| Background and objective: It is generally known that gastric cancer is popular in the digestive system.Its lower early diagnosis rate,higher malignancy and earlier metastasis are the possible causes of poor prognosis in gastric cancer.At present,the effect of traditional radiotherapy and chemotherapy to prolong the survival of gastric cancer is limited,and the commonly used CEA,CA-724 and CA-199 in clinical have no obvious significance in the diagnosis of early gastric cancer.Therefore,exploring new markers for early diagnosis and effective targets in signal pathway may be the methods to help patients prolong their survival.Micro RNAs(miRNAs)participate in many biological processes by negatively regulating the expression level of target genes which includes the occurrence,metastasis and angiogenesis of tumor cells and the types and expression levels of miRNAs in peripheral blood were significantly different with normal subjects.According to our previous study results,we found that there was a significant difference in serum miR-26a/b between gastric cancer patients and normal controls,but its specific regulatory mechanism is still under exploration.The purpose of this study is to to discuss the diagnostic value of miR-26a/b and relationship between micro RNA and clinicopathologic characteristics.Furthermore,to explore the possible pathway and potential mechanism of miR-26a/b affecting gastric cancer cell growth.Methods: 1.Clinical level experiment: the relationship between serum miR-26a/b expression and clinicopathologic characteristics in gastric cancer and its diagnostic value.(1)The peripheral blood samples of 121 patients with gastric adenocarcinoma and 116 healthy people were randomly selected in Tianjin Medical University Cancer Hospital from April to October 2016.All of 121 patients were primary patients without any treatment,including 58 males and 63 females.The median age was 63.5 10.0 years old.There were 66 males and 50 females in 116 healthy volunteers,with median age 35.8 7.1 years old and tumor disease excluded.Then the blood was extracted according to the instructions.QRT-PCR was used to detect the expression of miR-26a/b in serum of the two groups.(2)According to statistical methods,the relationship between serum miR-26a/b and clinicopathological characteristics of gastric cancer was analysed.(3)The diagnostic value of serum miR-26a/b as a biological marker of gastric cancer was evaluated by ROC curve.2.Tissue level experiment: to verify the expression of miR-26a/b in gastric cancer tissues and to explore downstream gene target.(1)16 pairs of gastric adenocarcinoma tissues and their matched adjacent normal tissues were collected from 121 patients and the expression of miR-26a/b was detected by Taqman probe method.(2)The downstream targeting of miR-26a/b was identified by bioinformatics software.(3)Western Blot and RT-qPCR were used to detect the expression of HGF in 16 pairs.of gastric adenocarcinoma tissues and their matched adjacent normal tissues.Make clear the negative regulation relation between miR-26a/b and HGF.3.In vitro: the effect of miR-26 a / b-HGF-VEGF signaling pathway on the biological behaviors of gastric cancer cell line.(1)HGF could mediate the expression of VEGF.Up-regulating or down-regulating the expression of miR-26a/b in MGC-803 gastric cancer cells verify the regulatory effect on HGF and VEGF.(2)The effects of miR-26a/b on proliferation,invasion and metastasis of gastric cancer cells were studied by means of cell function tests,including Edu proliferation test,transwell test and scratch test.(3)Lentivirus was used to transfect HGF and sh RNA down-regulated HGF to verify its effect on VEGF and its effect on the biological function of gastric cancer cells.4.In vivo: miR-26a/b-HGF-VEGF signaling pathway regulates tumor growth and angiogenesis in vivo.(1)MGC-803 overexpression HGF group,MGC-803 overexpressed miR-26 a,MGC-803 overexpression miR-26 b group and untreated MGC-803 cells were implanted into the axillary tumor of nude mice.(2)After 4 weeks,the mice were killed,the tumor was removed,the tumor size and weight were measured.Then the expression of HGF,VEGF,miR-26a/b and the angiogenesis of different groups were detected.Results: 1.Clinical level experiment: The level of miR-26a/b expression in serum of gastric cancer patients was significantly lower than that of normal controls by qRT-PCR.The expression of miR-26a/b was correlated with TNM stage,depth of gastric cancer invasion and lymph node metastasis.The later of TNM stage,the lower expression of miR-26a/b in serum.Thus miR-26a/b may act as tumor suppressor gene in gastric cancer.It can be used as a potential biological index to predict the clinical stage and prognosis of gastric cancer patients.Finally,the diagnostic value of serum miR-26a/b was evaluated by making ROC curve.The results showed that the AUC of miR-26a/b were 0.828 and 0.853,respectively.2.Tissue level experiments: Taqman probe assay showed that the content of miR-26a/b in gastric cancer tissues was lower than that in matched paracancerous tissues,consistent with the results in the serum.The "tumor suppressor gene" of miR-26a/b was further clarified.By using bioinformatics software,we identified the targeting effect of miR-26a/b on HGF and further detected the expression level of HGF in gastric cancer tissues.The results showed that HGF in cancer tissues was significantly higher than that in adjacent normal tissues,so there was a negative regulatory relationship between miR-26a/b and HGF.3.In vitro: After transfection of miR-26a/b analogues / inhibitory(mimics / inhibitors)in MGC-803 cells.The expression level of miR-26a/b was up-regulated / down-regulated.The results of Western Blot showed that the expression of HGF and VEGF protein decreased after up-regulation of miR-26a/b.Increased expression of HGF and VEGF protein after down-regulation of miR-26a/b.RT-qPCR results showed there was no significant change in m RNA level.Cell function tests confirmed that miR-26a/b inhibited the proliferation,invasion and metastasis of gastric cancer cells.In addition,lentivirus overexpression of HGF or transfection of sh RNA inhibited the expression of HGF in MGC-803 cells to affect cell proliferation and invasion,the expression of VEGF was consistent with that of HGF.4.In vivo: In miR-26a/b overexpression group,tumor growth was significantly inhibited,and the expression of HGF and VEGF protein decreased significantly,and miR-26a/b reduced the formation of tumor blood vessels in the body by inhibiting the expression of VEGF.Conclusion: Through clinical trials,we explored the relationship between miR-26a/b and clinicopathological features of gastric cancer patients and its potential diagnostic value.Then from tissue level,in vivo and in vitro animal experiments confirmed the possible signal pathway of miR-26a/b,that is targeting HGF to inhibit the growth,invasion,metastasis and angiogenesis of gastric cancer cells by weakening the expression of HGF-VEGF axis.. |
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