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Disruption of HPV16-E7 by CRISPR/Cas system induces apoptosis and growth inhibition in HPV16 positive human cervical cancer cells.

Hu Z, Yu L, Zhu D, Ding W, Wang X, Zhang C, Wang L, Jiang X, Shen H, He D, Li K, Xi L, Ma D, Wang H - Biomed Res Int (2014)

Bottom Line: By using clustered regularly interspaced short palindromic repeats- (CRISPR-) associated protein system (CRISPR/Cas system), a widely used genome editing tool in many organisms, to target HPV16-E7 DNA in HPV positive cell lines, we showed for the first time that the HPV16-E7 single-guide RNA (sgRNA) guided CRISPR/Cas system could disrupt HPV16-E7 DNA at specific sites, inducing apoptosis and growth inhibition in HPV positive SiHa and Caski cells, but not in HPV negative C33A and HEK293 cells.Moreover, disruption of E7 DNA directly leads to downregulation of E7 protein and upregulation of tumor suppressor protein pRb.Therefore, our results suggest that HPV16-E7 gRNA guided CRISPR/Cas system might be used as a therapeutic strategy for the treatment of cervical cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China.

ABSTRACT
High-risk human papillomavirus (HR-HPV) has been recognized as a major causative agent for cervical cancer. Upon HPV infection, early genes E6 and E7 play important roles in maintaining malignant phenotype of cervical cancer cells. By using clustered regularly interspaced short palindromic repeats- (CRISPR-) associated protein system (CRISPR/Cas system), a widely used genome editing tool in many organisms, to target HPV16-E7 DNA in HPV positive cell lines, we showed for the first time that the HPV16-E7 single-guide RNA (sgRNA) guided CRISPR/Cas system could disrupt HPV16-E7 DNA at specific sites, inducing apoptosis and growth inhibition in HPV positive SiHa and Caski cells, but not in HPV negative C33A and HEK293 cells. Moreover, disruption of E7 DNA directly leads to downregulation of E7 protein and upregulation of tumor suppressor protein pRb. Therefore, our results suggest that HPV16-E7 gRNA guided CRISPR/Cas system might be used as a therapeutic strategy for the treatment of cervical cancer.

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Disruption of the E7 gene could significantly enhance apoptosis monitored by FCM in SiHa and Caski cells, but not in C33A and HEK293 cells. gRNA-1/Cas9 ((a), (c)), gRNA-2/Cas9 ((b), (d)), gRNA-3/Cas9 ((e), (g)), and gRNA-4/Cas9 ((f), (h)) induced apoptosis rate in SiHa, Caski, C33A, and HEK293 cell lines (**P < 0.01 compared to blank control, per Student's t-test).
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fig3: Disruption of the E7 gene could significantly enhance apoptosis monitored by FCM in SiHa and Caski cells, but not in C33A and HEK293 cells. gRNA-1/Cas9 ((a), (c)), gRNA-2/Cas9 ((b), (d)), gRNA-3/Cas9 ((e), (g)), and gRNA-4/Cas9 ((f), (h)) induced apoptosis rate in SiHa, Caski, C33A, and HEK293 cell lines (**P < 0.01 compared to blank control, per Student's t-test).

Mentions: To determine whether the four groups of gRNAs/Cas9 could specifically induce cellular apoptosis in HPV16 positive cells, we introduced gRNAs/Cas9 plasmids into HPV16 positive SiHa and Caski cell lines, together with HPV negative C33A and HEK293 cell lines, respectively. Compared with the apoptosis rate of blank control group and con-gRNA group (apoptosis rates were less than 10%), the apoptosis rates induced by gRNA-1/Cas9, gRNA-2/Cas9, gRNA-3/Cas9, and gRNA-4/Cas9 were 50%, 40%, 47%, and 56%, respectively, in SiHa cells and 44%, 35%, 42%, and 48%, respectively, in Caski cells (Figure 3). On the other hand, apoptosis induced by the same four gRNA/Cas9 groups in HPV negative C33A and HEK293 cell lines were below 10%. Based on the data that gRNA-4/Cas9 treated groups displayed the highest rate of apoptosis both in SiHa and Caski cells, gRNA-4/Cas9 was selected for further experiments.


Disruption of HPV16-E7 by CRISPR/Cas system induces apoptosis and growth inhibition in HPV16 positive human cervical cancer cells.

Hu Z, Yu L, Zhu D, Ding W, Wang X, Zhang C, Wang L, Jiang X, Shen H, He D, Li K, Xi L, Ma D, Wang H - Biomed Res Int (2014)

Disruption of the E7 gene could significantly enhance apoptosis monitored by FCM in SiHa and Caski cells, but not in C33A and HEK293 cells. gRNA-1/Cas9 ((a), (c)), gRNA-2/Cas9 ((b), (d)), gRNA-3/Cas9 ((e), (g)), and gRNA-4/Cas9 ((f), (h)) induced apoptosis rate in SiHa, Caski, C33A, and HEK293 cell lines (**P < 0.01 compared to blank control, per Student's t-test).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4127252&req=5

fig3: Disruption of the E7 gene could significantly enhance apoptosis monitored by FCM in SiHa and Caski cells, but not in C33A and HEK293 cells. gRNA-1/Cas9 ((a), (c)), gRNA-2/Cas9 ((b), (d)), gRNA-3/Cas9 ((e), (g)), and gRNA-4/Cas9 ((f), (h)) induced apoptosis rate in SiHa, Caski, C33A, and HEK293 cell lines (**P < 0.01 compared to blank control, per Student's t-test).
Mentions: To determine whether the four groups of gRNAs/Cas9 could specifically induce cellular apoptosis in HPV16 positive cells, we introduced gRNAs/Cas9 plasmids into HPV16 positive SiHa and Caski cell lines, together with HPV negative C33A and HEK293 cell lines, respectively. Compared with the apoptosis rate of blank control group and con-gRNA group (apoptosis rates were less than 10%), the apoptosis rates induced by gRNA-1/Cas9, gRNA-2/Cas9, gRNA-3/Cas9, and gRNA-4/Cas9 were 50%, 40%, 47%, and 56%, respectively, in SiHa cells and 44%, 35%, 42%, and 48%, respectively, in Caski cells (Figure 3). On the other hand, apoptosis induced by the same four gRNA/Cas9 groups in HPV negative C33A and HEK293 cell lines were below 10%. Based on the data that gRNA-4/Cas9 treated groups displayed the highest rate of apoptosis both in SiHa and Caski cells, gRNA-4/Cas9 was selected for further experiments.

Bottom Line: By using clustered regularly interspaced short palindromic repeats- (CRISPR-) associated protein system (CRISPR/Cas system), a widely used genome editing tool in many organisms, to target HPV16-E7 DNA in HPV positive cell lines, we showed for the first time that the HPV16-E7 single-guide RNA (sgRNA) guided CRISPR/Cas system could disrupt HPV16-E7 DNA at specific sites, inducing apoptosis and growth inhibition in HPV positive SiHa and Caski cells, but not in HPV negative C33A and HEK293 cells.Moreover, disruption of E7 DNA directly leads to downregulation of E7 protein and upregulation of tumor suppressor protein pRb.Therefore, our results suggest that HPV16-E7 gRNA guided CRISPR/Cas system might be used as a therapeutic strategy for the treatment of cervical cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China.

ABSTRACT
High-risk human papillomavirus (HR-HPV) has been recognized as a major causative agent for cervical cancer. Upon HPV infection, early genes E6 and E7 play important roles in maintaining malignant phenotype of cervical cancer cells. By using clustered regularly interspaced short palindromic repeats- (CRISPR-) associated protein system (CRISPR/Cas system), a widely used genome editing tool in many organisms, to target HPV16-E7 DNA in HPV positive cell lines, we showed for the first time that the HPV16-E7 single-guide RNA (sgRNA) guided CRISPR/Cas system could disrupt HPV16-E7 DNA at specific sites, inducing apoptosis and growth inhibition in HPV positive SiHa and Caski cells, but not in HPV negative C33A and HEK293 cells. Moreover, disruption of E7 DNA directly leads to downregulation of E7 protein and upregulation of tumor suppressor protein pRb. Therefore, our results suggest that HPV16-E7 gRNA guided CRISPR/Cas system might be used as a therapeutic strategy for the treatment of cervical cancer.

Show MeSH
Related in: MedlinePlus