MiR-138-5p suppresses the progression of lung cancer by targeting SNIP1

Abstract

Background: MicroRNAs (miRNAs) play crucial roles in the development of various cancers. Here, we aimed to evaluate the roles of miR-138-5p in lung cancer progression and the value of miR-138-5p in lung cancer diagnosis.

Methods: Quantitative real-time PCR was performed to examine the expressions of miR-138-5p and smad nuclear interacting protein 1 (SNIP1) mRNA. The diagnostic value of miR-138-5p was analyzed using receiver operating characteristic (ROC) curve analysis, sensitivity, and specificity. We explored the effect of miR-138-5p on cell proliferation and metastasis by CCK-8, colony formation, wound healing and transwell assays. Western blot was employed to detect the protein expression of SNIP1 and related genes. Lung cancer cell growth was evaluated in vivo using xenograft tumor assay.

Results: MiR-138-5p was decreased in the serum of patients with non-small cell lung cancer (NSCLC) and in NSCLC cells and tissues. The area under the ROC curve of serum miR-138-5p in the diagnosis of NSCLC was 0.922. This finding indicates the high diagnostic efficiency for lung cancer. MiR-138-5p suppressed but its inhibitor promoted cell proliferation and migration compared with control treatment in vitro and in vivo. MiR-138-5p directly binds to the 3'-untranslated region of SNIP1 and negatively regulated the expression of SNIP1, thereby inhibiting the expression of cyclin D1 and c-Myc. Moreover, overexpression of SNIP1 rescues the miR-138-5p-mediated inhibition in NSCLC cells.

Conclusions: The results suggested that miR-138-5p suppressed lung cancer cell proliferation and migration by targeting SNIP1. Serum miR-138-5p is a novel and valuable biomarker for NSCLC diagnosis.

Keywords: SNIP1; biomarker; lung cancer; miR-138-5p; tumor suppressor.

FIGURE 1

MiR‐138‐5p is downregulated in lung cancer. (a) RT‐qPCR was performed to evaluate the expression of miR‐138‐5p in 18 lung cancer serum samples and matched adjacent normal serum samples. (b) The receiver‐operating curve (ROC) curve of serum miR‐138‐5p was analyzed in non‐small cell lung cancer (NSCLC) patients. (c) Relative expression of miR‐138‐5p in NSCLC cells. (d, e) miR‐138‐5p levels in lung adenocarcinoma (LUAD, n = 393) compared with normal controls (n = 43) and in lung squamous cell carcinoma (LUSC, n = 394) compared with normal controls (n = 45) from the cancer genome atlas (TCGA), respectively. All data are presented as the mean ± SD of three independent experiments (***p < 0.001).

FIGURE 2

MiR‐138‐5p inhibited the growth of lung cancer cells. (a, b) Relative expression of miR‐138‐5p in A549 and H1975 cells transfected with NC mimic, miR‐138‐5p mimic, NC inhibitor or miR‐138‐5p inhibitor. (c–f) The cell counting kit‐8 (CCK‐8) viability assay was performed at 24, 36, 48, and 96 h after the A549 and H1975 cells were transfected with miR‐138‐5p mimic, miR‐138‐5p inhibitor, or corresponding negative control RNA (NC mimic and NC inhibitor). (g–j) Colony formation of A549 and H1975 cells transfected with NC mimic, miR‐138‐5p mimic, NC inhibitor or miR‐138‐5p inhibitor. Left panel: Representative image. Right panel: Quantitative analysis. (c, d, g, h) Overexpression of miR‐138‐5p suppressed the growth of A549 and H1975 cells. (e, f, i, j) miR‐138‐5p inhibitor promoted the proliferation of A549 and H1975 cells. All data are presented as the mean ± SD of three independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001).

FIGURE 3

MiR‐138‐5p suppressed the migration of lung cancer cells. (a–d) Transwell analysis of A549 and H1975 cells transfected with miR‐138‐5p mimic, miR‐138‐5p inhibitor or corresponding negative control RNA (NC mimic and NC inhibitor). Left panel: Representative image. Right panel: Quantitative analysis. All data are presented as the mean ± SD of three independent experiments (*p < 0.05; **p < 0.01). (e, f) Wound healing assay of A549 and H1975 cells treated with equal amounts of miR‐138‐5p mimic, miR‐138‐5p inhibitor or corresponding negative control RNA (NC mimic and NC inhibitor). Top panel: Representative image. Bottom panel: Quantitative analysis of wound closure. All data are presented as the mean ± SD of three independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001). (a, b, e) Overexpression of miR‐138‐5p suppressed the migration of A549 and H1975 cells. (c, d, f) MiR‐138‐5p inhibitor promoted the migration of A549 and H1975 cells. (g, h) Expression levels of E‐cadherin, vimentin, cyclin D1, c‐Myc, and GAPDH in A549 and H1975 cells transfected with NC mimic, miR‐138‐5p mimic, NC inhibitor or miR‐138‐5p inhibitor.

FIGURE 4

SNIP1 is a target gene of miR‐138‐5p. (a) Graphical presentation of the miR‐138‐5p putative binding sites in the wild‐type and mutant type 3′UTR of SNIP1. (b, c) Luciferase reporter assay was performed following transfection with miR‐138‐5p mimic or NC mimic using a luciferase vector including the SNIP1 WT 3′UTR or the mutant 3′UTR in A549 or H1975 cells. (d, e) Luciferase reporter assay was performed following transfection with miR‐138‐5p inhibitor or NC inhibitor using a luciferase vector including the SNIP1 WT 3′UTR or the mutant 3′UTR in A549 or H1975 cells. Luciferase activity was measured 48 h after transfection. All data are presented as the mean ± SD of three independent experiments (**p < 0.01; ***p < 0.001). (f–i) After transfection of NC mimic, miR‐138‐5p mimic, NC inhibitor or miR‐138‐5p inhibitor in the A549 and H1975 cells, the mRNA expression of SNIP1 was detected by RT‐qPCR. All data are presented as the mean ± SD of three independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001). (j, k) Protein expression levels of SNIP1 were measured by Western blot after A549 and H1975 cells were transfected with NC mimic, miR‐138‐5p mimic, NC inhibitor or miR‐138‐5p inhibitor.

FIGURE 5

Overexpression of SNIP1 rescues the miR‐138‐5p‐mediated inhibition in non‐small cell lung cancer (NSCLC) cells. (a) Cell counting kit‐8 (CCK‐8) assays measured cell proliferation at the indicated time points in A549 cells transfected with NC mimic plus control vector (pcDNA3.1), miR‐138‐5p mimic plus control vector, or miR‐138‐5p mimic plus SNIP1 plasmid (pcDNA3.1‐SNIP1). (b) Colony formation assays in A549 cells expressing NC mimic plus control vector (pcDNA3.1), miR‐138‐5p mimic plus control vector, or miR‐138‐5p mimic plus SNIP1 plasmid (pcDNA3.1‐SNIP1) (left panel). Right panel: Quantification of colony formation assays. (c) Transwell analysis of A549 cells transfected with NC mimic plus control vector (pcDNA3.1), miR‐138‐5p mimic plus control vector, or miR‐138‐5p mimic plus SNIP1 plasmid (pcDNA3.1‐SNIP1). Left panel: Representative images of the migrated cells on the membrane. Right panel: Quantification of transwell migration assay. (d) Representative images of scratch wound healing assays in A549 cells expressing NC mimic plus control vector (pcDNA3.1), miR‐138‐5p mimic plus control vector, or miR‐138‐5p mimic plus SNIP1 plasmid (pcDNA3.1‐SNIP1) (left panel). Right panel: Quantitative analysis of wound closure. All data are presented as the mean ± SD of three independent experiments (**p < 0.01; ***p < 0.001)

FIGURE 6

MiR‐138‐5p suppresses the growth of A549 xenograft tumors. Tumorigenesis assay in vivo. The A549 cells (10 million cells per mouse) were treated with lentivirus which overexpressed miR‐138‐5p mimic or miR‐138‐5p inhibitor and were injected subcutaneously into 6‐week‐old female mice. The tumor volume was measured every 2 days. (a, f) Representative images of the excised tumors in the different groups. (b, g) Tumor volume of nude mice. (c, h) Quantitative analysis of the tumor weights on day 32. Data are expressed as median (interquartile range), **p < 0.01; Mann‐Whitney U test. (d, i) Relative expression of miR‐138‐5p in xenograft tumors. Values are presented as the mean ± SD of three independent experiments, **p < 0.01; ***p < 0.001. (e, j) Expression levels of SNIP1 in xenograft tumors. (k) Schematic diagram of miR‐138‐5p and SNIP1 regulating the proliferation and migration of lung cancer cells.