Objective: To investigate the mechanism of ursolic acid (UA) in inhibiting the growth and metastasis of breast cancer MDA-MB-231 ("231") cells by downregulating ANXA6. Methods: This study conducted relevant in vitro cytology and molecular biology experiments in the Department of Clinical Laboratory and Central Laboratory of Putuo Hospital, Shanghai University of Traditional Chinese Medicine from February 2023 to August 2024. Human breast cancer 231 cells were cultured in vitro, and the effects of different concentrations of UA on the proliferation and invasion and metastasis of 231 cells were detected by CCK-8 and Transwell assays. Western Blot was used to detect the effect of UA on the expression of ANXA6 and invasion and metastasis-related proteins MMP9, β-catenin and N-cadherin in 231 cells. The 231 cells that interfered with and overexpressed ANXA6 were constructed by lentivirus transfection to generate stable ANXA6 interfering and overexpressing 231 cells, which were divided into 231/KD-ANXA6 group, 231/KD-NC group, 231/OE-ANXA6 group, and 231/OE-NC group. CCK-8 assay and Transwell assay were used to detect the proliferation activity, invasion and metastasis ability of 231 cells after interference and overexpression of ANXA6 and the effect of UA on the proliferation ability of 231 cells after interference and overexpression of ANXA6. Western Blot and RT-PCR assays were used to detect the expression of invasion and migration biomarkers such as MMP9, β-catenin, and N-cadherin in 231 cells after interference and overexpression of ANXA6. Immunohistochemistry was used to detect the expression level of ANXA6 in breast cancer tissues, and the relationship between ANXA6 expression and clinicopathological features and prognosis of breast cancer was analyzed. Results: The CCK-8 assay results showed that compared with the control group (0 μmol/L UA, 100.00%±7.37%), the proliferative activity of 231 cells at UA concentrations of 2.5, 5, 10, 20 and 40 μmol/L (90.23%±1.76%, t=2.24, P<0.05; 85.19%±4.23%, t=3.02, P<0.05; 65.45%±0.35%, t=8.11, P<0.01; 37.79%±0.98%, t=14.50, P<0.001; 18.18%±0.15%, t=19.23, P<0.001) were significantly decreased. Furthermore, UA (10, 15, 20 μmol/L) inhibited the invasion and metastasis ability of 231 cells; Western Blot assay showed that compared with the control group (0 μmol/L UA), the protein expressions of MMP9 (1.07±0.03 vs 0.99±0.11, t=1.27, P>0.05), β-catenin (1.21±0.01 vs 0.99±0.07, t=5.47, P<0.05), N-cadherin (1.05±0.09 vs 0.90±0.03, t=2.65, P>0.05) at UA of 10 μmol/L; MMP9 (1.07±0.03 vs 0.79±0.09, t=5.26, P<0.001), β-catenin (1.21±0.01 vs 0.89±0.05, t=10.55, P<0.001), and N-cadherin (1.04±0.09 vs 0.68±0.10, t=4.59, P<0.05) at UA of 15 μmol/L; MMP9 (1.07±0.03 vs 0.52±0.07, t=12.50, P<0.001), β-catenin (1.21±0.01 vs 0.83±0.02, t=24.01, P<0.000 1) and N-cadherin (1.04±0.09 vs 0.49±0.11, t=6.70, P<0.01) at UA of 20 μmol/L. Interfering with ANXA6 inhibits the proliferation, invasion and migration of 231 cells, and overexpression of ANXA6 promotes the proliferation, invasion and migration of 231 cells. Western Blot assay showed that compared with the control group (KD-NC group), the protein expressions of MMP9 (1.07±0.01 vs 0.62±0.16, t=4.86, P<0.01), β-catenin (1.02±0.14 vs 0.64±0.15, t=3.20, P<0.05), N-cadherin (0.98±0.14 vs 0.67±0.12, t=2.85, P<0.05) were decreased expression; Compared with the control group (OE-NC group), the protein expressions of MMP9 (0.54±0.22 vs 1.06±0.08, t=3.90, P<0.05), β-catenin (0.92±0.07 vs 1.06±0.04, t=3.06, P<0.05) and N-cadherin (0.90±0.07 vs 1.06±0.01, t=3.75, P<0.05) were significantly increased. Interference with ANXA6 promoted the inhibitory effect of UA on the proliferation ability of 231 cells (P<0.05). Overexpression of ANXA6 weakened the inhibitory effect of UA on the proliferation of 231 cells (P<0.05).The results of immunohistochemistry assay showed that the expression level of ANXA6 in breast cancer tissue was significantly increased, and the expression of ANXA6 was related to tumor size (P<0.05), but not to age, T stage, N stage, pathological grade, AJCC stage, ER, PR and E-cad. Conclusion: The expression level of ANXA6 in breast cancer tissues is increased, and UA can inhibit the growth, invasion and metastasis of 231 cells by down-regulating the expression of ANXA6.
目的： 探讨熊果酸（UA）通过下调ANXA6抑制乳腺癌MDA-MB-231（简称“231”）细胞生长和转移的作用机制。 方法： 于2023年2月至2024年8月在上海中医药大学附属普陀医院检验科及中心实验室进行相关体外细胞学及分子生物学实验。体外培养人乳腺癌231细胞，CCK-8和Transwell实验检测不同浓度UA对231细胞增殖和侵袭转移的影响；蛋白质印迹检测UA对231细胞中ANXA6及侵袭转移相关蛋白基质金属蛋白酶9（MMP9）、β-连环蛋白（β-catenin）、N-钙黏蛋白（N-cadherin）表达的影响。通过慢病毒转染构建干扰和过表达ANXA6的231细胞，以产生稳定的ANXA6干扰和过表达231细胞，分为231/KD-ANXA6组、231/KD-NC组、231/OE-ANXA6组、231/OE-NC组。采用CCK-8实验及Transwell实验检测ANXA6 对231细胞增殖、侵袭和转移能力的影响及UA对干扰和过表达ANXA6后的231细胞的增殖能力的影响；采用蛋白质印迹和RT-PCR实验检测干扰和过表达ANXA6后，231细胞中MMP9、β-catenin、N-cadherin等侵袭转移生物标志物的表达。采用免疫组织化学方法检测乳腺癌组织中ANXA6的表达水平，并分析ANXA6 的表达与乳腺癌临床病理特征及预后的关系。 结果： CCK-8实验结果显示，与对照组（0 μmol/L UA，100.00%±7.37%）相比，UA浓度为2.5、5、10、20、40 μmol/L时231细胞的增殖活力（90.23%±1.76%，t=2.24，P<0.05；85.19%±4.23%，t=3.02，P<0.05；65.45%±0.35%，t=8.11，P<0.01；37.79%±0.98%，t=14.50，P<0.001；18.18%±0.15%，t=19.23，P<0.001）明显降低。此外，UA（10、15、20 μmol/L）也抑制231细胞的侵袭和转移；Western Blot实验显示，与对照组（0 μmol/L UA）相比，UA为10 μmol/L 时，MMP9（1.07±0.03 vs 0.99±0.11，t=1.27，P>0.05）、β-catenin（1.21±0.01 vs 0.99±0.07，t=5.47，P<0.05）、N-cadherin（1.05±0.09 vs 0.90±0.03，t=2.65，P>0.05）；UA为15 μmol/L 时，MMP9（1.07±0.03 vs 0.79±0.09，t=5.26，P<0.001）、β-catenin（1.21±0.01 vs 0.89±0.05，t=10.55，P<0.001）、N-cadherin（1.04±0.09 vs 0.68±0.10，t=4.59，P<0.05）；UA为20 μmol/L时，MMP9（1.07±0.03 vs 0.52±0.07，t=12.50，P<0.001）、β-catenin（1.21±0.01 vs 0.83±0.02，t=24.01，P<0.000 1）、N-cadherin（1.04±0.09 vs 0.49±0.11，t=6.70，P<0.01）表达量均降低。干扰ANXA6抑制231细胞的增殖、侵袭和转移，过表达ANXA6促进231细胞的增殖、侵袭和转移。Western Blot实验显示，与对照组（KD-NC组）比较，KD-ANXA6组231细胞中MMP9（1.07±0.01 vs 0.62±0.16，t=4.86，P<0.01）、β-catenin（1.02±0.14 vs 0.64±0.15，t=3.20，P<0.05）、N-cadherin（0.98±0.14 vs 0.67±0.12，t=2.85，P<0.05）表达降低；与对照组（OE-NC组）比较，OE-ANXA6组231细胞中MMP9（0.54±0.22 vs 1.06±0.08，t=3.90，P<0.05）、β-catenin（0.92±0.07 vs 1.06±0.04，t=3.06，P<0.05）、N-cadherin（0.90±0.07 vs 1.06±0.01，t=3.75，P<0.05）蛋白表达量增高。此外，干扰ANXA6后促进了UA对231细胞增殖能力的抑制作用（P<0.05）；过表达ANXA6后减弱了UA对231细胞增殖能力的抑制作用（P<0.05）。免疫组化实验显示ANXA6在乳腺癌组织表达水平显著增加；ANXA6的表达与肿瘤大小有关（P<0.05），而与年龄、T分期、N分期、病理分级、AJCC分期、ER、PR、E-cad的表达无关。 结论： ANXA6在乳腺癌组织中表达水平增加，UA可通过下调ANXA6表达抑制231细胞的生长、侵袭和转移。.