1.miR-17/20a Impedes Invasion And Metastasis Of Esophageal Cancer By Modulating TGF-β Pathway 2. MiR-92b Represses Invasion And Metastasis Of Esophageal Cancer By Targeting ITGAV

As small non-coding RNAs, microRNAs are capable of regulating tumor progression and metastasis via direct interaction with target genes. By chemotaxis model assay in vitro, we previously established two groups of esophageal squamous cell carcinoma (ESCC) sublines, namely 30-U/D and 180-U/D, with distinct motility capacity. Then we performed a miRNA microarray analysis of these four cell sublines. It is worth noting that miR-17 and miR-20a was overexpressed in the "U" sublines, which were less mobile, suggesting the correlation between the two microRNAs and ESCC cell migration. miR-17 and miR-20a are both located in miR-17-92 cluster, with the same seed sequence. Previous studies have revealed their diverse roles in other tumor types, but rarely reported in ESCC. In this study, we aimed at finding out the function and mechanism of miR-17/20a in ESCC.Firstly, we verified the outcome derived from microRNA chip. Compared with 30-D or 180-D, miR-17/20a showed a higher level in 30-U or 180-U cells. Subsequent functional experiments revealed that miR-17/20a dramatically inhibited the cell migration and invasion of ESCC without exerting influence on proliferation and apoptosis. Then, predicted by databases, numerous genes would probably be modulated by miR-17/20a. Among the candidates, TGFBR2 and SARA were confirmed to be the functional target genes of miR-17/20a, through the luciferase reporter assay, Western blot and transwell experiments, demonstrating that these genes were able to affect the migration and invasion of ESCC. Interestingly, TGFBR2 and SARA are both implicated in TGF-β pathway, which acts as either a tumor promoter or a tumor suppressor depending on cellular contexts. Our previous study showed that TGF-β treatment could promote ESCC migration. Therefore, we conducted further study to elucidate whether miR-17/20a could attenuate the activation of TGF-β pathway. We then found that miR-17/20a could weaken the phosphorylation of Smad2, which is crucial in the TGF-P signaling pathway. Simultaneously, with TGF-β treatment,30-D cells turned to be more mobile. These results indicated that miR-17/20a could suppress migration and invasion of ESCC by inhibiting TGF-P pathway. Moreover, miR-17/20a decreased pulmonary arrest of 30-D in an experimental model of lung metastasis, which further confirmed their negative impacts on ESCC metastasis. However, analysis of clinical samples and GEO archives concerned showed no significant differences in expressions of TGFBR2 and SARA between ESCC and adjacent normal tissues.In conclusion, our study identified for the first time that miR-17/20a impeded migration, even invasion-metastasis of ESCC cells while exerted little influence on proliferation and apoptosis. As the genuine target genes of miR-17/20a, TGFBR2 and SARA silencing hampered ESCC cell migration and invasion, although there were no significant differences of their expressions between ESCC and normal tissues. Moreover, miR-17/20a attenuated the TGF-β pathway to inhibit the migration and invasion of ESCC. Together, these results deepen our comprehension! of function and mechanism of miR-17/20a in ESCC local invasion and metastasis, and provided new insight into anti-cancer strategies and drug development.Integrins, a large family of transmembrane adhesion receptors for extracellular matrix (ECM), play crucial roles in migration and invasion of tumor cells, not only for physically tethering cells to ECM, but also for sending and receiving molecular signals that modulate these processes. The family consists of 26 members, including 18 a subunits and 8 β subunits, which form at least 24 distinct heterodimers on cell surface. Our previous study showed that miR-92b could repress invasion and metastasis of esophageal squamous cell carcinoma (ESCC) by targeting Integrin a6 (ITGA6). Further, in silico analysis revealed Integrin aV (ITGAV) was probably another target gene of miR-92b. In this study, we sought to find out the association between miR-92b and ITGAV, as well as their impacts on invasion and metastasis in ESCC.Firstly, through RT-qPCR experiment, we detected the expressions of miR-92b and ITGAV in 30-D, KYSE450 and KYSE510 cells with distinct motility capacities. The results showed that the expression of ITGAV correlated positively with cellular motility whereas negatively with the expression of miR-92b. Overexpression or knockdown of miR-92b led to decreased or increased ITGAV expression respectively. According to results from the ensuing luciferase reporter assay and functional experiments, we confirmed that ITGAV was the bona fide target of miR-92b, which affected the migration and invasion of ESCC.Then, we sought to reveal the underlying molecular mechanisms regulated by miR-92b/ITGAV in ESCC. Under chemotaxis condition, overexpression of miR-92b decreased FAK mediated activation of Rac1. Likewise, similar resultscould be observed in the ITGAV ablated 30-D cells. Besides, the motility capacity of 30-D cells exposed to a Racl inhibitor was dramatically decreased, indicating that Raclwas critical for motility of ESCC. These results together demonstrated that miR-92b-ITGAV-FAK-Raclwis of great importance for cell migration and invasion of ESCC.Additionally, miR-92b or ITGAV silencing reduced lung arrest of 30-D cells in the model of lung metastasis, further confirming their impacts on ESCC metastasis. Notably, knockdown of ITGAV has little influence on cell proliferation in vivo. Combined IHC results from ESCC specimens and statistical analysis of GEO archives showed that aberrant expression of ITGAV positively correlated with lymph node (LN) metastasis. Interestingly, our previous study elicited that the expression of miR-92b negatively correlated with ESCC lymph node metastasis, which confirmed that miR-92b impaired ESCC metastasis by targeting ITGAV.In summary, our study demonstrated that as a genluine target gene of miR-92b, ITGAV played an important role in the migration and invasion of ESCC, without influencing cell proliferation. miR-92b repressed invasion and metastasis of ESCC by modulating ITGAV-FAK-Racl pathway. ITGAV was expressed aberrantly in ESCC specimens and correlated positively with LN metastasis. All these results collectively deepen our comprehensions of function and mechanism of miR-92b and ITGAV in ESCC local invasion and metastasis, and provided new insights into anti-cancer strategies and clinical molecular diagnosis.