Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Jan 15;24(1):79.
doi: 10.1186/s12885-023-11798-y.

Lnc-PSMA8-1 activated by GEFT promotes rhabdomyosarcoma progression via upregulation of mTOR expression by sponging miR-144-3p

Lian Meng #  1 , Hao Shang #  1   2 , Qianqian Liu  1 , Zhenzhen Li  1 , Xiaomeng Wang  1 , Qianru Li  1 , Feng Li  1   3 , Zhenguo Zhao  4 , Chunxia Liu  5   6
Affiliations

Lnc-PSMA8-1 activated by GEFT promotes rhabdomyosarcoma progression via upregulation of mTOR expression by sponging miR-144-3p

Lian Meng et al. BMC Cancer. .

Abstract

Background: GEFT is a key regulator of tumorigenesis in rhabdomyosarcoma (RMS), and overexpression of GEFT is significantly correlated with distant metastasis, lymph node metastasis, and a poor prognosis, yet the underlying molecular mechanism is still poorly understood. This study aimed to investigate and validate the molecular mechanism of GEFT-activated lncRNAs in regulating mTOR expression to promote the progression of RMS.

Methods: GEFT-regulated lncRNAs were identified through microarray analysis. The effects of GEFT-regulated lncRNAs on the proliferation, apoptosis, invasion, and migration of RMS cells were confirmed through cell functional experiments. The target miRNAs of GEFT-activated lncRNAs in the regulation of mTOR expression were predicted by bioinformatics analysis combined with quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The expression of lnc-PSMA8-1, miR-144-3p, and mTOR was measured by qRT-PCR in RMS tissue samples and cell lines. The regulatory mechanisms of the lnc-PSMA8-1-miR-144-3p-mTOR signaling axis were verified by RNA-binding protein immunoprecipitation (RIP), a luciferase reporter assay, qRT-PCR analysis, Western blot analysis, and cell functional experiments.

Results: The microarray-based analysis identified 31 differentially expressed lncRNAs (fold change > 2.0, P < 0.05). Silencing the 4 upregulated lncRNAs (lnc-CEACAM19-1, lnc-VWCE-2, lnc-GPX7-1, and lnc-PSMA8-1) and overexpressing the downregulated lnc-FAM59A-1 inhibited the proliferation, invasion, and migration and induced the apoptosis of RMS cells. Among the factors analyzed, the expression of lnc-PSMA8-1, miR-144-3p, and mTOR in RMS tissue samples and cells was consistent with the correlations among their expression indicated by the lncRNA-miRNA-mRNA regulatory network based on the ceRNA hypothesis. lnc-PSMA8-1 promoted RMS progression by competitively binding to miR-144-3p to regulate mTOR expression.

Conclusion: Our research demonstrated that lnc-PSMA8-1 was activated by GEFT and that the former positively regulated mTOR expression by sponging miR-144-3p to promote the progression of RMS. Therefore, targeting this network may constitute a potential therapeutic approach for the management of RMS.

Keywords: GEFT; Rhabdomyosarcoma; ceRNA; lnc-PSMA8-1; mTOR; miR-144-3p.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Screening of GEFT-regulated lncRNAs in RMS cell lines. A: Heat map representation of microarray data about the expression levels of GEFT-related lncRNAsand GEFT-related-mRNA in GEFT-overexpression and -knockdown RMS cell lines. B: Expression levels of randomly selected 5 up-regulated lncRNAs and 5 down-regulated lncRNAs in GEFT-overexpression and -knockdown RMS cell lines as detected by qRT-PCR
Fig. 2
Fig. 2
Effects of lnc-CEACAM19-1, lnc-VWCE-2, lnc-GPX7-1, lnc-PSMA8-1, and lnc-FAM59A-1 on the proliferation and apoptosis of RMS cells. A: Effects on RD and RH30 cells transfected with siRNA (lnc-CEACAM19-1, lnc-VWCE-2, lnc-GPX7-1, or lnc-PSMA8-1) or lnc-FAM59A-1 overexpressed plasmid as detected by qRT-PCR. B: Proliferative capabilities of RD and RH30 cells transfected with siRNA (lnc-CEACAM19-1, lnc-VWCE-2, lnc-GPX7-1, or lnc-PSMA8-1) or lnc-FAM59A-1 overexpressed plasmid as determined through CCK-8 cell proliferation assay. C: Apoptosis rates of RD and RH30 cells transfected with siRNA (lnc-CEACAM19-1, lnc-VWCE-2, lnc-GPX7-1, or lnc-PSMA8-1) or lnc-FAM59A-1 overexpressed plasmid as determined through flow cytometry assay
Fig. 3
Fig. 3
Effects of lnc-CEACAM19-1, lnc-VWCE-2, lnc-GPX7-1, lnc-PSMA8-1, and lnc-FAM59A-1 on the invasion and migratory of RMS cells.:A: Invasion of RD and RH30 cells transfected with siRNA (lnc-CEACAM19-1, lnc-VWCE-2, lnc-GPX7-1, or lnc-PSMA8-1) or lnc-FAM59A-1 overexpressed plasmid as determined through transwell system. B: Migratory of RD and RH30 cells transfected with siRNA (lnc-CEACAM19-1, lnc-VWCE-2, lnc-GPX7-1, or lnc-PSMA8-1) or lnc-FAM59A-1 overexpressed plasmid as determined through transwell system
Fig. 4
Fig. 4
miR-144-3p may play a bridging role between lnc-PSMA8-1 and mTOR. A: Expression of mTOR in RD and RH30 cells transfected with siRNA (lnc-CEACAM19-1, lnc-VWCE-2, lnc-GPX7-1, or lnc-PSMA8-1) as detected by qRT-PCR. B: Lnc-PSMA8-1 expression in the nuclear and cytoplasmic RNA fractions obtained from RD and RH30 cells as shown by qRT-PCR. C: Predicted miR-144-3p binding sites in lnc-PSMA8-1 sequence and 3’-UTR of GEFT sequence and their induced mutations. D-F: Expression of lnc-PSMA8-1 (D) miR-144-3p (E) and mTOR (F) were detected in RMS samples (n = 20) and normal skeletal muscle samples (n = 10) by qRT-PCR. G-I: Correlation between lnc-PSMA8-1 and miR-144-3p, between miR-144-3p and mTOR, and between lnc-PSMA8-1 and mTOR as analyzed in 20 cases of RMS. J: Expression levels of lnc-PSMA8-1, miR-144-3p and mTOR in RD and RH30 cell lines as determined by qRT-PCR. K: Protein levels of mTOR and p-mTOR in RD and RH30 cell lines as determined by Western blot analysis. All the blots were cropped prior to hybridization with primary antibodies. The original blots are presented in Fig. S2
Fig. 5
Fig. 5
lnc-PSMA8-1 interacts with miR-144-3p and regulation of mTOR by miR-144-3p. A: RD and RH30 cells cotransfected with miR-144-3p mimics or negative control and luciferase reporter plasmid (lnc-PSMA8-1-WT/MUT/NC) were subjected to the luciferase reporter assay. B: Enrichment of lnc-PSMA8-1 bound to Ago2 or IgG in RD and RH30 cell lines as measured by qRT-PCR after RIP; IgG was used as a negative control. C: Expression levels of miR-144-3p in RD and RH30 cells transfected with lnc-PSMA8-1 siRNA or negative control as determined by qRT-PCR. D: Levels of lnc-PSMA8-1 in RD and RH30 cells transfected with miR-144-3p mimics or negative control as determined by qRT-PCR. E: Enrichment of mTOR bound to Ago2 or IgG in RD and RH30 cell lines as measured by qRT-PCR after RIP; IgG was used as a negative control. F: RD and RH30 cells cotransfected with miR-144-3p mimics or negative control and luciferase reporter plasmid (mTOR 3’UTR-WT/MUT/NC) were subjected to the luciferase reporter assay. G: Expression levels of mTOR in RD and RH30 cells transfected with miR-144-3p mimics or negative control as determined by qRT-PCR. H: Protein levels of mTOR and p-mTOR in RD and RH30 cells transfected with miR-144-3p mimics or negative control as determined by Western blot analysis. All the blots were cropped prior to hybridization with primary antibodies. The original blots are presented in Fig. S3 and Fig. S4
Fig. 6
Fig. 6
lnc-PSMA8-1 modulates mTOR expression via competitively binding to miR-144-3p. A: Expression of mTOR in RD and RH30 cells transfected with lnc-PSMA8-1 siRNA in combination with miR-144-3p inhibitor as detected by qRT-PCR. B: Protein levels of mTOR and p-mTOR in RD and RH30 cells transfected with lnc-PSMA8-1 siRNA in combination with miR-144-3p inhibitor as detected by Western blot analysis. C: RD and RH30 cells cotransfected with lnc-PSMA8-1 siRNA or negative control and luciferase reporter plasmid (mTOR 3’UTR-WT/MUT/NC) were subjected to the luciferase reporter assay. All the blots were cropped prior to hybridization with primary antibodies. The original blots are presented in Fig. S5
Fig. 7
Fig. 7
lnc-PSMA8-1 suppresses miR-144-3p function. A: Proliferative capability of RD and RH30 cells transfected with lnc-PSMA8-1 siRNA in combination with miR-144-3p inhibitor as determined through CCK-8 cell proliferation assay. B: Apoptosis rates of RD and RH30 cells transfected with lnc-PSMA8-1 siRNA in combination with miR-144-3p inhibitor as determined through flow cytometry assay. C-D: Invasion (C) and migratory (D) of RD and RH30 cells transfected with lnc-PSMA8-1 siRNA in combination with miR-144-3p inhibitor as determined through transwell system
Fig. 8
Fig. 8
mTOR mediates the biological function of lnc-PSMA8-1-miR-144-3p axis. A: Proliferation of RMS cells in RD and RH30 cells transfected with miR-144-3p inhibitor in combination with mTOR siRNA as detected by CCK-8 cell proliferation assay. B: Apoptosis of RMS cells in RD and RH30 cells transfected with miR-144-3p inhibitor in combination with mTOR siRNA as detected by flow cytometry assay. C-D: Invasion (C) and migratory (D) of RMS cells in RD and RH30 cells transfected with miR-144-3p inhibitor in combination with mTOR siRNA as detected by transwell system
Fig. 9
Fig. 9
Lnc-PSMA8-1 activated by GEFT promotes RMS progression via acting as a ceRNA for miR-144-3p to regulate mTOR
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Stuart A, Radhakrishnan J. Rhabdomyosarcoma. Indian J Pediatr. 2004;71(4):331–7. doi: 10.1007/BF02724100. - DOI - PubMed
    1. Shern JF, Yohe ME, Khan J. Pediatric Rhabdomyosarcoma. Crit Rev Oncog. 2015;20(3–4):227–43. doi: 10.1615/CritRevOncog.2015013800. - DOI - PMC - PubMed
    1. Hawkins DS, Spunt SL, Skapek SX. Children’s Oncology Group’s 2013 blueprint for research: soft tissue sarcomas. Pediatr Blood Cancer. 2013;60(6):1001–8. doi: 10.1002/pbc.24435. - DOI - PMC - PubMed
    1. Bompas E, Campion L, Italiano A, Le Cesne A, Chevreau C, Isambert N, Toulmonde M, Mir O, Ray-Coquard I, Piperno-Neumann S, et al. Outcome of 449 adult patients with rhabdomyosarcoma: an observational ambispective nationwide study. Cancer Med. 2018;7(8):4023–35. doi: 10.1002/cam4.1374. - DOI - PMC - PubMed
    1. Winkle M. Noncoding RNA therapeutics - challenges and potential solutions. Nat Rev Drug Discov. 2021;20(8):629–651. doi: 10.1038/s41573-021-00219-z. - DOI - PMC - PubMed

MeSH terms

LinkOut - more resources