasmid vector KLF6-pEGFP-C₁ containing anti-canncer gene KLF6.[Results] We extracted mRNA from normal human liver successfully, gained the cDNA of KLF6 by RT-PCR successfully. The cDNA of KLF6 was 852bp on electrophoresis site. We gained recombined plasmid vector KLF6-pEGFP-C₁.The cDNA of KLF6 was right.[Conclusions] We can gain the cDNA of KLF6 by RT-PCR properly andrecombined plasmid vector KLF6-pEGFP-C₁ successfully. The recombined plasmid can be transfected into cell and be identified and screened out.SECTION 2[Objective To copy, reproduce, purify and analyse the recombined plasmid vector KLF6-pEGFP-C₁. To transfect the recombined plasmid vector KLF6-pEGFP-C₁ into PC-3 cell and screen out the PC-3 cell with wt-KLF6.[Methods] We constructed receptible E.coli DH5a, mixed E.coli DH5a with plasmid vector KLF6-pEGFP-C₁, poured them into LB culture medium for 1 hour at 37*C, then inoculated them on plate culture medium with Amp. We harvested the live E.coli DH5a, extracted and prepared plasmid DNA by Alkaline Lysis with SDS largely, then purified and analysed them by endonuclease and electrophoresis. PC-3 cell was cultured in RPMI-1640 with 10% FCS and glutamine, then mixed with liposome LipofectAMINE2000 and plasmid vector KLF6-pEGFP-C₁ to transfect recombined plasmid vector KLF6-pEGFP-C₁ into PC-3 cell for 1 hour, then cultured them in the RPMI-1640 medium with 10% FCS. When we saw green fluorescence on PC-3 cell, then cultured them in RPMI-1640 with 10% FCS and G418 for 6 weeks.[ Results ] The recombined plasmid vector KLF6-pEGFP-C₁ was copied and reproduced largely in vitro. We purified and analysed the recombined plasmid vector KLF6-pEGFP-C₁, they were right. We transfected the recombined plasmid vector KLF6-pEGFP-C₁ into PC-3 cell successfully and screen out the single clone PC-3 cell with wt-KLF6. We got pure PC-3 cell containing KLF6 at last.[Conclusions] The recombined plasmid vector KLF6-pEGFP-C₁ can be copied and reproduced largely by receptible E.coli DH5a, be purified by Alkaline Lysis with SDS, be transfected into prostate cancer cell PC-3 with transfectionvector liposome IipofectAMINE2000.SECTION 3[Objective] To explore the effect of wt-KLF6 on the growth, proliferation, apoptosis and cell growth cycle phase of prostate cancer PC-3 cell, discuss the process mechanism of KLF6 in prostate cancer.[Methods] We observed the growth and proliferation of PC-3 cell containing KLF6-pEGFP-Q by MTT method, analysed the apoptosis and cell growth cycle phase of PC-3 cell containing KLF6-pEGFP-Ci by Flow Cytometry method.[Results] After transfected KLF6 into PC-3 Cell, wt-KLF6 enhanced the growth suppression of PC-3 cell[Suppression ratio=(30.0±5.4)%,P<0.01], induced the apoptosis of PC-3 cell [Apoptosis ratio=(24.3±2.3)%,P<0.01] significatively. Anti-cancer gene KLF6 also decreased the ratio of PC-3 cell phase S and G2/M, increased ratio of G0/G1 from (58.6±7.3)% to (76.0±9.8)%(P<0.05), inhibited the advance of PC-3 cell phase significatively, arrested and stopped PC-3 cell at G0/G1 phase.[Conclusions] Wild type KLF6 gene can enhance the growth suppression and induce apoptosis of PC-3 cell in vitro significatively. The mechanism of wt-KLF6 on prostate cancer may correlate with the growth suppression, inducement of apoptosis and inhibition and arrestment of PC-3 cell cycle. Anti-cancer gene KLF6 shows great potential for the gene therapy of androgen-independent carcinoma of prostate.SECTION 4I Objective] To explore the effect of wt-KLF6 on the expression of cyclin Dx and bcl-2 protein in prostate cancer PC-3 cell and discuss the process mechanism of wt-KLF6 in prostate cancer.EMethods] We observed the expression of cyclin Di and bcl-2 protein in prostate cancer PC-3 cell and in the PC-3 cell containing KLF6-pEGFP-Ci by immunohistochemical method, and compare the expression ratio of cyclin Di and bcl-2 protein in prostate cancer PC-3 cell with the expression ratio in PC-3 cell containing KLF6-pEGFP-Ci by f-test.(Results] When wt-KLF6 was transfected into PC-3 Cell, anti-cancer gene KLF6 down-regulated the expression of cyclin Di protein [Expression ratio=(25.3±3.7)%,P<0.05] , down-regulated the expression of bcl-2 protein [Expression ratio=(18.7±3.2)%,P<0.05] in PC-3 cells with anti-cancer gene wt-KLF6 significatively.[Conclusions] The expression of cyclin Di protein and bcl-2 protein down-regulate significatively in PC-3 cell which transfected with wild type KLF6 in vitro. The mechanism of anti-cancer gene KLF6 on prostate cancer may correlate with the down-regulation of the expression of cyclin Di and bcl-2 protein in prostate cancer PC-3 cell. Anti-cancer gene KLF6 shows great potential for the gene therapy of androgen-independent carcinoma of prostate.