探讨线粒体基质导入因子23（MIX23）影响三阴性乳腺癌（TNBC）的分子机制。

从癌症基因组图谱（TCGA）数据库下载乳腺癌患者的 RNA-Seq表达谱数据及其对应的临床信息，共纳入1 097例原发肿瘤组织和114例癌旁正常组织样本用于后续分析。从人类蛋白质图谱（HPA）数据库获取乳腺癌组织中MIX23的免疫组织化学染色图像（肿瘤组织样本11例，癌旁正常组织样本6例）并对蛋白表达水平进行定量分析。下载METABRIC乳腺癌队列（1 980例）的表达谱数据作为独立验证集。基于MIX23在乳腺癌患者中的表达均值，将TCGA数据与METABRIC数据各划分为高、低表达两组（TCGA：高表达480例，低表达534例；METABRIC：高表达969例，低表达1 011例）。采用Kaplan-Meier法绘制生存曲线，并通过Log-rank检验评估两组间生存率差异。通过RT-qPCR和Western blot检测TNBC细胞系 MDA-MB-231中MIX23的mRNA和蛋白表达。利用慢病毒载体构建稳定敲低MIX23的MDA-MB-231细胞（sh-MIX23组），将表达非靶向 scramble shRNA 慢病毒载体的细胞作为对照组。通过克隆形成、CCK-8及细胞划痕实验检测细胞增殖及迁移能力。基于TCGA数据进行差异基因分析，筛选下游靶点。

在TCGA队列中，与癌旁正常组织相比，MIX23 mRNA在乳腺癌组织中的表达水平显著升高（7.756±0.605比7.145±0.341，t=16.613，P<0.001）。HPA队列分析显示MIX23在肿瘤组织中的表达高于癌旁正常组织（43.978±4.158比55.211±7.339， t=-2.980， P=0.018）。MIX23在TNBC、HER-2阳性、Luminal A及Luminal B型不同亚型组织中的表达水平分别为8.154±0.743、7.858±0.628、7.613±0.535、7.744±0.477，4组间比较差异具有统计学意义（F=28.260，P<0.001）。Kaplan-Meier生存分析结果显示，在TCGA队列中，MIX23的高表达与无病间期缩短显著相关（HR=1.667，95%CI：1.087~2.555，P=0.018）。MIX23敲低后，与对照组相比，细胞克隆形成能力显著降低（184.500±9.576比110.200±6.976，t=12.578，P<0.001），增殖能力在24、48、72 h受到显著抑制（0 h：0.185±0.009比0.182±0.007，t=0.515，P=0.626；24 h：0.399±0.057比0.246±0.004，t=4.333，P=0.006；48 h：0.704±0.058比0.403±0.021，t=9.642，P<0.001；72 h：0.999±0.0416比0.452±0.031，t=21.110，P<0.000 1），24 h后的细胞相对迁移面积显著小于对照组（62.861±12.252比35.971±8.024，t=5.193，P<0.01）。与对照组比较，MIX23可以上调骨诱导因子（OGN）表达（1.195±0.254比1.958±0.223，t=-3.945，P=0.018），Akt、mTOR、EGFR表达明显下调（1.000±0.023比0.054±0.006，t=68.352，P<0.001；1.000±0.052比0.058±0.007，t=31.092，P<0.001；1.000±0.094比0.036±0.004，t=17.785，P<0.001）。

MIX23在乳腺癌组织中呈高表达且与预后不良相关，敲低MIX23可通过上调OGN并抑制下游EGFR/Akt/mTOR通路，进而抑制乳腺癌生长及转移。

To explore the molecular mechanism by which mitochondrial matrix import factor 23 (MIX23) affects triple-negative breast cancer (TNBC).

The RNA-Seq expression profile and corresponding clinical data of breast cancer patients were downloaded from The Cancer Genome Atlas (TCGA) database, including 1 097 primary tumor tissue samples and 114 adjacent normal tissue samples. Immunohistochemistry staining images of MIX23 in breast cancer tissues were obtained from the Human Protein Atlas (HPA) database for quantitative analysis of protein expression levels. Expression profile data of the METABRIC breast cancer cohort were downloaded as an independent validation set. Based on the mean expression level of MIX23 in breast cancer patients, the TCGA and METABRIC datasets were each stratified into high- and low-expression groups (TCGA: 480 cases in high-expression group, 534 in low-expression group; METABRIC: 969 in high-expression group, 1,011 in low-expression group). Kaplan-Meier method was used to plot survival curves, and log-rank test was used to evaluate the difference in survival rate between the two groups. RT-qPCR and Western blot were used to detect the mRNA and protein expression of MIX23 in TNBC cell line. Lentiviral vector was used to construct MDA-MB-231 cells with stable MIX23 knockdown (sh-MIX23 group), with the cells transfected with lentiviral vectors carrying non-targeted scramble shRNA as control group. Cell clone formation assay, CCK-8 assay and cell scratch assay were adopted to detect cell proliferation and migration abilities. Differential gene analysis was performed based on TCGA database to identify downstream target genes.

In the TCGA cohort, the mRNA expression level of MIX23 was significantly higher in breast cancer tissues than in adjacent normal breast tissues (7.756±0.605 vs 7.145±0.341, t=16.613, P<0.001). HPA cohort analysis also showed that MIX23 expression was significantly higher in tumor tissues than in normal tissues (43.978±4.158 vs 55.211±7.339, t=-2.980, P=0.018). The expression levels of MIX23 in TNBC, HER-2 positive, Luminal A and Luminal B subtypes were 8.154±0.743, 7.858±0.628, 7.613±0.535 and 7.744±0.477, respectively, with significant difference (F=28.260, P<0.0001). Kaplan-Meier survival analysis revealed that in the TCGA cohort, high MIX23 expression was significantly associated with shorter disease-free interval (HR=1.667, 95%CI: 1.087–2.555, P=0.018). After MIX23 knockdown, compared with control group, the colony formation ability of cells in the sh-MIX23 group was significantly decreased (184.5±9.576 vs 110.2±6.976, t=12.578, P<0.001), the cell proliferation was markedly inhibited at 24, 48 and 72 h (0 h: 0.185±0.009 vs 0.182±0.007, t=0.515, P=0.626; 24 h: 0.399±0.057 vs 0.246±0.004, t=4.333, P=0.006; 48 h: 0.704±0.058 vs 0.403±0.021, t=9.642, P<0.001; 72 h: 0.999±0.0416 vs 0.452±0.031, t=21.110, P<0.0001). The relative migration area of cells in the sh-MIX23 group was significantly smaller than that in the control group at 24 h after scratching (62.861±12.252 vs 35.971±8.024, t=5.193, P<0.01). Compared with the control group, MIX23 could up-regulate the expression of osteoglycin (1.195±0.254 vs 1.958±0.223, t=-3.945, P=0.018) and inhibit the expression of Akt, mTOR and EGFR (1.000±0.023 vs 0.054±0.006, t=68.352, P<0.0001; 1.000±0.052 vs 0.058±0.007, t=31.092, P<0.0001; 1.000±0.094 vs 0.036±0.004, t=17.785, P<0.0001).

MIX23 is highly expressed in breast cancer and correlated with poor prognosis. Knockdown of MIX23 affects the growth and metastasis of breast cancer by up-regulating osteoglycin and inhibiting the downstream EGFR/Akt/mTOR signaling pathway.