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. 2023 Apr;50(4):3557-3568.
doi: 10.1007/s11033-023-08259-x. Epub 2023 Feb 14.

LncRNA PCAT6 promotes proliferation, migration, invasion, and epithelial-mesenchymal transition of lung adenocarcinoma cell by targeting miR-545-3p

Chuyi Yang #  1 , Hongyu Huang #  1 , Yongpeng Li  2 , Ting Zhuo  1 , Lu Zhu  1 , Chenyang Luo  1 , Yanbin Wu  1 , Shouming Qin  3
Affiliations

LncRNA PCAT6 promotes proliferation, migration, invasion, and epithelial-mesenchymal transition of lung adenocarcinoma cell by targeting miR-545-3p

Chuyi Yang et al. Mol Biol Rep. 2023 Apr.

Abstract

Background: Lung cancer is a high incidence cancer on a worldwide basis and has become a major public health problem. Lung adenocarcinoma (LUAD) makes up approximately half of all lung cancers and is a threat to human health. Long non-coding RNAs (lncRNAs) is an important regulator of the development and progression of lung adenocarcinoma. In this manuscript we examined the role and potential mechanism of lncRNA PCAT6 in the development of LUAD.

Methods and results: Differences in lncRNA PCAT6 levels between LUAD samples and normal samples were first explored in the GEPIA database. We found that lncRNA PCAT6 expression was elevated, which was also validated in lung adenocarcinoma tissues and cell lines. Using western blotting, CCK-8, EdU, wound healing and transwell assays, we found that knockdown of lncRNA PCAT6 inhibited EMT, proliferation, migration, and invasion of LUAD cells. We noted a predicted a binding site for lncRNA PCAT6 and miR-545-3p through conducting bioinformatic analyses, and their binding was subsequently verified by a dual-luciferase reporter assay. Rescue experiments confirmed that miR-545-3p inhibitor partially abolished the inhibition function of lncRNA PCAT6 knockdown on LUAD cells. In addition, we predicted the downstream target genes of miR-545-3p and verified them by RT-qPCR. We found that EGFR was reduced in the silence of lncRNA PCAT6 and upregulated after miR-545-3p inhibition.

Conclusion: This study demonstrates that lncRNA PCAT6 promotes a more aggressive LUAD phenotype by sponging miR-545-3p. This finding may provide new ideas for the treatment of lung cancer.

Keywords: EMT; Lung adenocarcinoma; lncRNA PCAT6; miR-545- 3p.

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Conflict of interest statement

No potential conflict of interest was reported by the authors.

Figures

Fig. 1
Fig. 1
LncRNA PCAT6 was highly expressed in lung adenocarcinoma (a-b) GEPIA database showed abnormal expression of lncRNA PCAT6 in various tumors, and increased expression level in LUAD. (c) The expression level of lncRNA PCAT6 in lung adenocarcinoma samples and normal adjacent samples was detected by RT-qPCR. (d) The expression levels of lncRNA PCAT6 in three LUAD cell lines (A549, H1299 and H1975) and healthy BEAS-2B cells were detected by RT-qPCR. (e) Cytoplasmic-Nuclear fractionation was used to detect the proportion of PCAT6 in nucleus and cytoplasm. * p < 0.05, ** p < 0.01, *** p < 0.001
Fig. 2
Fig. 2
Silencing of lncRNA PCAT6 inhibited LUAD cell growth, migration, invasion and EMT (a) RT‑qPCR analysis of the transfection efficiency of three si-PCAT6 sequences. (b-c) EdU assays and CCK8 were performed to examin A549 and H1299 cell growth. (d-e) Scratch assays and transwell assays were performed to examine the effect of silencing of PCAT6 on LUAD cells. (f) WB assays tested the protein levels of EMT-related protein. *P < 0.05, **P < 0.01, ***P < 0.001 vs. si-NC group
Fig. 3
Fig. 3
MiR-545-3p can target lncRNA PCAT6 gene and negatively regulate its expression (a) The targeted miRNAs of PCAT6 were predicted by StarBase database. (b) RT-qPCR was used to detect the association between miRNAs and PCAT6. (c-d) The expression of miR-545-3p in LUAD samples and cells was observed by RT-qPCR. (e) Detection of miR-545-3p mimics and inhibitors by RT-qPCR. (f) The binding sites between PCAT6 with miR-545-3p. (g) Predicted spots were detected by dual-Luciferase reporter assay. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 4
Fig. 4
MiR-545-3p inhibitor partially reversed lncRNA PCAT6 silencing’s suppression on LUAD cell growth and migration (a-b) H1299 and A549 cell proliferation measured through EdU and CCK8 assays; (c) Scratch assays conducted to examine H1299 and A549 cell migration. *P < 0.05, **P < 0.01, ***P < 0.001 vs. si-NC group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. si-PCAT6#3 + inhibitor-NC group
Fig. 5
Fig. 5
MiR-545-3p inhibitor partially abolished lncRNA PCAT6 silencing inhibition of EMT, migration and invasion (a) Transwell assay was used to detect the migration and invasion of two lung adenocarcinoma cells. (b) Detection of EMT-related protein levels by WB assay. *P < 0.05, **P < 0.01, ***P < 0.001 vs. si-NC group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. si-PCAT6#3 + inhibitor-NC group
Fig. 6
Fig. 6
Predicted target mRNA of miR-545-3p (a)Schematic illustration to show the overlapping of the target mRNAs of miR-545-3p, as predicted by miRwalk, TargetScan and miRDB. (b) protein-protein interaction (PPI) network of the predicted target mRNAs of miR-545-3p. (c) KEGG enrichment analysis of the potential target mRNAs of miR-545-3p. (d) GO term enrichment for the potential target mRNAs of miR-545-3p. (e) The expression of EGFR was detected by RT-qPCR. *P < 0.05, **P < 0.01, ***P < 0.001 vs. si-NC group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. si-PCAT6#3 + inhibitor-NC group
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