| Objective:To investigate the expression of the new CD44 variant (CD44v17) in breast cancer and analyze the realation between CD44v17 and the size of tumor, clinical stage, lymph nodes metastasis, histological typing, ER, PR or cerbB-2. To analyze the correlation of the mRNA levels between CD44vl7 and MDRl.Methods:①The full length cDNA encoding CD44 was obtained by RT-PCR from the total RNA isolated from the MCF-7/Adr cells. In order to get CD44vl7 and MDRl gene, the specific real time PCR primers of the new CD44 variant (CD44v17) (Gene Bank NO.FJ216964) found in MCF-7/Adr in our laboratory and MDRl were designed. Treated the PCR product with 2% Agarose DNA gel electrophoresis, then reclaimed and purified the CD44vl7 and MDRl gene according to the directions.②After purification, the cDNA fragment was cloned into the pMD19-T simple vector. The recombinant plasmid pMD19-T-CD44v17 and pMD19-T-MDR1 were transformed into E.coli DH5a for amplification respectively, and then were digested with two restriction enzyme EcoR I and HindⅢafter being extracted with Plasmid Mini Preparation Kit. The nucleotides sequencing of the CD44v17 and MDR1 gene were confirmed by DNA sequencing.③The concentration of the plasmid containing the target gene was detected with Eppendof Bio-photometer and the plasmid copys was calculated according to the formula:C×10-8/(R+3000)×3.415x6.02×1023 (copies/μl). Then, the plasmid of standard was diluted from 108 to 104.④The expression of CD44vl7 at mRNA levels in 52 cases of breast cancer tissues and corresponding normal tissues was detected with the SYBR Green I real time fluorescent quantitative PCR (qRT-PCR) method according to standard curve of CD44V17 mRNA. The relationship between the CD44vl7 mRNA expression and biological characteristic of breast cancer was evaluated. The correlation between CD44v17 and MDR1 gene was analyzed.Results:①The significant difference of the CD44v17 mRNA expression levels was found between the group of breast cancer tissues (3.10±1.12) and the control group (2.31±0.84) (P＜0.01).②In addition, The expression levels of CD44v17 mRNA in the group of lymph nodes metastasis (4.07±1.13) were higher than that without lymph nodes metastasis (2.75±0.89) (P＜0.01); CD44v17 mRNA expression in the>5cm group (4.18±0.95) was significantly higher than that in the<5cm group (2.96±1.07) (P＜0.05);CD44v17mRNA expression in the carcinoma medull group (5.27±0.30) was higher than in the intraductal carcinoma (3.28±1.08) and infitrating ductal carcinoma group (2.92±1.04) (P<0.05); but there was no significant difference in CD44v17 mRNA expression between the intraductal carcinoma and infitrating ductal carcinoma group (P>0.05); CD44v17 mRNA expression in III clinical stage (4.18±0.92) was significantly higher than that in I (2.73±0.97) or II clinical stage (2.76±0.96) (P<0.05), but there was no significant difference in CD44vl7 mRNA expression between I clinical stage the and II clinical stage (P>0.05); however, there was no significant difference between CD44v17 mRNA and ER,PR or cerbB-2(P>0.05).③The significant difference of the MDR1 mRNA expression levels was found between the group of breast cancer tissues(2.37±0.98) and the control group(1.95±0.77)(P＜0.01).④There was a positive correlation between CD44vl7 and MDR1 (r=0.809, P<0.01)Conclusion:①CD44v17 mRNA is over-expressed in breast cancer. The expression of CD44vl7 mRNA may play important roles in tumorigenesis and progression of breast cancer. The expression of CD44v17 mRNA may be correlated with invasion, metastasis and prognosis of breast cancer.②There was a positive correlation between CD44v17 and MDR1. Over, expressed CD44 and MDR1 may be an important marker for poor prognosis of breast cancer.
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