The Study Of MicroRNA34a Expression And Effection In Choriocarcinoma Cell Line JEG-3

Objective:Choriocarcinoma (CC) is a high malignant tumor,at earlyperiod,it can hematogenous metastasis, spreading all over the body. Althoughthe effect of chemotherapy, but with the emergence of drug-resistant cases, thetreatment of choriocarcinoma have new difficulties. Therefore, how toimprove the cure rate and survival rate of these patients is a problem faced byclinicians. microRNA (miRNA) is a kind of non-coding single-stranded RNAmolecules,which is widespread in eukaryotes with about22nucleotidelength and through base pairing with the specificity target mRNA to causedegradation or inhibition of its target mRNA translation, then to regulate geneexpression at the posttranscriptional level. They are closely related to humangrowth and development, stem cell differentiation, self-renewal and tumor,heart disease, AIDS and other diseases, in particular, play an important role inthe tumor development process. mir-34is a conservative, non-coding miRNAwhich was first discovered in C. elegans, and keep homology in someinvertebrates. Human miR-34, including miR-34a, miR-34b and miR-34c,gene structure prediction showed that the mature mir-34a sequence is in theoriginal transcription exon2of its major gene, the position is located³0kb inthe downstream of this original transcription exon1. At the same time, themature mir-34b and mir-34c sequence in the original transcript of the samemajor gene intron1and exon2. According to the miRBase SequenceDatabase and ensemble database information, has-mir-34a located in the1p36.23,which sequence of the stem-loop structure is located on the1,9,134,314to9,134,423minus-strand of chromosome1,for a total of110nucleotide sequence; has-mir-34b, has-mir-34c are all located in11q23.1, thestem-loop structure sequences were located on chromosome11in the first110,888,873to110,888,956bits chain and the first110,889,374to 110,889,450positive-strand, respectively, a total of84and77nucleotidesequence. miR34have shown abnormal expression in a variety of tumors.miR34through p53activation, inhibition of the expression of E2F3, Bcl-2,c-myc, CDK4, CDK6,Cyclin D1, and Cyclin E2, to arrest the tumor cells inG1, inhibiting tumor cell growth and inducing tumor cell apoptosis,meanwhile,E2F3, of SIRT1and p53form a positive feedback loop, andconstantly enhance the role of itself and p53. Recent studies have confirmedthat miR34can be expressed in a variety of tumor cells: miR34a expression inhuman colon cancer development process down regulation, it's often missingin the in pancreatic cancer cells and neuroblastoma cell tumor; meanwhile,increase miR34a expression artificially can inhibit cell proliferation andactivate cell apoptotic pathway. These studies suggest that there is a linkbetween the miR34a expression down regulation or missing and tumorigenesisand cell proliferation, miR34a expression have important significance ininhibiting cell proliferation and activation of the apoptotic pathway, ValeryTarasovl et al. research confirms that miR34a is one of P53gene directlytarget, chromosome immunoprecipitation reaction and luciferase enzymeanalysis confirmed the P53gene activate miR34gene transcription, miR34family can promote cell growth arrest and death occurred with P53othertarget gene, such as P21and BAX under stress which can contribute tomalignancy. As a cell proliferation negative regulator, P53gene regulate cellgrowth and differentiation, to maintain the stability of the genomic DNA, theloss of P53function caused by P53mutation or deletion may plays animportant role in the tumor occurring and development. The study found thatP53gene related closely with the biological behavior of trophoblastic tumor.P53gene expression was related to the degree of mole trophoblastichyperplasia, and P53regulation of apoptosis level imbalance played animportant role to mole changing from benign to malignant. Also found thatetoposide can activate the p53gene in the MCF-7cell lines can induceoriginal transcription of mir-34a (pri-mir-34a) expression of3.7kb size.Therefore, this experiment through the use of etoposide administered in human choriocarcinoma cell line in JEG-3, in order to increase m icroRNA34a(miR34a) expression, and observing the expression of m iR34a withindifferent drug action time, detecting JEG-3choriocarcinoma cell proliferationand apoptosis at different time. And exploring the role of miR34a inchoriocarcinoma occurring and developing to provide a basis theory for thediagnosis and gene therapy of choriocarcinoma.Method:1. Culturing JEG-3cells with RPMI-1640which containing15%fetal bovine serum at37°C,5%CO₂cell incubator culture.2. Useclinical application chemotherapy drug etoposide raised the miR34aexpression of human choriocarcinoma JEG-3cells in vitro, the cultured cellsare divided into: control group (conventional training, did not carry out anyprocessing),6hours,12hours,24hours and48hours of dosing group.3.Under the inverted microscope to observe the cell morphological changes4.one step way Rrizol extracted total RNA, real-time PCR to detect miR34aexpression of different groups, the experiment was repeated three times.5.Four methyl thiazolyl tetrazolium (MTT) method detect cell proliferationability, the experimental set up four parallel hole6. flow cytometry detect cellapoptosis rate, the experiment was repeated three times.7All data werestatistically analyzed using SPSS13.0statistical software. Data presented asmean±standard differential (x±S), between the two groups, the number witht-test, interclass use one-way ANOVA or rank test, p>0.05for no significantdifference, p<0.05as has significantly difference, all the experimental resultswere repeated three times.Results:1choriocarcinoma JEG-3cell cultured in1640mediumcontaining15%(volume fraction) fetal bovine serum,,at37°C,5%(volumefraction) CO₂incubator. The cell is in good growth state, adherent growth,oval or round, tightly packed, uniform size, clear boundary, cell membraneintegrity, cell nucleus boundary is clear, visible secretory granules in thecytoplasm.2at6-24hour after administered：part of the adherent cells weredead, floating in the culture medium, the remaining cell morphology is stillgood；at48hour after administered：most of the adherent cells were dead, floating in the culture medium, the remaining cells form becoming irregular.the cells vary in size, some cells may have long pseudopodia, black particleincreased in the cytoplasm.3miR34a expressed in choriocarcinoma JEG-3cells. Control group miR34a expression level as1, the etoposide role6hour,12hour,24hour,48hour miR34a expression level is1.378±0.511,1.667±0.982,1.742±0.405, compared with the control group increases37.8%,66.7%,74.2%, but the difference was not have statistically significant (P﹥0.05);48hours miR34a expression level was5.423±3.611, compared withthe control group increase of442.3%, and the difference was statisticallysignificant (P <0.05).4.MTT method to detect the proliferation ability:6hoursdecreased by16.97%,12hours decreased by42.56%,24hours decreased by49.00%, decreased by70.57%in48hours, the differences were statisticallysignificant compared with the control group (P <0.05).5. flow cytometry todetect apoptosis rate:6hours increased4.92times,12hours increased7.46times,24hours increased8.76times,9.23times in48hours, compared withthe control group differences were statistically significant (P <0.05).Conclusion: MiR34a expressed in chorionic carcinoma JEG-3cells; miR34a expression can be up-regulated through chemotherapy drug etoposide,；microRNA34a maybe inhibits cell proliferation, but microRNA34a expressionand cell proliferation rate change are not in-phase,microRNA34a maybe onlyone of the inhibiting cell proliferation rate factor；microRNA34a maybeinducing cell apoptosis, but microRNA34a expression and cell apoptosis ratechange are not in-phase,microRNA34a maybe only one of the inducing cellapoptosis rate factor； microRNA34a may inhibits the occurring anddeveloping of choriocarcinoma.