Yanxiang Cheng

How does hypoxia inducible factor-1α participate in enhancing the glycolysis activity in cervical cancer?

The objective of this study is to explore the role of hypoxia inducible factor-1 (HIF-1) in glycolysis activity and its relationship with malignant biologic behaviors of cervical cancer. Immunohistochemistry was performed to study the protein expression and distribution of HIF-1α and glucose transport protein 1 (GLUT1) in cervical tissue of 158 cases, including 28 with normal cervical epithelium, 32 with cervical intraepithelial neoplasia, and 98 with invasive cervical cancer. Cobalt(II) chloride was used to induce hypoxia in Hela and Siha cells; the biologic behaviors of cells cultured in normal or hypoxic environments were monitored by colorimetric, Transwell, flow cytometry, and enzyme-linked immunosorbent assay; immunocytochemistry, Western blot, and reverse transcription-polymerase chain reaction were used to observe gene and protein expression of HIF-1α, GLUT1, and hexokinase II in cell lines during normoxia and hypoxia. The expression of HIF-1α and GLUT1 gradually increased from normal cervical tissue to cervical intraepithelial neoplasia, then to cervical cancer. There were significant differences among these groups (P < .05). HIF-1α was strongly associated with pathologic differentiation, clinic stage, magnitude of lesions, and patient age, whereas GLUT1 was associated with lymphatic metastasis (P < .05). HIF-1α was strongly associated with expression of GLUT1 (P < .05). In hypoxia, proliferation, invasion, resistance to apoptosis, and glycolysis of both Hela and Siha were enhanced compared with cells in normoxia (P < .05). Both gene and protein expressions of GLUT1 and hexokinase II were strengthened, whereas only the protein expression of HIF-1α was stronger in hypoxia than that in normoxia (P < .05). The results of Hela in normoxia and in hypoxia were similar to those of Siha (P > .05). HIF-1α plays a key role in cervical cancer both in vivo and in vitro. The role of HIF-1α can be implemented mainly by up-regulating its downstream gene, such as GLUT1, and the main mechanism may enhance glycolytic ability. Strong up-regulation and the role of HIF-1α suggest that HIF-1α could be an important factor in the onset and progression of cervical cancer and could be an attractive therapeutic molecular target for that type of cancer.

Knockdown of survivin contributes to antitumor activity in cisplatin-resistant ovarian cancer cells.

Survivin (SVV) is an important member of the inhibitor of apoptosis family. It is overexpressed in a number of cancer types, including human ovarian carcinomas. SVV promotes invasion, metastasis, growth and survival of malignant cells and confers resistance to specific chemotherapeutic drugs. The present study aimed to elucidate the role and possible mechanisms of SVV in cisplatin-resistant ovarian cancer cells (A2780/CP). Using a loss-of-function approach, we investigated the effects of adenovirus-mediated knockdown of SVV by small hairpin RNA (ad5-SVV) on the expression of pro-caspase-3, cleaved caspase-3, proliferating cell nuclear antigen (PCNA) and matrix metalloproteinase-2 (MMP-2) in A2780/CP cells by real-time PCR and western blot analysis. Proliferation was measured by MTT assay, invasive potential by Transwell, and cell apoptosis by FITC-Annexin V and propidium iodide for the functional analysis of A2780/CP cells following infection with ad5-SVV. As a result, knockdown of SVV downregulated the expression of PCNA and MMP-2 and upregulated the expression of pro-caspase-3 and cleaved caspase-3. In addition, knockdown of SVV enhanced cisplatin-induced proliferative activities, induced cell apoptosis and inhibited the invasive potential in A2780/CP cells. The present findings demonstrate that knockdown of SVV contributes to antitumor activity in cisplatin-resistant ovarian cancer cells via the downregulation of PCNA and MMP-2 expression and the upregulation of caspase-3 expression and indicate that SVV is a potential target for therapeutic anticancer drugs.

Single nucleotide polymorphisms of CTLA4 gene and their association with human cervical cancer

OBJECTIVE To evaluate the association between single nucleotide polymorphisms (SNPs) of cytotoxic T-lymphocyte-associated protein 4 (CTLA4) gene and susceptibility to cervical cancer. METHODS One hundred patients and 100 healthy controls from Hubei province were genotyped for 20 polymorphic loci using Sequenom. RESULTS The frequency of rs11571316 G allele and rs5742909 T allele, which are localized in the promoter region, and rs11571319 A allele, which is downstream of the gene, were significantly higher in patients than in controls. Luciferase assay showed that, as the previously reported rs5742909 T allele, rs11571316 G allele could significantly increase the expression of the reporter gene. CONCLUSION SNPs in the promoter region of (CTLA4) gene might increase the susceptibility to cervical cancer by increasing (CTLA4) gene expression.

Realgar-induced apoptosis of cervical cancer cell line Siha via cytochrome c release and caspase-3 and caspase-9 activation

ObjectiveTo explore the molecular mechanism of realgar-induced apoptosis of cervical cancer cells.MethodsThe cervical cancer cell line Siha was used to determine the cell viability and apoptosis after treatment with realgar using MTT assay and flow cytometry. The activities of caspase-3, -8, and -9 were detected by fluorescence resonance energy transfer technology and colorimetric assay, while the levels of Bcl-2, cytochrome c, and Bax were detected by Western blot method.ResultsInduction of apoptosis by realgar was detected in Siha cell line in a dose-dependent manner. The apoptosis was accompanied by a significant increase in cytochrome c release and activation of caspase-3 and caspase-9 but not caspase-8. Further, the realgar-induced apoptosis was inhibited by a broad-spectrum caspase inhibitor, a caspase-3 inhibitor, and a caspase-9 inhibitor but not by a caspase-8 inhibitor. Bcl-2 and Bax protein expressions were not changed by realgar.ConclusionThe induction of apoptosis by realgar is mediated through a cytochrome c-dependent pathway, which sequentially activates caspase-9 and caspase-3.

Aberrant expressions of endometrial Id3 and CTLA‐4 are associated with unexplained repeated implantation failure and recurrent miscarriage

Inhibitor of DNA‐binding protein 3 (Id3) is required for tumor angiogenesis and regulatory T‐cell generation. However, the involvement of Id3 in unexplained repeated implantation failure (RIF) and recurrent miscarriage (RM) remains poorly understood. Immunohistochemistry was used to identify Id3, CD34, CTLA‐4, and FOXP3 in the endometrium taken from the women with RIF (n=16), RM (n=16) and matched controls (n=8). The images were acquired and analyzed by the Vectra® automated quantitative pathology imaging system. Percentage of Id3+ cells was significantly higher in the endometrium of women with RIF and RM compared with controls. The numbers of Id3+ and CD34+ vessels in the endometrium were positively correlated in control but not in RIF or RM. Percentages of CTLA‐4+ cells, but not FOXP3+ cells, were significantly increased in the endometrium of RIF and RM women than those in controls. We found aberrant expressions of endometrial Id3 and CTLA‐4 in peri‐implantation endometrium of women with RIF and RM, suggesting the negative roles of these angiogenesis and immune tolerance markers involving in regulating endometrium receptivity.

Identification of differentially expressed genes and signaling pathways in ovarian cancer by integrated bioinformatics analysis

Background The mortality rate associated with ovarian cancer ranks the highest among gynecological malignancies. However, the cause and underlying molecular events of ovarian cancer are not clear. Here, we applied integrated bioinformatics to identify key pathogenic genes involved in ovarian cancer and reveal potential molecular mechanisms. Results The expression profiles of GDS3592, GSE54388, and GSE66957 were downloaded from the Gene Expression Omnibus (GEO) database, which contained 115 samples, including 85 cases of ovarian cancer samples and 30 cases of normal ovarian samples. The three microarray datasets were integrated to obtain differentially expressed genes (DEGs) and were deeply analyzed by bioinformatics methods. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichments of DEGs were performed by DAVID and KOBAS online analyses, respectively. The protein–protein interaction (PPI) networks of the DEGs were constructed from the STRING database. A total of 190 DEGs were identified in the three GEO datasets, of which 99 genes were upregulated and 91 genes were downregulated. GO analysis showed that the biological functions of DEGs focused primarily on regulating cell proliferation, adhesion, and differentiation and intracellular signal cascades. The main cellular components include cell membranes, exosomes, the cytoskeleton, and the extracellular matrix. The molecular functions include growth factor activity, protein kinase regulation, DNA binding, and oxygen transport activity. KEGG pathway analysis showed that these DEGs were mainly involved in the Wnt signaling pathway, amino acid metabolism, and the tumor signaling pathway. The 17 most closely related genes among DEGs were identified from the PPI network. Conclusion This study indicates that screening for DEGs and pathways in ovarian cancer using integrated bioinformatics analyses could help us understand the molecular mechanism underlying the development of ovarian cancer, be of clinical significance for the early diagnosis and prevention of ovarian cancer, and provide effective targets for the treatment of ovarian cancer.

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

SummaryThe 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 HPV16 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.