| Background and purposePapillary thyroid carcinoma,the most common pathological type of thyroid tumors,often metastasis to lymph node at early stage,which leads to high risk of postoperative recurrence,poor prognosis,and low survival rate.Previous studies show that the recurrence rate of lymph node metastasis in high-risk patients is 35%,and the recurrence rate of postoperative lymph node metastasis in low-risk patients can reach more than 13%.At present,the research of the mechanism of lymph node metastasis of PTC is not clear.Therefore,we need a reliable biological marker to predict the lymph node metastasis of PTC,and provide biological guidance for the treatment of it.Neuropilin 2,a member of the Neuropilins family,is a single transmembrane glycoprotein.NRP2 initiate the signal transduction of VEGF by binding with the VEGF receptor on endothelial cell.Regulates various physiological processes including neuronal development,lymphangiogenesis,and angiogenesis.Previous studies have confirmed the high expression of NRP2 in a variety of tumors,such as breast cancer,prostate cancer,colon cancer,gastric cancer,osteosarcoma,etc.The results of in vitro experiments show that NRP2 can promote lymphangiogenesis and angiogenesis in most tumors,down-expression of NRP2 can inhibit the proliferation of cancer cells,the migration and invasiveness.Therefore,NRP2 plays an important role in the development of malignant tumors.At present,there is no correlation study between papillary thyroid carcinoma and NRP2 in the world.Tumor lymph node metastasis is mainly related to the loose structure of the lymphatic endothelium and the poor continuity of the basement membrane.And lymph node metastasis is the main method of metastasis of papillary thyroid carcinoma.Recent studies have shown that certain factors in vascular endothelial cells such as VEGF-C can lead to the expansion of lymphatic vessels and increasethe number of tumor metastasis to lymph node.Because of the lack of expression in vascular endothelial cell,D2-40,a specifically marker of lymphatic endothelial cell,can be used as an indicator of microlymphatic density to investigate the mechanism of PTC lymphay node metastasis.Therefore,this experiment used immunohistochemistry to observe the expression of NRP2 in papillary thyroid carcinoma.labeling microlymphatic vessel density by D2-40.Analyzing the correlation between the expression of NRP2 and the lymph node metastasis of papillary thyroid carcinoma.Lymph node metastasis and the association between clinical parameters,including age,gender,and presence or absence of lymph node metastasis.We constructed an RNAi sequence that effectively inhibits the expression of NRP2,and detected the biological influence including proliferation,migration and invasion of B-CPAP cells after silencing the expression of NRP2,furthermore we will detect the mechanism of the lymph node metastasis of the papillary thyroid carcinoma,aiming to find a way which can improve the cure rate and the prognosis of the PTC.Methods1.Screened 60 cases of papillary thyroid carcinoma tissue and 30 cases of thyroid adenoma tissue between 2011 and 2016 from the department of pathology of the First Affiliated Hospital of Zhengzhou University..Immunohistochemistry was used to detect the protein expression of NRP2 in paraffin specimens in thyroid papillary carcinoma and normal thyroid tissue adjacent to thyroid adenoma.Labeling microlymphatic vessel density with monoclonal antibody D2-40.2.Designing and synthesizing siRNA sequences,screening one of the sequences that can most effectively inhibit the expression of NRP2,and transfecting the thyroid papillary carcinoma cells by lentiviral particles which packaged by Jikai Gene Company,and we divided the experimental group into LV-NRP2-RNAi and NRP2-siRNA-control.3.Detection of NRP2 protein expression after viral transfection by westernblotting.4.CCK-8 assay was used to detect the proliferation of PTC cells before and after transfection.5.Using wound-healing assay to observe the cells' migration ability in groups during different time periods.6.Transwell assay was used to detect the changes of cell invasive ability of the negative control group and the experimental group after PTC cell transfection.7.Statistical analysis,measurement data were expressed in the form of mean ± standard deviation(Mean ± SD),using chi-square test to analyze the correlation between the data.The test level is ?=0.05.Results1.The results of immunohistochemistry showed that NRP2 was positive and strongly positive in thyroid papillary carcinoma,and negative in normal thyroid tissue,the difference was statistically significant(P <0.05).Statistical analysis showed NRP2 expression and Lymph node metastasis of PTC was associated,and had no significant(P <0.05),correlation with age and gender(P > 0.05).The mean lymphatic vessel density of D2-40 in PTC with lymph node metastasis was 13.61±6.05,which was significantly different from MLD 8.26±4.32 in the papillary thyroid carcinoma without lymph node metastasis(P<0.05).The correlation coefficient between NRP2 and D2-40 was R=0.41(P<0.05).2.Western Blot results showed that papillary thyroid carcinoma cells were transfected 72 hours after virus transfection.The expression of LV-NRP2-RNAi protein in the transfection group was 0.43±0.08,0.90±0.08 in the negative control group and 0.98±0.15 in the untreated group.There was no significant difference between the untreated group and the negative control group,the difference was not statistically significant(P>0.05).Compared with the negative control group,the difference between the transfection group and the negative control group was statistically significant(P<0.05).The LV-NRP2-RNAi had lower than the NRP2-siRNA-control,and the inhibition rate reached 47.7%.3.The results of CCK-8 cell assay showed that the down-regulation of NRP2 in transfected B-CPAP cells significantly inhibited the proliferation of LV-NRP2-RNAi in the transfected group at 48 h,72h,96 h.Compared with the negative control group,the difference was statistically significant(P<0.05),while the negative control group had little difference with the untreated group,and there was no statistical significance,(P>0.05).4.The results of cell scratch test showed that the cell scratch healing rate of LV-NRP2-RNAi in the experimental group was lower than that in the negative control group at the same time of 1d,2d and 3d,and the difference was statistically significant(P<0.05).This indicates that the cell migration rate of thyroid papillary carcinoma B-CPAP is effectively inhibited after viral transfection of silencing NRP2 expression.5.The results of Transwell chamber showed that the number of transmembrane cells in the negative control group after transfection was 348.2±8.4,and the number of transmembrane cells in the LV-NRP2-RNA group of the experimental group was 174.3±12.3,(P<0.05).This indicates that after the expression of NRP2 was downregulated,and the cells' invasion ability of thyroid papillary carcinoma cell line B-CPAP was significantly decreased.Conclusion1.The expression of NPR2 is highly up-regulated in PTC,and the expression of NRP2 is closely related to lymphnode metastasis.So we think it could be used as an marker of the PTC's lymph node metastasis.2.Lentivirus-mediated specific NRP2 siRNA sequences can effectively inhibit the expression of NRP2 protein levels after transfection.3.Down-regulation of NRP2 can inhibit the proliferation of B-CPAP cells,reducing their cell migration and reduce invasion ability. |
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