The Study Of Quantitative Assessment Of The Gene Expression Of Human Organic Anion Transporting Polypeptide-E By Fluorescent Quantitative RT-PCR

[Objective) To investigate the expression of human organic anion transporting polypeptide-E mRNA in breast cancer lines MCF-7 and MCF-7/ADR and establish a method and an outer standard curve for detecting the gene expression of tumor cells' human organic anion transporting polypeptide-E by real-time fluorescent quantitative RT-PCR(FQ-RT-PCR). It will have great importance in basic research, clinicaltherapy and drug screening on human organic anion transporting polypeptide-E. [Materials and Methods]1. Cell cultivation: The breast cancer cell lines MCF-7 and MCF-7/ADR wereroutinely grown in RPMI1640 cultivation medium containing 10% fetal calf serum, penicillin and streptomycin in a humidified incubator at 37℃ and 5% CO₂.2. Extraction of total RNA: The total RNA of breast cancer cell lines MCF-7 and MCF-7/ADR were extracted by TRIzol, respectively.3. RT-PCR: Reverse transcription was performed using total RNA with Oligo dT as a template and M-MLV Reverse Transcriptase. PCR amplification used primers specific for OATP-E was performed then.4. Construction of recombined plasmid pGEM-OATP-E: cDNA fragments of OATP-E that were amplified by PCR were cloned into pGEM-T EasyVector. Sequence-verified clones were selected through blue-white screening and used for transformation into E.coli. JM109 competent cells.5. Establishment of the outer standard curve: The outer standard curve for detecting OATP-E mRNA by real-time fluorescent quantitative PCR was built up.6. The expression level of OATP-E mRNA in breast cancer cell lines MCF-7 and MCF-7/ADR were evaluated using the outer standard curve by real-time fluorescent quantitative PCR, respectively. The results were compared and analyzed by SPSS 11.5 software on computer.[Results] The expression of human organic anion transporting polypeptide-E was detected in breast cancer cell lines MCF-7 and MCF-7/ADR. Recombined plasmid pGEM-OATP-E was constructed and then transformed into E. coli. JM109 competent cells successfully. Aiter selecting of sequence-verified clones through blue-white stain the outer standard curve for detecting OATP-E mRNA by real-time fluorescent quantitative RT-PCR was established. The expression level of human organic anion transporting polypeptide-E mRNA in breast cancer cell lines MCF-7 and MCF-7/ADR were examined using the outer standard curve by real-time fluorescent quantitative RT-PCR, respectively. The OATP-E mRNA level in breast cancer cell line MCF-7/ADR is lower than that in breast cancer cell line MCF-7 (P< 0.05).[Conclusions] We developed a method for detecting the tumor cells' human organic anion transporting polypeptide-E mRNA by real-time fluorescent quantitative RT-PCR(FQ-RT-PCR) and established the outer standard curve. It will have great importance in basic research, clinical therapy and drug screening on human organic anion transporting polypeptide-E. With the method and the outer standard curve we found that the OATP-E mRNA level in breast cancer cell line MCF-7/ADR is lower than that in breast cancer cell line MCF-7 (P<0.05).