| Gastric carcinoma(GC)is one of the most common malignant tumors of the digestive tract in the past 40 years.The formation and existence of neovascularization in gastric cancer tissues provide material basis for the growth of gastric cancer cells.Therefore,it is of great value for us to identify the mechanism of angiogenesis in gastric cancer.Signaling proteins(Semaphorins,Semas)are a large family of secreted and membrane-bound molecules initially involved in nervous system development and axonal guidance.Recently,it has been found that they can regulate cell adhesion,cell movement,angiogenesis,immune response and tumor progression,but the specific mechanism of tumor progression and angiogenesis in gastric cancer has not been fully elucidated.As we all know,angiogenesis is a complex biological process involving multiple genes,which is involved in the occurrence and development of tumors.Micro ribonucleic acids(mi RNAs)are important regulators of angiogenesis and play an important role in the regulation of angiogenesis in tumor tissues.In this study,we predicted that mi R-30 b had binding sites with Sema3 A by using Targetscan database,and detected that mi R-30 b could target the 3 'UTR region of Sema3 A in the two-element luciferase report experiment,indicating the mutual targeting of mi R-30 b and Sema3 A.Next,we found that after transfection of mi R-30 b analogue into MKN28 cells,the m RNA and protein expression of Sema3 A could be significantly down-regulated.The results suggested that mi R-30 b might directly degrade the m RNA expression level of Sema3 A in gastric cancer cells,thus achieving the goal of targeted regulation of Sema3 A.In this study,collect the negative control group(NC group),mi R-30 b analog group(mi R-30 b Mimics group),mi R-30 b inhibitors group(mi R-30 b Inhibitor group),mi R-30 b inhibitors + sema3 A small interfering RNA group(mi R-30 b Inhibitor + si RNA group)and group sema3 A gastric cancer cell culture medium,applied to Human umbilical vein endothelial cells(Human umbilical vein endothelial cells.HUVECs).We found that angiogenesis was inhibited in mi R-30 b Inhibitor group and sema3 A group.Sema3 A expression was decreased in the mi R-30 b mimic group and the mi R-30 b inhibitor +sema3A si RNA group,but angiogenesis was promoted in the two groups.The expression level of pVEGFR2 was increased in the mi R-30 b mimic histone,but decreased in the mi R-30 b inhibitor group.The mi R-30 b mimics promoted angiogenesis and pVEGFR2 was blocked by the VEGFR2 inhibitor SKLB1002.These results suggest that mi R-30 b may regulate angiogenesis in the microenvironment of gastric cancer cell formation by targeting the regulation of sema3 A.This study has important theoretical and practical significance for the development of molecular targeted drugs for the treatment of gastric cancer in the future.Objective:In gastric cancer cell lines represented by MKN28,through exogenous increase in the down-regulation range of mi R-30 b,we detected whether mi R-30 b inhibited the expression of sema3 A by direct degradation or translation inhibition and their effect on angiogenesis,and further explored the biological molecular mechanism of its regulation of tumor angiogenesis.Methods:1.The Target Scan database and The Dual-Glo Luciferase Reporter Assay were used to predict The target genes in principle of base complementary pairing to verify The mutual target of mi R-30 b and sema3 A.2.The use of plasmid transfection technique and the mi R-30 b to simulate different concentrations and inhibitor transfection to cultivate good MKN28 gastric cancer cell line,the use of real-time quantitative reverse transcription polymerase chain reaction(RT-q PCR)technology to detect cells after transfection mi R-30 b m RNA expression of the situation,and find out mi R-30 b transfection optimal concentration,adopting the optimal concentration for follow-up experiments,and the use of plasmid transfection technology interference MKN28 sema3 A expression in gastric cancer cell line,In order to better explore the other biological functions of mi R-30 b in the development of human gastric cancer cells.3.Real-time reverse transcription quantitative polymerase chain reaction(RT-q PCR)was used to detect the m RNA expression of sema3 A in MKN28 gastric cancer cell lines in the two groups after transfection of mi R-30 b mimics and negative controls.Western Blotting was used to detect the protein expression level of sema3 A in MKN28 gastric cancer cell line.4.Plasmid transfection technique is used to change the negative control,mi R-30 b simulation,mi R-30 b inhibitors transfection to cultivate MKN28 gastric cancer cell line,and to interfere with the silence,sema3 A of gastric cancer cells in gastric cancer cell MKN28 con-transfection sema3 A interference RNA and mi R-30 b inhibitor,then using protein immunoblot technology(Western Blotting)in detection of the protein expression levels of sema3 A in MKN28 cells.5.The negative control group(NC group),the mir-30 b Mimics group(mir-30 b Mimics group),the mi R-30 b Inhibitor group(mi R-30 b Inhibitor+ si RNA group),the mi R-30 b Inhibitor+ sema3 A si RNA group(mi R-30 b Inhibitor+ si RNA group)and the gastric cancer cell medium exogenous added to the sema3 A group were collected and applied to human umbilical vein endothelial cells(HUVECs).Tube formation in HUVECs in each group was measured by Tube formation assay.6.Gastric cancer cell medium was collected from the negative control group(NC group),mi R-30 b Mimics group(mi R-30 b Mimics group)and mi R-30 b Inhibitor group(mi R-30 b Inhibitor group)and applied to human umbilical vein endothelial cells(HUVECs).Western Blotting was used to detect the expression of VEGFR and p-VEGFR in HUVECs.7.After the exogenous addition of SKLB1002,an inhibitor of p-VEGFR,to the mi R-30 b simulation group,protein expressions of p-VEGFR2 and VEGFR2 were first detected by Western Blotting,followed by Tube formation assay to detect the formation of small tubules in HUVECs in the mi R-30 b Mimics group and mi R-30 b Mimics+ SKLB1002 group.Results:1.The binding site of mi R-30 b to the 3 'UTR mutation site of sema3 A m RNA could be obtained from Target Scan database;The results showed that the relative luciferase activity in the WT+ Mimics group was significantly lower than that in the WT+NC,Mut+ NC and Mut+ Mimics group(P<0.05).2.MRNA expression of mi R-30 b and sema3 A in gastric cancer cells.Real-time reverse transcription quantitative PCR results showed that mi R-30 b mimics and inhibitors at different concentrations(0n M,1n M,10 n M,100 n M)could up-regulate(P<0.05)and down-regulate(P<0.05)the expression of mi R-30 b,respectively,with a concentration dependence.In addition,RTPCR was performed to detect the mi R-30 b simulation group and the negative control group in the MKN28 gastric cancer cell line,and the results showed that compared with the negative control(NC)group,the mi R-30 b simulation group could significantly reduce the expression of sema3 A m RNA(P<0.05).3.Comparison of tubular-forming capacity of HUVECs in each group(NC group,mi R-30 b Mimics group,mi R-30 b Inhibitor group,mi R-30 b Inhibitor+ si RNA group and sema3 A group).The results of in vitro tubular-formation test showed that compared with the NC group,the number of HUVECs tubules in the mi R-30 b Mimics group and the mi R-30 b Inhibitor+ si RNA group increased significantly with obvious lumen structure(P<0.05).Compared with the NC group,the number of tubules in the mi R-30 b Inhibitor group decreased significantly with no obvious lumen structure(P<0.05).4.Protein expression of sema3 A in gastric cancer cells.Western blot results showed that in the four experimental groups,compared with the NC group,the expression level of sema3 A in MKN28 gastric cancer cell line in the mi R-30 b simulation group was significantly decreased(P<0.05),while the expression level of sema3 A in the mi R-30 b inhibition group was significantly increased(P<0.05).Moreover,after exogenous addition of sema3 A si RNA to the mi R-30 b inhibitory group,the expression level of sema3 A decreased significantly compared with that of the inhibitory group(P<0.05).5.Protein expression of p-VEGFR2 and VEGFR2 in HUVECs(mi R-30 b Mimics group and mi R-30 b Inhibitor group).Western blot results showed that compared with the negative control group,the protein expression of p-VEGFR2 in the mi R-30 b mimic group was significantly up-regulated(p <0.05),while the protein expression of p-VEGFR2 in the mi R-30 b inhibitor group was significantly down-regulated(p <0.05).However,compared with the NC group,there was no significant difference in VEGFR2 protein expression between the two groups(P=0.8199).6.Protein expression of p-vegfr2 and VEGFR2 in HUVECs(Mimics group and Mimics+ SKLB1002 group).In the mi R-30 b Mimics group,exogenous addition of SKLB1002,an inhibitor of p-VEGFR,resulted in significant inhibition of p-VEGFR2 expression in Mimics+SKL1002 group compared with mi R-30 b Mimics group(p <0.05),while no significant difference in VEGFR2 expression in HUVECs(p =0.5850).7.After exogenous addition of SKLB1002,the small tubules of HUVECs in each group were observed.The results of the in vitro tubular-formation test showed that the number of tubules formed by HUVECs after the addition of the inhibitor was significantly reduced compared with that of the mi R-30 b simulation group,and the integrity of the lumen structure was also significantly reduced(P<0.05),and the angiogenesis capacity was significantly inhibited.Conclusion:1.In gastric cancer cells MKN28,mi R-30 b may regulate the expression of sema3 A by directly degrading m RNA.2.Sema3 A may be a key target gene of mi R-30 b regulating angiogenesis in MKN28 cells;3.In gastric cancer cells MKN28,mi R-30 b may regulate tumor angiogenesis through the Sema3A/ p-VEGFR2 axis. |
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