Ectodermal‑neural cortex 1 affects the biological function of lung cancer through the MAPK pathway

Abstract

Ectodermal‑neural cortex 1 (ENC1), a highly expressed protein in lung cancer tissues, was identified from the Cancer Genome Atlas (TCGA) database. The objective of the present study was to examine the effects of ENC1 on the biological functions of lung cancer cells. For this purpose, the expression of ENC1 was examined by RT‑qPCR to compare mRNA expression levels between 28 lung cancer tissue samples and para‑cancerous tissue samples. The association between ENC1 expression and clinicopathological features was evaluated between the 2 tissue types. Using RT‑qPCR and western blot analysis, the expression of ENC1 was investigated in a normal lung cell line (16HBE) and 2 lung cancer cell lines (A549 and H1299). The effect of siRNA targeting ENC1 (si‑ENC1) on the proliferation of A549 and H1299 cells was detected by CCK‑8 assay at the indicated time points. Transwell assay was used to measure the migration and invasion of A549 and H1299 cells following transfection with siRNA targeting ENC1 (si‑ENC1). The expression levels of several proteins related to migration and invasion were examined by western blot analysis. A mouse model of subcutaneous tumor xenotransplantation was established in nude mice to examine the effects of ENC1 downregulation on cancer cells. The results revealed that the expression of ENC1 in lung cancer tissues and lung cancer cells was significantly higher than that in para‑cancerous tissues and non‑cancer lung cells, respectively. The knockdown of ENC1 in the A549 and H1299 cells using si‑ENC1 significantly decreased cell proliferation, migration and invasion compared with the untransfected cells. The knockdown of ENC1 significantly downregulated the levels of matrix metalloproteinase (MMP)2, MMP9, N‑cadherin, p‑c‑Jun N‑terminal kinase (JNK), p‑extracellular signal‑regulated kinase (ERK) and p‑p38. The levels of E‑cadherin were upregulated. In the mouse lung tumor model, reduced levels of ENC1 inhibited the growth of lung tumors. On the whole, the present study demonstrates that ENC1 is involved in the proliferation, migration and invasion of lung cancer cells, and may thus be an effective diagnostic target for certain cancers. The inhibition or reduction of ENC1 activity may represent a breakthrough in the treatment of lung cancer.

Keywords: lung cancer; extracellular signal‑regulated kinase; c‑Jun N‑terminal kinase; ectodermal‑neural cortex 1.

Figure 1

High expression of ENC1 in lung cancer tissues and cells. (A and B) The expression of ENC1 between lung cancer and para-cancerous tissue from the TCGA database. (C) Kaplan-Meier survival plot of overall survival of lung cancer in TCGA database, categorized according to ENC1 gene expression (high vs. low, based on mean expression). (D) The expression of ENC1 in normal and tumor tissues by RT-qPCR. (E) The expression of ENC1 in tumor tissues between stage II and III by RT-qPCR. (F) The expression of ENC1 in normal and tumor tissues by immunohistochemical analysis. (G and H) The expression of ENC1 between normal cell line and lung cancer cell lines by RT-qPCR and western blot analysis. Scale bar, 50 µm; ^*P<0.05, ^***P<0.001 vs. normal group; **P<0.01 vs. 16HBE cell group; ENC1, ectodermal-neural cortex 1.

Figure 2

Gene expression profiling of si-ENC1-transfected A549. (A) Volcano plot of all genes detected in transcriptome analysis. (B) Heatmap represents the magnitude of gene expression of 1404 genes (P<0.05, LogFC ≥1 o r≤−1). (C and D) Relative fold change of genes representing key pathways identified by GO and KEGG analyses. (E and F) Heatmap represents magnitude of gene expression of cell adhesion and regulation of cell binding. ENC1, ectodermal-neural cortex 1.

Figure 3

Downregulation of ENC1 inhibits the migration and invasion of NSCLC cells. (A) Knockdown efficiency of ENC1 was quantified in A549 and H1299 by RT-qPCR. (B) Knockdown efficiency of ENC1 was quantified in A549 and H1299 by western blot analysis. (C-J) The effects of ENC1 deficiency on cell migration and invasion were investigated by Transwell assay. (K-M) The effects of ENC1 deficiency on the expression of N-cadherin, E-cadherin, MMP2 and MMP9 were investigated by western blot analysis. ^*P<0.05, ^**P<0.01, ^***P<0.001 vs. si-NC group. ENC1, ectodermal-neural cortex 1; NSCLC, non-small cell lung cancer.

Figure 4

Downregulation of ENC1 inhibits the proliferation of NSCLC cells. (A and B) Effects of ENC1 deficiency on the proliferation were investigated in A549 and H1299 by CCK-8 assay. (C-F) The effects of ENC1 deficiency on the proliferation were investigated in A549 and H1299 using an EdU detection kit. ^*P<0.05, ^**P<0.01 vs. si-NC group. ENC1, ectodermal-neural cortex 1; NSCLC, non-small cell lung cancer.

Figure 5

Downregulation of ENC1 affects the JNK and ERK pathways. (A-C) Effects of ENC1 deficiency on the expression of p-ERK, ERK, p-JNK, JNK p-p38 and p38 were quantified in A549 and H1299 cells by western blot analysis. ^*P<0.05 vs. si-NC group. ENC1, ectodermal-neural cortex 1.

Figure 6

Downregulation of ENC1 inhibits the growth of NSCLC in a mouse model. (A) Image of murine tissues from the 2 groups. (B) Tumor volume in the sh-ctrl and sh-ENC1 groups. (C) Tumor weight in the sh-ctrl and sh-ENC1 groups. (D) Tissue pathological changes in the sh-ctrl and sh-ENC1 groups, determined by H&E staining. (E-H) The expression levels of N-cadherin, E-cadherin, MMP2 and MMP9 were investigated and compared between the sh-ctrl and sh-ENC1 groups by immunohistochemistry. Scale bar, 50 µm; ^**P<0.01, vs. sh-ctrl. ENC1, ectodermal-neural cortex 1; NSCLC, non-small cell lung cancer.

Figure 7

Upregulation of ENC1 enhances the proliferation, migration and invasion of NSCLC cells. (A) The overexpression efficiency of ENC1 was quantified in A549 and H1299 by RT-qPCR. (B) The overexpression efficiency of ENC1 was quantified in A549 and H1299 by western blot analysis. (C) Effects of ENC1 overexpression on proliferation were investigated in A549 and H1299 cells by CCK-8 assay. (D) The effects of ENC1 overexpression on the proliferation were investigated in A549 and H1299 cells using an EdU detection kit. (E and F) The effects of ENC1 overexpression on cell migration and invasion were investigated by Transwell assay. ^*P<0.05, ^**P<0.01, ^***P<0.001 vs. OE-NC group. ENC1, ectodermal-neural cortex 1; NSCLC, non-small cell lung cancer.