| Cervical cancer is one of the most common gynecologic malignant tumors. Recently, the incidence rate of the disease has risen, with the trend happening in younger women. The prognosis of advanced or recurrent cervical cancer is poor. The clinical treatments are not satisfactory with severe side effects by the reasons of metastasis and recurrence, although the treatments, such as operation, chemotherapy or radiotherapy and other comprehensive treatments, are able to achieve a certain degree of clinical efficacy. Therefore, it is the hope of finding a new project for metastasis and recurrence of cervical cancer.Angiogenesis is an essential process in the growth and progression of cancer.Nitric oxide (NO) is one of the factors regulating vascularization. Inducible nitric oxide synthase (iNOS), as the key enzyme during NO synthesis, highly expressing in many tumor tissues, promotes blood vessel’s production and affects the growth of tumour cells. Researches have shown that iNOS is closely related to the invasion and metastasis of cancer.Similarly, the high expression of iNOS in cervical cancer is positively correlated with tumor staging and lymphatic metastasisVascular endothelial growth factor (VEGF) has been considered as the most important and strongest factor among so many cytokines which regulate vascularization so long. iNOS,mediated by NO level,can induce VEGF in the process of endothelial migration and vessel remodeling.RNA interference (RNAi) is a kind of gene manipulation technology which can suppress the expression of target gene, lentivirus is an ideal vector,carrying siRNA or shRNA,which has been widely used in RNAi.Thus, shRNA targeting against iNOS gene mediated by lentivirus could suppress the expression of VEGF and inhibit tumor angiogenesis, which may provide a new strategy for the treatment of cervical cancer in late stage.This study concludes three parts. Part1:Construction of the iNOS-shRNA lentivirus vector followed by transfection of cervical cancer cells in vitro. Part2and3:the effects of iNOS knockdown on VEGF expression. both in vitro and in vivo. Part Ⅰ Construction of iNOS shRNA lentivirus vector and transfection of cervical cancer cellsObjective Construct iNOS shRNA lentivirus vector and transfect cervical cancer cells.Methods Three pairs of iNOS-specific shRNA duplexes were selected and constructed into the frame of plasmid. After identification by PCR and gene sequencing, three duplexes were transfected into293T cell line. The efficiency of iNOS knockdown by different shRNA duplexes was evaluated by the expression of iNOS and the best one was chosen to transfect into cervical cancer cells SiHa and HeLa. The suppression effect was estimated by quantative RT-PCR and western.Results The successful construction were showed by PCR and sequencing, respectively, followed by lentiviral packaging and transfection of SiHa and HeLa cells, the expression of iNOS was down-regulated both on mRNA and protein level.Conclusions The expression of iNOS in SiHa and HeLa cells can be suppressed both on mRNA and protein level by iNOS-shRNA transfecion. Part II the influence on the growth and apoptosis of cervical cancer cells and VEGF expression in vitro after iNOS-shRNA transfecion.Objective to observe the influence on the growth and apoptosis of cervical cancer cells and VEGF expression in vitro after iNOS-shRNA transfection.Then,exogenous NO donor, sodium nitroprusside (SNP) with different concentration,applied to the cells to analyze the relation between the expression of iNOS, NO and VEGF.Methods SiHa and HeLa cells were divided into three groups respectively.Group1: transfected with iNOS-shRNA lentivirus vector.Group2transfected with scramble sequence lentivirus vector.Group3blank control Group. The proliferation ability and apoptosis occurrence were observed by MTT and Annexin V-FITC/PI test. Furthermore, the titer of supernatant VEGF and NO were determined by ELASA and Griess test, respectively. Finally, The expressions of iNOS, NO and VEGF were identified by RT-PCR and western blot.Results The growth ability of cervical cells after iNOS knockdown significantly declined and apoptosis ratio upgraded compared with negative control groups (P<0.05). In addition, the expressions of iNOS, VEGF, supernatant NO and VEGF markedly decreased (p<0.05).After supplementation of SNP with different concentration in all of the groups, VEGFmRNA and supernatant VEGF increased and both reached peak points when the concentration of NO was0.5mmol/L. However, when the concentration of NO was upgraded, VEGF level declined. Furthermore, there was not significant change to the expression of iNOS.Conclusions The growth of cervival cancer cells was inhibited after iNOS knockdown in concomitant with down regulation of NO and VEGF secretion in the supernatants. iNOS is relative to VEGF.The effect of iNOS on VEGF is mediated by NO. iNOS can regulate VEGF with lower concentration of NO. Part III The influence on the growth of cervical cancer cells in vivo after iNOS-shRNA transfectionObjective to investigate the influence on the growth of cervical cancer cells in vivo by xenograft formation after iNOS shRNA transfection and the expression of iNOS and VEGF in tumor tissues.Methods Vector control and iNOS-shRNA cells were injected subcutaneously into the flanks of nude mice (BALB/c-nu/nu) at the age of4weeks. The engrafted mice were inspected every day for tumor generation by visual observation and palpation. After25days, animals were sacrificed by cervical dislocation in compliance with regulations for use of vertebrate animal in research. the tumor sizes were measured by maximum length and width and prepared for tissues section. The expressions of iNOS and VEGF in the tumors were detected by immunohistochemical staining.Results The average size of xenograft tumor in iNOS knockdown groups was significantly smaller than that of control groups (P<0.05). Furthermore, both the expression of iNOS and VEGF were lower compared with control groups (P<0.05).Conclusions iNOS shRNA transfection can inhibit the growth of cervical cancer cells in vivo as well as decrease the expression of iNOS and VEGF. |
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