Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Apr 21:12:2685-2695.
doi: 10.2147/CMAR.S237181. eCollection 2020.

lncRNA DLX6-AS1 Promotes Proliferation of Laryngeal Cancer Cells by Targeting the miR-26a/TRPC3 Pathway

Yan Liu  1 , Xinyi Liu  1 , Xiaofeng Zhang  2 , Jinhu Deng  2 , Jian Zhang  2 , Hao Xing  3
Affiliations

lncRNA DLX6-AS1 Promotes Proliferation of Laryngeal Cancer Cells by Targeting the miR-26a/TRPC3 Pathway

Yan Liu et al. Cancer Manag Res. .

Abstract

Purpose: Laryngeal cancer is the most prevalent tumor type in head and neck cancers. Early diagnosis is considered as an important strategy for improving prognosis. The lncRNA DLX6-AS1 has been shown to modulate tumor phenotypes in several types of cancer, but the role of DLX6-AS1 in laryngeal cancer and its concrete mechanisms are not clear.

Methods: Tissue samples from laryngeal cancer patients and corresponding clinical data were used for detailed analysis. The laryngeal cancer cell lines HEp-2 and Tu-177 were studied. Cell proliferation, ROS production, mitochondrial respiratory function, intracellular and mitochondrial calcium influx were assessed. Western blotting, quantitative RT-PCR and luciferase assays were used to analyze the interactions. A xenografted tumor model was established to analyze the effects of DLX6-AS1 on tumor growth in vivo.

Results: lncRNA DLX6-AS1 had increased expression in tumor tissues compared with adjacent normal tissues and in higher clinical stages compared with lower stages, which was associated with poor prognosis. In detail, DLX6-AS1 knockdown decreased cell proliferation and affected key mitochondrial metabolic parameters in both HEp-2 and Tu-177 cells. Moreover, DLX6-AS1 knockdown suppressed TRPC3-mediated mitochondrial calcium uptake and ROS production. Furthermore, miR-26a functioned as a link between these two molecules, as it could be absorbed by DLX6-AS1 and thus regulated the levels of TRPC3. Finally, the DLX6-AS1/miR-26a/TRPC3 axis modulated laryngeal cancer proliferation both in vitro and in vivo.

Conclusion: This study provides new evidence that a novel lncRNA, DLX6-AS1, regulates mitochondrial calcium homeostasis, respiration and tumor proliferation via modulating the miR-26a/TRPC3 axis in laryngeal cancer.

Keywords: DLX6-AS1; TRPC3; laryngeal cancer; miR-26a; mitochondria.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflicts of interest in this study.

Figures

Figure 1
Figure 1
The expression levels of DLX6-AS1 correlated with the prognosis of laryngeal cancer patients. (A) The expression levels of lncRNA DLX6-AS1 was investigated in tumor and adjacent tissues in laryngeal cancer patients using Quantitative RT-PCR. **P<0.01. (B) The expression levels of DLX6-AS1 was measured in laryngeal cancer patients with stages I and II verses stages III and IV by qRT-PCR. *P<0.05. (C) The relative expression levels of DLX6-AS1 were measured via qRT-PCR and rated relative to patient No.1. (D) The effects of DLX6-AS1 on overall survivals were plotted using Kaplan-Meier survival analysis. *P<0.05. Date were presented as Mean ± SD.
Figure 2
Figure 2
The effects of DLX6-AS1 on cell proliferation and mitochondrial functions. (A) The effects of DLX6-AS1 specific siRNA on the levels of DLX6-AS1 using qRT-PCR in both HEp-2 and Tu-177 cells. **P<0.01. (B) The effects of DLX6-AS1 specific siRNA on cell proliferations. *P<0.05. (C, D) The effects of siDLX6-AS1 on cellular and mitochondrial ROS production in these two cells, using DHE and MitoSOX dyes, respectively. ***P<0.001. (E) The effects of siDLX6-AS1 on mitochondrial oxidative respiration using Seahorse respirometry. (F, G) The effects of DLX6-AS1 specific siRNA on ATP synthesis and H2O2 production in these two cells. **P<0.01, ***P<0.001. (H) The effects of mitochondrial specific ROS scavenger mitoTEMPO on cell proliferation in laryngeal cancer cells. *P<0.05, **P<0.01.
Figure 3
Figure 3
The effects of lncRNA DLX6-AS1-mediated TRPC3 upregulation on cell proliferation and calcium homeostasis. (A) The effects of DLX6-AS1 specific siRNA on the expression levels of TRPC3 using Western blotting. **P<0.01. (B) The effects of Pyr3 and mitoTEMPO on the laryngeal cancer cell proliferation treated with siNC and siTRPC3, respectively. *P<0.05, **P<0.01. (C, D) The effects of Pyr3 and TRPC3 specific siRNA on cellular and mitochondrial ROS production, compared with DMSO or siNC, respectively. *P<0.05, ***P<0.001. (E, F) The effects of siTRPC3 on cellular calcium uptake, induced by thapsigargin, compared with siNC. The laryngeal cancer cells were preincubated with Fura-2, with Fx/F0 indicated that the fluorescence values of certain dots versus the baseline of the measurements. **P<0.01. (G, H) The effects of TRPC3 inhibition on mitochondrial calcium uptake, induced by ATP in permeabilized cells. *P<0.05, **P<0.01, ***P<0.001.
Figure 4
Figure 4
MiR-26a functioned as a link between DLX6-AS1 and TRPC3. (A) The detailed binding sequence of miR-26a and wild-type TRPC3 3ʹ-UTR was presented, with the mutation sequence also presented. (B) The effects of miR-26a mimic and inhibitor on the expression levels of TRPC3 was demonstrated using Western blotting. *P<0.05, ***P<0.001. (C) The luciferase assay was conducted in HEp-2 cells co-transfected pmir-GLO-WT/MUT (TRPC3 3ʹ-UTR) plasmid and miR-NC/miR-26a-mimic. **P<0.01. (D) The binding sequence of both wild-type and mutant forms of DLX6-AS1 with miR-26a was presented. (E) The effects of DLX6-AS1 knockdown on the expression levels of miR-26a were demonstrated using qRT-PCR. *P<0.05, **P<0.01. (F) The luciferase assay was conducted in HEp-2 cells co-transfected pmir-GLO-WT/MUT (DLX6-AS1) plasmid and miR-NC/miR-26a-mimic. **P<0.01, ***P<0.001. (G) The effects of miR-26a-mimic and Pyr3 on mitochondrial calcium uptake in both HEp-2 and Tu-177 cells. **P<0.01, ***P<0.001. (H) The effects of miR-26a mimic on mitochondrial ROS production using MitoSOX dye. ***P<0.001.
Figure 5
Figure 5
The effects of the DLX6-AS1/miR-26a/TRPC3 axis on mitochondrial functions and cell proliferation. (A, B) The effects of DLX6-AS1 siRNA and miR-26a on the expression levels of TRPC3 using Western blotting in laryngeal cancer cells. ***P<0.001. (C) The effects of DLX6-AS1 siRNA and miR-26a on cell proliferations in both HEp-2 and Tu-177 cells. *P<0.05. (D) The effects of DLX6-AS1 siRNA and miR-26a on mitochondrial ROS productions in laryngeal cancer cells. **P<0.01, ***P<0.001. (E) The effects of miR-26a on mitochondrial oxidative respiration. *P<0.05, **P<0.01, compare with corresponding miR-NC group. *P<0.05, **P<0.01.
Figure 6
Figure 6
The effects of DLX6-AS1 siRNA on the tumor proliferation in vivo. (A) The effects of DLX6-AS1 siRNA on the tumor growth using nude mice models, with HEp-2 and Tu-177 cells pre-transfected with DLX6-AS1 specific siRNAs. The volume of each tumor was measured and analyzed. *P<0.05, **P<0.01. (B) The mitochondrial respiratory functions of grafted tumors were analyzed. *P<0.05, **P<0.01. (C) The enzymatic activities of mitochondrial complex I and complex II were measured using the tissues of grafted tumors. *P<0.05, **P<0.01. (D) The expression levels of TRPC3 in tumor tissues were detected by Western blotting. ***P<0.001.
See this image and copyright information in PMC

References

    1. Peller M, Katalinic A, Wollenberg B, Teudt IU, Meyer JE. Epidemiology of laryngeal carcinoma in Germany, 1998–2011. Eur Arch Otorhinolaryngol. 2016;273(6):1481–1487. doi: 10.1007/s00405-016-3922-8 - DOI - PubMed
    1. Ferlay J, Soerjomataram I, Dikshit R, et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015;136(5):E359–E386. doi: 10.1002/ijc.29210 - DOI - PubMed
    1. Marioni G, Marchese-Ragona R, Cartei G, Marchese F, Staffieri A. Current opinion in diagnosis and treatment of laryngeal carcinoma. Cancer Treat Rev. 2006;32(7):504–515. doi: 10.1016/j.ctrv.2006.07.002 - DOI - PubMed
    1. Hoffman HT, Porter K, Karnell LH, et al. Laryngeal cancer in the United States: changes in demographics, patterns of care, and survival. Laryngoscope. 2006;116(9Pt 2 Suppl 111):1–13. doi: 10.1097/01.mlg.0000236095.97947.26 - DOI - PubMed
    1. He HJ, Bing H, Liu G. TSR2 induces laryngeal cancer cell apoptosis through inhibiting NF-kappaB signaling pathway. Laryngoscope. 2018;128(4):E130–E134. doi: 10.1002/lary.27035 - DOI - PubMed