| Human hepatocellular carcinoma (HCC) is one of the most common malignant diseases, adjuvant chemotherapy plays an irreplaceable role on the prevention of the tumor recurrence and metastasis, but the successful treatment of HCC with chemotherapeutic drugs is often hampered by a marked drug resistance of this primary liver cancer. The Multi-Drug Resistance is always one of the key factors which limit the chemotherapy in tumor treatment. The possible drug-resistant mechanism includes pharmacological mechanism and cell mechanism. Among these it is highly concerned of the important role of ABC drug transporters played in the formation of the tumor multidrug resistance.ABC drug transporters also named ATP Binding Cassette Transporters is a special protein superfamily in the membrane transport protein families, these transporters can induce the transportation of many substrates through cell. It's found by recent research that the ABC drug transporters have pump function which is related to the decrease of medicament accumulation in the tumour cells, and it is the main reason that cause the tumor cells generate multidrug resistance (MDR), and this pump function also becomes the important defense mechanism which protect the tumour cells safe against the attack of chemo-treatment. According to the difference of the sequence homology and transmembrane topology domain, ABC transporter is divided into seven subfamily (ABCA-G), so far 48 ABC family members have been found in humans ,and ABCA2, ABCB1, ABCC1, ABCC2, ABCC3, ABCC5,and ABCG2 are closely related to the drug-resistance in present report.Cancer cells either are insensitive to drug treatment at the onset of therapy (intrinsic drug resistance) or acquire resistance after the first treatment with a chemotherapeutic agent (acquired drug resistance). This multidrug resistance (MDR) can be caused by expression of plasma membrane transporters belonging to the MDR P-glycoprotein family(ABCB) or the multidrug resistance protein (MRP) family (ABCC). These transporters mediate the ATP-dependent efflux of chemotherapeutic drugs out of cells. Expression of MDR1 mRNA and MDR1 P-glycoprotein in HCC has been demonstrated. Studies on cell lines derived from HCC indicated that the MDR phenotype is attributable not only to expression of the MDR1 P-glycoprotein gene but also to other mechanisms. Members of the MRP family, which are distinct from the P-glycoproteins, are integral membrane glycoproteins, some of which confer resistance to chemotherapeutic drugs. Recent studies have found that ABCC subfamily includes nine members (MRP1-9) at least, in which ABCC2, ABCC3, ABCC5 may also be involved in tumor resistance, but in domestic reports at present, it is not clear whether their expression in hepatocellular carcinoma cells is related to the liver drug resistance.Since ABC transporters has been widely studied for its function as to lead to multidrug resistance in tumor cells, the research of ABC drug transports protein in HCC is not common, and only several papers which research MDR1 and MRP1 from protein level using immunohistochemical method are reported, it is not clear what role ABC drug transport plays in the drug-resistance of HCC. Therefore, it is very significative to examine the expression level of ABC drug transporters which related to drug-resistance in hepatocarcinoma carcinoma cell lines and tissues to further study the mechanism of multidrug resistance in HCC.As the expression intensity of the resistance genes is related to their tolerance of chemotherapy drugs in tumor cells,so it is likely for us to found the genes that may be involved in the MDR through detecting their expression before and after induction. Since the MDR1 gene has been found, the new resistance-associated genes are found continuously, it is apparently better to detect the expression of many resistance-associated genes than to detect one single gene expression . To further understand the relationship between the multidrug resistance protein members and the drug resistance in HCC, and to further explore the mechanism of drug resistance about cancer cells, we propose to detect the mRNA expression of ABCC2, ABCC3 and ABCC5 genes which encode the multidrug resistance-associated protein (MRP)in hepatocellular carcinoma cell line by real-time PCR.The detection efficacys were very low such as cytomorphologic techniques,fluorescence in situ hybridization (FISH) and flow cytometry. Polymerase chain reaction (PCR) is able to detect frequencies of target cells as low as 10-6 to 10-5 ,but it can't detect the quantities of target cells accurately. A novel approach to quantitative PCR using real time detection had been developed recently.Quantitative Real time PCR(QRT-PCR) is a new technique which developed in recent years, it has proven to be a powerful method to detect the gene expression level. Here we use SYBR Green I as fluorescent dye,SYBR Green I is a dye that binds the minor groove of double strand DNA,As more double strand amplicons are produced, SYBR Green I dye signal will increase. Therefore, fluorescence signal intensity of the SYBR Green I is related to the quantity of double strand DNA. So based on the intensity of fluorescence signal we can know the quantity of double strand DNA in the PCR system. In quantitative real time PCR, the target gene can be relatively quantitatively analyzed, whose accuracy and sensitivity is higher than the conventional RT-PCR.In this study, we detect the expression level of ABCC2,ABCC3,ABCC5 genes using SYBR Greenâ… QRT-PCR and discuss the relationship between their expression and multidrug-resistance in hepatocarcinoma cell.OBJECTIVE1. To establish the method of fluorescence quantitative RT-PCR detecting the expression of target gene.2. To investigate the differential expression of genes encoding multidrug resistance-associated gene 2(ABCC2), multidrug resistance associated gene 3(ABCC3) and multidrug resistance-associated gene 5(ABCC5) in human normal cell line L-02, hepatocarcinoma cell line BEL and its Adriamycin-resistant counterpart cell line BEL/ADM.MATERIALS AND METHODS1.Specific primers were designed according to the nucleotide sequence of target gene .The cells were cultiated and their RNA were extracted. A known amount of RNA was added for transcription and amplification .2.The PCR products of beta-actin were purified and ligated into pGEM-T vector by T4 DNA ligase. The recombinants were transformed into DH-5α.The recombinant plasmids acted as quantitative standard templates. The quantitative standard templates were serially diluted and were amplified on PE7700 Sequence Detector. The method of FQ-RT-PCR detecting was established successfully, using this method ,we examined the expression of ABCC2,ABCC3,ABCC5 mRNA in the L-02 cell, BEL cell and BEL/ADM cell line, the method was evaluated such as sensitivity,stability, reproducibility at last.RESULTS:1. The quality of total RNA extracted from cells was well.The specific target gene had been amplified by common RT-PCR. Using the recombined plasmids as standard quantitative templates, we successfully established the method of FQ-RT-PCR detecting expression. When amplification reactions were over on PE7700 Sequence Detector, we could get "S"kinetics curves reflecting the progress of gene amplification and a quantitatives standard curve. Clear correlation was observed between the logarithm of initial template copies and the threshold cycle (CT) with the coefficient was 0.991 (p<0.01).2. The expression levels of ABCC2 ABCC3 ABCC5 genes in the L-02 cell, BEL cell and BEL/ADM cell line as follows (unit :copies/ul RNA):The expression of ABCC2 mRNA in the L-02 cell, BEL cell and BEL/ADM cell line averaged 7858±656. 8908±681 and 43920±4630,respectively . There was no significant variation in the expression of ABCC2 mRNA between L-02 and BEL cell line (P=0.397) ; but the expression of ABCC2 mRNA in the BEL/ADM cell line was significantly higher than that in the L-02 and BEL cell line. (P=0.000)The expression of ABCC3 mRNA in the L-02 cell, BEL cell and BEL/ADM cell line averaged 3767±320,6558±616 and 86110±4227,respectively . There was significant variation in the expression of ABCC3 mRNA among 3 cell lines. (P=0.018, P=0.000) .The expression of ABCC5 mRNA in the L-02 cell, BEL cell and BEL/ADM cell line averaged 13810±896,35060±1959 and 66370±1496,respectively. The expression of ABCC5 mRNA also showed statistically significant difference among 3 cell lines. (P=0.000) . CONCLUSIONS:1. The expression of ABCC2 mRNA in the BEL/ADM cell line was significantly higher than that in the L-02 and BEL cell line;however there was no significant variation in the expression of ABCC2 mRNA between L-02 and BEL cell line. ABCC2 might play a role on the intrinsic drug resistance. Whereas, the expression of ABCC3 and ABCC5 mRNA showed statistically significant difference among 3 cell lines. ABCC3 and ABCC5 may be related to the acquired drug resistance of hepatocellular carcinoma.2. The prokaryotic expression vector for production of beta-actin was successfully constructed,and the method of fluorescence quantitative RT-PCR detecting the expression of target genes was established successfully.3. The FQ-RT-PCR detecting gene was a sensitive,specific,reproducible,stable,rapid and accurate method. |
PDF Full Text Request