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. 2021 Apr;45(4):8.
doi: 10.3892/or.2021.7959. Epub 2021 Mar 2.

LINC00649 promotes bladder cancer malignant progression by regulating the miR‑15a‑5p/HMGA1 axis

Xuanyu Chen  1 , Song Chen  1
Affiliations

LINC00649 promotes bladder cancer malignant progression by regulating the miR‑15a‑5p/HMGA1 axis

Xuanyu Chen et al. Oncol Rep. 2021 Apr.

Abstract

The aim of the present study was to explore the effects of LINC00649 on the proliferation, migration and invasion of bladder cancer (BC) and identify possible mechanisms. Through TCGA database analysis of LINC00649 expression in bladder cancer and the association of LINC00649 with the BC patient prognosis, RT‑qPCR was employed for detecting LINC00649 expression in 60 clinical tissue specimens and cell lines of bladder cancer. The lentivirus stable transfection or small interfering RNA was used to increase or decrease the LINC00649 expression level in T24 and UM‑UC‑3 cells. CCK8 and clone formation assay were utilized to observe the effects of LINC00649 on the proliferation and colony formation of BC cells. Transwell experiment was performed to detect the effects of LINC00649 on the migration and invasion of bladder cancer. Bioinformatics database was used to identify the possible downstream targets of LINC00649 while RT‑qPCR, western blot analysis and dual luciferase reporter gene experiments were carried out to verify the possible molecular mechanism. The TCGA database analysis revealed a significantly high expression of LINC00649 in bladder cancer and an association of LINC00649 expression with overall survival rate of BC patients. As shown by RT‑qPCR detection, LINC00649 expression was notably upregulated in BC tissues and BC cell lines. In addition, statistical analyses unveiled that highly expressed LINC00649 was clearly associated with poor overall survival of bladder cancer. Based on the in vitro cell experiment, upregulated LINC00649 considerately enhanced the proliferation, migration and invasion of BC cells, as opposed to those in T24 and UM‑UC‑3 cells by suppressing LINC00649. Mechanically, LINC00649 may promote the malignant progression of bladder cancer by regulating miR‑15a‑5p to promote the HMGA1 expression axis. Overall, LINC00649 upregulates HMGA1 expression by binding to miR‑15a‑5p to enhance the proliferation, migration and invasion of BC cells. Thus, LINC00649 is a potential biomarker and therapeutic target for bladder cancer.

Keywords: bladder cancer; LINC00649; miR‑15a‑5p; HMGA1; malignant progression.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Figure 1.
Figure 1.
High LINC00649 expression in BC. (A) LINC00649 expression in BC tissues as analyzed by TCGA database. (B) Association of LINC00649 expression with the OS of BC patients as analyzed by TCGA database. (C) Correlation of LINC00649 expression with the DFS of BC patients as analyzed by TCGA database. (D) LINC00649 expression in BC tissues by RT-qPCR. (E) Analysis of the correlation between LINC00649 and the OS of BC patients. (F) LINC00649 expression in BC cells via RT-qPCR. *P<0.05; **P<0.01.
Figure 2.
Figure 2.
LINC00649 overexpression promoted the proliferation, migration and invasion of BC cells. (A) Stably highly expressed LINC00649 in T24 and UM-UC-3 cells as constructed by lentivirus stable transfection and transfection efficiency detected by RT-qPCR. (B) The influences of LINC00649 on T24 and UM-UC-3 cell proliferation by EdU experiment; magnification: ×200. (C) The influences of LINC00649 on the colony formation of T24 and UM-UC-3 cells via clone formation experiment. (D) The change of cell migration post LINC00649 overexpression in T24 and UM-UC-3 cells as detected by Transwell migration experiment; magnification, ×200. (E) Alteration of cell invasion post LINC00649 overexpression in T24 and UM-UC-3 cells as detected by Transwell invasion experiment; magnification, ×200. **P<0.01.
Figure 3.
Figure 3.
Inhibition of LINC00649 suppressed the proliferation, migration and invasion of BC cells. (A) Downregulation of LINC00649 expression in T24 and UM-UC-3 cells by using siRNAs and transfection efficiency by RT-qPCR. (B) The influence of LINC00649 suppression on the proliferation of T24 and UM-UC-3 cells through EdU experiment; magnification, ×200. (C) The influences of LINC00649 inhibition on the colony formation of T24 and UM-UC-3 cells as detected by clone formation experiment. (D) The change of cell migration post-LINC00649 downregulation in T24 and UM-UC-3 cells by Transwell migration experiment; magnification, ×200. (E) The alteration of cell invasion post LINC00649 downregulation in T24 and UM-UC-3 cells via Transwell invasion experiment; magnification, ×200. *P<0.05; **P<0.01; ***P<0.001.
Figure 4.
Figure 4.
LINC00649 bound to miR-15a-5p. (A) Subcellular localization of LINC00649 by nucleoplasm isolation experiment in T24 and UM-UC-3 cells. (B) Bioinformatics perdition of miRNAs that might be bound to LINC00649. (C) Expressions of miR-15a-5p, miR-15b-5p, miR-195-5p, miR-424-5p and miR-6838-5p via RT-qPCR post-overexpression of LINC00649 in T24 and UM-UC-3 cells. (D) The perdition of binding sites of LINC00649 to miR-15a-5p through bioinformatics website and the construction of LINC00649 wild-type plasmids and LINC00649 mutant plasmids. (E) miR-15a-5p bound to LINC00649 as indicated by the results of dual luciferase reporter gene. (F) miR-15a-5p expression in BC tissues via RT-qPCR. (G) Negative correlation of LINC00649 expression with miR-15a-5p expression in BC tissues. *P<0.05; **P<0.01.
Figure 5.
Figure 5.
miR-15a-5p targeted HMGA1. (A) Possible targets of miR-15a-5p as predicted by bioinformatics website and HMGA1 identified as a potential target by taking the intersection point. (B) HMGA1 expression in BC tissues as analyzed by TCGA database. (C) The perdition of binding sites of HMGA1 to miR-15a-5p through bioinformatics website and the construction of HMGA1 wild-type plasmids and HMGA1 mutant plasmids. (D) HMGA1 targeted by miR-15a-5p as shown by the results of dual luciferase reporter gene. (E) HMGA1 expression via RT-qPCR post-overexpression of both miR-15a-5p and LINC00649 in T24 and UM-UC-3 cells. (F) Protein expression of HMGA1 through western blot post-overexpression of both miR-15a-5p and LINC00649 in T24 and UM-UC-3 cells. *P<0.05; **P<0.01; #P<0.05.
Figure 6.
Figure 6.
miR-15a-5p suppressed the proliferation, migration and invasion of BC cells by inhibiting HMGA1. (A) HMGA1 expression detection by RT-qPCR post-overexpression of both miR-15a-5p and HMGA1 in T24 and UM-UC-3 cells. (B) Cell proliferation detection by EdU experiment post overexpression of miR-15a-5p and HMGA1 in T24 and UM-UC-3 cells; magnification, ×200. (C) Cell clone formation by colony formation experiment post-overexpression of miR-15a-5p and HMGA1 in T24 and UM-UC-3 cells. (D) The change of cell migration via Transwell migration experiment post-overexpression of miR-15a-5p and HMGA1 in T24 and UM-UC-3 cells; magnification, ×200. (E) The change of cell invasion via Transwell invasion experiment post overexpression of miR-15a-5p and HMGA1 in T24 and UM-UC-3 cells; magnification, 200. **P<0.01; ***P<0.001; #P<0.05; ##P<0.01.
Figure 7.
Figure 7.
LINC00649 promoted the proliferation, migration and invasion of BC cells by elevating HMGA1 expression. (A and B) HMGA1 expression detection by RT-qPCR and western blot analysis after co-transfection of LINC00649 siRNA and HMGA1 OE in T24 and UM-UC-3 cells. (C) Cell proliferation detection by EdU experiment after co-transfection of LINC00649 siRNA and HMGA1 OE in T24 and UM-UC-3 cells; magnification, ×200. (D and E) The change of cell migration and invasion via Transwell experiment after co-transfection of LINC00649 siRNA and HMGA1 OE in T24 and UM-UC-3 cells; magnification, ×200. **P<0.01; #P<0.05.
Figure 8.
Figure 8.
Knockdown of LINC00649 inhibited EMT of BC cells. (A and B) The expression of EMT markers were determined using RT-qPCR and Western blotting. Knockdown of LINC00649 increased the expression of E-cadherin and decreased the expression of N-cadherin and vimentin in BC cells, while miR-15a-5p could suppress EMT and HMGA1 showed the opposite effect.
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References

    1. Afonso J, Santos LL, Longatto-Filho A, Baltazar F. Competitive glucose metabolism as a target to boost bladder cancer immunotherapy. Nat Rev Urol. 2020;17:77–106. doi: 10.1038/s41585-019-0263-6. - DOI - PubMed
    1. Alifrangis C, McGovern U, Freeman A, Powles T, Linch M. Molecular and histopathology directed therapy for advanced bladder cancer. Nat Rev Urol. 2019;16:465–483. doi: 10.1038/s41585-019-0208-0. - DOI - PubMed
    1. Meng MV, Gschwend JE, Shore N, Grossfeld GD, Mostafid H, Black PC. Emerging immunotherapy options for bacillus calmette-guerin unresponsive nonmuscle invasive bladder cancer. J Urol. 2019;202:1111–1119. doi: 10.1097/JU.0000000000000297. - DOI - PubMed
    1. Soria F, D'Andrea D, Pohar K, Shariat SF, Lotan Y. Diagnostic, prognostic and surveillance urinary markers in nonmuscle invasive bladder cancer: Any role in clinical practice? Curr Opin Urol. 2018;28:577–583. doi: 10.1097/MOU.0000000000000545. - DOI - PubMed
    1. Dobruch J, Daneshmand S, Fisch M, Lotan Y, Noon AP, Resnick MJ, Shariat SF, Zlotta AR, Boorjian SA. Gender and bladder cancer: A collaborative review of etiology, biology, and outcomes. Eur Urol. 2016;69:300–310. doi: 10.1016/j.eururo.2015.08.037. - DOI - PubMed