Sodium new houttuyfonate suppresses metastasis in NSCLC cells through the Linc00668/miR-147a/slug axis

Abstract

Background: As most lung cancer patients present with invasive, metastatic disease, it is vital to investigate anti-metastatic treatments for non-small cell lung cancer (NSCLC). Houttuynia cordata is commonly used as a Chinese anticancer medicine in the clinic, and sodium new houttuyfonate (SNH), a main compound of this herb, has long been found to have antibiotic effects, although its anticancer effects have not been investigated. Here, we tried to address this lack of research from the perspective of the competing endogenous RNA (ceRNA) theory.

Methods: The effects of SNH on NSCLC cells were analysed with Cell Counting Kit-8 assays and colony formation assays. In addition, transwell assays and wound healing assays were used to determine the effects of SNH on migration and invasion in NSCLC cells. The levels of key genes and proteins were examined by quantitative real-time PCR, western blotting, immunofluorescence staining and IHC staining. Through transcriptome screening and digital gene expression profiling, Linc00668 was identified to be regulated by SNH. Dual-luciferase reporter assays and RNA immunoprecipitation assays verified the binding efficiency between miR-147a and Linc00668 or Slug.

Results: In the present study, SNH regulated NSCLC cells in multiple ways, the most prominent of which was suppressing the expression of Linc00668, which was indicated to promote migration and invasion in NSCLC cells. Functional studies demonstrated that Linc00668 acted as a ceRNA by sponging miR-147a to further regulate Slug mRNA levels, thereby influencing the progression of the epithelial-mesenchymal transition. Consistently, the results of in vivo animal models showed that SNH depressed Linc00668 and suppressed the metastasis of NSCLC.

Conclusions: SNH suppressed metastasis of NSCLC cells and the mechanism may involve with the Linc00668/miR-147a/Slug axis.

Keywords: Linc00668; Metastasis; NSCLC; Sodium new houttuyfonate (SNH); ceRNA; miR-147a.

Fig. 1

SNH beneficially reduces EMT progression in NSCLC cells. a. Chemical structure of SNH. b. CCK-8 proliferation assay of A549, NCI-H460 and NCI-H1299 cells treated with different concentrations of SNH for 24 H, 48 H or 72 H. c. Wound scratch assay of A549 and NCI-H1299 cells treated with SNH for 24 H. d. Transwell invasion assays showed that SNH decreased the invasion ability of A549 and NCI-H1299 cells. e. mRNA level of EMT markers in SNH-treated A549 and NCI-H1299 cells as determined by qRT-PCR. f. Expression of EMT markers in SNH-treated A549 and NCI-H1299 cells as determined by western blot analysis. g. Immunofluorescence staining of E-cadherin expression in SNH-treated A549 and NCI-H1299 cells. The bars and error bars indicate the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001

Fig. 2

Linc00668 is a potential target of SNH. a. Heat map of the differential expression of screened lncRNA and mRNA sequences in NCI-H1299 cells treated or not treated with SNH for 24 H. b. Bioinformatics analysis of Linc00668 expression in human lung adenocarcinoma and lung squamous cell carcinoma tumour tissues compared with normal tissues. c. Association of Linc00668 expression with NSCLC patient disease-free survival and overall survival. d. Linc00668 expression in 5 NSCLC cell lines, 1 human bronchial epithelial cell line (HBE) and 1 human renal epithelial cell line (293 T) as determined by qRT-PCR. e. Linc00668 expression in SNH-treated A549 and NCI-H1299 cells as determined by qRT-PCR. f. Validation of Linc00668 overexpression by p-Linc00668 in A549 and NCI-H1299 cells as determined by qRT-PCR. g. Validation of sh-RNA knockdown efficiency in NCI-H1299 cells as determined by qRT-PCR. The bars and error bars indicate the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001

Fig. 3

Linc00668 is required for the EMT in NSCLC cells. a. Linc00668 expression in SNH-treated A549 and NCI-H1299 cells after p-Linc00668 transfection as determined by qRT-PCR. b-c. Transwell invasion/migration assays showed that the metastatic ability of A549 and NCI-H1299 cells was increased after p-Linc00668 transfection but was diminished after the addition of SNH. d. mRNA levels of EMT markers in SNH-treated A549 and NCI-H1299 cells after p-Linc00668 transfection as determined by qRT-PCR. e-f. Expression of EMT markers in SNH-treated A549 (e) and NCI-H1299 (f) cells after p-Linc00668 transfection as determined by western blot analysis. g. Immunofluorescence staining of E-cadherin expression in SNH-treated A549 and NCI-H1299 cells after p-Linc00668 transfection. The bars and error bars indicate the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001

Fig. 4

Linc00668 functions as a ceRNA and sponges miR-147a in NSCLC cells. a. RIP assays of Linc00668 binding to Ago2 in A549 and NCI-H1299 cell extracts. b. Dual-luciferase reporter assays were used to determine the interaction between miRNAs and Linc00668. c. RIP assays of miR-147a binding to Ago2 in A549 and NCI-H1299 cell extracts. d. Predicted binding sites for miR-147a in the linc00668 sequence. e. RIP assays of Linc00668 binding to Ago2 in miR-147a mimic-transfected A549 and NCI-H1299 cell extracts. f. MiR-147a expression in p-Linc00668-transfected A549 and NCI-H1299 cells as determined by qRT-PCR. g. MiR-147a expression in sh-Linc00668–1-transfected NCI-H1299 cells as determined by qRT-PCR. h-i. Transwell invasion/migration assays showed that the metastatic ability of A549 (h) and NCI-H1299 (i) cells was diminished after miR-147a mimic transfection. j. MiR-147a expression in SNH-treated A549 and NCI-H1299 cells as determined by qRT-PCR. k. mRNA levels of EMT markers in miR-147a mimic-transfected A549 and NCI-H1299 cells as determined by qRT-PCR. L. Expression of EMT markers in miR-147a mimic-transfected A549 and NCI-H1299 cells as determined by western blot analysis. The bars and error bars indicate the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001

Fig. 5

MiR-147a directly targets Slug to mediate EMT progression in NSCLC cells. a. Predicted binding sites for miR-147a in the slug mRNA sequence. b. Bioinformatics analysis of Slug expression in human lung adenocarcinoma and lung squamous cell carcinoma tumour tissues compared with normal tissues. c-d. Slug expression in SNH-treated A549 and NCI-H1299 cells as determined by qRT-PCR (c) and western blot analysis (d). e-f. Slug expression in SNH-treated A549 and NCI-H1299 cells after p-Linc00668 transfection as determined by qRT-PCR (e) and western blot analysis (f). g-h. Slug expression in sh-Linc00668–1-transfected NCI-H1299 cells as determined by qRT-PCR (g) and western blot analysis (h). i-j. Slug expression in miR-147a mimic-transfected A549 and NCI-H1299 cells as determined by qRT-PCR (i) and western blot analysis (j). The bars and error bars indicate the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001

Fig. 6

Linc00668 regulated Slug expression indirectly via sponging miR-147a. a. Predicted binding sites for miR-147 on Linc00668 and a diagram depicting the construction of the wild type (WT) and mutant type (MUT) pmirGLO-Linc00668 plasmids. b-d. 293 T (b), A549 (c) and NCI-H1299 (d) cells were co-transfected with miR-147a mimics or N.C. mimics and pmirGLO or pmirGLO-Linc00668-WT or pmirGLO-Linc00668-MUT. Luciferase activity was detected 24 H after transfection using a dual-luciferase assay. e. Predicted binding sites for miR-147 on slug and a diagram depicting the construction of the WT and MUT pmirGLO-slug plasmids. f-h. 293 T (f), A549 (g) and NCI-H1299 (h) cells were co-transfected with miR-147a mimic or N.C. mimics and pmirGLO or pmirGLO-slug-WT or pmirGLO-slug-MUT. Luciferase activity was detected 24 H after transfection using a dual-luciferase assay. The bars and error bars indicate the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001

Fig. 7

Verification that SNH suppresses the EMT in vivo. a. Representative images of the lungs of nude mice that were injected with NCI-H1299-luc cells and treated with SNH. b. Representative images showing haematoxylin and eosin staining of lung samples from the different groups. c. Bioluminescent imaging and quantification of photon flux of different dose of SNH influenced groups with left lung parenchyma injection of luciferase-marked NCI-H1299 cells. d. Bioluminescent imaging and quantification of the photon flux of lungs from different groups. e. Linc00668 and slug expression in the tissue of lungs of the different groups as determined by qRT-PCR. f. IHC staining showing slug expression in the different groups. The bars and error bars indicate the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001

Fig. 8

Schematic diagram of mechanism on this research. SNH negatively regulates metastasis of NSCLC by Linc00668/miR-147a/Slug axis