RESUMO
AIMS: Long noncoding RNA (LncRNA) urothelial cancer associated 1 (UCA1) was dysregulated in colorectal cancers (CRC) and promoted tumor progression of CRC. The aims of this study are to further investigate the underlying mechanism. MAIN METHODS: Short hairpin RNAs (shRNAs) were applied for gene knockdown. microRNA mimic and pcDNA-UCA1 plasmids were transfected for miR-495 and UCA1 overexpression, respectively. MTT was applied to determine cell viability and sensitivity of 5-fluorouracil (FU). Transwell assays were performed to evaluate cell migration/invasion. Angiogenesis was evaluated by tube formation. Western blotting and quantitative PCR were utilized for protein and mRNA detection, respectively. The interaction of UCA1, miR-495 and SP1/SP3 were explored by dual-luciferase assay. RNA pulldown was adopted to determine the UCA1/miR-495 interaction. KEY FINDINGS: UCA1 was significantly upregulated in CRC tissues. UCA1 enhanced cell proliferation, migration/invasion, angiogenesis, epithelial-mesenchymal transition, and resistance to 5-FU in CRC cell lines. MiR-495 was inversely correlated to the expression of UCA1. The results indicated that UCA1 sponged miR-495, leading to the disinhibition of SP1/SP3 expression. SP1/SP3 induced the expression of DNA methyltransferases and, in turn, contributed to UCA1 mediated tumor-promoting actions. Reduction of SP1/SP3 exerted anti-cancer effects, which can be reversed by forced expression of UCA1. SIGNIFICANCE: UCA1-miR-495-SP1/SP3 axis is dysregulated in CRC and contributed to malignant phenotypes of CRC. UCA1-SP1/SP3 may form a positive feedback loop in CRC.
Assuntos
Neoplasias Colorretais/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , Adulto , Idoso , Apoptose/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , China , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , RNA Longo não Codificante/metabolismo , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3/genética , Fator de Transcrição Sp3/metabolismoRESUMO
BACKGROUNDS: Colorectal carcinoma (CRC) is a common malignant tumor. Increasing evidences indicated that CRC showed a resistance to 5-fluorouracil (5-FU) and further resulted in a poor prognosis. In this study, we aim to investigate the effect of long noncoding RNA nuclear paraspeckle assembly transcript 1 (LncRNA NEAT1) on cell viability, sensitivity to 5-FU, and autophagy of CRC cell lines. METHODS: MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-Htetrazolium bromide) was used to detect cell viability, immunofluorescent staining was used to detect autophagy puncta, and luciferase reporter system was used to determine binding ability between miR-34a and NEAT1 or putative targets. Additionally, indicated mRNAs and protein expressions were determined by qRT-PCR or western blotting, respectively. RESULTS: We found that NEAT1 expression was increased in CRC tissues and cells, which showed a negative correlation with miR-34a expression. In addition, NEAT1 knockdown noticeably inhibited the proliferation of CRC cells and enhanced 5-FU sensitivity. It revealed that NEAT1 knockdown suppressed the LC3 puncta and the expressions of Beclin-1, ULK1, and ratio of LC3II/I. Overexpression of miR-34a showed similar trends with NEAT1 knockdown. miR-34a was validated to target the putative binding sites in 3'-UTR of HMGB1, ATG9A, and ATG4B, which are involved in the activation of autophagy. Inhibition of miR-34a or overexpression of HMGB1 could effectively reverse elevated 5-FU sensitivity upon NEAT1 knockdown. In addition, 3-MA reversed NEAT1 overexpression-induced resistance in HT29 cells. CONCLUSION: These findings indicate that LncRNA NEAT1 could target miR-34a and promote autophagy to facilitate 5-FU chemoresistance in CRC.