ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
This study was aimed to elucidate the expression of costimulatory molecule CD80 and CD86 in HL-60 cells induced by proteasome inhibitor MG132 and its effect on allogeneic mixed lymphocyte reaction. Acute myelocytic leukemia cell line HL-60 and chronic myelocytic leukemia cell line K562 were cultured. The viability of the cells was measured by flow cytometry. Proteasome inhibitor MG132 at the concentrations of 2 or 3 µmol/L was used to stimulate the HL-60 cell cultured for 24 h and 48 h respectively, and the Annexin V/7-AAD staining and flow cytomotry were used to detect the apoptosis of the HL-60 cells. HL-60 and K562 cells were treated with 1 µmol/L MG132 for 24 h and 48 h respectively, then CD80 and CD86 antibodies were added, finally the expression of CD80 and CD86 was analysed by flow cytomery. The mRNA expression of CD86 in the HL-60 cells treated with 1 µmol/L MG132 was detected by RT-PCR. HL-60 and K562 cells were treated by 1 µmol/L MG132 and then underwent irradiation of 75 Gy (60)Co to kill the cells with their antigenicity preserved. Peripheral blood mononuclear cells (PBMNCs) of healthy volunteers, as reactive cells, were isolated and inoculated into the (60)Co irradiated HL-60 cells of different concentrations, as stimulating cells, CCK-8 was added and then the A value of absorbance was measured at the wave length of 450 nm in an enzyme labeling instrument. The results showed that the cell viability of the HL-60 cells treated with 1 µmol/L MG132 for 24 h an d 48 h was 92.95% and 85.87% respectively. The apoptotic rates of the HL-60 cells treated with MG132 increased in dose-and time-dependent manner. High-concentration of MG132 directly killed HL-60 cells. Before MG132 treatment K562 cells did not express CD86, but the CD86 expression of the HL-60 cells was up-regulated time-dependently after MG132 treatment (P < 0.01). The mRNA expression of CD86 in the HL-60 treated with MG132 was up-regulated time-dependently (P < 0.01). CCK-8 test showed that the proliferation level of PBMNC gradually increased along with the concentration of HL-60 cells treated with MG132 and reached its peak when the concentration of the HL-60 cells was 1×10(5) (P < 0.01). No remarkable proliferation of PBMNC was observed in the K562 groups no matter if the HL-60 cells had been treated with MG132. It is concluded that the high concentration of MG132 can directly kill HL-60 cells, low-concentration of MG132 can induce the expression of costimulatory molecule CD86 in HL-60 cells, also can improve the proliferation of PBMNC.
Subject(s)
Humans , Apoptosis , B7-2 Antigen , Allergy and Immunology , Cell Survival , Flow Cytometry , HL-60 Cells , K562 Cells , Leukocytes, Mononuclear , Leupeptins , Pharmacology , Lymphocyte Culture Test, Mixed , Proteasome Inhibitors , Pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Up-RegulationABSTRACT
The effects of tacrolimus postconditioning on protein-serine-threonine kinases (Akt) phosphorylation and apoptotic cell death in rats after spinal cord ischemia-reperfusion injury were investigated. Ninety male SD rats were randomly divided into sham operation group, ischemia-reperfusion group and tacrolimus postconditioning group. The model of spinal cord ischemia was established by means of catheterization through femoral artery and balloon dilatation. The spinal cord was reperfused 20 min after ischemia via removing saline out of balloon. The corresponding spinal cord segments were excised and determined for Akt activity in spinal cord tissue by using Western blotting at 5, 15, and 60 min after reperfusion respectively. Spinal cord tissue sections were stained immunohistochemically for detection of the phosphorylated Akt expression at 15 min after reperfusion. Flow cytometry was applied to assess apoptosis of neural cells, and dry-wet weights method was employed to measure water content in spinal cord tissue at 24 h after reperfusion. The results showed that the activities of Akt in tarcolimus postconditioning group were significantly higher than those in ischemia-reperfusion group at 5, 15, and 60 min after reperfusion (P<0.05, P<0.01). The Akt activities reached the peak at 15 min after reperfusion in ischemia-reperfusion group and tacrolimus postconditioning group. The percentage of apoptotic cells and water content in spinal cord tissue were significantly reduced (P<0.01) in tacrolimus postconditioning group as compared with those in ischemia-reperfusion group at 24 h after reperfusion. It is concluded that tacrolimus post-conditioning can increase Akt activity in spinal cord tissue of rats, inhibit apoptosis of neural cells as well as tissue edema, and thereby alleviate spinal cord ischemia-reperfusion injury.
ABSTRACT
The effects of tacrolimus postconditioning on protein-serine-threonine kinases (Akt) phosphorylation and apoptotic cell death in rats after spinal cord ischemia-reperfusion injury were investigated. Ninety male SD rats were randomly divided into sham operation group, ischemia-reperfusion group and tacrolimus postconditioning group. The model of spinal cord ischemia was established by means of catheterization through femoral artery and balloon dilatation. The spinal cord was reperfused 20 min after ischemia via removing saline out of balloon. The corresponding spinal cord segments were excised and determined for Akt activity in spinal cord tissue by using Western blotting at 5, 15, and 60 min after reperfusion respectively. Spinal cord tissue sections were stained immunohistochemically for detection of the phosphorylated Akt expression at 15 min after reperfusion. Flow cytometry was applied to assess apoptosis of neural cells, and dry-wet weights method was employed to measure water content in spinal cord tissue at 24 h after reperfusion. The results showed that the activities of Akt in tarcolimus postconditioning group were significantly higher than those in ischemia-reperfusion group at 5, 15, and 60 min after reperfusion (P<0.05, P<0.01). The Akt activities reached the peak at 15 min after reperfusion in ischemia-reperfusion group and tacrolimus postconditioning group. The percentage of apoptotic cells and water content in spinal cord tissue were significantly reduced (P<0.01) in tacrolimus postconditioning group as compared with those in ischemia-reperfusion group at 24 h after reperfusion. It is concluded that tacrolimus post-conditioning can increase Akt activity in spinal cord tissue of rats, inhibit apoptosis of neural cells as well as tissue edema, and thereby alleviate spinal cord ischemia-reperfusion injury.
Subject(s)
Animals , Male , Rats , Apoptosis , Immunosuppressive Agents , Pharmacology , Therapeutic Uses , Phosphorylation , Protein Serine-Threonine Kinases , Genetics , Metabolism , RNA, Messenger , Genetics , Metabolism , Rats, Sprague-Dawley , Reperfusion Injury , Drug Therapy , Metabolism , Spinal Cord , Metabolism , Pathology , Spinal Cord Ischemia , Drug Therapy , Metabolism , Tacrolimus , Pharmacology , Therapeutic Uses , Up-RegulationABSTRACT
To investigate the mechanism of lipid raft mediating chemotherapy resistance in cervical cancer. This experiment was carried out in the Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China from June 2010 to February 2011. Hela cells were divided into 6 groups: control group [Ctrl], cisplatin group [Cis], lipid raft interference agent group [MCD], NADPH oxidase inhibitor group [Apo], lipid raft interference agent combined with cisplatin group [MCD+Cis], and NADPH oxidase inhibitor combined with cisplatin group [Apo+Cis]. After the cervical cancer cells were treated with a correspondent agent for 24 hours, the number of surviving cells were measured utilizing cell counting kits-8 [CCK-8], and the hypoxia inducible factor-1alpha [HIF-1alpha levels were detected by Western blotting. Reactive oxygen species [ROS] levels were measured indirectly by detection of dichlorodihydrofluorescein fluorescence activity. The cell growth of MCD slowed down [survival cells was 62% compared with the Ctrl group], with the Apo group showing a similar effect [65% in the control group], and 49% for the Cis group, MCD+Cis was 21%, and Apo+Cis was 23%. While the level of HIF-1alpha protein and ROS of the MCD group, Apo group, Cis group, MCD+Cis group and Apo+Cis group were decreased significantly compared to the control group. The level of HIF-1alpha of MCD group decreased by 69.9%, Apo group by 60.2%, Cis group was 55.5%, MCD+Cis group by 21.1% and Apo+Cis group by 25.4%, while the level of ROS also decreased in the MCD group by 38.6%, Apo group by 35.3%, Cis group by 24%, MCD+Cis group by 12.3% and Apo+Cis group by 12.8%. Lipid raft may up-regulate ROS level and HIF-1alpha expression through activating NADPH oxidase, and thus promote chemotherapy resistance in cervical cancer.
ABSTRACT
<p><b>OBJECTIVE</b>To explore the molecular mechanism of realgar-induced apoptosis of cervical cancer cells.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>Induction 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.</p><p><b>CONCLUSION</b>The induction of apoptosis by realgar is mediated through a cytochrome c-dependent pathway, which sequentially activates caspase-9 and caspase-3.</p>
Subject(s)
Female , Humans , Apoptosis , Physiology , Arsenicals , Pharmacology , Caspase 3 , Metabolism , Caspase 8 , Metabolism , Caspase 9 , Metabolism , Caspase Inhibitors , Cell Line, Tumor , Cell Survival , Physiology , Cytochromes c , Metabolism , Dose-Response Relationship, Drug , Enzyme Activation , Enzyme Inhibitors , Pharmacology , Fluorescence Resonance Energy Transfer , Sulfides , Pharmacology , Uterine Cervical Neoplasms , Drug Therapy , Metabolism , PathologyABSTRACT
<p><b>OBJECTIVE</b>To evaluate the association between single nucleotide polymorphisms (SNPs) of cytotoxic T-lymphocyte-associated protein 4 (CTLA4) gene and susceptibility to cervical cancer.</p><p><b>METHODS</b>One hundred patients and 100 healthy controls from Hubei province were genotyped for 20 polymorphic loci using Sequenom.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>SNPs in the promoter region of (CTLA4) gene might increase the susceptibility to cervical cancer by increasing (CTLA4) gene expression.</p>
Subject(s)
Adult , Aged , Female , Humans , Middle Aged , Young Adult , Alleles , Antigens, CD , Genetics , CTLA-4 Antigen , Case-Control Studies , Gene Expression Regulation, Neoplastic , Genetics , Genetic Predisposition to Disease , Genotype , Polymorphism, Single Nucleotide , Uterine Cervical Neoplasms , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To observe the anti-tumor effect of silencing the expression of HIF-1alpha on cervical cancer in nude mice and to explore its mechanism of action.</p><p><b>METHODS</b>Human cervical cancer cell line Siha cells were divided into 3 groups: mock control group, control group transfected with scrambled sequence plasmid, and experimental group transfected with pU-HIF-1alpha-shRNA eukaryotic expression plasmid. Cultured cells of the three groups were inoculated in nude mice to establish cervical cancer-bearing nude mice. HIF-1alpha RNAi assay was performed to evaluate the tumor-suppressive effect of HIF-1alpha silencing on cervical cancer-bearing nude mice. Immunohistochemistry and Western blot were used to observe the distribution and protein expression of HIF-1alpha and GLUT1, while RT-PCR was adopted to detect the gene expression of HIF-1alpha, GLUT1 and HKII. The product of glycolysis (lactic acid) and apoptosis in tumor cells were detected by colorimetry and semi-quantitative TUNEL staining, respectively.</p><p><b>RESULTS</b>The tumor growth in experimental group was significantly slower than that in the two control groups (P < 0.05). On the 50th day after transplantation, the tumor weight in the experimental group was (1.90 +/- 0.28) g, significantly lower than (2.95 +/- 0.77) g in the control group and (2.54 +/- 0.56) g in the mock group (P < 0.01). In the experimental group, the gene and protein levels of HIF-1alpha were 0.45 +/- 0.04 and 1.25 +/- 0.92, and the levels of GLUT1 were 0.32 +/- 0.02 and 1.25 +/- 0.48, respectively. Both indicators in HIF-1alpha and GLUT1 were lower than that in the two control groups (P < 0.05). The expression levels of HKII gene and lactic acid in the experimental group were lower than that in the two control groups (P < 0.05), but the apoptotic cells were much more numerous in the experimental group than that in matched control groups (P < 0.01).</p><p><b>CONCLUSION</b>The gene therapy by siRNA targeted silencing of HIF-1alpha may down-regulate its downstream genes GLUT1 and HKII expression, therefore, to reduce the tumor glycolysis activity and promote tumor cell apoptosis, and exert a tumor-suppressing effect in vivo.</p>
Subject(s)
Animals , Female , Humans , Mice , Apoptosis , Cell Line, Tumor , Gene Silencing , Genetic Therapy , Glucose Transporter Type 1 , Genetics , Metabolism , Hexokinase , Genetics , Metabolism , Hypoxia-Inducible Factor 1, alpha Subunit , Genetics , Metabolism , Mice, Nude , Neoplasm Transplantation , Plasmids , RNA, Messenger , Metabolism , RNA, Small Interfering , Genetics , Random Allocation , Transfection , Tumor Burden , Uterine Cervical Neoplasms , Metabolism , Pathology , TherapeuticsABSTRACT
<p><b>OBJECTIVE</b>To study the growth-inhibitory and apoptosis-inducing effects of realgar nanometer suspension in human carcinoma cervical cell Siha line, and the effect on HPV16E6/E7 oncogene expression.</p><p><b>METHOD</b>A " micro-jet efflux" strategy was used for the preparation of realgar nanometer suspension. Siha cells were treated with various concentrations (6.25, 12.5, 25, 50 mg x L(-1)) of realgar nanometer suspension for different hours (12, 24, 48, 72 h). The effect of realgar nanometer suspension on Siha cell growth suppression was detected by MTT method. Special morphological changes of apoptosis were observed by light and transmission electron microscopy (TEM) and DNA fragments electrophoresis. The apoptotic rates were quantified by flow cytometry (FCM). The expression of HPV16E6/E7 mRNA was assayed by RT-PCR.</p><p><b>RESULT</b>After being treated with 25-50 mg x L(-1) realgar nanometer suspension for 48, 72 h, the survival of Siha cells decreased, and the rate of apoptosis markedly increased. With TEM and DNA electrophoresis, the special morphological changes were found. The apoptotic rates of Siha cells treated with realgar nanometer suspension were significantly higher than those in the control group (P < 0.01). G0-G1 phase arrest appeared following the treatment with realgar nanometer suspension in 25 and 50 mg x L(-1) 48 h. RT-PCR assay revealed that realgar nanometer suspension reduced HPV16E6/E7 gene expression.</p><p><b>CONCLUSION</b>Realgar nanometer suspension can inhibit the proliferation of human carcinoma cervical cell Siha line and induce the cell apoptosis. The mechanism may be related to the down-regulation of HPV16E6/E7 oncogene expression.</p>