miR-450a acts as a tumor suppressor in ovarian cancer by readjusting energy metabolism. miR-450a acts as a tumor suppressor in ovarian cancer by readjusting energy metabolism

Dysregulation of miRNA expression is associated with multiple diseases, including cancers where they can have oncogenic or tumor suppressive function. Here we investigated the potential tumor suppressive function of miR-450a, one of the most significantly downregulated miRNAs in ovarian cancer. RNAseq analysis revealed multipe genes involved in the epithelial-to-mesenchymal transition (EMT) were suppressed by miR-450a overexpression ovarian cancer cell line A2780. Consistently, miR-450a overexpression reduced tumor migration, invasion and increased anoikis in A2780 and SKOV-3 cell lines and reduced tumor growth in ovarian xenographic model. Combining AGO-PAR-CLIP and RNAseq analysis, we identified a panel of potential miR-450a targets of which many, including TIMMDC1, MT-ND2, ACO2 and ATP5B, regulate energetic metabolism. miR-450a expression indeed decreased mitochondrial membrane potential but increased glucose uptake and viability after glutamine withdrawal, characteristics of less invasive ovarian cancer cell lines, which are also less dependent on glutamine. In summary, we propose in this work that miR-450a acts as a tumor suppressor in ovarian cancer cells by modulating targets associated with glutaminolysis, which would lead to a decrease in the production of lipids, amino acids and nucleic acids, and also inhibition of signaling pathways associated with EMT Overall design: RNA-Seq from 3 different biological samples from SKOV3 and A2780 cell lines after miR-450a overexpression; small RNA-Seq from 3 different biological from A2780 cell line after miR-450a or miR-450b overexpression; AGO 4SU-PAR-CLIP from 2 to 4 biological samples from A2780 cells lines after miR-450a overexpression.

微小RNA（miRNA）表达失调与多种疾病存在密切关联，在癌症中，这类RNA可发挥致癌或抑癌作用。本研究针对卵巢癌中下调幅度最为显著的miRNA之一miR-450a，探究其潜在的抑癌功能。RNA测序（RNAseq）分析显示，在卵巢癌细胞系A2780中过表达miR-450a后，诸多参与上皮间质转化（EMT）的基因的表达受到抑制。功能验证实验结果一致显示：在A2780与SKOV-3细胞系中过表达miR-450a可抑制肿瘤迁移与侵袭，同时促进细胞失巢凋亡；在卵巢癌异种移植模型中则可延缓肿瘤生长。结合AGO-PAR-CLIP与RNAseq分析结果，本研究鉴定出一组miR-450a的潜在靶基因，其中诸多基因（如TIMMDC1、MT-ND2、ACO2及ATP5B）均参与能量代谢调控。实验证实，miR-450a的表达可降低线粒体膜电位，但在谷氨酰胺剥夺条件下可提升细胞的葡萄糖摄取能力与存活率，这恰好是侵袭性更低、对谷氨酰胺依赖程度更低的卵巢癌细胞系的特征。综上，本研究提出：miR-450a可通过靶向调控与谷氨酰胺分解相关的靶基因，在卵巢癌细胞中发挥抑癌功能；这一调控过程可减少脂质、氨基酸与核酸的合成，并抑制与上皮间质转化相关的信号通路。

实验整体设计：
1. 对过表达miR-450a的SKOV3与A2780细胞系，各取3份生物学重复样本进行RNA测序；
2. 对过表达miR-450a或miR-450b的A2780细胞系，取3份生物学重复样本进行小RNA测序（small RNA-Seq）；
3. 对过表达miR-450a的A2780细胞系，取2~4份生物学重复样本进行AGO 4SU-PAR-CLIP实验。