Effects Of Expression Silencing Of Bmi-1 On Senescence And Metastasis Of Human Gastric Caner Cell BGC823

Background and objectivesProtooncogene of Bmi-1 is a member of transcription inhibition factors of Polycomb family. Human Bmi-1 is located on chromosome 10p11.23 and composed by 10 exon, which cDNA total length up to 3568bp, including a relative molecular weight 45kD nucleoprotein with 326 amine acid coded by an open reading frame. Sequence homology comparison of Bmi-1 has demonstrated that the homology of Bmi-1 in human and mice is 86% and 98% with DNA sequence and amine acid sequence. It is essential to the extention of cell replication life and restraining p16 INK4a that Bmi-1 protein includes two domains, ring finger domain at C termini and H-T-H domain at central region. It has been shown that INK4a/ARF locus is downstream regulation locus of Bmi-1 and is negative controlled by Bmi-1 to affect cell multiplication and senescence. With up-regulation of Bmi-1, the expression of p16 INK4a is down-regulation and promotes cyclinD and CDK4/6 forming complex, and facilitates cell growth and multiplication by p16 INK4a/cyclin D/Rb passageway; the increasing expression of Bmi-1 also restrains p19ARF and prevents cell cycle stasis and cell apoptosis by p19ARF/MDM2/p53 passageway. The increasing expression of Bmi-1 is discovered in many tumors, which indicates a poor prognostic outcome. Research shows that Bmi-1 affect cell proliferation and cell senescence by acting on INK4a/ARF gene locus and the expression level of Bmi-1 is closely related to invasion and metastasis of the tumor. Therefore, in this study we plan to use RNA interference technology to silence the expression of Bmi-1 which was in gastric cancer cell BGC823. And meanwhile, we observe the effect of silence the expression of Bmi-1f on the senescent and metastasis of gastric cancer cell BGC823.MethodsAccording to the selection principle of RNAi aimed sequence, we use the siRNA sequence designing software siDESIGN Center (http://www.dharmacon.com/ designcenter/designcenterpage.aspx) provided by Dharmacon company to design Bmi-1 sequence. RNAi targeted sequences is selected and definited by BLAST homology analysis. Afterwards synthesizing two pairs of DNA oligonucleotides targeting directly Bmi-1 anneal into double strands hairpin DNA. The annealed products are purified and cloned into RNAi expression vector (pRNAT-U6.2) that has been cut by BamHI and Xhol restriction enzyme. Using pRNAT-U6.2 to appraise primer we can gain recons (pRNAT-U6.2-si1104 and pRNAT-U6.2-sil356) by PCR filtration. The insertion sequences of pRNAT-U6.2-si1104 and pRNAT-U6.2-si1356 are identified and confirmed by restrictive endonuclease digestion and DNA sequencing. According to Liposome LipofectAmineTM200, pRNAT-U6.2-si1104 and pRNAT-U6.2-sil356 are transfected into human gastric cancer (BGC823) cell, while empty vector group transfected empty vector pRNAT-U6.2 and untransfected group untransfected human gastric cancer cell BGC823 are taken as control groups. The expression of Bmi-1 gene mRNA and protein are examined by RT-PCR and Western blotting. Senescent cells staining and Transwell experiment are performed respectively to detect the effects of Bmi-1 expression which are in the slience BGC823 cell on the senescent cells and metastasis.Results1.We preliminarily bolt thirty RNAi as candidate. By homology searching and optimizing, we confirm Bmi-1 (NM₀05180) GGAGGAGGTGAATGATAAA (1104nt-1122nt)and GAGAGATGGACTGACAAAT(1356nt-1364nt)to be Bmi-1 siRNA targeted sequence.2.PCR amplification and bolt obtain recon pRNAT-U6.2-si1104 and pRNAT-U6.2-si1356, which insertion sequencing and design sequence are completely uniformity by DNA sequencing analysis.3.After Bmi-1 cell transfected by pRNAT-U6.2-si1104(group 1), pRNAT-U6.2-sil356 (group 2) respectively, the relative expression of Bmi-1 mRNA was 0.062±0.009(group 1) and 0.114±0.013(group 2); and that in empty vector control group and untransfected group are 0.314±0.041 and 0.321±0.038 respective. The relative expression of Bmi-1 mRNA in experimental groups (group 1-2) is dramatically lower than that of tow contrast group. There is significant difference between them (P<0.05).4. The Bmi-1 protein in cells untransfected gastric cancer BGC823 and cells transfected empty vector pRNAT-U6.2 is high expression, yet Bmi-1 protein in experimental groups'gastric cancer cells which is transfected targeted Bmi-1 (pRNAT-U6.2-si1104 and pRNAT-U6.2-sil356) is dramatically restrained. And among that, Bmi-1 protein in cells transfected by transfected is almost non-expression completely.5. The average cell senescent ratio in groupl-2 is 28.3%±3.9% and 25.9%±4.3% respectively, that in empty vector control group and untransfected group is 15.6%±2.7% and 17.2%±3.1%, which is dramatically higher than that of empty vector control group and untransfected group. There is significant difference between them (P<0.01).6.The average cell number of penetrating Matrigel in group1-2 is 22.4±4.2 and 33.6±5.5 respectively, that in empty vector control group and untransfected group is 74.7±9.3 and 68.9±10.1, which is dramatically lower than that of empty vector control group and untransfected group. There is significant difference between them (P<0.01).Conclusions1. Bmi-1 (NM 005180) GGAGGAGGTGAATGATAAA (1104nt-1122nt) and GAGAGATGGACTGACAAAT (1356nt-1364) are RNAi targeted sequence. That constructed siRNA pRNAT-U6.2-si1104 and pRNAT-U6.2-si1356 vector transfect into human gastric caner cell BGC823 can decrease the expression of Bmi-1 gene obviously.This RNAi targeted sequence is more ideal.2. Expression silencing of Bmi-1 by RNA interference could increase senescent cell rate and reduce metastasis of gastric caner cell effectively.