| Background and objective:Gastric cancer is one of the most common leading cause of cancer mortality inChina and even in Asia.Tumor malignancy is characterized by local invasion andmetastases,which are the main factors leafter radical surgery.ading to treatment failureand impact of long-term survival of cancer patients. Angiogenesis is important in theprocess of tumor invasion and metastasis.Blocking or reducing tumor vascular bloodsupply and inhibiting of tumor angiogenesis are important ways of controlling tumorgrowth and metastasis. In recent years, growing evidence indicates that microRNAplays an important role in the occurrence, development and prognosis of tumors. Astrong link between microRNA and tumor angiogenesis, metastasis or tumor stagehas also been found during past few years. Some research shows that microRNA-126(miR-126) is significantly expressed lower in gastric cancer tissue, which was relatedto patients’ prognosis.We predicted that miR-126may binding Vascular endothelialgrowth factor A(VEGFA)through complementary sequence by using bioinformaticsoftware,the target binding of miR-126to VEGFA was subsequently confirmed bydual-luciferase reporter assay. As we all know, VEAF family plays an important rolein tumor growth and metastasis by regulating vascular newborn,particularly,VEGFAplay an important role in tumor angiogenesis.The current study was aimedintent toestablish a stable miR-126overexpression or suppression expression in SGC-7901cell line and animal experiments by using tumor-bearing mice. Further clarified therelationship between the expression of miR-126and gastric cancer angiogenesis byanalyzing the association of miR-126with VEGFA.Methods:1.Stability-enhanced miRNA precursor mimicking miR-126and controlnonspecific miRNA precursor,Stability-slience miRNA precursor mimicking miR-126and control nonspecific miRNA precursor,were purchased from ShanghaiGENECHEM.The SGC-7901cells were divided into five experimental groups: miR-126group (over-expression of miR-126), Control-1group (over-expression ofmiR-126in the control group), Inhibit group (inhibit the expression of miR-126),Control-2group (inhibit the expression of miR-126in the control group), SGC-7901group (mock group). Gastric cancer cell lines SGC-7901were infected by recomb-inant lentivirus miR-126and miR-126inhibitor,with puromycin selection. Realtime-PCR was used to confirm the relative expression of miR-126. Western blot was usedto detected the expression level of VEGFA after the above pretreatment. Cellproliferation was assessed by MTT assay.2.The stable expression of miR-126in SGC-7901inoculated into nude mice,tumor formation was observed and measured tumor weight inhibition rate wascalculated.Immunohistochemical detection was used to determine the expression ofVEGFAand CD34in mice tumor xenograftResults：1.SGC-7901cells infected by recombinant lentivirus miR-126can over-expressmiR-126,and infected by recombinant lentivirus miR-126inhibitor can down-regulatethe expression of miR-126(P<0.05).Furthermore, the expression of inhibit group wassignificantly lower than the SGC-7901group (p=0.018).2.The effects of miR-126on the expression of VEGFA: According to WesternBlot Protein gray comparison, it is high levels expression of inhibit group comparedoverexpression group and blank group, and a2.9-fold than over-expression group (p<0.01).3.MTT assay cell proliferation: Overexpression of miR-126GC inhibited cellproliferation,24hours significantly inhibited.4.Ectopic expression of miR-126inhibits tumorigenicity in vivo:The averagetumourweight of mice inoculated with over-expression-miR-126-transfected SGC-7901cells at day42was0.57±0.21g, which was significantly lower (P <0.05) thanthat of mice inoculated with inhibit-transfected SGC-7901cells (2.79±0.31g) andmock group(2.01±0.34g).5.Immunohistochemical detection of VEGFA and CD34expression in eachexperimental group: VEGFA mainly expressed in the cytoplasm, stained yellow to peg yellow. The amount of VEGFA-antigen-positive was lower in the tumour derivedfrom over-expression-miR-126-transfected SGC-7901cells than that in the mockgroup and inhibit-transfected group.The Microvessel density (MVD) in miR-126overexpression group, inhibit group and SGC-7901group is:20.25±3.39,52.00±4.47,30.35±3.34. The amount of MVD was lower inthe tumour derived fromover-expression-miR-126-transfected SGC-7901cells than that in the mock groupand inhibit-transfected group.Conclusion：We presumed that miR-126regulates the expression of VEGFA andtumor angiogenesis in gastric cancer. |
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