Long Noncoding RNA PVT1 promoted Gallbladder Cancer proliferation Byepigenetically Suppressing mIR-18b-5p via DNA Methylation
Gallbladder cancer (GBC) accounts for 85–90% malignancies of the biliary tree worldwide. Considerable evidence has demonstrated that dysregulation of lncRNAs are involved in the progression of cancer. LncRNA PVT1 has been reported to play important roles in various cancers, but the role of PVT1 in gallbladder cancer remains unknown.
QRT-PCR was used to assess the expression of genes in different tissues or cells. Knockdown or overexpression of PVT1 combined with in vitro and in vivo assays were conducted to determine the effect of PVT1 on the GBC cells proliferation. We also conducted microarray analysis to screen out the miRNA that was regulated by PVT1. BSP was used to detect the methylation status of miR-18b-5p DNA promoter. RIP, ChIP, Co-IP and luciferase reporter assays were performed to validate the association between genes or proteins.
We found that PVT1 was upregulated in GBC tissues and cells, and its upregulation was related with poor prognosis in GBC patients. PVT1 promoted GBC cells proliferation and increased tumorigenicity in nude mice. Molecular experiments demonstrated that PVT1 recruited DNMT1 via EZH2 to the miR-18b-5p DNA promoter and suppressed the transcription of miR-18b-5p through DNA methylation. Moreover, HIF1A was proved to be the downstream target gene of miR-18b-5p and PVT1 regulated GBC cell proliferation via HIF1A.
Our studies clarified the PVT1/ miR-18b-5p/ HIF1A regulation axis and indicated that PVT1 could be a potential therapeutic target for GBC.
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This is a list of supplementary files associated with this preprint. Click to download.
Additional file 1: Supplementary Table 1.Information of the qRT-PCR primers, siRNAsand shRNA sequence involved in the present study.
Additional file 4: Supplementary Figure 1.The effect of DNMT1 on the expression of miR-18b-5p and the relation of PVT1 with DNMT1 detected by RIP assays.A, Relative expression of miR-18b-5p in GBC cells when DNMT1 was knockdown.B, Relative RIP assays examining the relation of PVT1 with DNMT1 inGBC-SD and SGC-996 cells by qRT-PCR.***p < 0.001.
Additional file 2: Supplementary Table 2.22 upregulated and 46downregulatedmicroRNAs in GBC-SD/si-PVT1-1 with pvalue< 0.05.
Additional file 3:Supplementary Table 3.The potential target genes of miR-18b-5p predicted by threemiRNA databases.
Posted 18 Jun, 2020
Long Noncoding RNA PVT1 promoted Gallbladder Cancer proliferation Byepigenetically Suppressing mIR-18b-5p via DNA Methylation
Posted 18 Jun, 2020
Gallbladder cancer (GBC) accounts for 85–90% malignancies of the biliary tree worldwide. Considerable evidence has demonstrated that dysregulation of lncRNAs are involved in the progression of cancer. LncRNA PVT1 has been reported to play important roles in various cancers, but the role of PVT1 in gallbladder cancer remains unknown.
QRT-PCR was used to assess the expression of genes in different tissues or cells. Knockdown or overexpression of PVT1 combined with in vitro and in vivo assays were conducted to determine the effect of PVT1 on the GBC cells proliferation. We also conducted microarray analysis to screen out the miRNA that was regulated by PVT1. BSP was used to detect the methylation status of miR-18b-5p DNA promoter. RIP, ChIP, Co-IP and luciferase reporter assays were performed to validate the association between genes or proteins.
We found that PVT1 was upregulated in GBC tissues and cells, and its upregulation was related with poor prognosis in GBC patients. PVT1 promoted GBC cells proliferation and increased tumorigenicity in nude mice. Molecular experiments demonstrated that PVT1 recruited DNMT1 via EZH2 to the miR-18b-5p DNA promoter and suppressed the transcription of miR-18b-5p through DNA methylation. Moreover, HIF1A was proved to be the downstream target gene of miR-18b-5p and PVT1 regulated GBC cell proliferation via HIF1A.
Our studies clarified the PVT1/ miR-18b-5p/ HIF1A regulation axis and indicated that PVT1 could be a potential therapeutic target for GBC.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8