Induction of SiHa Cells Apoptosis by Nanometer Realgar Suspension and Its Mechanism

The effects of nanometer realgar uterine cervix cancer cell line SiHa cells and suspension on proliferation and apoptosis of human oncogenic genes HPV16E6/E7 were investigated. A ＂micro-jet effiux＂ strategy was used for the preparation of nanometer realgar suspension. SiHa cells were treated with nanometer Realgar suspension in various concentrations （6.25, 12.5, 25 and 50 mg/L） for different durations （12, 24, 48 and 72 h）. The inhibitive effect of nanometer realgar suspension on growth of SiHa cells was detected by MTT method. Special morphological changes of apoptosis were observed by transmission electron microscopy （TEM） and DNA fragments electrophoresis. The apoptotic rate was quantified by flow cytometry （FCM）. The expression of HPV 16E6/E7 mRNA and protein was assayed by RT-PCR and Western blot respectively. The results showed after being treated with 25--50 mg/L nanometer realgar suspension for 48 h, the survival rate of SiHa cells was decreased, and apoptotic rate markedly increased in a time- and concentration-dependent manner. TEM and DNA electrophoresis revealed the special morphological changes of apoptosis. The apoptotic rate of SiHa cells treated with nanometer realgar suspension was significantly higher than in the control group （P〈0.01）, and G0/G1 phase arrest appeared following treatment with nanometer realgar suspension in 25 and 50 mg/L for 48 h. RT-PCR and Western blot assay indicated that nanometer realgar suspension reduced the HPV16E6/E7 gene expression. Nanometer realgar suspension could inhibit the proliferation and induce apoptosis of SiHa cells. The mechanism may be related to the down-regulation of the HPV16E6/E7 gene expression.

Effects of HPV Pseudotype Virus in Cutting E6 Gene Selectively in SiHa Cells

The objectives of this study were to investigate the effects of the CRISPR/Cas9 system mediated by the HPV pseudotype virus on SiHa cytobiology behavior by cutting the HPV16 E6 gene selectively and to explore the role of this system in the treatment of cervical cancer. After designing specific gRNA sequences targeting HPV 16 E6, generating hCas9-EGFP and E6-gRNA-RFP plasmids, and preparing the pseudovirus of HPV16 carrying E6-gRNA and Cas9 plasmids, we determined the titer of the pseudotype virus using the TCID50 method. We obtained the pseudotype virus of HPV16 carrying E6-gRNA and Cas9 plasmids to transfect cervical cancer SiHa cells. Experimental subjects were divided into control group, empty virus group, E6-gRNA transfected group, Cas9 transfected group and Cas9＋E6-gRNA transfected group. The molecular size of the cutting sequence was detected using the T7E1 enzyme digestion method and agarose gel electrophoresis, and the cleavage function of CRISPR/Cas9 on the E6 gene was determined at the same time. RT-PCR and Western blotting were performed to detect the mRNA and protein expression levels of E6 in all the groups; the Transwell cell migration assay was performed to detect the cell migration ability and metastasis in all groups. Heterotopic transplantation tumors were incorporated into mice and were used to investigate the effects of the CRISPR/Cas9 system mediated by the HPV pseudovirus on the tumorigenic ability of SiHa cells by selectively cutting HPV16 E6. The HPV16 pseudotype virus carrying E6-gRNA and Cas9 plasmids could successfully infect SiHa cells, and there were two cutting zones in the Cas9＋E6- gRNA transfected group. However, the empty virus group, E6-gRNA transfected group and Cas9 transfected group had no corresponding zone. Compared with those in the control group, the empty virus group, E6-gRNA transfected group and Cas9 transfected group, the mRNA and protein expression levels of E6 in SiHa cells were downregulated in the Cas9＋E6-gRNA transfected group （P〈0.01）. In addition, the proliferation and migration abilities of SiHa cells were significantly inhibited （P〈0.01）. There were no significant differences among the other groups. In contrast to the control group, the HPV pseudotype virus carrying E6-gRNA and Cas9 plasmids could significantly delay the growth of tumor cells of the ectopic tumor transplantation model （P〈0.01）. The CRISPR/Cas9 system mediated by the HPV pseudotype virus to knockout E6 gene expression exhibited a clear inhibitory effect on the biological function of SiHa cells, which indicated that knocking out the E6 gene using the CRISPR/Cas9 system mediated by the HPV pseudotype virus had a potential effect of eliminating HPV infection and inhibiting the growth of HPV-related tumors. Taken together, these findings provide insight into a new treatment strategy for the prevention and treatment of hr-HPV infected disease, particularly in HPV-related tumors.

Human Papillomavirus 16E6 Oncogene Mutation in Cervical Cancer

Objective： Cervical cancer （CC） is the second most common type of cancer in women worldwide, after breast cancer. High-risk human papillomaviruses （HR-HPVs） are considered to be the major causes of cervical cancer. HPV16 is the most common type of HR-HPVs and HPV16 E6 gene is one of the major oncogenes. Specific mutations are considered as dangerous factors causing CC. This study was designed to find mutations of HPV16 E6 and the relationship between the mutations and the happening of CC. Methods： The tissue DNA was extracted from 15 biopsies of CC. Part of HPV16 E6 gene （nucleotide 201-523） was amplified by polymerase chain reaction （PCR） from the CC tissue DNA. The PCR fragments were sequenced and analyzed. Results： The result of PCR showed that the positive rate of HPV16 E6 was 93.33% （14/13）. After sequencing ana analyzing, in the 13 out of 14 PCR fragments, 4 maintained prototype （30.77%）, 8 had a same 350G mutation （61.54%）, and 1 had a 249G mutation （7.69%）. Conclusion： This study suggest that there is a high infection rate of HPV in cervical cancer and most of the HPV16 E6 gene has mutations. Those mutations may have an association with the development of cervical cancer.