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HPV16 Oncoproteins Promote Cervical Cancer Invasiveness by Upregulating Specific Matrix Metalloproteinases

Kaewprag, Jittranan, Umnajvijit, Wareerat, Ngamkham, Jarunya, Ponglikitmongkol, Mathurose·2013·PLoS ONE
DOI 10.1371/journal.pone.0071611PMC PMC3742460
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Figure 1 In vitro invasion and MMP expression of HPV positive, HPV negative and HPV oncoprotein expressing C33A. (A) Quantitation of invasion using migration through filters coated with Matrigel for HPV positive (SiHa, Caski and HeLa) and negative (C33A and 293T) cells presenting as numbers of invading cells. (B) Relative expression of MMP-1, -2, -7, -9, -10, -11 and MT1-MMP measured in cell lines using qPCR. Data was normalized to C33A. *:p-value<0.05 relative to C33A. (C) Relative expression of MMP-2, -7 and MT1-MMP in C33A cells stably expressing different HPV16 (16E6, 16E6F, 16E6*I, 16E7, 16E6E7, 16E6*IE7), HPV18 (18E6E7) and low-risk HPV6 (6E6) oncoproteins as measured by qPCR and normalized to pcDNA3. *p-value<0.05 compared to pcDNA3 expressing cells. (D) The level of HPV transcripts compared to the control, HPRT, in each cell line as shown by RT-PCR. (E) P53 degradation activity of 16E6, 16E7 and 16E6E7 was compared by western blot analysis. GAPDH was used as loading control.
Figure 1 In vitro invasion and MMP expression of HPV positive, HPV negative and HPV oncoprotein expressing C33A. (A) Quantitation of invasion using migration through filters coated with Matrigel for HPV positive (SiHa, Caski and HeLa) and negative (C33A and 293T) cells presenting as numbers of invading cells. (B) Relative expression of MMP-1, -2, -7, -9, -10, -11 and MT1-MMP measured in cell lines using qPCR. Data was normalized to C33A. *:p-value<0.05 relative to C33A. (C) Relative expression of MMP-2, -7 and MT1-MMP in C33A cells stably expressing different HPV16 (16E6, 16E6F, 16E6*I, 16E7, 16E6E7, 16E6*IE7), HPV18 (18E6E7) and low-risk HPV6 (6E6) oncoproteins as measured by qPCR and normalized to pcDNA3. *p-value<0.05 compared to pcDNA3 expressing cells. (D) The level of HPV transcripts compared to the control, HPRT, in each cell line as shown by RT-PCR. (E) P53 degradation activity of 16E6, 16E7 and 16E6E7 was compared by western blot analysis. GAPDH was used as loading control.CC BY · PubMed Central
Figure 2 Invasion ability, MMP expression and activity of C33A cells stably expressing different HPV16 oncoproteins. (A) In vitro invasion and (B) wound healing assays for C33A cells expressing different HPV16 oncoproteins. (C) A representative gelatin zymographic gel showing MMP-2 activity normalized with equal amounts of loading protein (30 µg). Bands corresponding to the gelatinolytic activity of MMP-2 were quantified by densitometric analysis and compared with those obtained from control pcDNA3 expressing cells. (D) Western blot analysis of MT1-MMP using anti-MT1-MMP antibody (ab78738) at 1∶1,500 dilution with GAPDH as loading control. *p-value<0.05, **p-value<0.01.
Figure 2 Invasion ability, MMP expression and activity of C33A cells stably expressing different HPV16 oncoproteins. (A) In vitro invasion and (B) wound healing assays for C33A cells expressing different HPV16 oncoproteins. (C) A representative gelatin zymographic gel showing MMP-2 activity normalized with equal amounts of loading protein (30 µg). Bands corresponding to the gelatinolytic activity of MMP-2 were quantified by densitometric analysis and compared with those obtained from control pcDNA3 expressing cells. (D) Western blot analysis of MT1-MMP using anti-MT1-MMP antibody (ab78738) at 1∶1,500 dilution with GAPDH as loading control. *p-value<0.05, **p-value<0.01.CC BY · PubMed Central
Figure 3 HPV oncogenes, MMP transcripts and MMP protein levels in SiHa cells transfected with shRNA directed at E6 oncogene. (A) RT-PCR of 16E6F, 16E6*I, and 16E7 transcripts normalized to the control, HPRT. SiHa cells transfected with non-targeting shCtrl and vehicle control pSUPER were used as negative controls. shE6A and shE6B are two different shRNAs used in knock-down experiments. (B) qPCR of MMP-2, -7 and MT1-MMP transcripts and (C) MMP-2 activity as assayed by gelatin zymography, with equal amounts of protein (20 µg), in shRNA transfected cells. (D) MT1-MMP protein in knocked down cells as determine by western blot analysis with GAPDH as loading control. (E) Invasion ability of SiHa cells transfected with different shRNAs as determined by in vitro invasion and wound healing assays. Relative fold of cell invasion with untreated cells were set as 1 is reported. *p-value<0.05 compared to shCtrl cells.
Figure 3 HPV oncogenes, MMP transcripts and MMP protein levels in SiHa cells transfected with shRNA directed at E6 oncogene. (A) RT-PCR of 16E6F, 16E6*I, and 16E7 transcripts normalized to the control, HPRT. SiHa cells transfected with non-targeting shCtrl and vehicle control pSUPER were used as negative controls. shE6A and shE6B are two different shRNAs used in knock-down experiments. (B) qPCR of MMP-2, -7 and MT1-MMP transcripts and (C) MMP-2 activity as assayed by gelatin zymography, with equal amounts of protein (20 µg), in shRNA transfected cells. (D) MT1-MMP protein in knocked down cells as determine by western blot analysis with GAPDH as loading control. (E) Invasion ability of SiHa cells transfected with different shRNAs as determined by in vitro invasion and wound healing assays. Relative fold of cell invasion with untreated cells were set as 1 is reported. *p-value<0.05 compared to shCtrl cells.CC BY · PubMed Central
Figure 4 Inhibition of cell invasion by specific inhibitors for MMP-2 and MT1-MMP and assay of TIMP-2. (A) Cell invasion as determined using in vitro invasion and wound healing of HPV16E6E7 expressing cells and (B) SiHa cells treated with various concentrations of MMP-2 inhibitor, OA-Hy. (C) The same cell lines were also treated with anti-MT1-MMP antibody (20 µg/ml). Relative fold of cell invasion with pcDNA3 vehicle control or untreated cells set as 1 at φp<0.05 compare to pcDNA3 and *p-value<0.05 compare to untreated cells is shown. (D) TIMP-2 protein (normalized with equal loading protein of 5 µg) was detected using western blot analysis with an anti-TIMP-2 antibody (abcam, 1∶1,000 dilution) and TIMP-2 transcripts were measured by qPCR (normalized to HPRT) for HPV16 oncoprotein expressing cells.
Figure 4 Inhibition of cell invasion by specific inhibitors for MMP-2 and MT1-MMP and assay of TIMP-2. (A) Cell invasion as determined using in vitro invasion and wound healing of HPV16E6E7 expressing cells and (B) SiHa cells treated with various concentrations of MMP-2 inhibitor, OA-Hy. (C) The same cell lines were also treated with anti-MT1-MMP antibody (20 µg/ml). Relative fold of cell invasion with pcDNA3 vehicle control or untreated cells set as 1 at φp<0.05 compare to pcDNA3 and *p-value<0.05 compare to untreated cells is shown. (D) TIMP-2 protein (normalized with equal loading protein of 5 µg) was detected using western blot analysis with an anti-TIMP-2 antibody (abcam, 1∶1,000 dilution) and TIMP-2 transcripts were measured by qPCR (normalized to HPRT) for HPV16 oncoprotein expressing cells.CC BY · PubMed Central
Figure 5 Transcriptional activation activity of HPV16E6E7 on MMP promoters. (A) MMP-2 and (B) MT1-MMP promoter constructs in C33A cells expressing HPV16E6E7. The luciferase activities of three constructs of each promoter were compared with that of the pGL3-Basic vector (set as 1). *p-value<0.05 compared to pcDNA3.
Figure 5 Transcriptional activation activity of HPV16E6E7 on MMP promoters. (A) MMP-2 and (B) MT1-MMP promoter constructs in C33A cells expressing HPV16E6E7. The luciferase activities of three constructs of each promoter were compared with that of the pGL3-Basic vector (set as 1). *p-value<0.05 compared to pcDNA3.CC BY · PubMed Central
Figure 6 Representative phase contrast and fluorescent images of MMP-2 and MT1-MMP expression in cervical cancer tissues. Presence of MMP-2 and MT1-MMP are shown in red (Alexa Fluor 647) and cell nuclei in blue (Hoechst 33258). (A, B) no primary antibodies; (C, D) and (G, H) negative staining of MMP-2 and MT1-MMP respectively; (E, F) and (I, J) positive staining of MMP-2 and MT1-MMP respectively.
Figure 6 Representative phase contrast and fluorescent images of MMP-2 and MT1-MMP expression in cervical cancer tissues. Presence of MMP-2 and MT1-MMP are shown in red (Alexa Fluor 647) and cell nuclei in blue (Hoechst 33258). (A, B) no primary antibodies; (C, D) and (G, H) negative staining of MMP-2 and MT1-MMP respectively; (E, F) and (I, J) positive staining of MMP-2 and MT1-MMP respectively.CC BY · PubMed Central

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Cited by

Metadata

Authors
Kaewprag, J., Umnajvijit, W., Ngamkham, J., & Ponglikitmongkol, M.
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2013-08-13
Volume
8
Issue
8
Pages
e71611
ISSN
1932-6203
DOI
10.1371/journal.pone.0071611
PMC
PMC3742460
Cited by
35
References
0
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (unspecified)

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