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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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Effect of HPV16-E7 gRNA-4/Cas9 on cellular proliferation, DSB cleavage, and E7 and pRb proteins level. HPV16-E7 gRNA-4/Cas9 inhibited cellular viability in SiHa and Caski cells but not in C33A and HEK293 cells ((a)–(d)). Cellular viability of SiHa, Caski, C33A, and HEK293 cells were monitored by CCK-8 assay (*P < 0.05 compared to untreated group, per Student's t-test). T7E1 treatment of heteroduplex DNA in the gRNA-4/Cas9 group showed noncleaved products at 500 bp and cleaved products at 300 bp and 200 bp both in SiHa and Caski cells ((e), (f)). Black arrows indicate 500 bp, 300 bp, and 200 bp PCR products. Untreated represented cells transfected with only Cas9 plasmid and was used as a negative control. Con-gRNA meant cells treated with HPV16E6-gRNA-1/Cas9, which was proved to be inactive preexperimentally. M was 100 bp DNA marker. E7 protein was downregulated and pRb protein was upregulated both in SiHa and Caski cells ((g), (h)). Data were drawn from four independent experiments. β-Actin was used as an internal control.
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fig4: Effect of HPV16-E7 gRNA-4/Cas9 on cellular proliferation, DSB cleavage, and E7 and pRb proteins level. HPV16-E7 gRNA-4/Cas9 inhibited cellular viability in SiHa and Caski cells but not in C33A and HEK293 cells ((a)–(d)). Cellular viability of SiHa, Caski, C33A, and HEK293 cells were monitored by CCK-8 assay (*P < 0.05 compared to untreated group, per Student's t-test). T7E1 treatment of heteroduplex DNA in the gRNA-4/Cas9 group showed noncleaved products at 500 bp and cleaved products at 300 bp and 200 bp both in SiHa and Caski cells ((e), (f)). Black arrows indicate 500 bp, 300 bp, and 200 bp PCR products. Untreated represented cells transfected with only Cas9 plasmid and was used as a negative control. Con-gRNA meant cells treated with HPV16E6-gRNA-1/Cas9, which was proved to be inactive preexperimentally. M was 100 bp DNA marker. E7 protein was downregulated and pRb protein was upregulated both in SiHa and Caski cells ((g), (h)). Data were drawn from four independent experiments. β-Actin was used as an internal control.

Mentions: We next investigated whether disruption of E7 gene could inhibit cellular proliferation in HPV16 positive SiHa and Caski cells. At 72 h and 96 h after transfection, compared to untreated groups, there was a significant reduction in cell viability in gRNA-4/Cas9 treated SiHa and Caski cells (Figures 4(a) and 4(b)). However, the absorbance rate in gRNA-4/Cas9 treated HPV16 negative C33A and HEK293 cells did not show significant difference compared to untreated groups (Figures 4(c) and 4(d)). These results indicated that gRNA-4/Cas9 could specifically inhibit the growth of HPV16 positive cervical cancer SiHa and Caski cell lines, but not HPV16 negative cervical cancer cell C33A or normal cell line HEK293.


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)

Effect of HPV16-E7 gRNA-4/Cas9 on cellular proliferation, DSB cleavage, and E7 and pRb proteins level. HPV16-E7 gRNA-4/Cas9 inhibited cellular viability in SiHa and Caski cells but not in C33A and HEK293 cells ((a)–(d)). Cellular viability of SiHa, Caski, C33A, and HEK293 cells were monitored by CCK-8 assay (*P < 0.05 compared to untreated group, per Student's t-test). T7E1 treatment of heteroduplex DNA in the gRNA-4/Cas9 group showed noncleaved products at 500 bp and cleaved products at 300 bp and 200 bp both in SiHa and Caski cells ((e), (f)). Black arrows indicate 500 bp, 300 bp, and 200 bp PCR products. Untreated represented cells transfected with only Cas9 plasmid and was used as a negative control. Con-gRNA meant cells treated with HPV16E6-gRNA-1/Cas9, which was proved to be inactive preexperimentally. M was 100 bp DNA marker. E7 protein was downregulated and pRb protein was upregulated both in SiHa and Caski cells ((g), (h)). Data were drawn from four independent experiments. β-Actin was used as an internal control.
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Related In: Results  -  Collection

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

fig4: Effect of HPV16-E7 gRNA-4/Cas9 on cellular proliferation, DSB cleavage, and E7 and pRb proteins level. HPV16-E7 gRNA-4/Cas9 inhibited cellular viability in SiHa and Caski cells but not in C33A and HEK293 cells ((a)–(d)). Cellular viability of SiHa, Caski, C33A, and HEK293 cells were monitored by CCK-8 assay (*P < 0.05 compared to untreated group, per Student's t-test). T7E1 treatment of heteroduplex DNA in the gRNA-4/Cas9 group showed noncleaved products at 500 bp and cleaved products at 300 bp and 200 bp both in SiHa and Caski cells ((e), (f)). Black arrows indicate 500 bp, 300 bp, and 200 bp PCR products. Untreated represented cells transfected with only Cas9 plasmid and was used as a negative control. Con-gRNA meant cells treated with HPV16E6-gRNA-1/Cas9, which was proved to be inactive preexperimentally. M was 100 bp DNA marker. E7 protein was downregulated and pRb protein was upregulated both in SiHa and Caski cells ((g), (h)). Data were drawn from four independent experiments. β-Actin was used as an internal control.
Mentions: We next investigated whether disruption of E7 gene could inhibit cellular proliferation in HPV16 positive SiHa and Caski cells. At 72 h and 96 h after transfection, compared to untreated groups, there was a significant reduction in cell viability in gRNA-4/Cas9 treated SiHa and Caski cells (Figures 4(a) and 4(b)). However, the absorbance rate in gRNA-4/Cas9 treated HPV16 negative C33A and HEK293 cells did not show significant difference compared to untreated groups (Figures 4(c) and 4(d)). These results indicated that gRNA-4/Cas9 could specifically inhibit the growth of HPV16 positive cervical cancer SiHa and Caski cell lines, but not HPV16 negative cervical cancer cell C33A or normal cell line HEK293.

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