【摘要】：目的檢測E6-AP基因及膜聯(lián)蛋白A2(Annexin A2)在乳腺癌MDA-MB-231細(xì)胞中的表達(dá),探討其對癌細(xì)胞增殖、凋亡及浸潤轉(zhuǎn)移的影響。方法設(shè)計(jì)1條無關(guān)序列Negative-siRNA作為陰性對照組和針對E6-AP基因的3條特異性E6-AP-siRNAs片段轉(zhuǎn)染至MDA-MB-231細(xì)胞內(nèi)作為實(shí)驗(yàn)組,未經(jīng)處理細(xì)胞作為空白對照組,加入脂質(zhì)體處理的細(xì)胞為脂質(zhì)體組,利用RT-PCR檢測干擾E6-AP后在MDA-MB-231細(xì)胞中E6-AP和Annexin A2 mRNA相對表達(dá)水平。選擇出轉(zhuǎn)染效率最高的E6-APsiRNA1組及陰性對照組、空白對照組繼續(xù)行后續(xù)實(shí)驗(yàn)。Western blot檢測干擾E6-AP后E6-AP和Annexin A2在MDA-MB-231細(xì)胞中的蛋白的相對表達(dá)水平。利用CCK-8試劑盒法、流式細(xì)胞術(shù)、Transwell小室侵襲實(shí)驗(yàn)分別檢測干擾E6-AP后MDA-MB-231細(xì)胞的增殖、凋亡、侵襲能力�；虻膍RNA及蛋白表達(dá)水平、細(xì)胞凋亡率及細(xì)胞數(shù)的組間比較采用方差分析,兩兩比較采用LSD法,吸光度比較采用重復(fù)測量的方差分析。結(jié)果轉(zhuǎn)染72 h后,E6-AP基因干擾后各實(shí)驗(yàn)組(E6-AP-siRNA1組、E6-AP-siRNA2組、E6-AP-siRNA3組)及空白對照組、陰性對照組及脂質(zhì)體組中的E6-AP mRNA相對表達(dá)水平分別為0.159±0.003、0.325±0.006、0.229±0.007、0.593±0.031、0.594±0.012、0.612±0.016,Annexin A2 mRNA相對表達(dá)水平分別為0.929±0.017、1.013±0.082、0.992±0.024、1.341±0.037、1.323±0.010、1.326±0.012,差異均有統(tǒng)計(jì)學(xué)意義(F=850.792、417.447,P均0.050)。轉(zhuǎn)染72 h后,E6-AP-siRNA1組、空白對照組和陰性對照組中E6-AP及Annexin A2蛋白相對表達(dá)水平為分別為0.271±0.017、0.492±0.018、0.477±0.016及0.447±0.034、0.887±0.022、0.849±0.033,組間差異均有統(tǒng)計(jì)學(xué)意義(F=256.850、350.149,P均0.050)。轉(zhuǎn)染24、48、72、96 h后,E6-AP-siRNA1組、陰性對照組和空白對照組間比較,不同時間點(diǎn)之間比較,細(xì)胞吸光度差異均有統(tǒng)計(jì)學(xué)意義(F=524.828,P0.001;F=904.079,P0.001);分組與時間點(diǎn)存在交互作用(F=28.116,P0.001)。轉(zhuǎn)染72 h后,空白對照組、陰性對照組、E6-AP-siRNA1組的凋亡率分別為2.959±0.117、3.097±0.070、10.812±0.199,組間差異有統(tǒng)計(jì)學(xué)意義(F=3110.005,P0.050)。Transwell檢測E6-AP-siRNA1組、空白對照組、陰性對照組中細(xì)胞穿透Matrigel膠到達(dá)Transwell下室的細(xì)胞數(shù)分別為99±5、96±6、62±7,組間差異有統(tǒng)計(jì)學(xué)意義(F=55.404,P0.001)。結(jié)論干擾E6-AP基因可使Annexin A2表達(dá)下調(diào),同時可誘導(dǎo)MDA-MB-231細(xì)胞的凋亡,其增殖、侵襲能力也受到抑制。
[Abstract]:Objective to detect the expression of E6-AP gene and Annexin A2 in breast cancer MDA-MB-231 cells, and to investigate the effects of Annexin A2 on the proliferation, apoptosis, invasion and metastasis of breast cancer cells. Methods A sequence of unrelated Negative-siRNA was designed as negative control group and three specific E6-AP-siRNAs fragments for E6-AP gene were transfected into MDA-MB-231 cells as experimental group, untreated cells as blank control group and liposome treated cells as liposome group. The relative expression of E6-AP and Annexin A2 mRNA in MDA-MB-231 cells after interfering with E6-AP was detected by RT-PCR. The highest transfection efficiency of E6-APsiRNA1 group and negative control group were selected, and the control group continued to do the following experiment. Western blot was used to detect the relative expression level of E6-AP and Annexin A2 in MDA-MB-231 cells after interfering with E6-AP. The proliferation, apoptosis and invasiveness of MDA-MB-231 cells after interfering with E6-AP were detected by CCK-8 kit and flow cytometry. The expression level of mRNA and protein, cell apoptosis rate and cell number were compared by ANOVA, LSD method and absorbance analysis were used to compare the expression level of mRNA and protein, the rate of apoptosis and the number of cells. Results after 72 h of transfection, each experimental group (E6-AP-siRNA2 group) and blank control group (E6-AP-siRNA2 group) were transfected with E6-AP gene interference. The relative expression level of E6-AP mRNA in the negative control group and the liposome group was 0.159 鹵0.003n 0.325 鹵0.006U 0.229 鹵0.007 鹵0.593 鹵0.031U 0.594 鹵0.0121.0.612 鹵0.016Annexin A2 mRNA, respectively 0.929 鹵0.0171.013 鹵0.0820.92 鹵0.0241.341 鹵0.0371.323 鹵0.0101.323 鹵0.0101.323 鹵0.0101.323 鹵0.0101.3261.326 鹵0.0122.There were significant differences between the two groups (FG 850.792417.447P = 0.050). After 72 hours of transfection, the relative expression levels of E6-AP and Annexin A2 protein in the E6-AP-siRNA1 group, the blank control group and the negative control group were 0.271 鹵0.017, 0.492 鹵0.018, 0.477 鹵0.016 and 0.447 鹵0.034, 0.887 鹵0.022, 0.849 鹵0.033, respectively. There were significant differences between the two groups (F256.850350.149, P = 0.050). E6-AP-siRNA1 group, negative control group and blank control group had significant difference in cell absorbance at different time points (FF524.828P0.001, F904.079, P0.001), and there was interaction between the group and the time point (F28.116, P0.001) after transfection for 24 ~ 448 ~ 72H ~ (72) h after transfection, the difference of cell absorbance was statistically significant between the negative control group and the blank control group (F _ (524.828) P _ (0.001) F _ (904.079) P _ (0.001). 72 h after transfection, the apoptotic rate of control group and negative control group were 2.959 鹵0.117 鹵3.097 鹵0.070 ~ 10.812 鹵0.199, respectively. The difference was statistically significant (F _ (3110.005) P _ (0.050) .Transwell detection of E6-AP-siRNA1 group, blank control group, and control group, the apoptosis rate of E6-AP-siRNA1 group was 2.959 鹵0.117 鹵3.097 鹵0.070 鹵10.812 鹵0.199, respectively. In the negative control group, the number of cells penetrating the Matrigel gel to the lower chamber of Transwell was 99 鹵50.96 鹵60.62 鹵7, respectively, and the difference was statistically significant (FF55.404, P0.001). Conclusion interfering with E6-AP gene can down-regulate the expression of Annexin A2, induce apoptosis of MDA-MB-231 cells, and inhibit the proliferation and invasion of MDA-MB-231 cells.
【作者單位】： 川北醫(yī)學(xué)院附屬醫(yī)院甲狀腺乳腺外科;川北醫(yī)學(xué)院附屬醫(yī)院肝膽胰腸研究所;遂寧市中心醫(yī)院乳腺甲狀腺外科;第三軍醫(yī)大學(xué)附屬西南醫(yī)院乳腺外科;川北醫(yī)學(xué)院附屬醫(yī)院解剖學(xué)教研室;
【基金】：國家自然科學(xué)基金資助項(xiàng)目(81172496) 四川省教育廳重點(diǎn)項(xiàng)目(13ZA0228);四川省科技廳科技創(chuàng)新苗子工程項(xiàng)目(2016060)
【分類號】：R737.9
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本文編號：2148087