The Expression And Functional Study Of AQP1 In Human Breast Cancer

Part one: Expression of AQP1 in breast cancer and its relationship with clinicopathological characteristicsObjective:To investigate the difference of the expression level of AQP1 among breast cancer tissues,breast cancer tissues and breast benign lesions.And explore the relationships between AQP1 expression in breast cancer tissues with clinical and pathological characteristics,such as age,tumor size,histological type,histological grade,degree of lymphatic metastasis,distant metastasis,ER/PR status,HER-2 status.Methods:1.Human breast-related sample tissues: The study deal with 118 patients with breast cancer and 32 patients with breast benign lesion who were collected from January 2013 to January 2015 in Guangxi Medical University Affiliated Tumor Hospital.65 patients in whom the tissues adjacent to carcinoma were obtained from the patients with breast cancer.All tissues used were from the patients without any cancer treatment before.2.The expression level of AQP1 in breast cancer,the adjacent tissues and benign breast lesions was detected by immunohistochemistry.And the relationship between AQP1 expression in breast cancer and its clinicopathological characteristics were evaluated by statistical analysis.Results:1.AQP1 was expressed in breast cancer tissues,adjacent tissues and benign breast lesions.And AQP1 expressed lower in the juxtacancerous tissues(44.9% vs 13.4%,χ2 = 18.060,P = 0.000)and benign lesions(44.9% vs18.7%,χ2 = 7.222,P = 0.007)than that in breast cancer.But there was no significant difference in the expression of AQP1 between the juxtacancerous tissues and benign lesions(13.4% vs 18.7%,χ2 = 0.394,P = 0.530).2.The expression of AQP1 in ER,PR receptor or HER-2 negative breast cancer is higher than that in ER,PR receptor or HER-2 positive breast cancer patients(55.2 % Vs 35.0%,χ2 = 4.851,P = 0.028;53.6% vs 32.7%,χ2 =5.093,P = 0.024;52.1% vs 33.3%,χ2 = 3.944,P = 0.047).The expression of AQP1 was higher in breast cancer with lymph node metastasis or distant metastasis(59.0% vs 29.8%,χ2 = 5.027,P = 0.025;84.6% vs 40.0%,χ2 =9.307,P = 0.002).The expression of AQP1 was not significantly correlated with the histological type,histological grade,age and tumor size of breast cancer(all P> 0.05).3.The expression of AQP1 in triple-breast cancer(83.3%)was the highest,however,which was not significantly higher than HER-2-overexpressed subtype(83.3% vs 64.7%,χ2 = 1.872,P = 0.171).And the expression of AQP1 in Luminal A/B1 and Luminal B2 subtypeswas were significantly lower than that in Triple-negative(36.7% vs 83.3%,χ2 = 14.016,P =0.000;14.3% vs 83.3%,χ2 = 24.791,P = 0.000)and HER-2-overexpressed subtypes(36.7% vs 64.7%,χ2 = 4.009,P = 0.045;14.3% vs 64.7%,χ2 =12.101,P = 0.001).Conclusion:1.AQP1 expressed lower in the juxtacancerous tissues and benign lesions than that in breast cancer.But there was no significant difference between the juxtacancerous tissues and benign breast lesions2.The expression of AQP1 in estrogen receptor and progesterone receptor negative breast cancer was significantly higher than that in estrogen receptor and progesterone receptor positive ones.while AQP1 expression in HER-2negative breast cancer was slightly higher than HER-2 positive ones.3.The expression of AQP1 in patients with lymph node metastasis or distant metastatic breast cancer was higher than patients without lymphatic metastasis or distant metastasis.4.The expression of AQP1 in different molecular types of breast cancer was different: The expression of AQP1 was highest in the Triple-negative breast cancer,followed by HER-2-overexpressed subtype.And the two molecular subtypes expressed higher AQP1 than Luminal A/Luminal B1 and Luminal B2.Part two: The difference of AQP1 expression among different breast cancer cell linesObjective:To compare the difference of AQP1 expression among different breast cancer cell lines,and find the dominant expression one for the further study.Methods:1.The expression of AQP1 mRNA in MCF-7,MDA-MB-231,SK-BR-3 and MCF-10 A cells was detected by real-time PCR.2.The expression of AQP1 protein in MCF-7,MDA-MB-231,SK-BR-3 and MCF-10 A cells was detected by Western blotting.Results:1.The AQP1 mRNA relative expression in mammary epithelial cell MCF-10 A was significantly lower than human breast cancer cells MCF-7(1.272±0.337 vs2.639±0.653,P = 0.004),MDA-MB-231(1.272±0.337 vs 5.325±1.031,P =0.000),SK-BR-3(1.272±0.337 vs 4.240±0.714,P = 0.000).And the expression of AQP1 m RNA in MDA-MB-231 was were significantly higher than that in MCF-7(5.325±1.031 vs 2.639±0.653,P = 0.000)and SK-BR-3(5.325±1.031 vs4.240±0.714,P = 0.018).2.The expression of AQP1 protein in mammary epithelial cell MCF-10 A was significantly lower than human breast cancer cells MCF-7(0.178±0.028 vs0.381±0.038,P = 0.001),MDA-MB-231(0.178±0.028 vs 0.865±0.067,P =0.000),SK-BR-3(0.178±0.028 vs 0.810±0.038,P = 0.000).No significantly difference was found between MDA-MB-231 and SK-BR-3(0.865±0.067 vs0.810±0.038,P = 0.168).But the expression of AQP1 protein in MCF-7 was were significantly lower than that in MDA-MB-231(0.381±0.038 vs0.865±0.067,P = 0.000)and SK-BR-3(0.381±0.038 vs 0.810±0.038,P =0.000).Conclusion:1.The expression of AQP1 protein and mRNA in high invasive breast cancer cell lines(MDA-MB-231 and SK-BR-3)were higher than those in low-invasive breast cancer cell(MCF-7),and the expression is highest in MDA-MB-231.2.MDA-MB-231 cell were selected as the AQP1 dominant expression cell which will be used to evaluated the biological function of AQP1 in breast cancer in further study.Part three: The effects on proliferation,migration and invasion of MDA-MB-231 cell after AQP1 gene decreased by RNA interferenceObjective:To investigate the effect of proliferation,migration and invasion in the breast cancer cells MDA-MB-231 after AQP1 gene decreased by RNA interference.Methods:MDA-MB-231 cells were transfected with AQP1-sh RNA-LV,and then compared the expression of AQP1 m RNA and protein against MDA-MB-231cells(Blank control group)and cells transfected with NC-sh RNA-LV(Negative control group).CCK-8 kit and colony-forming assay were performed to detected the abilities of cell proliferation.And wound-healing,transwell migration and transwell invasion assay were performed to detected the abilities of cell migration and invasion.Results:1.The expression of AQP1 mRNA and protein in MDA-MB-231 cells transfected with AQP1-sh RNA-LV were significantly lower than that in blank control group and negative control group(all P <0.05).2.The ability of proliferation and the colony-forming rate were reduced in MDA-MB-231 cells after transfected with AQP1-sh RNA-LV.3.The ability of migration and invasion were reduced in MDA-MB-231 cells after transfected with AQP1-sh RNA-LV.Conclusion:AQP1-sh RNA-LV can effectively silence AQP1 gene,and successfully establish a stable transfected MDA-MB-231 cell line,lay the foundation for the next study of the function of AQP1.AQP1-shRNA-LV can significantly decreased the ability of proliferation,migration and invasion in MDA-MB-231 cells,and the interference of AQP1 expression may provide new ideas for the treatment of breast cancer,especially triple-breast cancer.Part four: The effects on xenograft and metastasis in nude mice of MDA-MB-231 cell line after down-regulation of AQP1 by RNA interferenceObjective To investigate the effects on xenograft and metastasis in nude mice of MDA-MB-231 cell line after AQP1 decreased by RNA interference.Methods MDA-MB-231 cells(the blank control group)and that transfected with AQP1-lentivirus AQP1-sh RNA-LV(the experimental group)or negative control lentivirus NC-sh RNA-LV(the negative group)were subcutaneously inoculated in breast fat pad of nude mice.The tumor formation time of subcutaneous xenografts was recorded.The volume and weight of each subcutaneous transplanted tumor were recorded every 5 days after tumor formation.The size and weight of the transplanted tumors were compared between the three groups after 35 days.And the lung metastases were observed in HE staining,.Results1.The growth of subcutaneous xenografts of nude mice in experimental group was significantly slower than that in negative control group and blank control group.The mean volume of the tumors in the blank control group(721.01 ±383.92mm3),the negative control group(656.07 ± 330.67mm3)and the experimental group(279.09 ± 110.10mm3)were significant different(F=8.895,P=0.001).The xenografts’ volume in experimental group were significantly smaller than that in the blank control group(P =0.002)and the negative control group(P =0.000).And there was significant difference among the weight of tumor in the blank control group(0.61 ± 0.34g),the negative control group(0.56 ± 0.28g)and the experimental group(0.27 ±0.11g)(F=7.124,P=0.002).The weight of tumor weight in the experimental group was significantly lighter than that in the negative control(P<0.05)or the blank control group(P<0.05).2.None nude mice suffer lung metastases in the experimental group.It seems that the emergence probability of pulmonary metastasis in the experimental group(0)was lower than that in the blank control group(3)and the negative control group(2).However,the differences were not statistically significant,P values were 0.222 and 0.481 respectively.Conclusion:1.Down-regulation of AQP1 gene expression by RNA interference can significantly inhibit the growth of transplanted tumor.2.Down-regulation of AQP1 gene expression by RNA interference may reduce the risk of spontaneous transfer in nude-mouse transplanted tumor model.