| Gastric cancer is one of the most common malignant tumor,the mortality in malignant tumors is in a second place in the world. China is a country of high incidence of gastric cancer, all cancer deaths among the first. Clinical manifestations of gastric cancer come on quickly, and prognosis of which is extremely poor. Early discovery,early diagnosis and early treatment is the most important method to decrease the mortality in malignant tumor. Unfortunately, most patients when doctor is advanced, the proportion of early gastric cancer less than 10%. The recent research show that the 2~4 percent of intramucosal carcinoma, the 10~15 percent of submucosalcarcinoma, the 80 percent of advanced gastric carcinoma have distant lymph node metastasis, the prognosis of gastric cancer and cancer invasion and whether there was lymph node metastasis. The recent research show that the form of the lymphatic related to carcinoma may help to the spread of the tumor and metastasis, so there are many researches focus on how to curb the form of the lymphatic related to carcinoma. VEGFR3 was firstly proved as a specific marker about lymphangiogenesis. VEGFR3 is specific receptors to lymphatic endodermis growth factor-VEGFC. VEGFC combination with its receptor VEGFR3 can cause lymphatic endothelial cell proliferation, migration and suppress apoptosis of endothelial cells, which play a role in the promotion of lymphangiogenesis.Neuropilins is a transmembrane protein, first found as a receptor of axon guidance molecule Semaphorin, plays an important regulatory role during the development of the nervous system. Further study demonstrate that NRPs also play a key role in tumor growth and metastasis. NRPs family include two members of NRP1 and NRP2. Maresa Caunt recently think that NRP2 involved the function of VEGFC induced lymphatic endothelial cell migration and lymphangiogenesis (in particular the new features of tumor-associated lymphatic vessels) . NRP2 blocking in tumor models also reduce tumor metastasis to sentinel lymph nodes and distant organs. Lymph node metastasis is an important biological characteristics of gastric cancer , occurrence of gastric cancer by local and distant lymph node metastasis in patients is one of the important factors leading to poor prognosis.Whether NRP2 involved in the development of gastric cancer invasion and metastasis and the mechanism are required to validate the idea of this issue.Objective: To detect the expression of NRP2 in different gastric cancer cell line. By the in vitro tumor cell experiment and the in vivo experiment using nude mice to know whether RNA interference expressing vector which we construct can interfere with the gene of NRP2 in gastric cancer cell and influence the biological characteristics of gastric cancer cell as well as the generation of lymphatic in nude mice transplanted tumor.Methods1.Using RT-PCR, Western blot method to detect the expression of NRP2 in gastric cancer cell line BGC823,SGC7901 on mRNA and protein levels.2.Using computer-aided design software, design and synthesis a specific oligonucleotide sequence for NRP2 gene, And cloned into the eukaryotic expression vector,get the recombinant plasmid shRNA-NRP2-1, shRNA-NRP2-2,shRNA-NRP2-3 and negative control plasmid shRNA-HK.3.Transfected into gastric cancer cell line SGC7901 with liposome, assay the NRP2mRNA and protein expression before-and-after transfection with semi-quantitative RT-PCR, western blot.choosing the best interference plasmid.4.Check out SGC7901 cell proliferation, cell cycle, apoptosis, invasion, migration before-and-after shRNA2-2 transfected by MTT, flow cytometry, invasion detected a small room experiments. Using semi-quantitative RT-PCR to detect the expression of CXCR4 of transfected SGC7901 cells before and after .5.Inoculat SGC7901 gastric cancer cells subcutaneously into nudnude rat, divided into the interference group, negative control group and blank control group, injected shRNA-NRP2-2, shRNAHK, PBS subcutaneously after tumor formation in nude mice. Determine differences in tumor volume and body weight of nude mice at different time points. Continuous observation for 6 weeks, tumor biopsy immunohistochemistry detect NRP2 protein changes and counting the lymphatic vessels density.Results1. Both cell lines express NRP2. The relative content of NRP2 protein in BGC823 cell line is 0.63±0.02, while which is 0.85±0.03 in SGC7901 cell line. it show significantly different between BGC823 cell line and SGC7901 cell line(P <0.05). The relative content of NRP2mRNA in BGC823 cell line is 0.56±0.01, and which is 0.86±0.03 in SGC7901 cell line.it show significantly different between BGC823 cell line and SGC7901 cell line(P <0.05). SGC7901 cells expressed higher levels of NRP2.2. SiRNA-NRP2 expression vector was successfully constructed which was proved by enzyme digestion analysis and DNA sequencing.3.Recombinant plasmid was transfected to SGC7901 cell line, and we found the absorbance ratio of NRP2/β-actin by western blot: plasmid NRP2-1 was 0.68±0.01, plasmid NRP2-2 was 0.34±0.03 and plasmid NRP 2-3 was 0.58±0.01, while HK group was 0.89±0.02 and blank group is 0.91±0.03. The inhibition ratio of three groups plasmid to the protein expression of NRP2 were 31.5%,63.2% and 49.6%. The absorbance ratio of NRP2/β-actins by RT-PCR is that: plasmid NRP2-1 was 0.63±0.02, plasmid NRP2-2 was 0.51±0.02 and NRP 2-3 was 0.61±0.01; and HK group was 0.95±0.01 and blank group was 0.97±0.01. The inhibition ratio of three groups plasmid to the mRNA of NRP2 were 36.3%, 57.4% and 37.2%. It was the best for the plasmid NRP 2-2 to inhibit the expression of NRP2, and it was significantly difference compared with plasmid NRP 2-1 and plasmid NRP 2-3 (P <0.05).3. The detection of growth cycle and apoptosis by flow cytometry (FCM) show that compared with the HK negative control group and blank control group the percent of cell in Go/G1 phase of shRNA NRP2 group increase obviously, and cell in S phase decrease while the rate of cell apoptosis increase. The detection of invasion and migration by Transwell show that the capacity of shRNA NRP2 group on invasion and migration is extremely lower than the HK negative control group and blank control group; The expression of CXCR4mRNA in shRNA NRP2 group is extremely lower than the HK negative control group and blank control group.4. In vivo experiment the expression of NRP2 protein in shRNA NRP2 group obviously reduces by the detection of Western bolt and Immunohistochemistry compared with the HK negative control group and the blank control group. The volume, weight and Microlymphatic vessel density of transplanted tumors in nude mice is inhibited obviously in accordance with the in vitro tumor cell experiment.Conclusions 1.NRP2-specific RNA interference inhibited SGC7901 cell proliferation, invasion, migration and induce apoptosis.2.Nude mice xenograft model of gastric cancer ,after treatment of shRNA2-2, can similarly silence the expression of NRP2.,while tumor volume, weight and MLD was also significantly inhibited.3. NRP2 could regulate the expression of CXCR4 to influence lymph node metastasis in gastric cancer. |
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