This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research
Zhiwei Quan
Shanghai Jiao Tong University School of Medicine
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background
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.
Methods
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.
Results
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.
Conclusions
Our studies clarified the PVT1/ miR-18b-5p/ HIF1A regulation axis and indicated that PVT1 could be a potential therapeutic target for GBC.
Keywords
lncRNAPVT1, Gallbladder cancer, proliferation, miR-18b-5p, DNA methylation
Figure 1
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This is a list of supplementary files associated with this preprint. Click to download.
- Highlights.docx
- SupplementaryTable1.xlsx
Additional file 1: Supplementary Table 1.Information of the qRT-PCR primers, siRNAsand shRNA sequence involved in the present study.
- SupplementaryFigure1.tif
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.
- SupplementaryTable2.xlsx
Additional file 2: Supplementary Table 2.22 upregulated and 46downregulatedmicroRNAs in GBC-SD/si-PVT1-1 with pvalue< 0.05.
- SupplementaryTable3.xlsx
Additional file 3:Supplementary Table 3.The potential target genes of miR-18b-5p predicted by threemiRNA databases.
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This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research
Zhiwei Quan
Shanghai Jiao Tong University School of Medicine
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 18 Jun, 2020
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Surgery
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background
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.
Methods
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.
Results
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.
Conclusions
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
This is a list of supplementary files associated with this preprint. Click to download.
- Highlights.docx
- SupplementaryTable1.xlsx
Additional file 1: Supplementary Table 1.Information of the qRT-PCR primers, siRNAsand shRNA sequence involved in the present study.
- SupplementaryFigure1.tif
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.
- SupplementaryTable2.xlsx
Additional file 2: Supplementary Table 2.22 upregulated and 46downregulatedmicroRNAs in GBC-SD/si-PVT1-1 with pvalue< 0.05.
- SupplementaryTable3.xlsx
Additional file 3:Supplementary Table 3.The potential target genes of miR-18b-5p predicted by threemiRNA databases.
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