See the published version of this preprint at Journal of Experimental & Clinical Cancer Research.
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Zhitao Jing
China Medical University Hospital
Corresponding Author
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Background
Glioma is the most common and malignant tumor of central nervous system. The tumor initiation, self-renewal, and multi-lineage differentiation abilities of glioma stem cells (GSCs) are responsible for glioma proliferation and recurrence. Although circular RNAs (circRNAs) play vital roles in the progression of glioma, the detailed mechanisms remain unknown.
Methods
qRT-PCR, western blotting, immunohistochemistry, and bioinformatic analysis were performed to detect the expression of circATP5B, miR-185-5p, HOXB5, and SRSF1. Patient-derived GSCs were established, and MTS, EDU, neurosphere formation, and limiting dilution assays were used to detect the proliferation of GSCs. RNA-binding protein immunoprecipitation, RNA pull-down, luciferase reporter assays, and chromatin immunoprecipitation assays were used to detect these molecules' regulation mechanisms.
Results
We found circATP5B expression was significantly upregulated in GSCs and promoted the proliferation of GSCs. Mechanistically, circATP5B acted as miR-185-5p sponge to upregulate HOXB5 expression. HOXB5 was overexpressed in glioma and transcriptionally regulated IL6 expression and promoted the proliferation of GSCs via JAK2/STAT3 signaling. Moreover, RNA binding protein SRSF1 could bind to and promote circATP5B expression and regulate the proliferation of GSCs, while HOXB5 also transcriptionally regulated SRSF1 expression.
Conclusions
Our study identified the SRSF1/circATP5B/miR-185-5p/HOXB5 feedback loop in GSCs. This provides an effective biomarker for glioma diagnosis and prognostic evaluation.
Keywords
Glioma stem cells, circATP5B, miR-185-5P, HOXB5, SRSF1, IL6, JAK2/STAT3 signaling
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This is a list of supplementary files associated with this preprint. Click to download.
- SupplementaryFigure1.tif
a Hematoxylin and eosin staining of the original patient tissues. b Immunofluorescence staining of CD133 and nestin in patient-derived GSCs. Scale bar = 50μm. c Representative images showing that GSCs were differentiated and adherent (above). Scale bar =20μm. Immunofluorescence showing differentiated GSCs expressing GFAP or βIII tubulin (middle and below). Scale bar = 50μm. d, e The expression of HOXB5 in different patient-derived GSCs (d) and non-GSCs (e) as detected by qRT-PCR. f, g The expression of HOXB5 in different patient-derived GSCs (f) and non-GSCs (g) as detected by western blotting. All data were expressed as the mean ± SD (three independent experiments). *p< 0.05; **p< 0.01; ***p< 0.001.
- SupplementaryFigure1.tif
a Hematoxylin and eosin staining of the original patient tissues. b Immunofluorescence staining of CD133 and nestin in patient-derived GSCs. Scale bar = 50μm. c Representative images showing that GSCs were differentiated and adherent (above). Scale bar =20μm. Immunofluorescence showing differentiated GSCs expressing GFAP or βIII tubulin (middle and below). Scale bar = 50μm. d, e The expression of HOXB5 in different patient-derived GSCs (d) and non-GSCs (e) as detected by qRT-PCR. f, g The expression of HOXB5 in different patient-derived GSCs (f) and non-GSCs (g) as detected by western blotting. All data were expressed as the mean ± SD (three independent experiments). *p< 0.05; **p< 0.01; ***p< 0.001.
- SupplementaryFigure2.tif
The expression of circATP5B, HOXB5, and SRSF1 in GSCs after lentiviral-based transfection. a,b The relative expression of circATP5B after circATP5B knockdown (a) or overexpression (b), as detected by qRT-PCR. c,d The relative expression of HOXB5 after HOXB5 knockdown (c) or overexpression (d), as detected by qRT-PCR. e,f The protein expression of HOXB5 after HOXB5 knockdown (e) or overexpression (f), as detected by western blotting. g,h The relative expression of SRSF1 after SRSF1 knockdown (g) or overexpression (h), as detected by qRT-PCR. i,j The protein expression of SRSF1 after SRSF1 knockdown (i) or overexpression (j), as detected by western blotting. All data were expressed as the mean ± SD (three independent experiments). **p< 0.01; ***p< 0.001.
- SupplementaryFigure2.tif
The expression of circATP5B, HOXB5, and SRSF1 in GSCs after lentiviral-based transfection. a,b The relative expression of circATP5B after circATP5B knockdown (a) or overexpression (b), as detected by qRT-PCR. c,d The relative expression of HOXB5 after HOXB5 knockdown (c) or overexpression (d), as detected by qRT-PCR. e,f The protein expression of HOXB5 after HOXB5 knockdown (e) or overexpression (f), as detected by western blotting. g,h The relative expression of SRSF1 after SRSF1 knockdown (g) or overexpression (h), as detected by qRT-PCR. i,j The protein expression of SRSF1 after SRSF1 knockdown (i) or overexpression (j), as detected by western blotting. All data were expressed as the mean ± SD (three independent experiments). **p< 0.01; ***p< 0.001.
- SupplementaryTable1.docx
- SupplementaryTable1.docx
- SupplementaryTable2.docx
- SupplementaryTable2.docx
- SupplementaryTable3.docx
- SupplementaryTable3.docx
- SupplementaryTable4.docx
- SupplementaryTable4.docx
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View the published article at Journal of Experimental & Clinical Cancer Research.
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A new preprint design is coming!
We have improved readability, clarity, accessibility, and opportunities for engagement.
See the published version of this preprint at Journal of Experimental & Clinical Cancer Research.
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.
Learn more about In Review
Research
Zhitao Jing
China Medical University Hospital
Corresponding Author
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: Published
View the published article at Journal of Experimental & Clinical Cancer Research.
Version 1
Posted 15 Dec, 2020
No community comments so far
Editorial decision: Major revision
On 29 Dec, 2020
Review #1 received
Received 28 Dec, 2020
Review #2 received
Received 28 Dec, 2020
Reviewer #2 agreed
On 18 Dec, 2020
Editor assigned
On 09 Dec, 2020
Reviewers invited
Invitations sent on 09 Dec, 2020
Reviewer #1 agreed
On 09 Dec, 2020
Submission checks complete
On 09 Dec, 2020
Editor invited
On 09 Dec, 2020
First submitted
On 08 Dec, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at Journal of Experimental & Clinical Cancer Research.
Background
Glioma is the most common and malignant tumor of central nervous system. The tumor initiation, self-renewal, and multi-lineage differentiation abilities of glioma stem cells (GSCs) are responsible for glioma proliferation and recurrence. Although circular RNAs (circRNAs) play vital roles in the progression of glioma, the detailed mechanisms remain unknown.
Methods
qRT-PCR, western blotting, immunohistochemistry, and bioinformatic analysis were performed to detect the expression of circATP5B, miR-185-5p, HOXB5, and SRSF1. Patient-derived GSCs were established, and MTS, EDU, neurosphere formation, and limiting dilution assays were used to detect the proliferation of GSCs. RNA-binding protein immunoprecipitation, RNA pull-down, luciferase reporter assays, and chromatin immunoprecipitation assays were used to detect these molecules' regulation mechanisms.
Results
We found circATP5B expression was significantly upregulated in GSCs and promoted the proliferation of GSCs. Mechanistically, circATP5B acted as miR-185-5p sponge to upregulate HOXB5 expression. HOXB5 was overexpressed in glioma and transcriptionally regulated IL6 expression and promoted the proliferation of GSCs via JAK2/STAT3 signaling. Moreover, RNA binding protein SRSF1 could bind to and promote circATP5B expression and regulate the proliferation of GSCs, while HOXB5 also transcriptionally regulated SRSF1 expression.
Conclusions
Our study identified the SRSF1/circATP5B/miR-185-5p/HOXB5 feedback loop in GSCs. This provides an effective biomarker for glioma diagnosis and prognostic evaluation.
Figure 1
Figure 1
Figure 2
Figure 2
Figure 3
Figure 3
Figure 4
Figure 4
Figure 5
Figure 5
Figure 6
Figure 6
Figure 7
Figure 7
Figure 8
Figure 8
This is a list of supplementary files associated with this preprint. Click to download.
- SupplementaryFigure1.tif
a Hematoxylin and eosin staining of the original patient tissues. b Immunofluorescence staining of CD133 and nestin in patient-derived GSCs. Scale bar = 50μm. c Representative images showing that GSCs were differentiated and adherent (above). Scale bar =20μm. Immunofluorescence showing differentiated GSCs expressing GFAP or βIII tubulin (middle and below). Scale bar = 50μm. d, e The expression of HOXB5 in different patient-derived GSCs (d) and non-GSCs (e) as detected by qRT-PCR. f, g The expression of HOXB5 in different patient-derived GSCs (f) and non-GSCs (g) as detected by western blotting. All data were expressed as the mean ± SD (three independent experiments). *p< 0.05; **p< 0.01; ***p< 0.001.
- SupplementaryFigure1.tif
a Hematoxylin and eosin staining of the original patient tissues. b Immunofluorescence staining of CD133 and nestin in patient-derived GSCs. Scale bar = 50μm. c Representative images showing that GSCs were differentiated and adherent (above). Scale bar =20μm. Immunofluorescence showing differentiated GSCs expressing GFAP or βIII tubulin (middle and below). Scale bar = 50μm. d, e The expression of HOXB5 in different patient-derived GSCs (d) and non-GSCs (e) as detected by qRT-PCR. f, g The expression of HOXB5 in different patient-derived GSCs (f) and non-GSCs (g) as detected by western blotting. All data were expressed as the mean ± SD (three independent experiments). *p< 0.05; **p< 0.01; ***p< 0.001.
- SupplementaryFigure2.tif
The expression of circATP5B, HOXB5, and SRSF1 in GSCs after lentiviral-based transfection. a,b The relative expression of circATP5B after circATP5B knockdown (a) or overexpression (b), as detected by qRT-PCR. c,d The relative expression of HOXB5 after HOXB5 knockdown (c) or overexpression (d), as detected by qRT-PCR. e,f The protein expression of HOXB5 after HOXB5 knockdown (e) or overexpression (f), as detected by western blotting. g,h The relative expression of SRSF1 after SRSF1 knockdown (g) or overexpression (h), as detected by qRT-PCR. i,j The protein expression of SRSF1 after SRSF1 knockdown (i) or overexpression (j), as detected by western blotting. All data were expressed as the mean ± SD (three independent experiments). **p< 0.01; ***p< 0.001.
- SupplementaryFigure2.tif
The expression of circATP5B, HOXB5, and SRSF1 in GSCs after lentiviral-based transfection. a,b The relative expression of circATP5B after circATP5B knockdown (a) or overexpression (b), as detected by qRT-PCR. c,d The relative expression of HOXB5 after HOXB5 knockdown (c) or overexpression (d), as detected by qRT-PCR. e,f The protein expression of HOXB5 after HOXB5 knockdown (e) or overexpression (f), as detected by western blotting. g,h The relative expression of SRSF1 after SRSF1 knockdown (g) or overexpression (h), as detected by qRT-PCR. i,j The protein expression of SRSF1 after SRSF1 knockdown (i) or overexpression (j), as detected by western blotting. All data were expressed as the mean ± SD (three independent experiments). **p< 0.01; ***p< 0.001.
- SupplementaryTable1.docx
- SupplementaryTable1.docx
- SupplementaryTable2.docx
- SupplementaryTable2.docx
- SupplementaryTable3.docx
- SupplementaryTable3.docx
- SupplementaryTable4.docx
- SupplementaryTable4.docx
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