Multiple miRNAs have been identified as potential diagnostic biomarkers and therapeutic targets for NSCLC. For example, expression of miR-128-3p was significantly up-regulated in NSCLC tissues by comparison with adjacent normal tissues; its overexpression remarkably induced the migration and invasion of A549, Calu-3 and H520 cells [17]. In the present research, miR-199a-5p was found to be dramatically down-regulated in NSCLC tissues, serum samples of NSCLC patients and multiple NSCLC cell lines. Gain-of-function and loss-of-function experiments further illustrated that miR-199a-5p constrained the proliferation, migration and invasion, and facilitated the apoptosis of NSCLC cells, indicative of a prospective value regarding miR-199a-5p in NSCLC therapeutics. Some other studies also demonstrate that miR-199a-5p represses the malignancy of NSCLC cell lines, such as H1299, A549 and SPCA-1 cells, which is consistent with our demonstrations [18, 19]. The underexpression of miR-199a-5p in NSCLC has been reported by several previous reports [6, 7, 18, 19]. “competitive endogenous RNA (ceRNA)” mechanism can probably contribute to the dysregulation of miR-199a-5p in NSCLC. It is reported that, LINC01123 sponges miR-199a-5p as a ceRNA to negatively regulate its expression in NSCLC cells [18]; similarly, in NSCLC cells, miR-199a-5p is reported to be adsorbed and repressed by lncRNA PVT1 [19]. Interestingly, in cardiomyocytes, pSTAT3 is able to bind to the promoter region of miR-199a-2 gene, and can repress its transcription and down-regulate its expression, which is more significant under hypoxic conditions, suggesting miR-199a-5p is modulated by STAT3 signaling and HIF-1α signaling [12, 20]. In the present study, for the first time, we demonstrated that overexpression of STAT3 or HIF-1α increased the expression of miR-199a-5p in NSCLC cells, which indicated that the hypoxic tumor microenvrionment and activation of STAT3 signaling contributed the dysregulation of miR-199a-5p in NSCLC cells.
HIF-1α, recognized as oxygen-labile subunit of HIF-1, contains the transactivation domains responsible for HIF-1α transcriptional activity [21, 22]. Despite the fact that HIF-1α is merely highly expressed during hypoxic condition, it is also detectable in normoxic condition. Hypoxia is a common characteristic in the microenvironment of various types of solid tumors, promoting HIF-1α expression [22–24]. In turn, HIF-1α is conducive to the adjustment of tumors to hypoxia through transcriptional activation of more than 100 downstream genes including LEP, EPO, PKM, etc; in this regard, HIF-1α further promotes proliferation and growth of tumor cells [23]. In NSCLC, HIF-1α is up-regulated in tumor tissues and cell lines, and its overexpression facilitates the proliferation, migration and invasion of cancer cells; what’s more, the 5-year survival rate of patients with low expression level of HIF-1α is higher than those with high expression level of HIF-1α [25–27]. For shedding more light upon the underlying mechanism by which miR-199a-5p exhibited its tumor-suppressive role in NSCLC, miRmap, miRanda and TargetScan databases were employed for prediction of target gene of miR-199a-5p in this study. Interestingly, the gene of HIF-1α, HIF1A, was validated as a target gene of miR-199a-5p in NSCLC in the present study. Our data suggested that the dysregulation of HIF-1α in NSCLC is not only due to the hypoxic microenvrionment, but also partly resulted from the down-regulation of upstream miRNAs.
STAT3, a member of signal transducer and activator of transcription (STAT) family, regulates the expressions of genes relevant to cell cycle, cell survival, and immune response. In recent years, STAT3 has been found to be constitutively activated in multiple types of human cancers, indicating that STAT3 is a valuable target for cancer therapy [28]. STAT3 has been identified to be abnormally increased in NSCLC tissues and cell lines, and knockdown of STAT3 can induce apoptosis, reduce proliferation, migration and invasion of A549 and H1975 cells [29]. Consistently, we demonstrated that transfection of STAT3 overexpression plasmids notably induced proliferation, migration and invasion of NSCLC cells. It is noteworthy that HIF-1α is previously reported to activate STAT3 by means of repressing miR-34a in colorectal cancer [11]. Furthermore, in head and neck squamous cell carcinoma, with static inhibition of STAT3 activation, expression in HIF-1α was repressed [30]. In the present work, we demonstrated that HIF-1α overexpression activated STAT3, and STAT3 overexpression repressed the expression of miR-199a-5p, but promoted the expression of HIF-1α. Altogether, our study presented a novel positive feed-back loop, which was formed by miR-199a-5p, HIF-1α and STAT3 in NSCLC, and this positive feed-back loop is crucial to clarify the mechanism of hyperactivation of HIF-1α pathway and STAT3 signaling in NSCLC tissues.
At the last part of our present study, we analyzed the sensitivity of A549 cells to Bevacizumab and found the positive feed-back loop also contributed to Bevacizumab sensitivity. Bevacizumab, possessing high efficacy and safety, is a monoclonal anti-VEGF antibody and an encouraging target drug for NSCLC, particularly advanced NSCLC patients [31, 32]. There remains little study investigating the molecular mechanism regarding Bevacizumab sensitivity in NSCLC at the present. We found the positive feed-back loop miR-199a-5p-HIF-1α-STAT3 also exists after Bevacizumab treatment and miR-199a-5p enhanced sensitivity of A549 cells to Bevacizumab. Our study provides a new thought to further improve outcomes for Bevacizumab therapy. miR-199a-5p may have the potential to improve prognosis for NSCLC patients in advanced stages.