The Inhibition Effect Of Dihydromyricetin On Choriocarcinoma Cell JAR Growth By Targeting MiR373

MicroRNA is a highly conserved,endogenous non coding small RNA in the evolutionary process.It is widely distributed in eukaryotes,and plays a very important role in the regulation of eukaryotic gene expression.In recent years,it has been found that the abnormal expression of Micro RNA can regulate the process of tumor development.With the development of targeted therapy,many of the molecules in MicroRNA have been found to be the target of tumor therapy.Using Microarray,we investigated the chorionic carcinoma(referred to as choriocarcinoma)differentially expressed in tissues and normal villi in MicroRNA.It found that 63 upregulation of MicroRNA,74 downregulation of MicroRNA in choriocarcinoma.Among them,MicroRNA373 down-regulated in choriocarcinoma tissues significantly.MicroRNA373 plays an important role in the two-side regulation of multiple tumor development as oncogene or anti-oncogene.In this study,we investigated the role of MicroRNA373 on choriocarcinoma cell growth and the expression of MicroRNA373 in choriocarcinoma.Applying the real-time quantitative PCR,we detected MicroRNA373 expression in choriocarcinoma cells.The expression of MicroRNA373 in choriocarcinoma JAR and JEG cells were much lower than that in normal chorion HTR-8 cells.CCK-8 cell proliferation assay found the proliferation of choriocarcinoma JAR cell was inhibited significantly after transfected with MicroRNA373,which showed that MicroRNA373 can inhibit the growth of choriocarcinoma cells.DMY is extracted from the herb Ampelopsis stems and leaves.It has the functions of eliminating free radical,anti inflammation and bacteriostasis.In recent years,it has been found that DMY also has anti-tumor effect,and can inhibit the growth of various tumors in vivo and in vitro.In this study,we found that DMY can promote the expression of MicroRNA373.With increasing concentration of DMY,expression level of MicroRNA373 in JAR cell line was increased.At the same time,the density of JAR cells decreased,the cell retracted,cells’ size differenced,the intercellular space became larger,and the nuclear color deepened.These results suggested that DMY may affect the growth of choriocarcinoma cells.The activity of choriocarcinoma JAR cells with different concentrations of DMY were further detected by CCK 8 method.We found that with increasing concentration of DMY the proliferation inhibition rate of choriocarcinoma JAR cells increased gradually and the cell activity decreased.The above studies showed that DMY can promote the expression of MicroRNA373 and inhibit the proliferation in choriocarcinoma JAR cells.This study indicated that MicroRNA373 was an effective target for inhibition of Chorioepithelioma cell growth in vitro,and proved that DMY inhibited the growth of choriocarcinoma cells by acting on the target MicroRNA373.It has important significance for exploring the pathogenesis and treatment target of choriocarcinoma.It also provides a theoretical basis for the application of DMY in clinical treatment of choriocarcinoma.Objective:1.Explicit the low expression of MicroRNA373 in choriocarcinoma cell and the transfection of MicroRNA373 could inhibit the proliferation of choriocarcinoma cells.2.Explicit DMY can promote the expression of MicroRNA373 in choriocarcinoma cell,and inhibit the growth of choriocarcinoma cellsMethods:1.Real-time quantitative PCR(Real-time PCR)were used to detect MicroRNA373 differential expressions between normal chorion HTR-8 cells and choriocarcinoma JEG,JAR cells.2.Enhance the expression of MicroRNA373 in choriocarcinoma JAR cells by transient transfection.3.The transfection efficiency of MicroRNA373 in choriocarcinoma JAR cells was observed by inverted fluorescence microscopy.4.Real-time quantitative PCR(Real-time PCR)technique was used to verify the expression change of MicroRNA373 after transfection.5.CCK8 method was used to detect the proliferation of JAR cells with overexpression of MicroRNA373 in choriocarcinoma.6.MicroRNA373 expression in choriocarcinoma JAR cells after treated with DMY was detected by Real-time quantitative PCR(Real-time PCR)technique.7.The mrphological changes of choriocarcinoma JAR cells after treated with DMY were observed by Iverted microscopy.8.CCK8 method was used to detect the proliferation of JAR cells in different concentrations of DMY.Results:1.The expression of MicroRNA373 in choriocarcinoma cell JAR and JEG were lower than that in normal trophoblast cells HTR-8.The difference was statistically significant(P < 0.05).2.After transfected with MicroRNA373,the growth of JAR cells was found to be poor,and some of them died.The expression of green fluorescent protein was observed under fluorescence microscope.The green fluorescence was showed in Choriocarcinoma JAR cells,and the cell outline can be identified clearly.The transfection efficiency was 85%.3.The results of Real-time quantitative PCR showed that compared with the negative control group,MicroRNA373 expression was significantly increased after transient transfection of human choriocarcinoma cell line JAR,the difference was statistically significant(P < 0.05).4.The results of CCK8 showed that the OD value of JAR cells was lower,the number of viable cells was decreased and the proliferation inhibition rate was increased after MicroRNA373 transient transfection,(P<0.05).It is suggested that the increase of MicroRNA373 expression can reduce the proliferation of JAR cells effectively.5.Real-time quantitative PCR results showed that DMY promoted the expression of MicroRNA373 in human choriocarcinoma cell line JAR.Furthermore,the expression of MicroRNA373 in choriocarcinoma JAR cells increased gradually with the increasing concentration of DMY,having a positive correlation with dose concentration(P<0.05).6.Inverted microscope observation showed that the JAR cells were compact adherent cells with clear contour and uniform size before treated with DMY.With the increasing concentration of DMY,the density of choriocarcinoma JAR cells decreased,cells shrinkaged,cells’ size became different,the gap became large,the color deepened,the mass of cells formatted.7.The results of CCK-8 showed that the activity of JAR cells decreased after treated with DMY,and the cells’ activity decreased with the increasing concentration of DMY.The difference was significant statistically(P < 0.05).The results indicated that DMY Inhibited the activity of Choriocarcinoma JAR cells,which dependented on its concentration.Conclusion:1.MicroRNA373 is an effective target for inhibiting the growth of choriocarcinoma cells in vitro.2 DMY inhibited Chorioepithelioma cells’ growth by acting on the target MicroRNA373.