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A Study On The Mutations Of ARIDIA Gene And The Correlation Of ARID1A And ER, PR, P53mRNA In The Endometrial Carcinoma Cell Lines

Posted on:2014-02-08Degree:MasterType:Thesis
Country:ChinaCandidate:H F YangFull Text:PDF
GTID:2234330398991744Subject:Obstetrics and gynecology
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Objective: Endometrial carcinoma is one of the common malignanttumors of female reproductive tract malignancy, which is accounted for aboutof half of the gynecological cancers in the United States. The incidence ofendometrial cancer in female malignancy is the fouth in our country. Researchdata shows that the incidence is significantly increased year by year at homeand abroad in recent years, but its incidence etiology and pathogenesisremains unclear. The main measures for the treatment of endometrial cancerwere surgery. Therefore, through a variety of basic research to explore thecauses and pathogenesis of endometrial cancer incidence is contribute to earlydetection, early diagnosis and early treatment, at the same time, it has apositive meaning for assessing endometrial cancer prognosis. Molecularpathogenesis is contribute to study the etiology of endometrial cancer.ARID1A (AT-rich interactive domain) is a recently identified tumorsuppressors gene, which is located in the chromosome1p36. ARID1A is animportant integral member of the SWI/SNF complex. ARID1A encodesBAF250a protein, which shows low expression in a variety of cancer and thelevel of expression is related to the pathology pathological stage of cancertissue. Mutations of ARID1A gene analysis showed46(6%) of759kinds ofmalignant tumors. Various studies have shown that loss of protein expressionwas associated with inactivating mutations of ARID1A. Our previous studiesshowed that the expression levels of ARID1A mRNA and BAF250a inendometrial carcinoma tissue were lower than those in normal endometrialtissue, moreover it was gradually decreased in poorly differentiatedendometrial adenocarcinoma. It indicates that ARID1A is correlated withpathological grading of endometrial carcinoma. Our previous studies also showed that there was negative correlation of BAF250a with ER in poorlydifferentiated endometrial cancer, and there was negative correlation ofBAF250a with P53protein. But the mutation of ARID1A gene in endometrialcancer has not been reported, we selected three endometrial cancer cell lines(Ishikawa, HEC-1A, KLE) to culture. Polymerase chain reaction (PCR) wasemployed to studied whether there was a mutation in ARID1A, so to explorethe possible reason of endometrial carcinoma and to explore the pathogenesisof ARID1A gene in endometrial cancer cell lines. The expression of ARID1A,ER, PR, P53mRNA in the endometrial cancer cell lines and the correlation ofARID1A and ER, PR, P53mRNA were detected by RT-PCR.Methods:1Culture cell lines. Application DMEM/F-12completely prudential andHyclone fetal bovine serum to culture cell lines and select logarithmic phasecell lines, which concentration was4×105/ml.2A study on the mutations of ARID1A gene in endometrial cancer celllines by direct sequencing test. Genomic DNA was extracted from the threecell lines by genomic DNA purification Kit of Promega Company. Afterapplication of ARID1A DNA sequence of exon regions from NCBI, designedand synthesized primers to amplify destination genetic segments byPolymerase chain reaction. After getting the destination gene by PCR andconfirming through agarose gel electrophoresis, the destination gene was sentto Shanghai SangGong biological engineering Co.LTD to obtain the sequenceof the amplification products by direct sequencing test. Compared with thehuman genome genetic sequence to analyze the results to find whether therewas a mutation.3The expression of ARID1A ER, PR and P53mRNA in endometrialcancer cell lines were detected by RT-PCR amplification. After obtainingcDNA sequence of ARID1A, ER, PR and P53through GenBank, thendesigned and composed primers. Used RT-PCR amplifications with theprimers to amplify destination genetic segments and β-actin as an internalreference. Agarose gel electrophoresis was used to confirm amplification effects. Employ gel-pro analysis3.1to test these.4Used SPSS13.0statistical analysis software to analyze experimentaldata. P<0.05indicates that the difference has a statistical significance.Results:1The DNA, which were extracted from endometrial cancer cell lines,were amplified through polymerase chain reaction. After electrophoresis strips,the products emerged destination bands. By the direct sequencing test, wefounded:(1) Same sense mutation: the mutation of ARID1A gene in Ishikawawas transition of G to A at codon1644; the mutation of ARID1A gene inHEC-1A was transversion of C to A at codon3969.(2) Missense mutation: themutation of ARID1A gene in Ishikawa were transversion of G to C at codon833,the mutations of ARID1A gene in HEC-1A were transversion of A to T atcodon1212and transversion of G to T at codon5281; the mutation ofARID1A gene in KLE were transversion of G to C at codon833.(3)Nonsense mutation: the mutation of ARID1A gene in HEC-1A was transitionof C to T at codon5503; the mutation of ARID1A gene in KLE was transitionof C to T at codon6343.(4) Frameshift mutation: the mutation of ARID1Agene in KLE was deletion of C at codon2272.2The expression of ARID1A, ER, PR mRNA in Ishikawa were higherthan the expression of ARID1A, ER, PR mRNA in HEC-1A and KLE(P<0.05). The expression of P53mRNA in Ishikawa were lower than theexpression of P53mRNA in HEC-1A and KLE (P<0.05).3There was no significant correlation of ARID1A mRNA with ER, PRmRNA in Ishikawa and HEC-1A(P>0.05). There was positiver correlation ofARID1A mRNA with ER, PR mRNA in KLE(P<0.05). There was negativecorrelation of ARID1A mRNA with P53mRNA in Ishikawa, HEC-1A andKLE(P<0.05).Conclusions:1We founded mutations of the ARID1A gene in endometrial cancer celllines (Ishikawa, HEC-1A, KLE). Missense mutation and nonsense mutationonly was found in HEC-1A and KLE, and Frameshift mutation only was found in KLE.2With the reduction of the cell lines differentiation, the expressions ofARID1A, ER, PR mRNA were gradually increased. There was positivercorrelation of ARID1A mRNA with ER, PR mRNA in KLE. There wasnegative correlation of ARID1A mRNA with P53mRNA in Ishikawa,HEC-1A and KLE.3We believed that the cause of endometrial cancer may be due to genemutation and we could think that the low expression of ARID1A may relate toARID1A gene mutation.
Keywords/Search Tags:Endometrial cancer cell lines, ARID1A, direct sequencingtest, genetic mutation, RT-PCR
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