Expression And Functional Significance Analysis Of LRP16 Gene On Human Breast Cancer

Objective: â‘  To probe the possible clinical significance of LRP16 gene mRNA expression level in patients with breast cancer. â‘¡ The invasive abilities of MCF-7 cells with different LRP16 expression level were further investigated, and the possibly involved mechanism was also studied. â‘¢Furtherly, the effects of different LRP16 expression level on the therapeutic reponse were explored in MCF-7 cells.Methods: â‘  The LRP16 mRNA level in carcinoma and the matched paratumor tissues from 22 breast cancer patients was detected by using Northern Blot method. â‘¡ Three MCF-7 human breast cancer cell lines with different LRP16 expression level established by siRNA-expressing strategy, M-pL374(suppressive rate 90%), M-pL668(suppressive rate 60%) and M-pLGFPi(negative control) were used as model cells. The invasive abilities of the model cells were determined by using transwell assays and the FVB mice in vivo experiment. Western blot and/or immunohistochemical method were used to campare the protein levels of E-cadherin, MMPs in model cells. â‘¢The effect of the anti-estrogenic agent(ICI 182780) on the LRP16 expression level in MCF-7 cells were detected also by Northern Blot assay. The death assays on M-pL374, M-pL668, M-pLGFPi cell lines treated with irradiation or chemotherapy were performed to analyze the correlation between LRP16 gene expression and the kill effect.Results: â‘ The LRP16 mRNA was overexpression in 9 out of 22 patients(40%). In the 9 patients, 2~3 times in 4 cases, 3~5 times in 4 cases and 6 times only 1 cases. â‘¡As far as the overexpression of LRP16 gene, there had only 1 out of 9 patients which the tumor diameter were less than 3 centimeter, while it was 8 out of 13 patients which the tumor diameter ranged from 3 to 5 centimeter, there was statistically difference (Fish's test, P=0.031). Similarly there had 8 out of 12patients detected metastases in axillary lymphoid nodes, while only 1 out of 10 negative patients, so this showed significant difference (Fish's test, P=0.011). The clinical stages of 9 patients of overexpression: 1 cases in stage I , 4 cases in stage II, 4 cases in stage III. ?Immunohistochemical analysis revealed that Ki-67 was detected in 82% of 22 patients, TOPOII a in 72.7%, but only 2 patients were negative simultaneously and showed unoverexpression of LRP16 mRAN. A positive correlation was observed between LRP16 overexpression and Ki-67 super- positive dyeing(marked index more than 50%) (P=0.046). The immunoblot positive percent of ER(estrogen recepter) and PR(progestin recepter) was the same as 59%( 13/22). ALL of 9 patients with high LRP16 mRAN level had 7 ER(+), while 13 patients with low LRP16 mRAN level owed 6 ER(+), but the difference was not significant {P=0.1380). There were 8 PR(+) patients in all 9 patients of overexpression LRP16, versus 5 PR(+) in all 13 patients of unoverexpression LRP16, the difference was statistically significant {P-0.0180). ?The results of cell adhensive assay: 60 min following plating of cells, 80% M-pLGFPi cells attached to matrigel basement membrane, whereas less than 60% M-pL374 cells was attached. At 120 min, the either cell lines adhered more than 95% to wells.The difference showed statistically significant from 30min to 90min. At cell scratch wound assay, after cells were scratch-wounded and postincubated for a further 48h, M-pLGFPi cells flatted and spread around the blank spaces, but did not of M-pL374 cells. At the cell migration assay, the numbers of M-pLGFPi cells migrated through transwell membrane were 45% more than M-pL374 cells at 24h following the incubating the cells. Furthermor, the numbers of M-pLGFPi cells infiltrated through transwell matrigel basement membrane were more than twofold of M-pL374 cells at invasion assay. The numbers of tumor metastasis nodes on FVB mouse lung surface: 21.8+4.45 of M-pLGFPi cells, 9.6 + 2.08 of M-pL374 cells. ?The expression level of E-cadherin mRNA or protein in M-pL374 or M-pL668 cells was 2~5 times as high as it in M-pLGFPi cells. A reverse correlation was observed between LRP16 mRNA levels and E-cadherin...