摘要
目的研究CD83对三阴性乳腺癌细胞增殖、凋亡、周期、迁移和侵袭过程的影响,探讨其调控三阴性乳腺癌细胞生物学行为的机制。方法从TCGA数据库下载822个乳腺癌组织样本和572个正常组织样本,分析CD83在乳腺癌组织中的表达差异。并通过Western blot检测人正常乳腺上皮细胞MCF-10A和乳腺癌细胞MCF-7、BT-549、MDA-MB-231这4种细胞中CD83的蛋白表达量。使用CRISPR/Cas9方法建立稳定敲除CD83的MDA-MB-231细胞株sg-CD83-#1、sg-CD83-#2和sg-CD83-#3,提取细胞蛋白检验敲除效率,根据检测结果选取敲除效果最好的细胞株(sg-CD83)进行后续实验。采用细胞计数试剂盒8(CCK-8)、划痕愈合实验和Transwell法检测sg-CD83组的增殖、迁移和侵袭能力,sg-ctrl组作为CD83敲除的对照。通过Co-IP分析CD83与ITGB1之间的分子相互作用。通过质粒转染建立CD83过表达的MDA-MB-231细胞株,采用划痕愈合实验和Transwell法检测ov-CD83组的增殖、迁移和侵袭能力,vector组作为对照。通过敲低CD83过表达细胞中的ITGB1,检测ITGB1对CD83功能的影响,si-NC组作为ITGB1敲低的对照。定量资料的组间比较采用独立样本t检验,多组间比较采用单因素方差分析,多组两两比较采用LSD法。结果TCGA数据库的生物信息学分析发现在Luminal A型(4.63±0.79)、Luminal B型(4.57±0.77)、HER-2过表达型(4.62±0.75)、TNBC(5.40±1.13)和正常组织(3.30±1.17)中的CD83的mRNA表达量比较,差异有统计学意义(F=196.802,P<0.001)。TNBC中CD83表达高于其他3种类型(P均<0.001)。MCF-10A、MCF-7、BT-549、MDA-MB-231细胞中CD83的蛋白表达量分别为1.00±0.01、1.44±0.02、1.71±0.02、1.89±0.07,差异有统计学意义(F=353.070,P<0.001)。sg-ctrl、sg-CD83-#1、sg-CD83-#2、sg-CD83-#3组MDA-MB-231细胞中CD83的蛋白表达量分别为1.00±0.02、0.79±0.18、0.32±0.03、0.23±0.05,差异有统计学意义(F=48.758,P<0.001)。sg-CD83-#3组敲除效果最好,因此选取sg-CD83-#3组细胞株作为敲除组用于后续实验(sg-CD83)。sg-ctrl组和sg-CD83组的细胞在24、48、72和96 h时450 nm处的吸光度分别为0.63±0.05比0.51±0.03、0.92±0.03比0.70±0.06、1.27±0.02比0.90±0.09、1.54±0.14比1.06±0.09,差异有统计学意义(P均<0.050)。sg-ctrl组和sg-CD83组凋亡率[(5.96±0.06)%比(12.82±0.07)%;t=-128.877,P<0.001]、G_(0)/G_(1)期细胞比例[(31.87±0.76)%比(44.75±1.50)%;t=-13.229,P<0.001]、细胞划痕愈合率[(34.47±0.87)%比(16.63±0.70)%;t=27.554,P<0.001]、迁移细胞数(143.67±4.16比62.67±1.53;t=31.636,P<0.001)、侵袭细胞数(123.67±3.06比26.33±2.52;t=42.593,P<0.001)比较,差异均有统计学意义。sg-ctrl组和sg-CD83组中ITGB1表达量分别为1.00±0.02、0.34±0.02,差异有统计学意义(t=40.417,P<0.001);黏着斑激酶(FAK)表达量分别为1.00±0.01、0.79±0.07,差异有统计学意义(t=5.144,P=0.033);磷酸化FAK(p-FAK)表达量分别为0.99±0.03、0.35±0.05,差异有统计学意义(t=19.395,P<0.001)。Co-IP实验中,CD83与ITGB1被共同沉淀。vector组和ov-CD83组细胞划痕愈合率[(8.40±2.73)%比(45.08±1.51)%;t=-20.340,P<0.001]、迁移细胞数(151.00±2.65比337.67±3.51;t=-73.532,P<0.001)、侵袭细胞数(120.33±4.16比288.67±4.51;t=-47.506,P<0.001)比较,差异均有统计学意义。si-NC组和si-ITGB1组细胞划痕愈合率[(44.63±2.11)%比(13.93±2.90)%;t=14.839,P<0.001]、迁移细胞数(339.67±3.06比181.67±2.31;t=71.458,P<0.001)、侵袭细胞数(289.00±5.29比170.33±5.03;t=28.144,P<0.001)比较,差异均有统计学意义。结论CD83通过ITGB1/FAK信号轴调控三阴性乳腺癌细胞迁移和侵袭,可能作为潜在的乳腺癌治疗靶点。
Objective To investigate the effect of CD83 on the proliferation,apoptosis,cell cycle progression,migration,and invasion of triple negative breast cancer cells,and to explore the mechanisms underlying its regulation of the biological behaviors of these malignant cells.Methods To evaluate CD83 expression in breast cancer,we retrieved 822 breast cancer and 572 normal tissue samples from TCGA.This dataset enabled analysis of CD83 expression differences in breast cancer.We also used Western blot to measure CD83 protein levels in MCF-10A(normal breast epithelial cells)and breast cancer cells(MCF-7,BT-549,MDA-MB-231).This method facilitated direct CD83 protein level comparison between normal and cancerous breast cells,enhancing insights into CD83’s role in breast cancer.Using the CRISPR/Cas9,we established stable CD83 knockout MDA-MB-231 cell lines(sg-CD83-#1,sg-CD83-#2,sg-CD83-#3)and selected sg-CD83 for further study based on protein extraction and CD83 disruption efficiency.Functional assays including CCK-8,scratch wound healing,and Transwell were conducted on sg-CD83 versus sg-ctrl,sg-ctrl as the negative control for CD83 knockout.Co-IP was used to examine CD83-ITGB1 interactions.Establishment of MDA-MB-231 cell line overexpressing CD83 through plasmid transfection,and created an ov-CD83 group to study CD83 overexpression effects,assessing proliferation,migration,and invasion via scratch and Transwell assays against vector,which as a control for CD83 overexpression.The impact of ITGB1 on CD83 function was assessed by silencing ITGB1 in CD83-overexpressing cells,using the si-NC as a control for ITGB1 knockdown.Statistical analysis involved independent t-tests for intergroup comparisons and one-way ANOVA with LSD post hoc tests for multiple groups,ensuring robustness of findings.Results TCGA database analysis revealed significant CD83 mRNA expression differences among breast cancer subtypes and normal tissue.CD83 levels were 4.63±0.79 in lunimal A subtype,4.57±0.77 in luminal B subtype,4.62±0.75 in HER-2 overexpression subtype and 5.40±1.13 in TNBC subtype and 3.30±1.17 in normal tissue.Statistical analysis showed highly significant differences(F=196.802,P<0.001),with TNBC showing notably higher CD83 expression than other subtypes(P<0.001).This suggests CD83’s potential as a TNBC biomarker and therapeutic target,highlighting the need for further functional studies in this breast cancer subtype.The protein expression levels of CD83 in MCF-10A,MCF-7,BT-549,and MDA-MB-231 cells were 1.00±0.01,1.44±0.02,1.71±0.02,and 1.89±0.07,respectively,showing statistical significance(F=353.070,P<0.001).The sg-CD83-#3 group showed the best CD83 knockout efficiency.Thus,it was selected as the CD83 knockout model(sg-CD83)for further experiments.The protein expression of CD83 in MDA-MB-231 cells in the four groups of sg-ctrl,sg-CD83-#1,sg-CD83-#2,and sg-CD83-#3 were 1.00±0.02,0.79±0.18,0.32±0.03,and 0.23±0.05,respectively,with statistical significance(F=48.758,P<0.001).The absorbance at 450 nm for the sg-ctrl group and the sg-CD83 group at 24,48,72,and 96 hours was as follows:0.63±0.05 vs 0.51±0.03,0.92±0.03 vs 0.70±0.06,1.27±0.02 vs 0.90±0.09,and 1.54±0.14 vs 1.06±0.09.These differences were statistically significant(all P<0.050).The apoptosis rates in the sg-ctrl group and sg-CD83 group were[(5.96±0.06)%vs(12.82±0.07)%,(t=-128.877,P<0.001)],the G_(0)/G_(1)phase cell ratio was[(31.87±0.76)%vs(44.75±1.50)%,(t=-13.229,P<0.001)];There were statistically significant differences in cell scratch healing rate[(34.47±0.87)%vs(16.63±0.70)%,(t=27.554,P<0.001)],the number of migrating cells(143.67±4.16 vs 62.67±1.53,t=31.636,P<0.001),and the number of invasive cells(123.67±3.06 vs 26.33±2.52,t=42.593,P<0.001).The expression levels of ITGB1 were 1.00±0.02 in the sg-ctrl group and 0.34±0.02 in the sg-CD83 group,showing statistical significance(t=40.417,P<0.001).Similarly,FAK expression was 1.00±0.01 in the sg-ctrl group and 0.79±0.07 in the sg-CD83 group(t=5.144,P=0.033).The p-FAK levels were 0.99±0.03 and 0.35±0.05,respectively,with a statistically significant difference(t=19.395,P<0.001).In the co-immunoprecipitation(Co-IP)assay,CD83 and ITGB1 were co-precipitated.There were statistically significant differences in scratch wound healing rates[(8.40±2.73)%vs(45.08±1.51)%,t=-20.340,P<0.001],migratory cell counts(151.00±2.65 vs 337.67±3.51,t=-73.532,P<0.001),and invasive cell counts(120.33±4.16 vs 288.67±4.51,t=-47.506,P<0.001)between the vector group and ov-CD83 group.There were statistically significant differences in scratch wound healing rates[(44.63±2.11)%vs(13.93±2.90)%,t=14.839,P<0.001],migratory cell counts(339.67±3.06 vs 181.67±2.31,t=71.458,P<0.001),and invasive cell counts(289.00±5.29 vs 170.33±5.03,t=28.144,P<0.001)between the si-NC group and si-ITGB1 group.Conclusion CD83 regulates the migration and invasion of triple negative breast cancer cells through the ITGB1/FAK signaling pathway and may serve as a potential target for breast cancer.
作者
马庆东
万韦君
罗东林
Ma Qingdong;Wan Weijun;Luo Donglin(Department of Breast and Thyroid Surgery,Army Medical Center,Chongqing 400042,China;National Key Laboratory of Trauma and Chemical Poisoning,Army Medical Center,Chongqing 400042,China;Department of Stem Cell and Regenerative Medicine,Army Medical Center,Chongqing 400042,China)
出处
《中华乳腺病杂志(电子版)》
2025年第3期145-153,共9页
Chinese Journal of Breast Disease(Electronic Edition)
基金
重庆市自然科学基金项目(CSTB2023NSCQ-BHX0199)
军事医学重点临床学科建设(普通外科)项目。
关键词
乳腺肿瘤
肿瘤侵袭
CD83
Breast neoplasms
Neoplasm invasiveness
CD83
