Long noncoding RNA LINC00968 inhibits proliferation, migration and invasion of lung adenocarcinoma through targeting miR-22-5p/CDC14A axis

Abstract

Lung adenocarcinoma (LUAD) is a high aggressive human cancer which usually diagnosed at advanced stages. Accumulating evidences indicate that long noncoding RNAs (lncRNAs) are crucial participants in LUAD progression. In the present study, we found that lncRNA LINC00968 was significantly down-regulated in LUAD tissues and cell lines. LINC00968 level was positively correlated to survival rate, and negatively correlated to tumor node metastasis (TNM) stage, tumor size and lymph node metastasis of LUAD patients. We over-expressed LINC00968 in LUAD cells using lentivirus, inhibited proliferation and cell cycle arrest at G1 phase were detected. LINC00968 over-expression also suppressed migration, invasion and epithelial mesenchymal transition. We further validated that LINC00968 localized in cytoplasm and acted as an upstream regulator of microRNA miR-22-5p, which was up-regulated in LUAD tissues and cell lines. Besides, elevated miR-22-5p expression abolished the effect of LINC00968 over-expression on LUAD progression including in vivo tumor growth. In addition, we first validated that cell division cycle 14A (CDC14A), which was down-regulated in LUAD tissues, was a downstream target of miR-22-5p. We over-expressed CDC14A in LUAD cells and miR-22-5p induced LUAD progression was partially reversed. In conclusion, our study demonstrated that LINC00968 inhibited proliferation, migration and invasion of LUAD by sponging miR-22-5p and further restoring CDC14A. This novel regulatory axis might provide us with promising diagnostic and therapeutic target in LUAD treatment.

Keywords: CDC14A; LINC00968; Lung adenocarcinoma progression; miR-22-5p.

Fig. 1

LINC00968 was down-regulated in LUAD tissues and cell lines. A Expression of LINC00968 in Normal tissues (blue) and LUAD, LUSC tissues (red) in TCGA database. B LINC00968 level in LUAD tissues and adjacent non-tumor tissues in 60 LUAD patients was measured using quantitative real-time PCR. C Analysis of correlation between LINC00968 level and overall survival rate and time of LUAD patients. D Relative LINC00968 level in normal lung epithelial cell line BEAS2B and four LUAD cell lines was detected using quantitative real-time PCR. LINC00968: long intergenic non-protein coding RNA 968; LUAD: lung adenocarcinoma; LUSC: lung squamous cell carcinoma. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 2

LINC00968 over-expression inhibited proliferation, migration and invasion of LUAD cells. A549 and H1975 cells were infected with LV-VC and LV-LINC00968. A Quantitative real-time PCR was performed to measure LINC00968 level in A549 and H1975 cells. B Cell counting kit-8 assay was carried out to evaluate cell proliferation. C Cell cycle of A549 and H1975 cells was detected using flow cytometry. D Wound healing assay was performed to assess the cell migration ability. E Cell invasion capacity was assessed using transwell assay. F Protein levels of E-cadherin, N-cadherin, TWIST and SNAIL were detected using western blotting. LINC00968: long intergenic non-protein coding RNA 968; LUAD: lung adenocarcinoma; LV-NC: negative control lentivirus; LV-LINC00968: LINC00968 over-expression lentivirus; *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

LINC00968 was a putative regulator of miR-22-5p. A Relative miR-22-5p level in LUAD tissues and adjacent non-tumor tissues was measured using quantitative real-time PCR. B Binding site of miR-22-5p on LINC00968 is shown in Fig. 3B. C Interaction between LINC00968 and miR-22-5p was validated by luciferase reporter assay. D Relative miR-22-5p level in A549 and H1975 cells after LINC00968 over-expression was measured using quantitative real-time PCR. E The subcellular localization of LINC00968 was displayed using FISH assay. F Enrichment of LINC00968 and miR-22-5p on Ago2 complex was determined using RNA immunoprecipitation assay. G Enrichment of LINC00968 using biotinylated miR-22-5p probe was detected using quantitative real-time PCR.LINC00968: long intergenic non-protein coding RNA 968; LUAD: lung adenocarcinoma; FISH: fluorescent in situ hybridation; **p < 0.01, ***p < 0.001, ns: no significant

Fig. 4

LINC00968 inhibited proliferation, migration and invasion of LUAD cells through regulating miR-22-5p. A549 and H1975 were infected with LV-LINC00968/LV-NC and transfected with miR-22-5p/NC agomir. A Cell proliferation was evaluated using cell counting kit-8 assay. B Cell cycle was detected using flow cytometry. C Wound healing assay was performed to assess cell migration. D Transwell assay was carried out to assess cell invasion. E Protein levels of E-cadherin, N-cadherin, TWIST and SNAIL was determined using western blotting. F A549 cells were subcutaneously injected to mice to form tumor xenograft model, and tumor volumes were analyzed. G Expression of Ki67 in mouse tumor tissues was determined by immunohistochemistry. LINC00968: long intergenic non-protein coding RNA 968; LUAD: lung adenocarcinoma; LV-NC: negative control lentivirus; LV-LINC00968: LINC00968 over-expression lentivirus; *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

miR-22-5p promoted proliferation, migration and invasion of LUAD cells through targeting CDC14A. A Relative CDC14A mRNA level in LUAD tissues and adjacent non-tumor tissues were measured using quantitative real-time PCR. B CDC14A protein level in LUAD and adjacent non-tumor tissues was detected by western blotting. C Correlation between miR-22-5p level and CDC14A level in LUAD tissues was analyzed using linear regression. D Binding site of miR-22-5p on CDC14A was shown, and their interaction was validated using luciferase reporter assay. E Relative mRNA level of CDC14A after miR-22-5p/NC agomir was determined using quantitative real-time PCR. A549 and H1975 cells were co-transfected with miR-22-5p/NC agomir and pcDNA3.1-CDC14A/pcDNA3.1. F Cell counting kit-8 assay was performed to evaluate cell proliferation. G Cell cycle was detected using flow cytometry. H Wound healing assay was performed to evaluate cell migration. I Transwell assay was carried out to assess cell invasion. J Protein levels of E-cadherin, N-cadherin, TWIST and SNAIL was determined using western blotting.LINC00968: long intergenic non-protein coding RNA 968; LUAD: lung adenocarcinoma; CDC14A: cell division cycle 14A; NC: non-specific control; *p < 0.05, **p < 0.01, ***p < 0.001