Study Of The Expression Analysis Between MiR-27a And E-cadherin In The Gastric Carcinoma With High Peritoneal Metastasis Potential Cell Line GC9811-P

Background：Gastric cancer（GC）is the common malignant which is endanger to the healthyof the people in the world.It is usually that peritoneal metastasis happen in the half ofpatients with advanced gastric cancer,and low efficency in the clinicaltreatment.MicroRNAs are a class of small non-coding RNA including more than20nucleotides that regulate their target mRNA expression at the post-transcriptionallevel by binding to complementary sequences found in the3’-untranslated region（UTRs）of the target mRNAs,which are related to the process of tumors．The recentstudy revealed the expression of miR-27a（microRNA-27a）is high in the Gastriccancer tissue, and verifed that promoted the invasion and metastasis ability of tumorthrough dramatically decreasing the E-cadherin′s expression levels.The presentstudys revealed that methylation of the E-cadherin gene promoter is contribute to thegene inactivation. Compared to Gastric cancer tissue, the expression of theE-cadherin gene in the gastric carcinoma tissue with peritoneal metastasis is lower.So,It is possible that the expression of miR-27a in the gastric carcinoma with peritonealmetastasis is higher than primary gastric cancer from the above researches,Whichcsuse that the expression of the E-cadherin gene in the gastric carcinoma tissue withperitoneal metastasis is lower.The study discusss the overexpression of the miR-27ain the gastric carcinoma with high peritoneal metastasis potential cell lines GC9811-Pat the first time,and preliminarily clarify related that the expression analysis betweenmiR-27a and E-cadherin.We detect the possibility that miR-27a could become a targetof the antisence gene therapy the gastric carcinoma with high peritoneal metastasis,itis providing the experimental evidence that studying the research in the future.Objective：1、To select the cell lines of miR-27a over-expression from the three gastriccarcinoma cells,such as SGC7901(the gastric adenocardnomacell)、MKN-28(the poorly differentialated stomach gland cancer cell line)、 GC9811-P(the gastriccarcinoma with high peritoneal metastasis potential cell line).2、To investigate the expression relation of miR-27a、E-cadherin and E-cadheringene promoter methylation in the gastric carcinoma with high peritoneal metastasispotential cell lines GC9811-P.To explore whether it influence the ability ofproliferation and apoptosis in vitro after down-regulate miR-27a expression.Methods：1、To detect respectively the expression of E-cadherin mRNA and protein、E-cadherin gene promoter methylation、miR-27a in the SGC7901、MKN-28、GC9811-P cell lines by RT-PCR、WesternBlot、MSP、real-time-PCR;comparison theexpression difference among the measured data.2、There are three experimental groups,such as the blank control group (onlyadd the culture medium), negative control group (transfect100nmol／L miRNA-angagomir negative control-FAM),transfect group (transfect100nmol／LmiR27a-antagomir-FAM).3、 the miR27a antagomir-FAM was transfected into GC9811-P cell linesmomently;24h later,observe the efficiency of transfection under the invertedmicroscope;we should detect the expression of E-cadherin mRNA and protein、E-cadherin gene promoter methylation、miR-27a in GC9811-P cell lines among thethree Experimental groups, and comparison the expression difference among the threeExperimental groups.4、The proliferation of GC9811-P cell lines treated with miR27a-antagomir weredisplayed by MTT experiment,the early apoptosis cells were explored by the flowcytometry.Results：1、Real-time-PCR display that the expression level of miR-27a in the GC9811-Pwas the highest among the three gastric cancer cell lines(P<0.05).RT-PCR displaythat the expression of E-cad mRNA and protein in the GC9811-P were the lowestamong the three gastric cancer cell lines(P <0.05).MSP display that the state of E-cadherin gene promoter region methylation in the GC9811-P was the most obviousamong the three gastric cancer cell lines(P <0.05).2、The efficiency of antagomir transient transfect into the gastric carcinoma withhigh peritoneal metastasis potential cell line GC9811-P,which was tested about60%;the comparison among the three Experimental groups,we find that the expression ofmiR-27a was down-regulation apparently,by contrary,up-regulation of E-cadherinmRNA and protein in the transfer group(P <0.05),the state of E-cadherin genepromoter methylation got the reversion(P <0.05).3、MTT assay displayed the slower proliferations rate in the transfer group bycontrast with the negative control group(P<0.05),the maximum CPIR(cellularproliferation inhibition rate)of the transfection group was36h after transfection.4、 Flow cytometry showed miR27a-antagomir dramatically induced theapoptosis of GC9811-P cell lines compared with the negative control group (P<0.05).Conclusion：1、Among the three gastric cancer cell lines,the expression level of miR-27a inthe GC9811-P was the highest,The expression level of E-cadherin in the GC9811-Pwas the lowest,the state of E-cadherin gene promoter methylation in the GC9811-Pwas the most obvious.2、If decreasing the miR-27a level through miR27a-antagomir, the state ofE-cadherin gene methylation was reversed, E-cadherin will up-regulation;miR-27aparticipated in the process of E-cadherin gene promoter region methylation todownregulate expression of E-cadherin.3、If we inhibit the expression of miR27a,it would inhibit the GC9811-P′sproliferation ability and induce the apoptosis;It is providing that miR-27a could be atarget of the antisence gene therapy in the gastric carcinoma with peritonealmetastasis.