| Objective:The purpose of this study was to analyze the content of mi R-1246 in the serum of patients with cervical squamous cell carcinoma,cervical squamous intraepithelial lesion(SIL)and healthy women who underwent C2 hysterectomy,and to analyze mi R-1246 as serum Tumor markers,the diagnostic value of cervical squamous cell carcinoma,and the clinical significance in patients’postoperative recovery and relapse monitoring.To explore the effect of mi R-1246 on the growth of human cervical squamous cell carcinoma xenografts in nude mice,the correlation between mi R-1246 and vascular endothelial growth factor VEGF,and the effect on tumor microangiogenesis.Methods:(1)Real-time fluorescence quantitative polymerase chain reaction was used to detect the expression level of mi R-1246 in serum of 80 patients with cervical squamous cell carcinoma,30 patients with cervical squamous intraepithelial lesion,and 30 healthy women.(2)Make ROC curve and evaluate serum mi R The diagnostic value of-1246 for cervical squamous cell carcinoma.(3)Analyze the correlation between the expression of mi R-1246 and the clinicopathological features of the tumor.(4)Follow up 60 cases of surgically treated cervical squamous cell carcinoma patients with postoperative serum mi R-1246 content and 8 relapsed patients with serum mi R-1246 content,evaluate serum mi R-1246 as a tumor marker,and restore postoperative recovery of cervical squamous cell carcinoma patients.The significance of postoperative relapse monitoring.(5)Lentiviral vectors were used to transfect human cervical squamous cell carcinoma Si Ha cells.The four groups of lentiviral vectors were mi R-1246 lentiviral overexpression group(mi R-1246-LV),overexpression control group(LV-NC),The mi R-1246 lentivirus silent group(mi R-1246-IN)and the silent control group(IN-NC).RT-PCR was used to verify the transfection results.(6)Inject the successfully transfected cells under the skin of the axilla of the mice,observe the tumor formation under the skin,record the tumor volume every 5 days and draw the growth curve.After 30 days,the nude mice were anesthetized,and the nude mice were sacrificed by cervical dislocation and stripped.Subcutaneous tumor and measure tumor volume.(7)Immunohistochemical technique was used to compare the expression levels of vascular endothelial growth factor VEGF in tumor tissues of each group,and the expression of microvessel count index CD34 was observed to evaluate the microvessel density.(8)Western blot was used to detect the expression of vascular endothelial growth factor VEGF protein in the tumor volume of each group.Results:(1)RT-q PCR results showed that the content of mi R-1246 in serum of cervical squamous cell carcinoma patients group(0.322±0.383)was significantly higher than that of cervical squamous intraepithelial lesion patients group(0.096±0.130)or healthy physical examination women group(0.038±0.028),the difference is statistically significant(p=0.000442,p=0.000013).There was no statistically significant difference between the cervical squamous intraepithelial lesion group(0.096±0.130)and the healthy women group(0.038±0.028)(p=0.421).(2)The area under the ROC curve is0.791(95%CI:0.709-0.872).When the cut-off value is 0.517,the sensitivity is67%and the specificity is 85%.ROC analysis shows that serum mi R-1246 is used in vitro Diagnosis of tumors has good specificity and sensitivity.(3)Analysis of clinical data shows that the high expression level of mi R-1246 is associated with FIGO stage(p=0.017),tumor size(p=0.001),lymph node metastasis(p=0.021),and vascular infiltration in cervical squamous cell carcinoma patients(p=0.028)is closely related to the patient’s age(p=0.601),depth of tumor infiltration(p=0.098),nerve invasion(p=0.085),positive margin(p=0.554),parauterine infiltration(p=0.301)Irrelevant.(4)In patients with cervical squamous cell carcinoma who underwent extensive C2 hysterectomy+pelvic lymph node dissection,the expression level of mi R-1246 in serum changed from 1 week after operation(0.601±0.462)to 1 month after operation(0.053±0.032)Gradually decreased,there was no statistically significant difference between the first month after operation and healthy women(p>0.05),indicating that the patient returned to the level of healthy women after one month after surgery;some patients with cervical squamous cell carcinoma relapsed after surgery(1.122±0.355),The expression level of mi R-1246(1.122±0.355)in serum of patients with recurrent cervical squamous cell carcinoma was significantly higher than that of healthy women(0.075±0.027),and the difference was statistically significant(p=0.000067).(5)Observe the transfection under a microscope,and compare the field of view with normal light and fluorescence to determine that the transfection rate is above 80%.Real-time fluorescence quantitative PCR method was used to detect the content of mi R-1246 in transfected cells.The relative expression of mi R-1246 in mi R-1246 lentiviral overexpression group(mi R-1246-LV)was 13.83±0.96,and the overexpression control group(LV-NC)The relative expression level of mi R-1246 was 1.13±0.03,the difference was statistically significant(p=0.00002);the relative expression level of mi R-1246 in the mi R-1246lentivirus silencing group(mi R-1246-IN)was 0.78±0.21,the relative expression level of mi R-1246 in the silent control group(IN-NC)was 1.19±0.01,the difference was statistically significant(p=0.029).Prove that the transfection was successful.(6)After the nude mice were sacrificed at 30 days,the tumor volume of the nude mice was measured.The tumor volume of the mi R-1246-LV group was 1537.26±166.79 mm~3,and the tumor volume of the LV-NC group was860.27±118.29 mm~3.The difference was statistically significant(p=0.001);the tumor volume of the mi R-1246-IN group was 396.25±263.52 mm~3,and the tumor volume of the IN-NC group was 854.51±119.34 mm~3.The difference was statistically significant(p=0.014).(7)Immunohistochemical method was used to detect the relevant indexes in the transplanted tumor tissue of nude mice.The expression of VEGF in mi R-1246-LV group was 84.67±14.01%,and the expression of VEGF in LV-NC group was 41.33±3.51%.The difference between the two was statistically significant(p=0.007);the expression of VEGF in the mi R-1246-IN group was 22.67±4.51%,and the expression of VEGF in the IN-NC group was 48.33±3.06%.The difference between the two was statistically significant Academic significance(p=0.001).The MVD of the mi R-1246-LV group was 69.67±8.02,and the MVD of the LV-NC group was15.33±3.05.The difference between the two was statistically significant(p=0.0004).The MVD of the mi R-1246-IN group was 4.33±2.52,and the MVD of the IN-NC group was 18.67±6.02.The difference between the two was statistically significant(p=0.02).(8)Western Blot method was used to detect the expression of VEGF protein in transplanted tumors of nude mice.The gray analysis value of VEGF in mi R-1246-LV group was 0.86±0.07,and the gray analysis value of LV-NC group was 0.62±0.76,(p=0.016,p<0.05),the difference is statistically significant;the grayscale analysis value of VEGF in the mi R-1246-IN group is 0.69±0.065,and the grayscale analysis value of the IN-NC group is 1.13±0.70,(p=0.001,p<0.05),The difference was statistically significant.Conclusion:(1)The tumor volume of the mi R-1246-LV group is always greater than that of the LV-NC,while the tumor volume of the mi R-1246-IN group is always smaller than that of the IN-NC group.Overexpression of mi R-1246 can promote the growth of transplanted tumors in nude mice,and silence of mi R-1246 can inhibit the growth of transplanted tumors in nude mice.(2)Overexpression of mi R-1246 can promote the expression of VEGF,silencing mi R-1246 can inhibit the expression of VEGF,and mi R-1246 can positively regulate the expression of VEGF.(3)Overexpression of mi R-1246 can increase the density of microvessels and promote microangiogenesis.Silencing mi R-1246 can reduce the density of microvessels and inhibit the formation of microvessels.mi R-1246 can promote tumor angiogenesis.(4)The tumor volume of the mi R-1246-LV group is always greater than that of the LV-NC,while the tumor volume of the mi R-1246-IN group is always smaller than that of the IN-NC group.Overexpression of mi R-1246 can promote the growth of transplanted tumors in nude mice,and silence of mi R-1246 can inhibit the growth of transplanted tumors in nude mice.(5)Overexpression of mi R-1246can promote the expression of VEGF,silencing mi R-1246 can inhibit the expression of VEGF,and mi R-1246 can positively regulate the expression of VEGF.(6)Overexpression of mi R-1246 can increase the density of microvessels and promote microangiogenesis.Silencing mi R-1246 can reduce the density of microvessels and inhibit the formation of microvessels.mi R-1246 can promote tumor angiogenesis. |
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