Establishment Of A Breast Cell Line Stably Transfected By Silence MTDH Gene And Study Of The Proliferation And Invasion On The Cell

Objective:using the recombinant plasmid(pcDNA6.2-GW/EmGFPmiR-MTDH hereinafterreferred to as miRNA-MTDH) which MTDH expression is suppressed by miRNA tostably transfect into human breast carcinoma cell line MDA-MB-231, to establish anew breast cancer cell lines stable don’t express MTDH, and study of the effects ofmiRNA-MTDH on the cell proliferation and invasion. Our research aims to researchthe function and mechanism of MTDH furtherly in breast cancer, and provide a cellmodel and experimental basis for in vivo subsequently, and also to provide theevidence for breast cancer which are targetly treated by miRNA-MTDH.Methods:Establishment of a Breast Cell Line MDA-MB-231Stably Transfe-cted bySilence MTDH gene1. Stably transfect miRNA-MTDH into human breast carcinoma cell lineMDA-MB-231by lipofectin medium, after screened with blasticidin4-5weeks, weestablish a new breast cancer cell lines stable don’t express MTDH (Named forMDA-MB-231/P). Using the MDA-MB-231cell lines which stably transfectednegative plasmid as controls (named for MDA-MB-231/P0). The efficiency of stablytransfection through by detecting the express of the green fluorescent protein GFP.2. The groups of the experiment: No treatment group (MDA-MB-231cell group);The control group (MDA-MB-231/P0cell group); Interference group(MDA-MB-231/P cell group).Use RT-PCR,Western blot methods to detect theexpression of MTDH mRNA and protein of MDA-MB-231, MDA-MB-231/P0and MDA-MB-231/P to verifiy the stable cell line obtain.Study of the Proliferation and invasion of Effects of the Stably TransfectionCell1. The groups of the experiment: No treatment group (MDA-MB-231cellgroup);The control group (MDA-MB-231/P0cell group); Interference group(MDA-MB-231/P cell group).2. Study the proliferation ability of MDA-MB-231, MDA-MB-231/P0andMDA-MB-231/P by using cell growth curve to compare the proliferation capacitychange of MDA-MB-231/P cell.3. Study the invasion ability of MDA-MB-231, MDA-MB-231/P0andMDA-MB-231/P by using transwell assay to compare the invasion ability change ofMDA-MB-231/P cell.Results:1. Get about strip the same size of purpose (about6KB). miRNA-MTDHconcentration984μ g/ml, OD260/OD280value is1.956; The negative controlplasmid concentration598μ g/ml, OD260/OD280value is1.942, the ratio is in thereasonable range. Prove extraction of plasmid is successed, and plasmid is high purityto transfect.2. Through detecting the fluorescent protein GFP, the stably transfected efficiencyof MDA-MB-231/P0and MDA-MB-231/P is90%.3. Result of RT-PCRshow that: Compared to the no treatment group, MTDHmRNAexpression level of MDA-MB-231/P cell are significantly dropped71.72%(P <0.05),but no treatment group compared to the control group with no significant difference (P>0.05).4. Result of Western blot show that: Compared to the no treatment group, MTDHprotein expression level of MDA-MB-231/P cell are significantly dropped73.08%(P<0.05), but no treatment group compared to the control group with no significantdifference (P>0.05). 5. Results of MTT show that: Compared to the no treatment group, cellproliferation of MDA-MB-231/P dropped dramatically (P <0.05), but no treatmentgroup compared to the control group with no significant difference (P>0.05).6. Results of Transwell experimental show that: Compared to the no treatmentgroup, cell invasion of MDA-MB-231/P dropped dramatically (P <0.05), but notreatment group compared to the control group with no significant difference (P>0.05).Conclusions:1. Successfully established of a breast Cell Line name as MDA-MB-231/P whoseMTDH gene is stably silenced.2. After stably transfected the miRNA-MTDH, the MTDH expression of the breastcancer MDA-MB-231cells can significantly inhibited.3. Breast cancer cell MDA-MB-231proliferation and invasion is inhibitedeffectively after stably transfected by miRNA-MTDH...