| Objective: Endometrial cancer is a group of primary endometrialepithelial malignancy, and the most type is endometrial glandsadenocarcinoma, the most common crowd is perimenopausal andpostmenopausal women. As one of the most common malignant tumor offemale reproductive system, and its incidence accounted for7%in femalemalignant tumor,20%~30%in the female genital tract malignant tumor. Inrecent years, its incidence shows ascendant trend and younger according to theforeign epidemiological survey. But the etiology and pathogenesis is still notclear. Although biopsy is the only gold standard in the diagnosis ofendometrial carcinoma, it is an invasive procedures. Today, we still need anearly diagnosis of endometrial carcinoma method. MicroRNA is a kind ofsingle non coding RNA, which is22nt of long and exists in widely eukaryoticcells, and it can make target mRNA degradation or inhibit its translationthrough it and miRNA complementary completly or incompletly, thus it cancontrol the expression of target gene, and affect cell proliferation,differentiation and apoptosis. MiRNA can control the occurrence,development and transformation process of tumor through the similar cancergene, tumor-suppressor genes or other ways. The study of miR-21is mostpopular. The present study found that it participate in the differentiation,proliferation and apoptosis of the cells, and related to the occurrence of tumorsclosely. P53is the most widely research in human tumor suppressor genes.The combination of P53protein and DNA polymerase, which can make thecopy initial complex inactivation, and inhibite tumor cell proliferationexcessively. The mutations of P53can lead to the gene excessive expression,and this kind of abnormal excessive expression often closely related to theclinical stage, the organization classification and the erosion degree inmuscle layer of the endometrial cancer. This study aims to observe the proliferation and apoptosis of endometrial carcinoma Ishikawa cells afterinhibition miR-21, and the expression of P53in endometrial carcinomaIshikawa cells, and to explore their relationship in endometrial carcinoma, tofind the role of miRNA-21in the occurrence and development process ofendometrial cancer, and provide the new targets for its diagnosis andtreatment.Methods:1Cultivate endometrial carcinoma cell line of Ishikawa.2The group of experiments:①though the Lipofectamine2000liposometransfection reagent, the treatment group was transfected with miR-21specificinhibitor antisense oligonucleotides;②the negative control group wastransfected with irrelevant miR-21specific inhibitor nucleotidesequence;③the liposome control group was transfected with Lipofectamine2000liposome transfection reagent;④the blank control group that is notransfection group.3The cell multiplication of the endometrial carcinoma cell was assayedby CCK-8.4Fluorescence activated cell sorting(FACS) was used to analyze theendometrial carcinoma cell apoptosis.5The miR-21and P53expression in the endometrial carcinoma cells wasdetected by real-time quantitative RT-PCR.6The P53protein expression in the endometrial carcinoma cells wasmeasured by Western blot analysis.7The above tests were performed in SPSS (version13.0; SPSS Inc,Chicago, IL, USA). P<0.05for a significant difference. All the experimentsare repeated3times.Results:1The transfection efficiency was observed by laser confocal microscopafter24h and48h using Lipofectamine2000liposome transfection methodtransfection. The test group and the negative control group were weakfluorescent expression, and the fluorescence expression was enhanced after 48h (transfection rate70%~85%).2The CCK-8experiments detected: the four groups of cells wereproliferated on the whole. The blank control group and the liposome controlgroup had similar growth and slightly faster than the treatment group and thenegative control group from0to24h; the blank control group, the liposomecontrol group and the treatment group were growed quickly from24to48hours; the growth rate from48to72h: the liposome control group>blankcontrol group>treatment group>negative control group; the four groups ofcells continued to maintain growth condition and achieved logarithmic phasefrom72to96h, the blank control group, liposome control group and treatmentgroup slightly faster. There was no significant difference betweengroups(P>0.05).3The influence of transfection cell apoptosis was tested by FCM after3days. The experimental group compared with blank control group, liposomesgroup and the negative control group accounts for a strikingly low proportionof early apoptosis, the comparison between groups had significant difference(P<0.05). Experimental group compared with blank control group, liposomesgroup and the negative control group had the similar late apoptosis cellpercentage, the comparison between groups there was no significantdifference (P>0.05).4The expression levels of miRNA-21and P53mRNA were detected byReal-time RT-PCR. Compared with the blank control group, the miR-21expression quantity of the experimental group is reduced about40%, andsignificantly lower than other groups, compared with each group hadsignificant difference (P<0.05). Compared with the blank control group andliposome control group, the expression quantity of treatment group andnegative group P53mRNA reduced significantly, and it had significantdifference (P<0.05).5P53protein expression was tested by the Western Blot. The P53proteinexpression is obviously higher than that of the negative, blank and liposomecontrol group, and it has significant difference (P<0.05). Conclusions:1miR-21was expressed in endometrial carcinoma Ishikawa cells,miR-21inhibitor can effectively inhibit its expression in the cell.2miR-21can promote the apoptosis of endometrial carcinoma Ishikawacells, but it has no obvious influence for the proliferation of endometrialcarcinoma Ishikawa cells.3P53protein has high expression in carcinoma of endometrium Ishikawacells.4miR-21didn’t play a significant role for P53gene regulation inendometrial carcinoma Ishikawa cells, but it can inhibit the P53proteinexpression in endometrial carcinoma Ishikawa cells. And it affects theoccurrence, development and prognosis of endometrial carcinoma. |
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