LncRNA DHRS4-AS1 Inhibits the Stemness of NSCLC Cells by Sponging miR-224-3p and Upregulating TP53 and TET1

Abstract

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death. This study aimed to examine the roles of DHRS4-AS1/miR-224-3p signaling in the cancer cell stemness of NSCLC. Real-time PCR showed that DHRS4-AS1 was downregulated in cancerous tissues, and bioinformatics analysis revealed that high DHRS4-AS1 expression indicated a good prognosis for NSCLC patients. Sphere and colony formation assays showed that DHRS4-AS1 overexpression significantly suppressed NSCLC cell colony formation and stem cell-like properties. DHRS4-AS1 also abrogated the expression of OCT4, SOX2, CD34, and CD133, markedly inhibited the expression of epithelial-mesenchymal transition (EMT)-related factors, N-cadherin, ZEB1, and Vimentin, and increased E-cadherin expression in spheres. Furthermore, luciferase reporter assays and real-time PCR analysis demonstrated that DHRS4-AS1 and miR-224-3p were antagonistically repressed in NSCLC cells. RNA immunoprecipitation (RIP) analysis revealed that DHRS4-AS1 interacted with miR-224-3p. DHRS4-AS1 partially reversed the miR-224-3p-decreased TP53 and TET1, resulting in the inhibition of tumor growth in vivo. Finally, TP53 and TET1 were antagonistically regulated by DHRS4-AS1 and miR-224-3p in NSCLC cells. In conclusion, TP53- and TET1-associated DHRS4-AS1/miR-224-3p axis is an essential mechanism by which NSCLC modulates cancer cell stemness.

Keywords: DHRS4-AS1; TET1; TP53; cancer cell stemness; miR-224-3p; non-small cell lung cancer.

Figure 1

Detection of DHRS4-AS1 expression in NSCLC and its clinical significance. (A) The survival rate of NSCLC was analyzed on the GEPIA web portal by DHRS4-AS1 expression. (B) Histopathological pictures shown the adjacent normal and cancer tissues. (C) Real-time PCR analysis of DHRS4-AS1 expression in adjacent lung (N = 83) and cancer (N = 83) tissues; #p < 0.01 vs. adjacent tissues. (D) Real-time PCR analysis of DHRS4-AS1 expression in lung cancer cell lines and normal lung epithelial cell line; #p < 0.01 vs. BEAS-2B cells. (E) Real-time PCR analysis of DHRS4-AS1 expression in parental and sphere-forming A549 and Calu-3 cells; #p < 0.01 vs. parental cells. All the results are expressed as the mean ± SD from three independent experiments, and each sample was repeated in triplicate.

Figure 2

DHRS4-AS1 suppressed colony formation and stem cell-like properties in vitro. (A) Real-time PCR analysis of DHRS4-AS1 expression after 48 h of transfection in A549 and Calu-3 cancer stem cells; #p < 0.01 vs. pcDNA. (B,C) Colony formation analysis of A549- and Calu-3-derived stem cells after overexpressing DHRS4-AS1 for 10 days, at which point the colonies were captured and counted; #p < 0.01 vs. pcDNA. (D,E) Representative images of spheres formed by cancer stem cells (D) and quantification of cancer stem cell spheres; #p < 0.01 vs. pcDNA. (F,G) Real-time PCR analysis of the expression of cancer stemness-related genes after 72 h of transfection in A549 and Calu-3 cancer stem cells; #p < 0.01 vs. pcDNA. (H,I) Real-time PCR analysis of the expression of EMT-related factors in tumor spheres; #p < 0.01 vs. pcDNA. All the results are expressed as the mean ± SD from three independent experiments, and each sample was repeated in triplicate.

Figure 3

Antagonistic repression between DHRS4-AS1 and miR-224-3p in NSLC cancer cells. (A) Bioinformatics analysis of miR-224-3p expression in the GSE74190 dataset as determined by GEO2R analysis. (B) Real-time PCR analysis of miR-224-3p expression in lung cancer (N = 83) and adjacent (N = 83) tissues; #p < 0.01 vs. adjacent tissues. (C) Real-time PCR analysis of miR-224-3p expression in parental cells and A549 and Calu-3 stem cell spheres; #p < 0.01 vs. parental cells. (D) Schematic representation of the predicted miR-224-3p binding sites on the DHRS4-AS1 sequence according to the StarBase analysis. (E) Real-time PCR analysis of miR-224-3p expression after 24 h of transfection in A549 and Calu-3 cells; #p < 0.01 vs. scramble. (F,G) Luciferase assay showing pGLO-DHRS4-AS1-WT (wild type) and pGLO-DHRS4-AS1-Mut (Mut) after 24 h of transfection in mock-, miR-224-3p- or anti-miR-224-3p-transfected A549 and Calu-3 cells; #p < 0.01 vs. scramble. All data was normalized to wildtype control group (WT Scramble) (H) Real-time PCR analysis of DHRS4-AS1 expression after 24 h of transfection in A549 and Calu-3 cells; #p < 0.01 vs. scramble. (I) Real-time PCR analysis of miR-224-3p expression in A549 and Calu-3 cells after 24 h of DHRS4-AS1 overexpression; #p < 0.01 vs. pcDNA. (J) RIP analysis showed that DHRS4-AS1 and miR-224-3p could directly bind to AGO2 in A549 and Calu-3 cells; #p < 0.01 vs. IgG. All the results are expressed as the mean ± SD from three independent experiments, and each sample was repeated in triplicate.

Figure 4

DHRS4-AS1 knockdown reversed the anti-miR-224-3p-mediated inhibition of colony formation and stem cell-like properties in vitro. (A,B) Colony formation analysis of cancer stem cell proliferation after 20 days of transfection, at which point the colonies were captured and counted; #p < 0.01 vs. scramble+si-NC, ^φp < 0.01 vs. anti-miR-224-3p+si-NC. And the DHRS4-AS1 levels were determined by using real-time PCR after 48 h transfection. (C) Quantification of cancer stem cell spheres after 72 h of transfection; #p < 0.01 vs. scramble+si-NC, ^φp < 0.01 vs. anti-miR-224-3p+si-NC. (D,E) Real-time PCR analysis of the expression of cancer stemness-related genes after 72 h of transfection; #p < 0.01 vs. scramble+si-NC, ^φp < 0.01 vs. anti-miR-224-3p+si-NC. (F,G) Real-time PCR analysis of the expression of EMT-related factors in tumor spheres; #p < 0.01 vs. scramble+si-NC, ^φp < 0.01 vs. anti-miR-224-3p+si-NC. All the results are expressed as the mean ± SD from three independent experiments, and each sample was repeated in triplicate.

Figure 5

Knockdown of DHRS4-AS1 attenuated anti-miR-224-3p-mediated tumor growth inhibition in vivo. Calu-3 cells were transfected with lenti-viruses meidatescramble, anti-miR-224-3p, si-NC, or si-DHRS4-AS1 for 48 h. The infected Calu-3 cells were collected and injected into nude mice (n = 3/group). (A) Tumor volumes were measured at the indicated times; #p < 0.01 vs. scramble+si-NC, ^φp < 0.01 vs. anti-miR-224-3p+si-DHRS4-AS1. Tumors were collected (B) and measured (C) after injection on the 15th day. #p < 0.01 vs. scramble+si-NC, ^φp < 0.01 vs. anti-miR-224-3p+si-DHRS4-AS1. (D,E) Western blot detected stemness-related and EMT-related markers in xenograft tumors; the optical densities of proteins were determined with ImageJ software; #p < 0.01 vs. scramble+si-NC. All results are expressed as the mean ± SD.

Figure 6

Tumor suppressors, TP53 and TET1 were antagonistically regulated by DHRS4-AS1 and miR-224-3p. (A) Schematic representation of the predicted miR-224-3p binding sites on TP53 and TET1 according to the StarBase prediction. (B) Co-expression analysis of DHRS4-AS1 with TP53 or TET1 according to the StarBase analysis. (C) Luciferase assay of pGLO-TP53-3′-UTR and pGLO-TET1-3′-UTR luciferase activity in transfected Calu-3 cells; #p < 0.01 vs. scramble+pcDNA, ^φp < 0.01 vs. miR-224-3p+pcDNA, ^δp < 0.01 vs. miR-224-3p+DHRS4-AS1. All data was normalized to wild type control group (Scramble+pcDNA) (D) Real-time PCR analysis of TP53 and TET1 expression in transfected Calu-3 cells; #p < 0.01 vs. scramble+pcDNA, ^φp < 0.01 vs. miR-224-3p+pcDNA, ^δp < 0.01 vs. miR-224-3p+DHRS4-AS1. (E,F) Western blot analysis of TP53 and TET1 expression in transfected Calu-3 cells; the optical densities of protein were determined with ImageJ software, #p < 0.01 vs. scramble+pcDNA, ^φp < 0.01 vs. miR-224-3p+pcDNA, ^δp < 0.01 vs. miR-224-3p+DHRS4-AS1. All data are expressed as the mean ± SD from three independent experiments, and each sample was repeated in triplicate.