Formononetin Promotes The Apoptosis Of Prostate Cancer PC-3 Cells By Regulating The Expression Of LncRNAH19

FORMONONETIN INDUCES APOPTOSIS OF PC-3 HUMAN PROSTATE CANCER CELLSBackground and objective:Formononetin(FN),the main active constituent of red clover,is also found in angelica,astragalus and other traditional Chinese herbal medicines.FN has been proved to have a variety of biological activities,including antioxidation,antiviral and protective effects,which received extensive attention in the research of anticancer drugs.Nevertheless,it is still controversial whether formononetin is effective for clinical application.Here,we investigated the toxic effects and the potential molecular mechanism of Formononetin in PC-3 prostate cancer cells.In the present study,we measured the expression of lncRNA H19 and apoptosis-related factors Bax and Bcl-2 in PC-3 cells after the treatment of formononetin,to futherly investigated the pharmacological effects of formononetin.Methods:1.100 mmol/L drug storage solution was prepared and diluted to the final concentration with RPMI-1640 cell medium.After the treatment of formononetin at different concentrations(0,20,40,60,80 and 100 ?mol/L)for 24h and 48 h respectively,cell viability of PC-3 cells in each group was measured by CCK8 assay.2.PC-3 cells were treated with different concentrations(0,25,50 and 100 ?mol/L)of formononetin for 48 h,then Hoechst 33258 nuclear staining test was conducted to observe the nuclear morphology of each group under fluorescence microscope.3.After 48 h'treatment with different concentrations of formononetin(0,25,50 and 100 ?mol/L),the expression of lncRNAH19,Bax and Bcl-2 mRNA in PC-3 cells were detected by real?time quantitative PCR and the expression of apoptosis-related proteins Bax and Bcl-2 were detected by Western-blot.Results:1.The results of CCK8 assay suggested that formononetin could significantly inhibit the activity of PC-3 cells.After the treatment,viability of PC-3 cells in experimental groups(20,40,60,80 and 100?mol/L)was decreased in turn with the increasing of drug dose or treatment time.2.The results of Hoechst 33258 staining showed that after 48 h,the concentrating and fragmentation changes in nucleus dose-dependently raised.3.It was observed in real-time quantitative PCR and Western blot that formononetin could down-regulate the expression of lncRNAH19 and Bcl-2,while up-regulate the expression of Bax in a dose-dependent way.Conclusion:Our experiment presented evidence that formononetin induces apoptosis of prostate cancer PC-3 cells in a time-dose dependent way.This effect may be realized by regulation of lncRNA H19 and the mitochondrial apoptosis pathway.H19 HAS A POSITIVE REGULATORY EFFECT ON THE EXPRESSION OF IGF-1R IN PC-3 CELLSBackground and objective:LncRNA H19 is an important epigenetic factor,which may promote the malignant progression of prostate cancer.Developing H19-tagerted drugs for the treatment of prostate cancer is a promising research content.Our results in the first part have shown that formononetin could promote the apoptosis of PC-3 cells by reducing the expression of lncRNA H19,but the underlying mechanism was not completely elucidated.In this part,we investigated the interreaction between H19 and IGF-1R in PC-3 cells.This will help to find the downstream target of H19 and futherly explore the underlying molecular mechanisms of formononetin in PC-3 cells.Methods:H19 overexpression lentivirus and H19 silencing lentivirus were constructed and co-cultivated with PC-3 cells respectively for 96 h.Then total RNA in each group was extracted.The infection effect was observed by detecting fluorescent labels and relative expression of lncRNA H19.Real-time quantitative PCR was used to detect the relative expression of IGF-1R mRNA.Results:As expected,the overexpression of H19 increased IGF-1R mRNA expression in PC-3 cells,whereas the inhibition of H19 decreased IGF-1R mRNA expression in PC-3 cells.Compared with the control group,when the expression of lncRNA H19 was increased to 21.11±2.10 times,relative expression of IGF-1R mRNA was increased to 1.24±0.13 times subsequently.When the expression of IncRNA H19 was decreased to 0.37±0.08 times,relative expression of IGF-1R mRNA was decreased to 0.71±0.03 times,the differences were statistically significant.(P<0.05).Conclusion:According to the results,IGF-1R is likely to be a downstream molecule of H19 in prostate PC-3 cells.Formononetin could induce the apoptosis of PC-3 cells by regulating the H19/IGF-1R pathway.