Cancer Res Treat.  2021 Oct;53(4):1042-1056. 10.4143/crt.2020.1208.

Inhibition of lncRNA KCNQ1OT1 Improves Apoptosis and Chemotherapy Drug Response in Small Cell Lung Cancer by TGF-β1 Mediated Epithelial-to-Mesenchymal Transition

• Li D
• Tong Q
• Lian Y
• Chen Z
• Zhu Y
• Huang W
• Wen Y
• Wang Q
• Liang S
• Li M
• Zheng J
• Liu Z
• Liu H
• Guo L

• KMID: 2521580
• DOI: http://doi.org/10.4143/crt.2020.1208

Abstract

Purpose
Drug resistance is one of the main causes of chemotherapy failure in patients with small cell lung cancer (SCLC), and extensive biological studies into chemotherapy drug resistance are required.
Materials and Methods
In this study, we performed lncRNA microarray, in vitro functional assays, in vivo models and cDNA microarray to evaluate the impact of lncRNA in SCLC chemoresistance.
Results
The results showed that KCNQ1OT1 expression was upregulated in SCLC tissues and was a poor prognostic factor for patients with SCLC. Knockdown of KCNQ1OT1 inhibited cell proliferation, migration, chemoresistance and promoted apoptosis of SCLC cells. Mechanistic investigation showed that KCNQ1OT1 can activate transforming growth factor-β1 mediated epithelial-to-mesenchymal transition in SCLC cells.
Conclusion
Taken together, our study revealed the role of KCNQ1OT1 in the progression and chemoresistance of SCLC, and suggested KCNQ1OT1 as a potential diagnostic and prognostic biomarker in SCLC clinical management.

Keyword

LncRNA; KCNQ1OT1; Small cell lung cancer; Prognostic; Chemotherapy

Figure

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  Fig. 1 High level of KCNQ1OT1 was a poor prognosis factor for patients with small cell lung cancer (SCLC). The expression of KCNQ1OT1 in H69 and H69AR (A), in H446 and H446DDP (B) cells. (C) The expression of KCNQ1OT1 in SCLC tissues and non-tumoral lung tissues. (D) The role of KCNQ1OT1 in the prognosis of SCLC patients. **p < 0.01.

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  Fig. 2 Knockdown of KCNQ1OT1 expression inhibited proliferation, migration and invasion of small cell lung cancer cells. (A) siRNA-mediated knockdown of KCNQ1OT1 expression in H69, H69AR, H446, and H446DDP cells. (B) shRNA-mediated knockdown of KCNQ1OT1 expression in H69, H69AR, H446, and H446DDP cells. The effect of KCNQ1OT1 knockdown on proliferation in H69 (C), H446 (D), H69AR (E), and H446DDP (F) cell using Cell Counting Kit 8 assay. The effect of KCNQ1OT1 knockdown on colony formation (G), migration (H), and invasion (I) in H446, H446DDP, and H69AR cells. *p < 0.05, **p < 0.01.

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  Fig. 3 Knockdown of KCNQ1OT1 expression promoted small cell lung cancer cell apoptosis. Flow cytometric analysis was used for cell apoptosis detection after KCNQ1OT1 knockdown in H69, H69AR, H446, and H446DDP cells. (A) A bar graph demonstrating the rate of apoptosis using flow cytometry analysis. (B) Representative flow cytometry analysis graphs. PI, propidium iodide. *p < 0.05, **p < 0.01.

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  Fig. 4 Knockdown of KCNQ1OT1 expression resulted in increased chemosensitivity in small cell lung cancer cells. Cells were treated with the indicated dose of drug. IC50 values are as showed for ADM, DDP, VP-16 in H69, H69-NC, H69-si1, and H69-si2 cells (A), in H69AR, H69AR-NC, H69AR-si1, and H69AR-si2 cells (B), in H446, H446-NC, H446-si1, and H446-si2 cells (C), in H446DDP, H446DDP-NC, H446DDP-si1, and H446DDP-si2 cells (D). *p < 0.05, **p < 0.01.

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  Fig. 5 Knockdown of KCNQ1OT1 increased apoptosis of cells treated with chemotherapy drugs. Flow cytometric analysis was used for cell apoptosis detection for KCNQ1OT1 knockdown cells treated with chemotherapy drugs. A bar graph demonstrating the rate of apoptosis using flow cytometry analysis in KCNQ1OT1 knockdown H69 (A), H69AR (B), H446 (C), and H446DDP (D) cells. *p < 0.05, **p < 0.01.

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  Fig. 6 Knockdown of KCNQ1OT1 expression inhibited small cell lung cancer cell chemoresistance and tumorigenesis in vivo. (A) Excised tumors’ image from tumor bearing nude mice in H69AR and H446DDP group, volume change curve of H69AR (B) and H446DDP (C) group measured on the indicated days. (D) Excised tumors image from tumor bearing nude mice in H69AR+Chemo and H446DDP+Chemo group, volume change curve of H69AR+Chemo (E) and H446DDP+Chemo (F) group measured on the indicated days. *p < 0.05, **p < 0.01.

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  Fig. 7 KCNQ1OT1 may promote small cell lung cancer chemoresistance by regulating transforming growth factor-β and epithelial-to-mesenchymal transition signaling pathway. Analysis of mRNA expression of KCNQ1OT1, TGFB1, SMAD2, SMAD3, CDH1, TWIST1, ZEB-2, and VIM in H69AR (A) and H446DDP (D) cells, the protein expression of TWIST1, ZEB-2, VIM, E-CAD, TGFB1, SMAD2, and SMAD3 in H69AR-si (B) and H446DDP-si (E) cells were measured with western blotting and presented as histogram (C, F). *p < 0.05, **p < 0.01.

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  Fig. 8 Exogenous TGFB1 increased KCNQ1OT1 expression and activated epithelial-to-mesenchymal transition pathway in small cell lung cancer cells. Exogenous TGFB1 stimulated H69 cell lines, and the mRNA level of KCNQ1OT1, SMAD2, SMAD3, TWIST1, ZEB-2, VIM, and CDH1 were detected at 12 hours (A), 36 hours (B), and 60 hours (C), respectively. Exogenous TGFB1 stimulated H446 cell lines, and the mRNA level of KCNQ1OT1, SMAD2, SMAD3, TWIST1, ZEB-2, VIM, and CDH1 were detected at 12 hours (D), 36 hours (E), and 60 hours (F), respectively. *p < 0.05, **p < 0.01.

Reference

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