探讨微RNA(miRNA)-3178和三羟异黄酮(genistein, Gen)在三阴性乳腺癌化疗敏感性中的作用。

(1)分别用不同浓度的单药多柔比星(DOX, 0.125、0.250、0.500 μmol/L)、紫杉醇(PTX, 0.20、0.40、0.80 μmol/L)或联合Gen(2.5 μmol/L)处理MDA-MB-231细胞后，用MTT法检测细胞生长情况，并计算药物的IC₅₀；(2)采用小分子干扰RNA(siRNA)干扰技术，将miRNA-3178小干扰片段瞬时转染MDA-MB-231细胞(miRNA-3178 siRNA组)，以转染无义siRNA的细胞作为阴性对照组，再用real-time PCR法检测干扰结果，并分别用不同浓度DOX或PTX处理miRNA-3178 siRNA组及阴性对照组细胞，检测2组细胞间化疗药物敏感性的差异；(3)分别用0.250 μmol/L DOX、0.40 μmol/L PTX及2.5 μmol/L Gen处理MDA-MB-231细胞，再用real-time PCR法检测所有处理组与未处理组细胞miRNA-3178表达量的差异。2组细胞间生长抑制率及miRNA-3178表达量的比较采用t检验，多组细胞间miRNA-3178表达量的比较采用单因素方差分析，组间两两比较采用LSD法。

(1)MTT结果显示：用0.125、0.250、0.500 μmol/L DOX单药处理MDA-MB-231细胞48 h后，细胞生长抑制率分别为16.7%±0.4%、25.9%±0.1%及44.9%±0.1%，联合2.5 μmol/L Gen处理后细胞生长抑制率均显著增加，分别为29.8%±0.3%、45.3%±0.4%及68.5%±0.4%，组间比较，差异均有统计学意义(t＝38.12、61.57、82.70，P均< 0.001)；用0.20、0.40、0.80 μmol/L PTX单药处理MDA-MB-231细胞48 h后，细胞生长抑制率分别为15.3%±0.3%、27.9%±0.5%及39.2%±0.1%，联合2.5 μmol/L Gen处理后细胞生长抑制率也显著增加，分别为32.7%±0.7%、48.3%±0.1%及63.3%±0.2%，组间比较，差异也均有统计学意义(t＝34.41、58.63、213.91，P均<0.001)。DOX单独作用于细胞的IC₅₀为1.230 μmol/L，联合2.5 μmol/L Gen后，IC₅₀降低为0.440 μmol/L；PTX单独作用于细胞的IC₅₀为0.64 μmol/L，联合2.5 μmol/L Gen后，IC₅₀降低为0.27 μmol/L。(2)在不同浓度DOX(0.125、0.250、0.500 μmol/L)或PTX(0.20、0.40、0.80 μmol/L)的作用下，miRNA-3178 siRNA组MDA-MB-231细胞生长抑制率均明显低于其阴性对照组(转染无义siRNA)(12.3%±0.6%比16.7%±0.4%，21.2%±0.9%比25.9%±0.1%，27.2%±0.9%比44.9%±0.1%，t＝8.99、7.33、27.34，P均<0.050；8.8%±0.5%比15.3%±0.3%，13.4%±1.1%比27.9%±0.5%，20.2%±0.9%比39.2%±0.1%，t＝16.80、17.57、30.48，P均< 0.001)。(3)所有处理组及未处理组细胞间miRNA-3178表达量的差异具有统计学意义(F＝ 66.57，P<0.001)。组间两两比较显示，与未处理组相比，DOX(0.250 μmol/L)及PTX(0.40 μmol/L)处理组MDA-MB-231细胞miRNA-3178表达量无明显变化(P＝0.611、0.235)，而Gen(2.5 μmol/L)可显著增加MDA-MB-231细胞miRNA-3178的表达量(11.10±0.33比5.77±0.21，P<0.010)。

Gen和miRNA-3178可能增加三阴性乳腺癌细胞MDA-MB-231对PTX和DOX化疗的敏感性。

To investigate the effect of miRNA-3178 and genistein on chemosensitivity of triple negative breast cancer MDA-MB-231 cells.

(1) MDA-MB-231 cells were treated by doxorubicin (0.125, 0.250, 0.500 μmol/L) and paclitaxel (0.20, 0.40, 0.80 μmol/L) alone or combined with genistein (2.5 μmol/L). MTT assay was performed to detect the cell growth. IC₅₀ was also determined. (2) Using small interfering RNA (siRNA) technique, MDA-MB-231 cells were transfected with small interference fragment miRNA-3178 (miRNA-3178 siRNA group), and the cells transfected with nonsense siRNA served as negative control. The real-time PCR was used to detect the interference results. The miRNA-3178 siRNA group and negative control group were treated with different concentrations of doxorubicin or paclitaxel respectively to detect the chemosensitivity of cells between two groups. (3) MDA-MB-231 cells were treated with 0.250 μmol doxorubicin, 0.40 μmol/L paclitaxel and 2.5 μmol/L genistein, respectively, and the expression of miRNA-3178 in all treated and untreated groups was detected by real-time PCR. The t test was used to compare the growth inhibition rate of cells and miRNA-3178 expression between two groups. One-way analysis of variance was used to compare the expression of miRNA-3178 among multiple groups. The LSD method was used for pairwise comparison.

(1) After being treated with 0.125, 0.250, 0.500 μmol/L doxorubicin for 48 h, the growth inhibition rate of MDA-MB-231 cells was 16.7%±0.4%, 25.9%±0.1% and 44.9%±0.1%, respectively. If combined with 2.5 μmol/L genistein, the growth inhibition rate was 29.8%±0.3%, 45.3%±0.4% and 68.5%±0.4%, respectively, indicating a significant difference (t=38.12, 61.57, 82.70, P<0.001). After MDA-MB-231 cells were treated with 0.20, 0.40, 0.80 μmol/L paclitaxel for 48 h, the growth inhibition rate was 15.3%±0.3%, 27.9%±0.5% and 39.2%±0.1% respectively. If combined with 2.5 μmol/L genistein, the growth inhibition rate was 32.7%±0.7%, 48.3%±0.1% and 63.3%±0.2%, respectively, indicating a significant difference (t=34.41, 58.63, 213.91, all P<0.001). The IC₅₀ was 1.230 μmol/L when MDA-MB-231 cells were treated with doxorubicin alone, and decreased to 0.440 μmol/L if combined with 2.5 μmol/L genistein. The IC₅₀ was 0.64 μmol/L when MDA-MB-231 cells were treated with paclitaxel alone, and decreased to 0.27 μmol/L if combined with 2.5 μmol/L genistein. (2) Under different concentrations of doxorubicin (0.125, 0.250, 0.500 μmol/L) or paclitaxel (0.20, 0.40, 0.80 μmol/L), the growth inhibition rate of MDA-MB-231 cells in miRNA-3178 siRNA group was significantly lower than that in negative control group (transfected with non-sense siRNA) (12.3%±0.6% vs 16.7% ± 0.4%, 21.2% ± 0.9% vs 25.9% ± 0.1%, 27.2% ± 0.9% vs 44.9% ± 0.1%, t=8.99, 7.33, 27.34, all P<0.050; 8.8%±0.5% vs 15.3%±0.3%, 13.4%±1.1% vs 27.9%±0.5%, 20.2%±0.9% vs 39.2%±0.1%, t=16.80, 17.57, 30.48, all P<0.001). (3) The difference in miRNA-3178 expression was statistically significant between treatment groups and untreated groups (F=66.57, P< 0.001). The results of pairwise comparison showed that compared with untreated group, the expression of miRNA-3178 in MDA-MB-231 cells treated with 0.40 μmol/L paclitaxel and 0.250 μmol/L doxorubicin treatment group presented no significant difference (P=0.611, 0.235), and the combination with 2.5 μmol/L genistein significantly increased the expression of miRNA-3178 in MDA-MB-231 cells (11.10±0.33 vs 5.77±0.21, P<0.010).

miRNA-3178 combined with genistein may increase the chemosensitivity of triple negative breast cancer MDA-MB-231 cells to paclitaxel and doxorubicin.