MiR-9Inhibits Ovarian Serous Carcinoma Cell Proliferation, Migration And Invasion Via Targeting TLN1

Epithelial ovarian carcinoma (EOC) is the sixth leading cancer and the most lethal gynecologic malignancy in women worldwide. Although great progress has been made in surgery and chemotherapy, analysis of trends in overall5-year survival rates for women with EOC indicats only modest improvement from37%to46%in the past30years. One of the greatest obstacles to improving EOC outcome is the poor understanding of the molecular underpinnings of EOC pathogenesis and progression. EOC is a highly heterogeneous disease, with ovarian serous carcinoma (OSC) being the most common histologic subtype. microRNAs (miRNAs) are a large family of non-coding small RNAs that post-translationally regulate gene expression by mediating target mRNA cleavage or translational inhibition through binding with the3’untranslated region (3’UTR) of the target mRNA. Recent evidence indicates that miRNAs can function as oncogenes or tumor suppressor genes. Increasing amounts of studies show that miRNAs are aberrantly expressed in EOC and may be involved in EOC tumorigenesis and progression. TLN1encodes a focal adhision protein (talinl) which can interact with multiple cytoskeleton proteins and play essential roles in cell adhesion and motility. Recent studies demonstrate that TLN1is upregulated in cancers of the prostate, liver and oral cavity, and that TLN1overexpression is correlated with cancer progression and metastasis. Our previous study found elevated serum TLN1protein levels in both the initiation and progression phases of chemically induced OSC in rats. This finding hints that TLN1may play a role in OSC.To investigate the clinical relevance of TLN1in OSC, human OSC specimens were subjected to immunoprofiling for TLN1expression. Staining intensity was quantitated by Fromowitz’s standard to determine the cytoplasmic levels of TLN1. Results show that TLN1was overexpressed in OSC tissues. In addition, the intensity of TLN1immunoreactivity in lymph node metastasis was significantly higher compared with primary tumors. Interestingly, TLN1was differentially expressed between low-grade and high-grade OSC, i.e., TLN1level in high-grade tumors was increased compared with low-grade tumors. These findings suggest that TLN1overexpression may contribute to the aggressive phenotype of OSC and promote cancer metastasis.The mechanism underlying the upregulation of TLN1is largely unknown. The present study hypothesized that miRNAs might be associated with the aberrent expression of TLNl in OSC. To elucidate the role of miRNAs in TLN1upregulation in OSC cells, a putative miRNA--miR-9, which is downregulated in OSC, was computationally predicted as a possible regulatory miRNA for TLN1. Subsequent experiments demonstrated that miR-9inhibited TLN1expression at both protein and mRNA levels. Furthermore, luciferase reporter assay showed that miR-9directly interacted with TLN13’UTR. Thus TLN1was identified as a novel target of miR-9.To reveal the function of miR-9in OSC cells and the underlying mechanism, effects of miR-9on cell viability, colony formation, apoptosis,migration and invasion were tested. Since it was demonstrated that miR-9expression was down-regulated in SKOV3, CAOV3and OVCAR3OSC cells compared with the HOSEpiC surface epithelial cell line, a "gain-of-function" approach by exogenous miR-9transfection was used. Overexpression of miR-9inhibited OSC cell proliferation, migration and invasion, but did not affect OSC cell apoptosis. Knockdown of TLN1in OSC cells mimicked the effect observed when miR-9was overexpressed by decreasing cell proliferation, migration and invasion. Furthermore, TLN1-regulated FAK/AKT signaling pathway was downactivated by miR-9overexpression. These results indicate that miR-9exerts anti-proliferative, anti-migratory and anti-invasive activity in OSC, and that direct targeting of TLN1may contribute to the tumor-suppressor role of miR-9.In summary, the present study identified a focal adhesion protein, TLN1,whose expression correlated with OSC aggressiveness and progression, as a novel target of miR-9. miR-9functions as a tumor suppressor in OSC via targeting TLN1and downactivating the FAK/AKT signaling pathway. These findings might be useful for understanding OSC pathogenesis and providing a new target for OSC therapeuitics.