LncRNA KCNQ1OT1 regulates proliferation and cisplatin resistance in tongue cancer via miR-211-5p mediated Ezrin/Fak/Src signaling

Abstract

Numerous findings have demonstrated that long noncoding RNA (lncRNA) dysregulation plays a key role in many human neoplasms, including tongue squamous cell carcinoma (TSCC), yet the potential mechanisms of lncRNAs in chemo-resistance remain elusive. Our research showed that the lncRNA KCNQ1OT1 was upregulated in chemo-insensitive TSCC tissues compared with chemo-sensitive TSCC specimens. Meanwhile, high KCNQ1OT1 expression was closely correlated with poor prognosis. Furthermore, KCNQ1OT1 promoted TSCC proliferation and conferred TSCC resistance to cisplatin-induced apoptosis in vitro and in vivo. Using online database analysis, we predicted that the lncRNA KCNQ1OT1 facilitates tumor growth and chemo-resistance by acting as a competing endogenous RNA (ceRNA) to modulate the expression of miR-211-5p. And miR-211-5p upregulation significantly impaired TSCC proliferation and resumed TSCC chemo-sensitivity, which is contrary to the function of lncRNA KCNQ1OT1. Luciferase experiments confirmed that miR-211-5p harbor binding sites for the 3'-UTRof Ezrin mRNA, and Ezrin/Fak/Src signaling was activated in cisplatin-resistant TSCC cells. Finally, miR-211-5p inhibition in sh-KCNQ1OT1-expressing TSCC cells rescued the suppressed cell proliferation and cisplatin resistance induced by KCNQ1OT1 knockdown. In summary, our study has elucidated the role of the oncogenic lncRNA KCNQ1OT1 in TSCC growth and chemo-resistance, which may serve as a new target for TSCC therapy.

Fig. 1. KCNQ1OT1 is identified as a cisplatin resistant tongue-cancer-related lncRNA, predicts disease prognosis and mainly locates in cytoplasm.

aThe differentially expressed lncRNAs between three chemo-insensitive tissues and three chemo-sensitive tissues were detected using a microarray. b,c The results from microarray analysis were validated in cisplatin-resistant tongue cancer cells and their parental cells by RT-qPCR. d The expression of KCNQ1OT1 in adjacent normal tissues, chemo-sensitive samples and chemo-insensitive TSCC tissues was examined by RT-qPCR. e The progression-free survival rates of 102 TSCC patients were compared in the KCNQ1OT1-low and KCNQ1OT1-high groups. f Representative images of in situ hybridization for KCNQ1OT1in paraffin-embedded TSCC tissues and adjacent normal tissues (scale bar: 100 μM). g The proportion of KCNQ1OT1 in CAL27-res and SCC9-res cells was detected by RT-qPCR

Fig. 2. Knockdown of KCNQ1OT1 inhibits tongue cancer cell proliferation.

a Efficiency of KCNQ1OT1 knockdown in CAL27-res/CAL27 and SCC9-res/SCC9 cells by two siRNAs was verified by RT-qPCR. b Influence of KCNQ1OT1 knockdown on cell viability of CAL27-res and CAL27 cells was measured by the MTS assay. c Effect of KCNQ1OT1 knockdown on flat colony formation was measured in SCC9 and CAL27 cells. d Effect of KCNQ1OT1 knockdown on cell viability of SCC9-res and SCC9 cells was measured by the MTS assay. e CAL27 and SCC9 cells were transfected with KCNQ1OT1 siRNAs or control siRNA and the cell viability were examined by EDU assay

Fig. 3. Downregulation of KCNQ1OT1 represses chemo-resistance of tongue cancer cells.

a,b CAL27-res/CAL27 cells (a) and SCC9-res/SCC9 cells (b) transfected with KCNQ1OT1 siRNAs or control siRNA were treated with different concentration of cisplatin for 24 h and the cell survival rate was analyzed by MTS assay. c,d The CAL27 (c) and SCC9 (d) cells transfected with control or KCNQ1OT1 siRNAs were treated with 4 μM cisplatin (for CAL27 cells) or 8 μM cisplatin(for SCC9 cells) for 24 h. The percentage of apoptotic cells was analyzed by flow cytometer. e,f KCNQ1OT1-depleted CAL27 (e) and SCC9 (f) cells were treated with either DMSO or 4 μM cisplatin(for CAL27 cells) and 8 μM cisplatin(for SCC9 cells) for 24 h, and the expression of cleaved PARP and cleaved caspase-3, -7, and -9 were detected by Western blot analysis

Fig. 4. KCNQ1OT1 binds to miR-211-5p and represses their expression.

a Differentially expressed miRNAs were validated in CAL27-res/CAL27 cells and SCC9-res/SCC9 cells by RT-qPCR. b Anti-AGO2 RIP was performed in CAl27 and SCC9 cells transiently overexpressing miRNA mimics, followed by RT-qPCR to detect lncRNA KCNQ1OT1 associated with AGO2. c Schematic illustration of the predicted binding sites between KCNQ1OT1 and miR-211-5p and mutation of potential miR-211-5p binding sequence in KCNQ1OT1. d Luciferase assays in 293T cells transfected with wild-type or mutant KCNQ1OT1 and miR-211-5p. e The expression of miR-211-5p was inhibited in CAL27 and SCC9 transfected with miR-211-5p inhibitors or miR-control by RT-qPCR. f The expression of miR-211-5p was upregulated in CAL27 and SCC9 transfected with KCNQ1OT1 siRNAs or control siRNA by RT-qPCR. g The expression of KCNQ1OT1 was inhibited in CAL27 and SCC9 transfected with miR-211-5p inhibitors or miR-control by RT-qPCR

Fig. 5. miR-211-5p suppress TSCC proliferation and chemo-resistance by targeting Ezrin/Fak/Src signaling.

a Target sequence of miR-211-5p in Ezrin 3′-UTR predicted by TargetScan and miRanda as well as the mutated sequence. b Luciferase assays in 293 T cells transfected with wild-type or mutant Ezrin and miR-211-5p mimics. c Cell proliferation was detected by MTS assays in CAL27 cells transfected with or without miR-211-5p inhibitors. d Cell proliferation was detected by MTS assays in CAL27-res cells transfected with or without miR-211-5p mimics. e Cell survival capacity was examined by MTS assays under cisplatin pressure in CAL27 cells transfected with or without miR-211-5p inhibitors. f Cell survival rate was examined by MTS assays under cisplatin pressure in CAL27-res cells transfected with or without miR-211-5p mimics. g Clones formation was detected in CAl27 cells transfected with miR-211-5p inhibitors. h The expression of Ezrin, p-Fak, total Fak, p-Src, and total Src were detected in miR-211-5p-depleted CAL27 cells and miR-211-5p overexpressed CAL27-res cells by Western blot analysis

Fig. 6. KCNQ1OT1 promotes TSCC progression via miR-211-5p-mediated Ezrin/Fak/Src signaling.

a Cell survival rate of CAL27 cells transfected with siR-KCNQ1OT1 or siR-control simultaneously with miR-211-5p inhibitors or miR-control inhibitors under cisplatin pressure were examined with MTS assays. b Cell proliferation ability of CAL27 cells transfected with siR-KCNQ1OT1 or siR-control simultaneously with miR-211-5p inhibitors or miR-control inhibitors under cisplatin pressure were examined with MTS assays. c The expression of Ezrin, p-Fak, total Fak, p-Src, and total Src were detected in CAL27 cells transfected with siR-KCNQ1OT1 or siR-control simultaneously with miR-211-5p inhibitors or miR-control inhibitors by Western blot analysis. d Correlation analysis between miR-211-5p and KCNQ1OT1 was performed in TSCC tissues. e Clones formation capacity was detected in CAL27 cells transfected with siR-KCNQ1OT1 or siR-control simultaneously with miR-211-5p inhibitors or miR-control inhibitors

Fig. 7. Downregulation of KCNQ1OT1 suppresses tumorigenicity and chemo-resistance of tongue cancer cells in vivo.

a Construction of KCNQ1OT1 stable knockdown CAL27 cells. b The volume of tumors was measured every 3 days. c The weight of tumors was measured after the tumors were surgically dissected. d Photographs of surgically dissected tumors of each group. e The expression of Ki67 in the xenografts was examined by IHC. f The apoptosis in the xenografts was detected by TUNEL assay. The histograms showed the score of IHC and proportion of TUNEL-positive cells in each group. The percentage in the histograms indicates the decreased or increased percentage in the cisplatin group compared with the PBS group