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Primary research
Xuejun Chen
The Second Affiliated Hospital of Zhejiang University School of Medicine
Corresponding Author
ORCiD: https://orcid.org/0000-0002-9323-9461
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Objective: Ovarian cancer (OVC) is the fifth leading cause of cancer-related deaths in women and has a significant impact on physical and mental health of women. This study explores the molecular mechanism of miR-636 acting as a tumor suppressor in OVC in vitro and in vivo, and provides new insight into the treatment of OVC.
Methods: Protein-protein interaction (PPI) analysis was performed to identify the hub gene in Hedgehog (Hh) pathway. TargetScan database was used to predict the upstream regulatory miRNAs of Gli2 to obtain the target miRNA. qRT-PCR was performed to test the expression of miR-636, while Western blot were conducted to detect the expression of Hh and EMT (epithelial-mesenchymal transition) related genes in OVC cell lines. MTT assay and wound healing assay were used to measure the effect of miR-636 on OVC cell proliferation and migration. Flow cytometry was carried out to examine the effect of miR-636 on cell cycle, and Western blot was used for identification of changes in expression of Hh and EMT related proteins. Dual-luciferase reporter gene assay was implemented to detect the targeted relationship between miR-636 and Gli2. The xenotransplantation model was used to detect the effect of miR-636 on OVC cell proliferation in vivo.
Results: PPI interaction analysis found that Gli2 was the hub gene in Hh pathway. Based on TargetScan and GEO databases, Gli2 was found to be targeted regulated by the upstream miR-636. In vitro experiments discovered that miR-636 was significantly lowly expressed in OVC cell lines. Overexpressing miR-636 significantly inhibited HO-8910PM cell proliferation and migration abilities as well as induced cell cycle arrest in G0/G1 phase, while the inhibition of miR-636 promoted cell proliferation and migration abilities. Dual-luciferase reporter gene assay revealed that Gli2 was a target gene of miR-636. Besides, overexpressing miR-636 decreased protein expression of Gli2, while the inhibition of miR-636 increased protein expression of Gli2. Furthermore, the overexpression and inhibition of miR-636 both affected the expression of proteins related to Hh signaling pathway and EMT. Rescue experiments verified that overexpression of Gli2 reversed the inhibitory effect of miR-636 on HO-8910PM cell proliferation and migration abilities, and attenuated the blocking effect of miR-636 on HO-8910PM cell cycle. The xenotransplantation model suggested that miR-636 inhibited cell growth of OVC by decreasing Gli2 expression. Besides, overexpressing Gli2 potentiated the EMT process in OVC via decreasing E-cadherin protein expression and increasing Vimentin protein expression, and it reversed the inhibitory effect of miR-636 on OVC cell proliferation and migration abilities in vivo.
Conclusion: miR-636 inhibits the Hh pathway activation via targeted binding to Gli2, thus inhibiting EMT, cell proliferation and migration in OVC.
Keywords
miR-636, Gli2, ovarian cancer, cell proliferation and migration
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Peer Review Timeline
CURRENT STATUS: Published
View the published article at Cancer Cell International.
No community comments so far
Editorial decision: Accept
On 17 Dec, 2020
Reviewers invited
Invitations sent on 28 Nov, 2020
Reviewer #1 agreed
On 28 Nov, 2020
Review #1 received
Received 28 Nov, 2020
Editor assigned
On 25 Nov, 2020
Submission checks complete
On 25 Nov, 2020
Editor invited
On 25 Nov, 2020
Version 1
Posted 05 Aug, 2020
No comments provided
Editorial decision: Major revision
On 27 Aug, 2020
Review #1 received
Received 26 Aug, 2020
Review #2 received
Received 25 Aug, 2020
Reviewers invited
Invitations sent on 11 Aug, 2020
Reviewer #1 agreed
On 11 Aug, 2020
Reviewer #2 agreed
On 11 Aug, 2020
Reviewer #3 agreed
On 11 Aug, 2020
Editor assigned
On 05 Aug, 2020
Submission checks complete
On 04 Aug, 2020
Editor invited
On 04 Aug, 2020
First submitted
On 02 Aug, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
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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 Cancer Cell International.
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
Primary research
Xuejun Chen
The Second Affiliated Hospital of Zhejiang University School of Medicine
Corresponding Author
ORCiD: https://orcid.org/0000-0002-9323-9461
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 Cancer Cell International.
No community comments so far
Editorial decision: Accept
On 17 Dec, 2020
Reviewers invited
Invitations sent on 28 Nov, 2020
Reviewer #1 agreed
On 28 Nov, 2020
Review #1 received
Received 28 Nov, 2020
Editor assigned
On 25 Nov, 2020
Submission checks complete
On 25 Nov, 2020
Editor invited
On 25 Nov, 2020
Version 1
Posted 05 Aug, 2020
No comments provided
Editorial decision: Major revision
On 27 Aug, 2020
Review #1 received
Received 26 Aug, 2020
Review #2 received
Received 25 Aug, 2020
Reviewers invited
Invitations sent on 11 Aug, 2020
Reviewer #1 agreed
On 11 Aug, 2020
Reviewer #2 agreed
On 11 Aug, 2020
Reviewer #3 agreed
On 11 Aug, 2020
Editor assigned
On 05 Aug, 2020
Submission checks complete
On 04 Aug, 2020
Editor invited
On 04 Aug, 2020
First submitted
On 02 Aug, 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 Cancer Cell International.
Objective: Ovarian cancer (OVC) is the fifth leading cause of cancer-related deaths in women and has a significant impact on physical and mental health of women. This study explores the molecular mechanism of miR-636 acting as a tumor suppressor in OVC in vitro and in vivo, and provides new insight into the treatment of OVC.
Methods: Protein-protein interaction (PPI) analysis was performed to identify the hub gene in Hedgehog (Hh) pathway. TargetScan database was used to predict the upstream regulatory miRNAs of Gli2 to obtain the target miRNA. qRT-PCR was performed to test the expression of miR-636, while Western blot were conducted to detect the expression of Hh and EMT (epithelial-mesenchymal transition) related genes in OVC cell lines. MTT assay and wound healing assay were used to measure the effect of miR-636 on OVC cell proliferation and migration. Flow cytometry was carried out to examine the effect of miR-636 on cell cycle, and Western blot was used for identification of changes in expression of Hh and EMT related proteins. Dual-luciferase reporter gene assay was implemented to detect the targeted relationship between miR-636 and Gli2. The xenotransplantation model was used to detect the effect of miR-636 on OVC cell proliferation in vivo.
Results: PPI interaction analysis found that Gli2 was the hub gene in Hh pathway. Based on TargetScan and GEO databases, Gli2 was found to be targeted regulated by the upstream miR-636. In vitro experiments discovered that miR-636 was significantly lowly expressed in OVC cell lines. Overexpressing miR-636 significantly inhibited HO-8910PM cell proliferation and migration abilities as well as induced cell cycle arrest in G0/G1 phase, while the inhibition of miR-636 promoted cell proliferation and migration abilities. Dual-luciferase reporter gene assay revealed that Gli2 was a target gene of miR-636. Besides, overexpressing miR-636 decreased protein expression of Gli2, while the inhibition of miR-636 increased protein expression of Gli2. Furthermore, the overexpression and inhibition of miR-636 both affected the expression of proteins related to Hh signaling pathway and EMT. Rescue experiments verified that overexpression of Gli2 reversed the inhibitory effect of miR-636 on HO-8910PM cell proliferation and migration abilities, and attenuated the blocking effect of miR-636 on HO-8910PM cell cycle. The xenotransplantation model suggested that miR-636 inhibited cell growth of OVC by decreasing Gli2 expression. Besides, overexpressing Gli2 potentiated the EMT process in OVC via decreasing E-cadherin protein expression and increasing Vimentin protein expression, and it reversed the inhibitory effect of miR-636 on OVC cell proliferation and migration abilities in vivo.
Conclusion: miR-636 inhibits the Hh pathway activation via targeted binding to Gli2, thus inhibiting EMT, cell proliferation and migration in OVC.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
This is a list of supplementary files associated with this preprint. Click to download.
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