The Research On Regulation Effects Of Induction Of Heparanase Gene Promoter Methylation Targeted By Short Interfering RNAs In Human Gastric Cancer Invasion And Metastasis

Heparanase plays a key role in promoting tumor angiogenesis,invasiveness andmetastasis.This predominant enzyme is primarily responsible for cleaving heparin sulphate,the main polysaccharide constituent of extracellular matrix and basement membrane,thushaving become a novel target of tumor therapy.The change of status of methylation in thepromoter CpG island epigenetic relates to the transcription condition of coherent gene inthe process of tumor angiogenesis and development.Methylation could play an importantrole in detecting the tumor at early stage,and preventing and treating the tumor as anepigenetic molecules marker.Heparanase is overexpressed in gastric carcinomas and significantly associated withthe tumor invasion and matsatasis.This study is to examine the expression of heparanase inthe gastric carcinomas specimens and diverse differential gastric celllines,and to explorethe status of methylation in the promoter CpG island,screen the siRNA sequence that couldinduce methylation of the promoter of heparanase,set up the tumor specific transduction system of siRNA mediated by tumor-specific promoter,identify the regulation effect totumor invasiveness and metastasis of siRNA inducing methylation targeting the promoterof heparanase in cell and integrity level,provide a new visual field to regulate tumorinvasiveness and metastasis by using epigenetic methods.The alteration of status of methylation in the promoter CpG island was detected bynMSP and BSP assays.The change of heparanase expression of gastric cancer cell linesafter siRNA interfere was investigated by Western Blot,real time PCR.The invasiveability was detected by Transwell assay,the adhesiveness ability was evaluated byadherence test.The expressions of heparanase mRNA and protein were significantly descend aftersiRNA interfering each group by pGenesil-1_site1,_site2,the other sites were notsignificantly descend meanwhile.The change tendency was not identical with thedifferentiation of the tumor cells,but was identical with the change of frequency ofmethylaiton in each group.The status of methylation in the promoter CpG island ofheparanase was hypomethylation,the frequence of methylation in the region of Thetranscription factor,early growth response 1 (EGR1)is associated with the inducibletranscription of the heparanase gene of diverse differentiation degree,however,the specificsite and the frequence of methylation was not identical.The frequence of methylation ingastric cancer cells lines were identical with the status of gastric tissues,so it was toestablish rationale to investigate the effects of methylation of promoter CpG island ofheparanase to gastric oncobiology.The status of methylation in heparanase gene promoter region was significantlycorrelated with expression of heparanase.It reveal that the the status of methylation play animportant role in tumor invasiveness and metastasis.By using siRNA,the invasive abilitywas weaken in each cell groups,and the adherence ability was also suppressed.Thisresearch demonstrate the methylation in heparanase gene promoter could depress theexpression of heparanase,thus,it could effectively inhibit the tumor invasiveness and metastasis and provide a novel rationale and strategy for tumor therapy and preventingmetastasis and detecting tumor in the early stage. PartⅠRetrospective Study on Expression of Heparanase in Gastric Carcinomaand Clinical SignificanceObjective:To investigate the expressions of heparanase in the gastric carcinomasspecimens and diverse differential gastric cell lines,and to explore theircorrelation to clinical pathological features of gastric carcinoma.Methods:The expressions of heparanase proteins in 72 cases of gastric carcinoma anddiverse differential gastric cancer cell lines were detected by SABCimmunohistochemistry and immunofluorescence.Analyses the relationshipbetween the heparanase and gastric clinicpathological features,and evaluatethe effects of heparanase in tumor invasiveness and metastasis by concordanceat gene and protein level.Results:56 of the 72 colorectal carcinoma specimens (77.8%)were positive inheparanase by immunohistochemistry and 93.1% (67/72)by RT-PCR and83.3% (60/72)by Western Blot,the expression of heparanase was correlatedwith invasion depth and lymph node metastasis and TNM stage of gastriccarcinoma(P＜0.05).Heparanase expression was detected significantly in all ofgastric cancer cell lines,the order of heparanase expression is TMK-1＞SGC-7901＞MKN-28(P＜0.05).Conclusion:The expression of the heparanase in gastric carcinoma tissues and diversedifferential gastric cancer cell lines may be associated with its progression andmetastasis and differentiation degree and TNM stage but not with otherclinicpathological features.Molecular targets of heparanase may also beimportant factors in the metastasis of gastric cardinoma and it could provideidentifying evidence to gastric diagnose and treat. PartⅡAnalysis of the Status of Methylation in the Region of Heparanase Promoter inGastric CarcinomaObjective:To explore the status of methylation in the promoter CpG islands of heparanase,and to screen the CG sites for methylation.Methods:Extract the DNA of genome of gastric carcinoma and diverse differentialgastric cancer cell lines,hydrosulfite modify the nucleic acid,detect the statusof methylation in the promoter CpG island by MSP and BSP methods.Results:It was hypomethylation in the promoter CpG islands of heparanase by usingMSP detecting the region of the promoter,and significant relevance was foundbetween the status change and the differentiation degree.The order offrequency of methylaiton was TMK-1＞SGC-7901＞MKN-28(P＜0.05).Theresults of DNA sequencing were identical with this tendency.The frequency ofmethylaiton was not at equal pace in the diverse differentiation tissues and thesite of methylaiton was neither fixed in one place.Conclusion:The status of methylation in the promoter CpG island of heparanase washypomethylation,the frequence of methylation in the region of Thetranscription factor,early growth response 1 (EGR1)is associated with theinducible transcription of the heparanase gene of diverse differentiation degree,however,the specific site and the frequence of methylation was not identical.The frequence of methylation in gastric cancer cells lines were identical withthe status of gastric tissues,so it was to establish rationale to investigate theeffects of methylation of promoter CpG island of heparanase to gastriconcobiology. PartⅢThe study of the effects of siRNA targeting heparanase gene promoter to methylationin gastric cancer cell linesObjective:To screen the siRNA sequence that could induce methylation of the promoter ofheparanase,set up the tumor specific transduction system of siRNA mediatedby tumor-specifi-promoter.Methods:The shRNA oligonucleotides targeting for heparanase gene promoter weresynthesized and cloned into pGenesil-1 to generate shRNA eukaryoticexpression vectors.The recombinant named as pGenesil-1_site1,_site2,_site3,_site4,and _site5.shRNA expression plasmids were identificated bysequencing.The eukaryotic expression vectors were transfected into diversedifferentiation cells:MKN-28(weI1),SGC-7901(moderately),TMK-1(poor).The green fluorescent protein (GFP)was detected by fluorescence microscope,and the silencing effects of the recombinant vectors were determined byRT-PCR.Screen the siRNA sequence sites for constructing the specifictransduction system and investigate the effects of heparanase in gastriccarcinoma subsequently.Results:The recombinant sequence identified by sequencing was the same as thetargeting one.In the MKN-28,SGC-7901,TMK-1 cells transfected with therecombinant vectors,the expression of green fluorescent protein (GFP)wasdetected,the silencing effect of pGenesil-1_site1,_site2 was more evident thanother recombinant vectors.The methylaiton status of promoter was changedsignificant in pGenesil-1_site1,_site2 at SGC-7901,TMK-1 cells groups,andMKN-28 cells group was only significant in pGenesil-1_site2,the other groupsand control group were not significant in the changes of methylaiton status ofpromoter. Conclusion:shRNA recombinant vector was established successfully by RNAi techniqueand effectively transfected into 3 diverse differentiation degree gastric cancercells lines.Screen the effectively siRNA sequence targeting differentmethylation sites to induce methylation of promoter.It provided the rationalefor siRNA effects of inducing methylation of DNA in gene promoter regionwhile not effects of the silencing gene expressing by targeting the encodingregion of DNA. PartⅣThe study of the effects of siRNA targeting heparanase gene promoter to theexpression of heparanase in gastric cancer cell linesObjective:To investigate the role of siRNA targeting heparanase gene promoter inregulating the expression of heparanase and the tumor invasion and matsatasis.Methods:The alteration of status of methylation in the promoter CpG islands wasdetected by nMSP and BSP assays.The change of heparanase expression ofgastric cancer cell lines after siRNA interfere was investigated by Western Blot,real time PCR.The invasive ability was detected by Transwell assay,theadhesiveness ability was evaluated by adherence test.Results:The cell growth curve show that the growth was inhibited by siRNA inpGenesil-1_site1,_site2 sequence in each cells groups,and the order of the cellgrowth was_site1,_site2＞_site3,_site4＞_site5,the control group.In cellsinvasive test,cells of _site1,_site2 groups migrated into the gap more thanother groups and control group at same time-points after inducing the lesion.The counts of parental cells penetrating through membrane were 113±7,significantly more than 48±4 in control group (P＜0.05).The adherence cells of_site1,_site2 groups was 0.83±0.02,significantly higher than 0.38±0.01 incontrol group (P＜0.05).Conclusion:siRNA suppress the expression of heparanase in the differentiation gastriccancer cells lines,and the change was not identical with the differentiation ofthe tumor cells,the research results show that inducing methylation of DNA inheparanase gene promoter region could suppress the expression of heparanase,but it was not significantly correlated with the frequency of methylation.Theactual reason need subsequently research to define.