Quantitative Study On MRNA And Protein Expressions Of Organic Anion Transporting Polypeptide B And D In Lung Cancers And Its Significance

[Objective] To build a method for detecting expressions of OATP-B and OATP-DmRNA in lung cancers using real-time FQ-RT-PCR and to identify their protein expressions by Immunohistochemistry. To analysis the relationship between mRNA/protein expressions and the clinical pathological data and to evaluate the clinical significance.[Materials and Methods]1. Establishment and optimization of the standard curve:(1) Extraction of total RNA: To extract the total RNA of 40 samples including primary lung cancers and normal lung tissues with Tiozol respectively according to the manufacturer's instructions.(2) Reverse Transcription: To perform the reverse transcription using total RNA with Oligo dT as a template and M-MLV Reverse Transcriptase in a sterile RNase-free microcentrifuge tube.(3) Polymerase Chain Reaction and Consruction of recombined plasmid of OATP: PCR amplification used primers specific for OATP-B and OATP-D was performed . To gel-purify and subclone the PCR product into the PGEM-T Easy vector according to the protocol. To transform the recombinant plasmid into E.coli.JM109 competent cells and select sequence-verified clones through blue-white screening .(4) Establishment and optimization of the standard curve: To optimize the reaction conditions including the concentrations of primers and SYBRGREEN and Mg²⁺ concentration on the basis of data reports and to build up the standard curve for detecting OATP-B and OATP-D mRNA by fluorescent quantitative PCR.2. Immunohistochemistry analysis: The protein expressions of OATP-B and OATP-D were available by S-P IHC with anti-OATP-Band anti-OATP-D as antibodies. To analyse the IHC result with Image Pro Plus 4.0 software.3. Analysis of clinical pathological data: To analyse the relationship between mRNA/protein expressions and the clinical pathological data such as different pothological types,sex,age,size of tumor,with/without lymph metastasis.4. Statistical analysis: To analyse all data using the SPSS11.5 statistics package, and all data expressed as means±S.E.M. F test was performed to compare multi-group means while statistical analysis was performed with q test between multi-group means, taking p< 0.05 as the criterion of significance.[Results] To subclone the OATP PCR product into the PGEM-T easy vector to construct the recombinant plasmid and transform the recombinant plasmid into E.coli.JM109 competent cells, and to select sequence-verified clones through blue-white screening. After optimizing the thermcycling conditions, a quantitative PCR assay to quantify OATP-B and OATP-D mRNA expression was successfully built and the standard curve was successfully established. The expressions of OATP-B and OATP-DmRNA were all signicantly up-regulated in different pathological type lung cancers compared to normal lung tissues. There was also a significant difference in the expressions of OATP-B and OATP-DmRNA between lung cancers with lymph metastasis and those without lymph metastasis(P<0.005). Immunohistochemistry analysis showed higher expressions of OATP-B and OATP-D protein both in lung cancers and in cancers with lymph metastasis compared to those without lymph metastasis(P<0.005).Otherwise, the mRNA/protein expressions of OATP-B and OATP-D has no statistical siginifice in samples with different pothological types,sex,age or size.[Conclusions] 1. To build and optimize the fluorescent quantitative PCR reaction system for detecting OATP-B and OATP-D mRNA. 2. The mRNA/protein expressions of OATP-B and OATP-D were all signicantly up-regulated both in lung cancers and in cancers with lymph metastasis. The stusy cued an important role that OATP-B and OATP-D had played in the genesis and development of lung cancers ,which may pose new molecule mechanism theories for tumorigenesis and drugtreatment in one aspect, and provide a fast and reliable fluorescent quantitative PCR method to detect mRNA expressins of multioncogenes on large scale in clinic in another aspect.