MicroRNA-26a suppresses angiogenesis in human hepatocellular carcinoma by targeting hepatocyte growth factor-cMet pathway
Potential conflict of interest: Nothing to report.
Supported by China National Key Projects for Infectious Disease (2008ZX10002-021, 2012ZX10002-012), the National Key Basic Research Program of China (2013CB910500; 2009CB521701), Program for Changjiang Scholars and Innovative Research Team (the Ministry of Education of China; IRT1118), Program of Shanghai Chief Scientist (08XD14008), China National Natural Science Foundation (81120108016; 30600589), Program for Outstanding Medical Academic Leader (LJ10010).
MicroRNA (miR)-26a can suppress tumor growth and metastasis of hepatocellular carcinoma (HCC). Since angiogenesis is important for tumor growth and metastasis, we investigated the possible roles of miR-26a in tumor angiogenesis. Down-regulation of miR-26a was found to correlate with an increased angiogenic potential of HCC. Through gain- and loss-of-function studies, miR-26a was demonstrated to significantly inhibit vascular endothelial growth factor A (VEGFA) expression in HCC cells and then suppress the promoting effects of HCC cells on in vitro proliferation, migration, and capillary tube formation of endothelial cells, as well as in vivo tumor angiogenesis of HCC. Hepatocyte growth factor (HGF) was identified as a target of miR-26a. HGF simulation antagonized the effects induced by miR-26a up-regulation. In contrast, silencing HGF induced similar effects to miR-26a. We further found that miR-26a exerted its antiangiogenesis function, at least in part, by inhibiting HGF-hepatocyte growth factor receptor (cMet) and its downstream signaling pathway, in turn, suppressing VEGFA production in HCC cells and impairing VEGFR2-signaling in endothelial cells. HCC patients who had high miR-26a, low HGF, low VEGFA, or low microvessel density (MVD) in tumor tissues had a better prognosis with longer overall survival (OS) and time to recurrence (TTR). In multivariate analysis, miR-26a, or in combination with HGF, was demonstrated to be an independent prognostic indicator for OS and TTR of HCC patients. Conclusion: miR-26a could suppress tumor angiogenesis of HCC through HGF-cMet signaling, and it is a new hopeful therapeutic target and prognostic marker for HCC. (Hepatology 2014;59:1874–1885)
Hepatocellular carcinoma (HCC) is a highly vascularized tumor with frequent intrahepatic and extrahepatic metastasis, which is responsible for the rapid recurrence and poor survival of HCC.[1-3] Therefore, it is urgent to explore the molecular mechanisms of underlying metastasis and tumor angiogenesis of HCC, which would provide potential effective therapeutic targets to improve the survival of HCC patients. MicroRNAs (miRNAs) are involved and play critical roles in a variety of biological processes through suppressing the expression of target messenger RNAs (mRNAs). Deregulation of miRNAs has been shown to result in aberrant gene expression, and contributes to invasion and metastasis of many kinds of human tumors including HCC.[2, 5-9]
There are still controversies about the roles of miR-26a in human malignancies. Many studies show that miR-26a serves as a potential tumor suppressor in several distinct cancer types including HCC,[10-15] but some others indicate that it exhibits oncogenic properties in glioma, lung cancer, and cholangiocarcinoma.[16-18] It is possible that miR-26a plays organ-specific roles, in part due to the different cellular contexts of tumors. Recently, we found that miR-26a could suppress tumor growth and metastasis of HCC through IL-6-Stat3 signaling. In this study, using both gain- and loss-of-function analyses, we further find the suppressive function of miR-26a on tumor angiogenesis. Moreover, we reveal that Hepatocyte growth factor (HGF) is a target of miR-26a, and miR-26a exerted its antiangiogenesis function, at least in part, by inhibiting the HGF-hepatocyte growth factor receptor (cMet) signaling pathway. Our findings provide the underlying mechanism of miR-26a in angiogenesis of HCC and implicate miR-26a as a potential therapeutic target for HCC.
Active tumor angiogenesis is confirmed to associate with invasion and metastasis of HCC.[1-3, 30-33] It has been reported that the dysfunction of miRNAs in tumor cells affects tumor angiogenesis, including in HCC.[2, 9] In the present study, using in vitro and in vivo assays, for the first time we identify the antiangiogenic function of miR-26a. The level of miR-26a expression was inversely associated with MVD in HCC tissues. Through up- and down-regulating miR-26a in HCC cells, we confirm that miR-26a could inhibit the abilities of in vitro proliferation, migration, and capillary tube formation of HUVECs, as well as suppress in vivo tumor angiogenesis in nude mice models bearing human HCC.
We found that the inhibitory effects of miR-26 on tumor angiogenesis of HCC might be due to the decreased expression of VEGFA, an important proangiogenic factor secreted by tumor cells.[29, 33] Cancer cells can secrete VEGF into the microenvironment to promote tumor angiogenesis to meet their need for a blood supply.[34, 35] Blocking VEGF leads to the regression of vascular network and inhibition of tumor growth and metastasis. MiR-195 was reported to suppress angiogenesis of HCC by directly inhibiting the expression of VEGF, and miR-27b targets VEGFC to inhibit tumor angiogenesis of colorectal cancer.[9, 37] In this study, we found that miR-26a could inhibit the expression of VEGFA. However, VEGF is not an effective target gene of miR-26a. Therefore, it is crucial to elucidate the mechanisms of how miR-26a in tumor cells affects VEGFA expression.
Through the correlation analysis in both HCC tissues and cell lines, together with both in vitro and in vivo functional studies, we confirm that miR-26a inhibits tumor angiogenesis at least in part by suppressing HGF. An inverse correlation was found between the mRNA levels of miR-26a and HGF mRNA levels or protein levels in HCC tissues. This correlation is further confirmed by evaluating the effects of up- and down-regulating miR-26a on HGF expression in HCC cells. Moreover, HGF down-regulation in HCC cells could induce similar effects as those of miR-26a on HUVECs, and HGF stimulation could antagonize the effects of miR-26a. This suggests that HGF serves as a downstream mediator of miR-26a function in angiogenesis of HCC.
It is well known that HGF plays important roles in cell proliferation, motility, metastasis, and angiogenesis of HCC, mainly through activating cell surface receptors such as cMet.[19-22, 38] HGF can stimulate cancer cells to produce VEGF,[20, 39] which can be antagonized by blocking c-Met activation. Meanwhile, its downstream PI3K/Akt/mTOR/S6K and HIF-1α-VEGF signal pathways are considered to involve in HGF-inducing VEGF expression.[23-25, 27] In this study, the solid data of both in vitro and in vivo assays indicate that miR-26a significantly decreases the expression levels of the proteins related to PI3K/Akt/mTOR/S6K and HIF-1α-VEGF signal pathways, and moreover, these effects are induced through the HGF-cMet pathway. These provide further evidence to support that miR-26a decreases VEGFA expression by suppressing HGF-cMet pathway.
Another interesting finding of this study is that miR-26a down-regulation in HCC cells increases VEGFA levels in the tumor microenvironment through the HGF-cMet pathway, which induces the activation of VEGFR2 signaling in endothelial cells and promotes tumor angiogenesis in HCC. We found that the decreased VEGFA induced by miR-26a through HGF-cMet pathway results in significant decreases in the expression levels of p-VEGFR2, p-Akt, and p-ERK in HUVECs, and subsequently inhibits activities of ECs. VEGF stimulates cellular responses by activating tyrosine kinase receptors (VEGFRs) on the cell surface. As a pivotal activator of the VEGFR2 pathway, tumor-secreted VEGF binds to and induces the phosphorylation and activation of VEGFR2 on endothelial cells, and then results in phosphorylation of the extracellular signal-regulated kinase (ERK) and AKT, and promotes the proliferation, migration, and survival of endothelial cells.[29, 33]
Consistent with our previous study that miR-26a is a novel prognostic marker and therapeutic target for HCC, in our present study we further confirm that miR-26a, HGF, their combination, MVD, and VEGFA are independent prognostic indicators for OS and TTR in HCC patients. The prognostic performance of the combination of miR-26a and HGF is much better than that of miR-26a or HGF alone.
miR-26a has been reported to regulate hepatocyte proliferation during liver regeneration, block G1/S transition,[12, 15, 43] and suppress in vivo tumor growth and metastasis of HCC.[14, 44] This study provides further evidence to support that miR-26a is a promising prognostic marker and therapeutic target for HCC. As anti-VEGF therapies are important strategies for cancer treatment including for HCC, miR-26a could be a novel antiangiogenic upstream target of VEGF, and therefore has potential clinical significance.