LINC00968 can inhibit the progression of lung adenocarcinoma through the miR-21-5p/SMAD7 signal axis

Abstract

Background: Long non-coding RNAs (LncRNAs) have been associated with several types of cancer. However, little is known about their role in lung adenocarcinoma (LUAD).

Results: LINC00968 was significantly differentially expressed in LUAD tissues. Downregulated LINC00968 was associated with clinicopathological features of LUAD. LINC00968 inhibited cell growth and metastasis by regulating the Hippo signaling pathway We demonstrated that LINC00968 acts as a ceRNA to consume miR-21-5p, enhancing the accumulation of SMAD7, a miR-21-5p target.

Conclusions: LINC00968 limits LUAD progression via the miR-21-5p/SMAD7 axis and may serve as a prognostic biomarker and therapeutic target for LUAD.

Methods: We conducted comprehensive data mining on LINC00968 based on the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) database. The expression of LINC00968 in LUAD cells was determined using in situ hybridization. We detected LINC00968 function in LUAD cells using the MTT, clone formation, and transwell assays, and tumor xenografts. Label-free quantitative proteomics, western blotting, a dual-luciferase reporter assay, immunofluorescence, and RNA immunoprecipitation assays were used to determine the correlations among LINC00968, miR-21-5p, and SMAD7. Gain- and loss-function approaches were used to explore the effects of LINC00968, miR-21-5p, and SMAD7 on cell proliferation, migration, and invasion.

Keywords: LINC00968; SMAD7; lung adenocarcinoma (LUAD); metastasis; prognosis.

Figure 1

Decreased LINC00968 is related to the patient's pathological grade and prognosis in LUAD. (A) Identification of common significantly differentially expressed lncRNAs from four independent LUAD microarray analyses using the GEO2R analysis online tools. (B) The levels of LINC00968 expression were downregulated in tumor tissues compared with that of non-tumor lung tissues (GSE19188, GSE27262, GSE40791, and GSE30219). (C) LINC00968 expression in LAC and non-tumor tissues based on the data from the GSE18842 and TANRIC databases. (D) The levels of LINC00968 expression were validated by in situ hybridization histochemistry in tissue biopsies. (E) The area under the curve (AUC) of LINC00968 levels in LAC patients based on a microarray, ranged from 0.9501 to 1.000, proving that LINC00968 was an effective diagnostic molecular marker for LAC patients. (F) Low LINC00968 expression was associated with advanced disease stages in the GSE50081 and GSE30219 datasets (clinical stages, N stages, and T stages). (G) Based on the TANRIC database, the relationship between LINC00968 expression and advanced disease stages was further validated (clinical stages, N stages, and T stages). (H) Kaplan-Meier overall survival curves and Kaplan-Meier estimates of time to the first progression for patients with LAC classified according to relative LINC00968 expression level. (I) Low expression of LINC00968 was associated with shorter overall survival and disease-free survival time.

Figure 2

LINC00968 inhibits malignant phenotype in tumor cells. (A) After treatment with LINC00968 overexpressing or control plasmids, the LINC00968 expression levels were validated by qTR-PCR. (B) Cell proliferation was detected at the indicated time points. (C) Representative images of cell forming colonies and colony formation efficiency of each group. (D) The migration ability of LINC00968 overexpressing cells in each group. The histogram of the migration cell number is on the right. (E) The representative images of cells moving across the Matrigel membranes. (F) Tumor xenografts from nude mice. (G) Upregulation of LINC00968 significantly decreased tumor weight. (H) Representative images of the lung metastatic foci on the surface area. Arrows indicate the lung metastatic foci (left). The lung weights in each group are listed to the right. (I) Representative images of lung metastatic foci after HE-staining (left). The number of metastatic foci (right). *P<0.05, **P<0.01, ***P<0.001.

Figure 3

LINC00968 regulates the SMAD-mediated Hippo signaling pathway. (A) The workflow of random peptide arrays used to screen downstream proteins specifically regulated by LINC00968 in LUAD. (B) Volcano plots of the differentially expressed lncRNAs (266 upregulated and 208 down-regulated proteins) in LINC00968 overexpressing and control A549 cells. (C) The most frequent proteins and relevant signaling pathways regulated by LINC00968. (D) SMAD and STK3 expressions were increased whereas YAP1 was decreased after LINC00968 upregulation as detected by western blotting. (E) YAP1 expression (red), validated by immunofluorescence following LINC00968 overexpression in LUAD cells.

Figure 4

LINC00968 inhibits tumor development by regulating the miR-21-5p/SMAD7 axis. (A) FISH detection of subcellular localization of LINC00968 in A549 and H1299 cells. (B) The miRNACancerMAP datasets suggested that there was a significant positive correlation between LINC00968 and SMAD7. (C) The miRNAs negatively related to LINC00968 and SMAD7 most frequently were summarized by miRNACancerMAP. (D) Correlation between LINC00968 and the top 12 most frequent miRNAs. (E) Correlation between SMAD7 and the top 12 most frequent miRNAs. (F) qRT-PCR quantification of LINC00968. LINC00968 was more enriched in the cells treated with the Ago2 antibody compared with those treated with the IgG negative control antibody. (G) The complementary sites of LINC00968, miR-21-5p, and SMAD7. (H) The expression of miR-21-5p was decreased and increased in LINC00968 overexpressing and knockdown LUAD cells, respectively. (I) LINC00968 expression was decreased in the miR-21-5p mimics group and increased in the miR-21-5p inhibitor group. (J) H1299 and A549 cells co-transfected with a miR-21-5p mimic or negative control (NC) and luciferase vectors containing either wild type (LINC00968-WT) or mutated (LINC00968-mut) miR-21-5p-binding sites. The luciferase activity was measured after 48 h. (K) and (L) SMAD7 and YAP1 protein expression following LINC00968, SMAD7 and/or miR-21-5p modulation in A549 and H1299 cells. *P<0.05, **P<0.01, ***P<0.001.

Figure 5

LINC00968 inhibits tumor development via sponging miR-21-5p/SMAD7 axis. The miR-21-5p mimics significantly increased cell proliferation (A), colony-forming ability (C), invasion, and migration (E), and also partially attenuated the inhibitory effects of LINC00968 over-expression. SMAD7 knockdown enhanced cell proliferation (B), colony-forming ability (D), invasion, and migration (F), and also partially attenuated the inhibitory effects of LINC00968 over-expression. **P<0.01, ***P<0.001.

Figure 6

The expression level and clinical value of miR-21-5p and SMAD7 in LUAD. (A) The miR-21-5p expression was significantly increased in LUAD tissues compared with that of non-tumor tissues from the LUAD chip GSE63805 dataset (A) and XENA database (B). (C) The survival curve analyses indicated that high expression of miR-21-5p was associated with poor clinical outcomes. SMAD7 expression decreased in tumor tissues compared with non-tumor lung tissues from the GSE63459 dataset (D) and XENA database (E). The Kaplan Meier-plotter (F) and XENA (G) database demonstrated that the low SMAD7 expression was associated with significantly poor patient survival. (H) SMAD7 protein expression was downregulated in human LUAD tissue biopsies as evidenced by immunohistochemical staining from the Human Protein Atlas Database.