State Key Laboratory of Molecular Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China.
Department of Breast Surgery, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China.
Chin Med J (Engl). 2023 Sep 5;136(17):2086-2100. doi: 10.1097/CM9.0000000000002569.
Metastasis is the main cause of tumor-associated death and mainly responsible for treatment failure of breast cancer. Autophagy accelerates tumor metastasis. In our work, we aimed to investigate the possibility of microRNAs (miRNAs) which participate in the regulation of autophagy to inhibit tumor metastasis.
MiRNA array and comprehensive analysis were performed to identify miRNAs which participated in the regulation of autophagy to inhibit tumor metastasis. The expression levels of miR-3653 in breast cancer tissues and cells were detected by quantitative real-time polymerase chain reaction. In vivo and in vitro assays were conducted to determine the function of miR-3653. The target genes of miR-3653 were detected by a dual luciferase reporter activity assay and Western blot. The relationship between miR-3653 and epithelial-mesenchymal transition (EMT) was assessed by Western blot. Student's t -test was used to analyze the difference between any two groups, and the difference among multiple groups was analyzed with one-way analysis of variance and a Bonferroni post hoc test.
miR-3653 was downregulated in breast cancer cells with high metastatic ability, and high expression of miR-3653 blocked autophagic flux in breast cancer cells. Clinically, low expression of miR-3653 in breast cancer tissues (0.054 ± 0.013 vs . 0.131 ± 0.028, t = 2.475, P = 0.014) was positively correlated with lymph node metastasis (0.015 ± 0.004 vs . 0.078 ± 0.020, t = 2.319, P = 0.023) and poor prognosis ( P < 0.001). miR-3653 ameliorated the malignant phenotypes of breast cancer cells, including proliferation, migration (MDA-MB-231: 0.353 ± 0.013 vs . 1.000 ± 0.038, t = 16.290, P < 0.001; MDA-MB-468: 0.200 ± 0.014 vs . 1.000 ± 0.043, t = 17.530, P < 0.001), invasion (MDA-MB-231: 0.723 ± 0.056 vs . 1.000 ± 0.035, t = 4.223, P = 0.013; MDA-MB-468: 0.222 ± 0.016 vs . 1.000 ± 0.019, t = 31.050, P < 0.001), and colony formation (MDA-MB-231: 0.472 ± 0.022 vs . 1.000 ± 0.022, t = 16.620, P < 0.001; MDA-MB-468: 0.650 ± 0.040 vs . 1.000 ± 0.098, t = 3.297, P = 0.030). The autophagy-associated genes autophagy-related gene 12 ( ATG12 ) and activating molecule in beclin 1-regulated autophagy protein 1 ( AMBRA1 ) are target genes of miR-3653. Further studies showed that miR-3653 inhibited EMT by targeting ATG12 and AMBRA1 .
Our findings suggested that miR-3653 inhibits the autophagy process by targeting ATG12 and AMBRA1 , thereby inhibiting EMT, and provided a new idea and target for the metastasis of breast cancer.
转移是肿瘤相关死亡的主要原因，也是导致乳腺癌治疗失败的主要原因。自噬加速肿瘤转移。在我们的工作中，我们旨在研究参与自噬调控以抑制肿瘤转移的 microRNAs（miRNAs）的可能性。
通过 miRNA 阵列和综合分析来鉴定参与自噬调控以抑制肿瘤转移的 miRNAs。通过实时定量聚合酶链反应检测乳腺癌组织和细胞中 miR-3653 的表达水平。进行体内和体外实验以确定 miR-3653 的功能。通过双荧光素酶报告基因活性测定和 Western blot 检测 miR-3653 的靶基因。通过 Western blot 评估 miR-3653 与上皮-间充质转化（EMT）的关系。采用 Student's t 检验比较任意两组之间的差异，采用单因素方差分析和 Bonferroni 事后检验分析多组之间的差异。
miR-3653 在高转移能力的乳腺癌细胞中下调，高表达 miR-3653 阻断了乳腺癌细胞中的自噬流。临床上，乳腺癌组织中 miR-3653 的低表达（0.054±0.013 比 0.131±0.028，t=2.475，P=0.014）与淋巴结转移（0.015±0.004 比 0.078±0.020，t=2.319，P=0.023）和不良预后（P<0.001）呈正相关。miR-3653 改善了乳腺癌细胞的恶性表型，包括增殖、迁移（MDA-MB-231：0.353±0.013 比 1.000±0.038，t=16.290，P<0.001；MDA-MB-468：0.200±0.014 比 1.000±0.043，t=17.530，P<0.001）、侵袭（MDA-MB-231：0.723±0.056 比 1.000±0.035，t=4.223，P=0.013；MDA-MB-468：0.222±0.016 比 1.000±0.019，t=31.050，P<0.001）和集落形成（MDA-MB-231：0.472±0.022 比 1.000±0.022，t=16.620，P<0.001；MDA-MB-468：0.650±0.040 比 1.000±0.098，t=3.297，P=0.030）。自噬相关基因自噬相关基因 12（ATG12）和 Beclin 1 调节自噬蛋白 1（AMBRA1）是 miR-3653 的靶基因。进一步的研究表明，miR-3653 通过靶向 ATG12 和 AMBRA1 抑制 EMT。
我们的研究结果表明，miR-3653 通过靶向 ATG12 和 AMBRA1 抑制自噬过程，从而抑制 EMT，为乳腺癌的转移提供了新的思路和靶点。
