Research On The Effects Of MTDH Gene Transfecting Into Breast Cancer Cell Line MDA-MB-231on Chemotherapy Sensitivity

Objective：Breast cancer is the most common malignant tumor in women.The incidence rate of breast cancer is the first of female malignant tumors inchina, and an upward trend year by year. It has seriously threatened to thewomen’s health. The activation of proto-oncogene and deactivation ofsuppressor gene lead to breast cancer, whose invasion and metastasis are themain causes of death in patients.MTDH is called LYRIC, AEG-1and3D3, which is a new-foundmulti-functional proto-oncogene in2002. MTDH is over expressed in avariety of malignant tumors, such as breast cancer, esophageal cancer,melanoma, liver cancer, ovarian cancer, non-small cell lung cancer, prostatecancer, malignant gliomas and neuroblastoma etc. Its over-expression isclosely related to the occurrence development and metastasis of tumors. Theamplification of MTDH gene can obviously increase lung metastasis of breastcancer and reduce survival time of patients. The siRNA knock-out MTDHgene can decrease40%of the expression of MTDH in breast cancer cell and80%of lung metastasis of patients with breast cancer. The high expression ofMTDH gene can enhance anchorage-independent growth of neuroblastomaand invasiveness of malignant tumor cells, and the application of siRNA cansignificantly inhibit infiltration of neuroblastoma. MTDH can resistant tochemotherapeutic drugs. The inhibition of MTDH expression can enhance thesensitivity of breast cancer cells to chemotherapeutic drugs and stressor, butthe high expression of MTDH can cause the drug resistance of cancer cells.The survival time of patients with high expression of MTDH is shorter thanthe survival time of patients with low expression in malignant tumor, and therecurrence rate of patients with high expression is higher. It indicates that thelevel of MTDH expression can be an important biological index for the evaluation of the prognosis of malignant tumor.The present study is focus on the molecular characteristics, function andmechanism of MTDH, but the study on the relationship between thetransfection of MTDH gene and chemotherapeutic drug sensitivity of breastcancer cell MDA-MB-231is less. The purpose of this study is thedetermination of the levels of MTDH gene and protein expression in breastcancer cell MDA-MB-231and the chemotherapeutic drug sensitivity beforeand after transfection for judging the relationship between MTDH and thedrug sensitivity and researching a single evaluation for the prognosis andtreatment of breast cancer.Methods: A plasmid containing MTDH gene was transformed intoE.coli.DH5α and amplified. Then the plasmid containing high activity ofMTDH gene was transfected into breast cancer MDA-MB-231cells byliposome method after the lowest expression of MDTH gene has been provedin these cells in early tests. The levels of mRNA and protein expression weredetected by realtime PCR and Western Blot methods before and aftertransfection and valuated the effect. After successful transfection, the drugsensitivity of breast cancer cell MDA-MB-231of pre and post transfectionwas detected by MTT assays in24hours. Breast cancer cell was treated with1mg/L of adriamycin and8mg/L of taxol, respectively, that had beenconfirmed in early tests. Apoptosis induced by adriamycin and taxol beforeand after transfection was detected by flow cytometry for proving therelationship between MTDH and the drug sensitivity of adriamycin and taxol,further. The expression level of MTDH and the drug sensitivity of breastcancer cell were detected by SPSS13.0statistical software for confirmingsome differences.Results:1The detection of levels of MTDH mRNA and protein expression inbreast cancer cell MDA-MB-231before and after transfectionThe levels of MTDH gene and protein expression in breast cancer cellstransfected with0.8ug of MTDH gene were detected by realtime PCR and Western Blot method after48hours. The expression levels of mRNA in breastcancer cells of pre and post transfection were1.00±0.00and3.25±0.02,respectively, and the expression levels of MTDH protein in breast cancer cellsof pre and post transfection were0.48±0.07and1.27±0.08. The statisticalanalysis showed that there were some differences of MTDH gene and proteinexpression in breast cancer cells before and after transfection. Differencesbetween two groups were of statistical significance (P <0.05).2The detection of drug sensitivity of human breast cancer cellMDA-MB-231to adriamycin before and after transfectionThe inhibition rate of1mg/L of adriamycin to breast cancer cell detectedby MTT method before and after transfection in24hours: the inhibition ratesof1mg/L of adriamycin to breast cancer cell were60.72±0.01%and41.68±0.03%before and after transfection in24hours. The apoptosis rate of1mg/L of adriamycin to breast cancer cell detected by flow cytometry beforeand after transfection in24hours: the apoptosis rates of1mg/L of adriamycinto breast cancer cell were39.76±0.44%and20.59±0.59%before and aftertransfection in24hours. The statistical analysis showed that there were somedifferences of inhibition rate and apoptosis rate of adriamycin to breast cancercell before and after transfection. Differences between two groups were ofstatistical significance (P <0.05).3The detection of drug sensitivity of human breast cancer cellMDA-MB-231to taxol before and after transfectionThe inhibition rate of8mg/L of taxol to breast cancer cell detected byMTT method before and after transfection in24hours: the inhibition rates of8mg/L of taxol to breast cancer cell were58±0.38%and40.52±0.03%beforeand after transfection in24hours. The apoptosis rate of8mg/L of taxol tobreast cancer cell detected by flow cytometry before and after transfection in24hours: the apoptosis rates of8mg/L of taxol to breast cancer cell were24.88±0.37%and13.97±0.50%before and after transfection in24hours. Thestatistical analysis showed that there were some differences of inhibition rateand apoptosis rate of taxol to breast cancer cell before and after transfection. Differences between two groups were of statistical significance (P<0.05).Conclusions:1The transient transfection of MTDH gene into MDA-MB-231cells canobviously enhance the expression of MTDHmRNA and protein in breastcancer cells.2The inhibition rate and apoptosis rate of adriamycin to breast cancercell line MDA-MB-231are lower with the higher expression of MTDH. Itindicates that the high expression of MTDH may be related to the drugsensitivity of breast cancer cells to adriamycin.3The inhibition rate and apoptosis rate of taxol to breast cancer cell lineMDA-MB-231are lower with the higher expression of MTDH. It indicatesthat the high expression of MTDH may be related to the drug sensitivity ofbreast cancer cells to taxol.