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Long Non-Coding RNA CCDC183-AS1 Acts as a miR-589-5p Sponge to Promote The Progression of Hepatocellular Carcinoma Through Regulating SKP1 Expression
Huifang Liang
Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology
Corresponding Author
ORCiD: https://orcid.org/0000-0001-6874-3634
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Hepatocellular carcinoma (HCC) is a common type of malignant human cancer with high morbidity and poor prognosis, causing numerous deaths per year worldwide. Growing evidence has been demonstrated that long non-coding RNAs (lncRNAs) are closely associated with hepatocarcinogenesis and metastasis. However, the roles, functions, and working mechanisms of most lncRNAs in HCC remain poorly defined.
Methods: Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression level of CCDC183-AS1 in HCC tissues and cell lines. Cell proliferation, migration and invasion ability were evaluated by CCK-8 and transwell assay, respectively. Animal experiments were used to explore the role of CCDC183-AS1 and miR-589-5p in vivo. Bioinformatic analysis, dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were performed to confirm the regulatory relationship between CCDC183-AS1, miR-589-5p and SKP1.
Results: Significantly upregulated expression of CCDC183-AS1 was observed in both HCC tissues and cell lines. HCC patients with higher expression of CCDC183-AS1 had a poorer overall survival rate. Functionally, overexpression of CCDC183-AS1 markedly promoted HCC cell proliferation, migration and invasion in vitro and tumor growth and metastasis in vivo, whereas the downregulation of CCDC183-AS1 exerted opposite effects. MiR-589-5p inhibitor counteracted the proliferation, migration and invasion inhibitory effects induced by CCDC183-AS1 silencing. Mechanistically, CCDC183-AS1 acted as a ceRNA through sponging miR-589-5p to offset its inhibitory effect on the target gene SKP1, then promoted the tumorigenesis of HCC.
Conclusions: CCDC183-AS1 functions as an oncogene to promote HCC progression through the CCDC183-AS1/miR-589-5p/SKP1 axis. Our study provided a novel potential therapeutic target for HCC patients.
Keywords
Hepatocellular carcinoma, CCDC183-AS1, miR-589-5p, SKP1
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This is a list of supplementary files associated with this preprint. Click to download.
- SupplementaryTable1.xls
- SupplementaryFig.1.jpg
Supplementary Fig. 1 CCDC183-AS1 promoted HCC cell proliferation and metastasis in vitro and in vivo. a-b. The overexpression (a) and knockdown (b) efficiency of CCDC183-AS1 were examined by qRT-PCR. c-d. Representative images of transwell assays in CCDC183-AS1 overexpressed (c) or knockdown (d) HCC cells. e. Representative images of the liver of CCDC183-AS1 overexpressing group and control group. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001; ns, no significance.
- SupplementaryFig.2.jpg
Supplementary Fig. 2 CCDC183-AS1 acted as a ceRNA to sponge miR-589-5p in HCC cells. a. Relative expression levels of miR-589-5p were evaluated by qRT-PCR in 97H cells transfected with the miR-589-5p mimics or inhibitor, respectively. b-c. The relative luciferase activities were detected in 97H cells after co-transfection with CCDC183-AS1-WT or CCDC183-AS1-MUT and mimics, inhibitor or NC, respectively. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001; ns, no significance.
- SupplementaryFig.3.jpg
Supplementary Fig. 3 miR-589-5p inhibited HCC cell proliferation and metastasis in vitro and in vivo. a-b. Representative images of transwell assays in HLF (a) and 97H (b) cells transfected with miR-589-5p mimics or inhibitor, respectively. c. The expression levels of miR-589-5p were examined by qRT-PCR in HLF and 97H cells with stable miR-589-5p overexpression or knockdown. d. Representative images of the liver of miR-589-5p overexpressing group and control group. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001; ns, no significance.
- SupplementaryFig.4.jpg
Supplementary Fig. 4 SKP1 is a direct target of miR-589-5p. a. Venn diagrams showing the number of target genes of miR-589-5p predicted by mirDIP, MicroT-CDS, TargetScan, miRWalk and miRDB. b-d. Expression levels of candidate genes were measured by qRT-PCR after CCDC183-AS1 overexpression (b-c) or knockdown (d). e-f. qRT-PCR analysis of the expression of candidate genes in HLF (e) and 97H (f) cells when treated with miR-589-5p mimics or inhibitor. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001; ns, no significance.
- SupplementaryFig.51.jpg
Supplementary Fig. 5 SKP1 promoted HCC cell proliferation and metastasis. a-b. CCK-8 assays were used to evaluate 97H cells proliferation after SKP1 overexpression (a) or knockdown (b). c-d. Cellular migratory and invasive capabilities were assessed by transwell assays in 97H cells after SKP1 overexpression (c) or knockdown (d). e-f. Representative images of transwell assays in HLF and 97H cells after SKP1 overexpression (e) or knockdown (f). g. Western blot analysis to determine expression level of the cell cycle and EMT relative marker in SKP1 overexpressed and knockdown 97H cells. h. The expression levels of SKP1 in HCC and adjacent non-cancer tissues were evaluated by western blot(n=80). Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001; ns, no significance.
- SupplementaryFig.52.jpg
Supplementary Fig. 5 SKP1 promoted HCC cell proliferation and metastasis. a-b. CCK-8 assays were used to evaluate 97H cells proliferation after SKP1 overexpression (a) or knockdown (b). c-d. Cellular migratory and invasive capabilities were assessed by transwell assays in 97H cells after SKP1 overexpression (c) or knockdown (d). e-f. Representative images of transwell assays in HLF and 97H cells after SKP1 overexpression (e) or knockdown (f). g. Western blot analysis to determine expression level of the cell cycle and EMT relative marker in SKP1 overexpressed and knockdown 97H cells. h. The expression levels of SKP1 in HCC and adjacent non-cancer tissues were evaluated by western blot(n=80). Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001; ns, no significance.
- SupplementaryFig.6.jpg
Supplementary Fig. 6 CCDC183-AS1 promotes HCC progression through the CCDC183-AS1/miR-589-5p/SKP1 axis. a-b. Representative images of transwell assays in HLF (a) and 97H (b) cells transfected with indicated NC, siCCDC183-AS1, miR-589-5p inhibitor or SKP1, respectively.
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This preprint is under consideration at Journal of Experimental & Clinical Cancer Research. A preprint is 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.
Learn more about In Review
Research
Long Non-Coding RNA CCDC183-AS1 Acts as a miR-589-5p Sponge to Promote The Progression of Hepatocellular Carcinoma Through Regulating SKP1 Expression
Huifang Liang
Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology
Corresponding Author
ORCiD: https://orcid.org/0000-0001-6874-3634
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 1
Posted 21 Dec, 2020
No community comments so far
Review #1 received
Received 26 Dec, 2020
Editorial decision: Minor revision
On 26 Dec, 2020
Review #2 received
Received 22 Dec, 2020
Review #3 received
Received 21 Dec, 2020
Reviewer #3 agreed
On 18 Dec, 2020
Reviewer #2 agreed
On 18 Dec, 2020
Reviewers invited
Invitations sent on 13 Dec, 2020
Reviewer #1 agreed
On 13 Dec, 2020
Editor assigned
On 12 Dec, 2020
Submission checks complete
On 12 Dec, 2020
Editor invited
On 12 Dec, 2020
First submitted
On 10 Dec, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Cancer Biology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Hepatocellular carcinoma (HCC) is a common type of malignant human cancer with high morbidity and poor prognosis, causing numerous deaths per year worldwide. Growing evidence has been demonstrated that long non-coding RNAs (lncRNAs) are closely associated with hepatocarcinogenesis and metastasis. However, the roles, functions, and working mechanisms of most lncRNAs in HCC remain poorly defined.
Methods: Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression level of CCDC183-AS1 in HCC tissues and cell lines. Cell proliferation, migration and invasion ability were evaluated by CCK-8 and transwell assay, respectively. Animal experiments were used to explore the role of CCDC183-AS1 and miR-589-5p in vivo. Bioinformatic analysis, dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were performed to confirm the regulatory relationship between CCDC183-AS1, miR-589-5p and SKP1.
Results: Significantly upregulated expression of CCDC183-AS1 was observed in both HCC tissues and cell lines. HCC patients with higher expression of CCDC183-AS1 had a poorer overall survival rate. Functionally, overexpression of CCDC183-AS1 markedly promoted HCC cell proliferation, migration and invasion in vitro and tumor growth and metastasis in vivo, whereas the downregulation of CCDC183-AS1 exerted opposite effects. MiR-589-5p inhibitor counteracted the proliferation, migration and invasion inhibitory effects induced by CCDC183-AS1 silencing. Mechanistically, CCDC183-AS1 acted as a ceRNA through sponging miR-589-5p to offset its inhibitory effect on the target gene SKP1, then promoted the tumorigenesis of HCC.
Conclusions: CCDC183-AS1 functions as an oncogene to promote HCC progression through the CCDC183-AS1/miR-589-5p/SKP1 axis. Our study provided a novel potential therapeutic target for HCC patients.
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