Clinical Significance Of SuPAR In The Detection Of Cervical Cancer And The Study Of The Gene Therapy

Cervical cancer is one of the most important diseases threatening women's health. Epidemiological investigation has revealed that the incidence of cervical cancer is getting higher yearly and the age of the patient affected tends to be younger. Since the screening system has not been well developed, the incidence of cervical cancer in China was still six times as much that in the developed countries, in which 80% of cases has developed to the invasive cancer when they were diagnosed. At present operation is the major and effective therapeutic measure for early cases, while 10~15% of patients eventually developed to recurrent or metastasis diseases. It has become an important strategy to screen the patients with high risk of recurrence and metastasis and set up individual therapeutic regime to improve the survival rate and life quality in gynecological oncology field. It is also very important to find an effective approach to prevent the cervical cancer from invasion and metastasis.It has been considered that the tumor development, growth, invasion and metastasis are a complicated process, and UPA/uPAR system plays an important role in all these tumor bioactivities. suPAR in blood may be a more convenient and reliable index to reflect the actions of uPA system. The current study intended to investigate the relationship between uPAR and the invasion, metastasis and vascular formation of cervical cancer, and explore the clinical significance of detecting suPAR in patients with earlier cervical cancer, as well as its possibility to be a biological biomarker in cancer micrometastasis. To further detect the function of suPAR in inhibiting the growth of cervical cancer, suPAR plasmid (pcDNA3.1-suPAR) was constructed and transfered into Hela cells, and the expression of suPRA and tumor growth in nude mice were examined. Our study will provide effective theoretical basis for the treatment of cervical cancer by using suPAR gene target strategy.PartⅠThe expression and clinical significance of uPAR and VEGF-C in cervical squamous cancerObjectives: To analyze the expression of uPAR in cervical carcinoma, the relationship between VEGF-C expression and cancer cell invasion, cell metastasis and angiogenesis, and to find a more sensitive biological indicator for cancer micrometastasis.Methods: The expressions of uPAR and VEGF-C in 40 cases of early cervical squamous cancer tissues (stage I and stage II) were examined by using immunohistochemistry method and the roles of uPAR and VEGF-C in the infiltration and metastasis of early cervical squamous cancer were detected by retrospectively analyzing the clinical data.Results: 1 Immunohistochemical results of uPAR and VEGF-C in cervical carcinomaThe positive staining rates of uPAR were 85% in both stage I and stage II cervical carcinoma tissues, and the expression rates of VEGF-C in stage I and stage II cervical carcinoma tissues were 80% and 90%, respectively. The expressions of uPAR and VEGF-C in cervical carcinoma of both stageⅠandⅡwere higher than those in CIN and normal tissues (P<0.05), while no statistical difference (P>0.05) was showed between stage I and stage II tissues. The expressions of uPAR and VEGF-C were Significant between G1 and G2, as well as G1 and G3, but no significant difference between G2 and G3 groups (P>0.05).2 uPAR, VEGF-C and prognosis of cervical carcinoma40 cases of cervical carcinoma were followed -up. In uPAR positive group, 23 out of 34 patients recurred (57.50% of recurrence rate), 11 cases had recurrent disease (27.50%), 3 cases developed distant metastasis (7.50%) and 9 cases died (22.50%). In VEGF-C positive group, 17 out of 34 patients recurred (42.50% of recurrence rate), 7 cases had recurrent disease (17.50%), 1 case developed distant metastasis (2.50%) and 9 cases died (22.50%).There was no significant difference between the recurrent/metastatic/dead rates of the two groups (P >0.05), while the relativity analysis was significant between two groups.Conclusions: 1 The expressions of uPAR and VEGF-C were tremendously increased in patients with cervical cancer, and were correlated with increased grade change, indicating that uPAR and VEGF-C play an important role in the processes of tumor growth and invasion in patients with cervical cancer.2 The group with higher expressions of uPAR and VEGF-C showed stronger invasiveness and poorer prognosis in patients with cervical carcinoma,3 The detection of uPAR and VEGF-C showed clinical significance for early stage of cervical carcinoma, which might help to evaluate the prognosis and guide treatment, reduce the distant metastasis and improve survival.Part II Clinical value of detecting plasma suPAR in patients with early cervical squamous cancerObjectives: To detect the expressions of suPAR in tissues of normal cervix, cervical intraepithelial neoplasia and cervical carcinoma in order to explore the related clinical significance.Methods: ELISA was used to analyze suPAR, SccAg values in 60 patients with cervical squamous carcinoma compared with normal cervical tissues from 38 healthy women as the control group. SuPAR and SccAg in normal cervix, cervical intraepithelial neoplasia, cervical cancer and advanced carcinoma were detected and compared. The clinical significance of suPAR and SccAg was discussed.Results: 1 The Plasma suPAR level in healthy womensuPAR level was 0.8967±0.0411 Log10(ng/ml) in 38 cases of healthy women, and the reference range was 0.8297~0.9581 Log10(ng/ml)2 The plasma level of suPAR in patients with cervical carcinoma and CINSuPAR levels in cervical carcinoma and CIN were statistically different from that in control group (P<0.01). Furthermore, the suPAR level increased with the grade, the higher the grade, the higher the suPAR level. 3 Detection of plasma suPAR in patients with stageⅡb～Ⅳcervical carcinoma before and after chemotherapy and radio therapy.The suPAR level in patients with invasive cervical carcinoma was significantly decreased after chemotherapy and radio therapy (P<0.05).4 Comparison of sensitivity, specificity of uPAR and SccAgThe sensitivity, specificity, positive predictive value and negative predictive value of SuPAR were 98.43%, 66.67%, 91.30% and 92.30%, respectively, while those for SccAg were 57.81%, 94.44%, 97.40% and 38.60% respectively. The area of ROC of suPAR and SccAg were 0.858 and 0.805, respectively. SuPAR was superior to SccAg in diagnosing accuracy.Conclusions: 1 Plasma suPAR and SccAg increased with the increased degree of cervical lesions, the depth of myometrial invasion and clinical stage. suPAR were obviously associated with loading dose of turmor.2 Since SuPAR had higher sensitivity, specificity and accuracy, it was an useful indicator for monitoring tumor progress and evaluating prognosis in patients with cervical cancer.PartⅢThe construction of subcutaneously implanted tumor with uPAR suPAR gene stablely expressed Hela cells and study on its mechanism of gene targeting therapyObjectives: To construct a plasmid carrying suPAR gene and transfect Hela cells with pcDNA-suPAR and to carry out an in-depth study of the effect of suPAR gene on Hela cells and to elucidate suPAR gene expression, cancer invasion and metastasis changes, providing effective theoretical basis for the treatment of cervical cancer.Methods: RT-PCR was used to obtain 849bp gene fragment from HepG2 hepatoma cell line. ELISA was used to detect the expression of suPAR gene and its relationship with cell infiltration and metastasis. Ukaryotic expression pc-suPAR vector was constructed and restriction enzyme digestion was confirmed. ELISA was used to detect the effective expression of suPAR in eukaryotic cell Hela. A stable transfection of suPAR was also carried out in Hela cells. Tumor was inoculated in nude mice to observe tumor growth curve. The relationship of gene transcriptional regulation and cell invasion and metastasis was obtained by analyzing the level of suPAR gene transcription.Results: 1 SuPAR gene was obtained successfullyRT-PCR was successfully used to clone a full-length gene for the 849bp fragment of suPAR from human hepatoma cell line HepG2.2 suPAR carrying eukaryotic expression vector was obtained successfully2.1 psuPAR enzyme digestionsuPAR gene RT-PCR product and plasmid with pcDNA3.1/V5 were digested by XbaI and EocRI enzyme and ligated, then transformed to TOPO10 bacteria. The above-mentioned dual-enzyme digestion was used to verify. Electrophoresis results revealed two bands with approximately 849bp and 5500bp, which correlated with expectation.2.2 psuPAR sequencingThe recombinant psuPAR vector was sequenced. Results also showed that the recombinant psuPAR vector carried suPAR nucleotide sequence and its encoding amino acid sequence is fully consistent with GeneBank human suPAR sequences (1-849bp) .3 The effective expression of suPAR in eukaryotic cells of HelaFor the detection of the expression of suPAR in eukaryotic cells, psuPAR was transfected into Hela cells. 72h later, the expression of suPAR was examined by the ELISA from the supernatant of transfected cells. Expression of suPAR from the supernatant of transfected cell was mucher higher than that from not transfected (p<0.05). This result suggests that psuPAR plasmid was well expressed in Hela cells.4 Hela cells with stable transfected suPAR gene was obtained successfullyInsert SuPAR gene into the eukaryotic expression vector pcDNA3.1-V5/His containing the neomycin gene (neo) resistance gene. Then the recombinant expression plasmid pcsuPAR was obtained. The expression plasmid was transfected into Hela cells by G418 (800μg/ml) complete medium pressure screening to obtain positive clones. A concentration of 400μg/ml of G418 was used to maintain screening. SuPAR stable gene expression in Hela cell clones was identified by ELISA methods. OD value of each clone from ElISA was converted to all cloning ng/ml value in accordance with the standard curve y = 0.197x-0.291.5 In vivo results for suPAR gene inhibition of tumor growth in subcutaneously transplanted cervical cancer nude miceHela cells stably expressing SuPAR gene and wild type Hela cells were transplanted subcutaneously into nude mice respectively. On the 10th day, a touchable tumor block was found in nude mice transplanted with wild-type Hela cells but not in Hela cells stably expressing SuPAR gene. Mice were sacrificed on the 30th day after subcutaneous transplanting. Tumor block was weighted, and results showed that the weight of Hela/ pcDNA3.1-suPAR group was significantly lower than that of Hela group. Hela/pcDNA3.1-suPAR Group showed condensed nuclear staining, nuclear dense staining, decreased nuclear mitotic and significantly reduced cancer infiltration by Paraffin HE.Conclusions:1 suPAR gene expression level was closely related to tumor invasion and metastasis ability. Higher level of suPAR was related to stronger invasion and metastasis as well as poorer prognosis;2 suPAR gene was obtained successfully;3 Eukaryotic expression vector pc-suPAR construction and restriction enzyme digestion were successfully obtained;4 Hela cell line stably transfected with suPAR gene was obtained;5 Successfully transferred to the pcDNA3.1-suPAR cervical cancer animal model of nude mice;6 pcDNA3.1-suPAR gene vaccination nude suPAR gene showed significant anti-tumor effect, indicating that suPAR gene transfer was an effective strategy for the treatment of cervical cancer.