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Ya-ling Tang
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Background: Human papillomavirus (HPV)-positive oral squamous cell carcinoma (OSCC) is increasing worldwide with typically higher grade and stage. Studies suggested that microRNAs (miRNAs) play a critical role in cancer; However, their role in HPV-positive OSCC progression remains unclear.
Methods: miRNA microarray was performed to identify differentially expressed miRNAs. qRT-PCR and FISH were performed to determine the relative expression of miR-550a-3-5p. CCK-8, Flow cytometry, Wound healing, Cell invasion assays and xenograft experiments were conducted to analyze the biological roles of miR-550a-3-5p. Tumor-associated macrophages (TAMs) generation, co-culturing of cancer cells with TAMs, Western blot, Dual-luciferase reporter gene assay, Immunohistochemistry and animal studies were performed to explore the mechanisms underlying the functions of miR-550a-3-5p.
Results: In this study, we identified 19 miRNAs differentially expressed in HPV-positive OSCC specimens. One of these, miR-550a-3-5p, was down-regulated in HPV-positive OSCC. This down-regulation correlated with higher tumor size and nodal metastasis. Biofunctional investigations revealed that miR-550a-3-5p inhibited tumor growth and progression in nude mice models without altering the in vitro migration, invasion and EMT of HPV-positive OSCC cells. After co-culturing cancer cells with tumor-associated macrophages (TAMs), we found that the effects of miR-550a-3-5p on suppressing migration, invasion and EMT of HPV-positive OSCC cells were dependent on decreasing M2 macrophages polarization. Moreover, we identified that miR-550a-3-5p, down-regulated by E6 oncoprotein, inhibited M2 macrophages polarization by YAP/CCL2 signaling, which in turn abrogating EMT program in HPV-positive OSCC cells. Using YAP inhibitor, verteporfin (VP) in a HPV-positive OSCC model of transgenic mice also showed that tumors were less progressive when compared to those in Vehicle group. In both xenografts and clinical HPV-positive OSCC samples, miR-550a-3-5p levels were inversely associated with YAP, CCL2 expressions and the number of M2 macrophages.
Conclusions: E6/miR-550a-3-5p/YAP/CCL2 signaling induces M2 macrophages polarization to enhance EMT and progression, revealing a novel crosstalk between cancer cells and immune cells in HPV-positive OSCC microenvironment.
Keywords
Human papillomavirus, OSCC, microRNA, EMT, microenvironment
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Received 21 May, 2020
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On 21 May, 2020
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Received 21 May, 2020
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On 20 May, 2020
Reviewers invited
Invitations sent on 20 May, 2020
Reviewer #1 agreed
On 20 May, 2020
Submission checks complete
On 19 May, 2020
Editor invited
On 19 May, 2020
Version 1
Posted 23 Apr, 2020
No comments provided
Review #2 received
Received 29 Apr, 2020
Editorial decision: Major revision
On 29 Apr, 2020
Review #1 received
Received 23 Apr, 2020
Reviewer #2 agreed
On 21 Apr, 2020
Reviewer #3 agreed
On 21 Apr, 2020
Editor assigned
On 21 Apr, 2020
Reviewers invited
Invitations sent on 21 Apr, 2020
Reviewer #1 agreed
On 21 Apr, 2020
First submitted
On 20 Apr, 2020
Submission checks complete
On 20 Apr, 2020
Editor invited
On 20 Apr, 2020
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Subject Areas
Cancer Biology
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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
Ya-ling Tang
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: Under Review
No community comments so far
Review #1 received
Received 21 May, 2020
Reviewer #2 agreed
On 21 May, 2020
Review #2 received
Received 21 May, 2020
Editor assigned
On 20 May, 2020
Reviewers invited
Invitations sent on 20 May, 2020
Reviewer #1 agreed
On 20 May, 2020
Submission checks complete
On 19 May, 2020
Editor invited
On 19 May, 2020
Version 1
Posted 23 Apr, 2020
No comments provided
Review #2 received
Received 29 Apr, 2020
Editorial decision: Major revision
On 29 Apr, 2020
Review #1 received
Received 23 Apr, 2020
Reviewer #2 agreed
On 21 Apr, 2020
Reviewer #3 agreed
On 21 Apr, 2020
Editor assigned
On 21 Apr, 2020
Reviewers invited
Invitations sent on 21 Apr, 2020
Reviewer #1 agreed
On 21 Apr, 2020
First submitted
On 20 Apr, 2020
Submission checks complete
On 20 Apr, 2020
Editor invited
On 20 Apr, 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
The most recent version of this article is available here.
Background: Human papillomavirus (HPV)-positive oral squamous cell carcinoma (OSCC) is increasing worldwide with typically higher grade and stage. Studies suggested that microRNAs (miRNAs) play a critical role in cancer; However, their role in HPV-positive OSCC progression remains unclear.
Methods: miRNA microarray was performed to identify differentially expressed miRNAs. qRT-PCR and FISH were performed to determine the relative expression of miR-550a-3-5p. CCK-8, Flow cytometry, Wound healing, Cell invasion assays and xenograft experiments were conducted to analyze the biological roles of miR-550a-3-5p. Tumor-associated macrophages (TAMs) generation, co-culturing of cancer cells with TAMs, Western blot, Dual-luciferase reporter gene assay, Immunohistochemistry and animal studies were performed to explore the mechanisms underlying the functions of miR-550a-3-5p.
Results: In this study, we identified 19 miRNAs differentially expressed in HPV-positive OSCC specimens. One of these, miR-550a-3-5p, was down-regulated in HPV-positive OSCC. This down-regulation correlated with higher tumor size and nodal metastasis. Biofunctional investigations revealed that miR-550a-3-5p inhibited tumor growth and progression in nude mice models without altering the in vitro migration, invasion and EMT of HPV-positive OSCC cells. After co-culturing cancer cells with tumor-associated macrophages (TAMs), we found that the effects of miR-550a-3-5p on suppressing migration, invasion and EMT of HPV-positive OSCC cells were dependent on decreasing M2 macrophages polarization. Moreover, we identified that miR-550a-3-5p, down-regulated by E6 oncoprotein, inhibited M2 macrophages polarization by YAP/CCL2 signaling, which in turn abrogating EMT program in HPV-positive OSCC cells. Using YAP inhibitor, verteporfin (VP) in a HPV-positive OSCC model of transgenic mice also showed that tumors were less progressive when compared to those in Vehicle group. In both xenografts and clinical HPV-positive OSCC samples, miR-550a-3-5p levels were inversely associated with YAP, CCL2 expressions and the number of M2 macrophages.
Conclusions: E6/miR-550a-3-5p/YAP/CCL2 signaling induces M2 macrophages polarization to enhance EMT and progression, revealing a novel crosstalk between cancer cells and immune cells in HPV-positive OSCC microenvironment.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
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