Novel regulation of miR-34a-5p and HOTAIR by the combination of berberine and gefitinib leading to inhibition of EMT in human lung cancer.

HOTAIR is an important carcinogenic lncRNA and involves in tumorigenesis, and invasion. MiR-34a-5p functions as a tumour suppressor. However, the underlying mechanism of HOTAIR regulation especially in association with miR-34a-5p in non-small-cell lung cancer (NSCLC) has not been explored. Herein, we performed series of in vitro experiments, including viability, migration, invasion, apoptosis and in vivo xenograft model, and identified that HOTAIR was remarkably elevated in NSCLC cells. Enforced HOTAIR expression promoted migration and invasion, while depleted HOTAIR diminished the ability of migration and invasion of NSCLC cells. We also observed that miR-34a-5p was dramatically inhibited in NSCLC cells and the binding correlation between HOTAIR and miR-34a-5p was confirmed by dual-luciferase reporter and RNA immunoprecipitation assays. We also showed that induction of miR-34a-5p and reduction of HOTAIR, and the interaction between miR-34a-5p and HOTAIR resulted in the suppression of epithelial-mesenchymal transition (EMT) as illustrated by induction of key epithelial markers E-cadherin expression, reduction of vimentin and EMT-inducing transcription factor snail. Excessive expression of snail resisted miR-34a-5p-inhibited cell growth. Snail binds to E-cadherin promoter and regulates E-cadherin expression. There was a synergy in combination of berberine and gefinitib in this process. Similar findings were also observed in a tumour xenograft model. Collectively, this is the first report demonstrating reciprocal interaction of miR-34a-5p- and HOTAIR-mediated regulation of snail resulting in inhibition of EMT process by the combination of berberine and gefitinib suggesting that regulation of miR-34a-5p- and HOTAIR-mediated inhibition of EMT may provide novel treatment paradigms for lung cancer.

Novel reciprocal interaction of lncRNA HOTAIR and miR-214-3p contribute to the solamargine-inhibited PDPK1 gene expression in human lung cancer.

Solamargine (SM) has been shown to have anti-cancer properties. However, the underlying mechanism involved remains undetermined. We showed that SM inhibited the growth of non-small cell lung cancer (NSCLC) cells, which was enhanced in cells with silencing of long non-coding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR), while it overcame by overexpression of HOTAIR. In addition, SM increased the expression of miR-214-3p and inhibited 3-phosphoinositide-dependent protein kinase-1 (PDPK1) gene expression, which was strengthened by miR-214-3p mimics. Intriguingly, HOTAIR could directly bind to miR-214-3p and sequestered miR-214-3p from the target gene PDPK1. Intriguingly, overexpression of PDPK1 overcame the effects of SM on miR-214-3p expressions and neutralized the SM-inhibited cell growth. Similar results were observed in vivo. In summary, our results showed that SM-inhibited NSCLC cell growth through the reciprocal interaction between HOTAIR and miR-214-3p, which ultimately suppressed PDPK1 gene expression. HOTAIR effectively acted as a competing endogenous RNA (ceRNA) to stimulate the expression of target gene PDPK1. These complex interactions and feedback mechanisms contribute to the overall effect of SM. This unveils a novel molecular mechanism underlying the anti-cancer effect of SM in human lung cancer.

Reciprocal interaction of HOTAIR and SP1 together enhance the ability of Xiaoji decoction and gefitinib to inhibit EP4 expression.

ETHNOPHARMACOLOGICAL RELEVANCE: The Chinese herbal prescription Xiaoji decoction (XJD) has long been used for cancer treatment. However, the molecular mechanisms underlying the effects of this medicine, particularly to enhance the efficiency of EGFR-TKI in the treatment of lung cancer have not been well elucidated. MATERIALS AND METHODS: Cell viability and cell cycle distribution were detected by MTT assay and flow cytometry, respectively. The phosphorylation of ERK1/2 and protein levels of SP1 and EP4 were determined by Western blot. The expression of the HOX transcript antisense RNA (HOTAIR) was measured by qRT-PCR. Transient transfection experiments were used to overexpress the HOTAIR, SP1 and EP4 genes. The interaction between HOTAIR and SP1 were further examined via RNA immunoprecipitation (RIP) assay. A tumor xenograft model was used to confirm the in vitro findings. RESULTS: We showed that XJD inhibited growth and induced cell arrest of human non-small cell lung cancer (NSCLC) cells. We also found that XJD increased the phosphorylation of ERK1/2 and inhibited levels of HOTAIR and SP1, EP4 proteins, which were blocked by inhibitor of MEK/ERK. There was reciprocal interaction between HOTAIR and SP1. Silencing of HOTAIR reduced EP4 protein levels and repressed the growth of NSCLC cells, while overexpression of HOTAIR and SP1 overcame XJD-reduced EP4 protein expression. Additionally, excessive expressed EP4 reversed the effect of XJD on cell growth. Importantly, there was synergy of XJD with another cancer treatment drug, EGFR-TKI gefitinib, in this process. We also found that XJD inhibited tumor growth in a xenograft nude mice model. CONCLUSIONS: Our results show that XJD inhibits NSCLC cell growth via ERK1/2-mediated reciprocal repression of HOTAIR and SP1 protein expression, followed by reduced EP4 gene expression. XJD and gefitinib exhibit synergy in this process. The in vitro and in vivo study provides a novel mechanism by which XJD enhances the growth inhibitory effect of gefitinib in gefitinib-resistant NSCLC cells.