FOXA1-induced LINC00621 promotes lung adenocarcinoma progression via activating the TGF-β signaling pathway

Abstract

Background: Lung adenocarcinoma (LUAD) is highly malignant and associated with poor prognoses in patients worldwide. There has been widespread recognition that lncRNAs are tightly linked to LUAD tumorigenesis and development. Here, we identified that the LINC00621 level was increased in LUAD tissues and concerned with the poor prognoses in LUAD patients.

Methods: Bioinformatical analysis and RT-qPCR determined the level of LINC00621 in LUAD tissues and cell lines. The admeasurement of the proliferation, migration, and invasion abilities of LUAD cells was utilized in the CCK8 and Transwell formulas. Luciferase reporter assay was used to corroborate the downstream target genes of LINC00621. The phosphorylated SMAD3 protein was tested by Western blotting assay. The impression of LINC00621 knockdown on LUAD tumor growth and metastasis put into effect by murine models. ChIP-qPCR assay was carried out to verify the transcriptional regulation by FOXA1 on LINC00621.

Results: In vitro, the knockdown of LINC00621 significantly reduced the proliferative, migrating, and invasive abilities, the same was true for tumorigenesis and metastasis in vivo. MiR-34a-5p as a straight target of LINC00621 was ascertained, and LUAD patients with inferior miR-34a-5p levels had undesirable prognoses. Furthermore, TGFBR1 is an immediate and functional connection site of miR-34a-5p. Collectively, LINC00621 can sponge miR-34a-5p and upregulate TGFBR1 levels, which further sensitized TGF-β signaling pathway. Finally, it was revealed that FOXA1 transcriptionally upregulated LINC00621.

Conclusion: This study uncovered that FOXA1-induced LINC00621 promotes LUAD progression via the miR-34a-5p/TGFBR1/TGF-β axis, and is one novel therapeutic target that may be used in LUAD treatment.

Keywords: FOXA1; LINC00621; MiR-34a-5p; TGF-β; lung adenocarcinoma.

FIGURE 1

LINC00621 is aberrantly overexpressed in lung adenocarcinoma (LUAD) and is associated with poor prognosis. (a, b) LINC00621 expression in tumor and normal tissues included paired and unpaired from TCGA. (c) The relative LINC00621 expression was analyzed by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) in the tumor and adjacent normal tissues (n = 10). *(d) The relative LINC00621 expression analyzed in nonmetastatic LUAD patient tissue samples (n = 16) and metastatic LUAD patient tissue samples (n = 14) by qRT‐PCR. (e) The pan‐cancer analysis of LINC00621 expression. (f, g) Kaplan–Meier analysis showed that elevated expression of LINC00621 was associated with overall survival and progression‐free interval in LUAD patients. Data shown are mean ± SD (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 2

LINC00621 facilitates lung adenocarcinoma (LUAD) cell proliferation in vitro and in vivo. (a) Proliferation enrichment analysis of LINC00621. (b) The expression of LINC00621 was knocked down by two different shRNAs in both HCCC827 and A549 cells. (c, d, e) The proliferation ability of HCC827 and A549 cells after knockdown LINC00621 expression was determined by cell counting kit‐8 (CCK‐8) and colony formation assays. (f) A549 cells treated with sh‐NC lentivirus were inoculated subcutaneously into the left flanks and treated with sh‐1 lentivirus into the right flanks of nude mice (n = 5). (g) Tumor volume. (h) Tumor weight. Data shown are mean ± SD (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, compared with the normal control group.

FIGURE 3

LINC00621 knockdown inhibited the migration and invasion of lung adenocarcinoma (LUAD) cells in vitro and in vivo. (a) Enrichment analysis of LINC00621 metastasis. (b) A transwell assay was used to quantify the migration and invasion of A549 and HCC827 cells with LINC00621 knockdown. (c) Imaging of lung metastasis in mice and lung tissue section. (d) Metastasis count analysis of sh‐1 and NC group. Data shown are mean ± SD (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

FIGURE 4

MiR‐34a‐5p was a direct LINC00621 target. (a, b) We analyzed the subcellular localization of LINC00621 in lung adenocaqrcinoma (LUAD) cells using nucleocytoplasmic separation assays and RNA FISH results. (c) MiRNA target by LINC00621 was predicted by LncBase. (d) Survival analysis of miR‐34a‐5p in LUAD. (e) The relative miR‐34a‐5p level in sh‐1, sh‐2 and NC group. (f) Putative target sequence of miR‐34a‐5p on the 3′‐UTR of LINC00621 and detection of luciferase activity by luciferase reporter assay. Data shown are mean ± SD (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

FIGURE 5

LINC00621 activated TGF‐β signaling via miR‐34a‐5p/TGFBR1. (a) The enrichment of the TGF‐β signaling pathway. (b) TGF‐β signal activity was downregulated by LINC00621 knocked and upregulated by miR‐34a‐5p inhibitor in A549 and HCC827 cells. (c) Western blot experiment showed that protein expression level of p‐SMAD3 repressed by low expression of LINC00621 could be promoted by miR‐34a‐5p inhibitor in A549 and HCC827 cells. There was no change in the level of SMAD protein. P84 protein was used as an internal control. (d) Schematic representation of the predicted binding site between miR‐34a‐5p and TGFBR1 in wild‐ and mutant‐types is shown. (e) Correlation between TGFBR1 and miR‐34a‐5p expression levels. (f, g) TGFBR1 protein and mRNA level of A549 and HCC827 were fortified miR‐34a‐5p mimics. (h) Luciferase reporter activities of wild‐type TGFBR1 and mutant TGFBR1 reporters in A549 and HCC827 cells after adding miR‐34a‐5p mimics. (i, j) Kaplan–Meier analysis showed that elevated expression of TGFBR1 was associated with overall survival and progression‐free interval in lung adenocarcinoma (LUAD). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

FIGURE 6

FOXA1 might be the transcription factor of LINC00621. (a) The top five transcription factors bound to LINC00621 promotor are exhibited. (b) The expression of FOXA1 in tumor and normal tissues of lung adenocarcinoma (LUAD) patients in TGGA. (c) The mRNA level of LINC00621 of A549 and HCC827 cells with FOXA1 knockdown. (d) DNA motif of FOXA1 and the binding sites of FOXA1 on the promotor of LINC00621 are shown. (e) ChIP‐qPCR, chromatin immunoprecipitation‐quantitative polymerase chain reaction (ChIP‐qPCR) determined the binding sites of FOXA1. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.