| Objective:This study focused on the key scientific issue that Qi Lan Decoction affects the proliferation and apoptosis of prostate cancer cells and plays an anti-cancer role.Taking the miR-1297/PTEN/PI3K/AKT signal pathway as the breakthrough point,we explored the mechanism of Qi Lan Decoction on prostate cancer by studying the effect of Qi Lan Decoction on the proliferation and apoptosis of prostate cancer cells and the expression of key cytokines in the miR-1297/PTEN/PI3K/AKT signaling pathway.It is of great significance for clarifying the scientific connotation of Qi Lan Decoction by revealing its target of regulating prostate cancer.Methods:1.Screening and validation of prostate cancer cell lines and miRNA.Firstly,three different prostate cancer cells(DU145,LNCaP,PC-3)and normal prostate RWPE-1 cells were prepared,and the lyophilized powder of Qi Lan Decoction was made.Then four groups of PC-3,DU145,LNCaP and RWPE-1 cells were set up.The expression of miRNA in four groups of cell lines was detected by RT-PCR and the expression of PTEN in four groups of cell lines was detected by Western Blot.After the screening of prostate cancer cell lines,the target cell lines(DU145 cells)were treated with the lyophilized powder of Qi Lan Decoction.The expression of miR-1297,miR-153-5p and miR-21-5p in each group was detected by RT-PCR.Finally,searching the miRDB and Target Scan Human databases,retrieving the binding degree of miR-1297,miR-153-5p,miR-21-5p and PTEN,and considering the effect of Qi Lan Decoction on the expression of three miRNAs in DU145 cells,so we screened the target miRNA for subsequent experiments.2.In vitro and vivo study on the effect of Qi Lan Decoction on proliferation and apoptosis of DU145 cells.The CCK-8 experiment was used to observe the proliferation inhibition effect of different concentrations of Qi Lan Decoction on DU145 cells.Therefore,the high,medium and low concentrations of Qi Lan Decoction were selected for subsequent experiments.Flow cytometry was used to detect the effect of high,medium and low concentrations of Qi Lan Decoction on cell cycle and apoptosis of DU145 cells.Western blot was used to detect the expression of Cyclin D1,Bax,Bcl-2,and Cleaved-caspase 3,which are related to cell cyle and apoptosis,in order to further clarify the proliferation inhibition and apoptosis induction of DU145 cells by high,medium and low concentrations of Qi Lan Decoction.Then the effect of high,medium and low concentrations of Qi Lan Decoction on the growth of prostate cancer in vivo was observed by the tumorigenesis experiment of implantation in situ in nude mice.HE staining was used to observe the pathological changes of tumor tissue in each group.Western blot was used to detect the levels of Cyclin D1,Bax,Bcl-2,and Cleaved-caspase 3 in tumor tissue of each group.3.To explore the mechanism of Qi Lan Decoction on regulating the proliferation and apoptosis of prostate cancer cells in vitro based on miR-1297/PTEN/PI3K/AKT Signaling Pathway.This part was divided into six groups: negative control group(NC mimics),Qi Lan Decoction group(NC mimics+QLF),miR-1297 over-expression group(miR-1297 mimics),miR-1297 over-expression group+Qi Lan Decoction(miR-1297 mimics+QLF),AKT activator group(SC79),AKT activator group+Qi Lan Decoction(SC79+QLF).DU145 cells were transfected with miR-1297 mimics and sampled at 8h,24 h,48h and 72 h respectively.The protein expression level of PTEN at different time points after overexpression was detected by Western blot to determine the effective action time of miR-1297 mimics.Western blot was used to detect the expression of p-AKT in SC79 groups at different concentrations to determine the effective concentration and time of SC79 action.Then,RT-PCR and Western blot were used to detect the expression of miR-1297/PTEN/PI3K/AKT signaling pathway related genes and proteins in DU145 cells in each group.Finally,CCK-8,Ed U test and flow cytometry were used to detect the cell proliferation,cycle,apoptosis and other phenotypes in each group,and the expression level of phenotype-related proteins was also detected and analyzed.Results:1.Screening and validation of prostate cancer cell lines and miRNA:(1)Expression of miRNA in different cell lines.Compared with normal prostate RWPE-1 cells,the expression of miR-1297,miR-153-5p and miR-21-5p in prostate cancer DU145 and PC-3 cells showed statistical differences(P<0.05),and the expression of three miRNAs in PC-3 cells increased or decreased the most significantly.Compared with RWPE-1 cells,there was no statistical difference in the expression of miR-1297 on LNCaP cells of prostate cancer(P>0.05),but there was a statistical difference in the expression of miR-153-5p and miR-21-5p(P<0.05).Inter-group comparison showed that the expression of miR-1297 and miR-21-5p in PC-3 cell group increased or decreased significantly than that in DU145 cell group(P<0.05).(2)Expression of PTEN in different cell lines.PTEN protein was not expressed in PC-3 cells.PTEN protein was expressed differently in DU145,LNCaP and RWPE-1 cells,and the highest expression of PTEN was found in RWPE-1 cells.Compared with RWPE-1 cells,the expression of PTEN protein in DU145 and LNCaP cells decreased significantly(P<0.01).Compared with DU145 cells,the expression of PTEN protein in LNCaP cells was significantly increased(P<0.01).(3)Screening results of prostate cancer cells.According to the expression of miRNA and PTEN in different cell lines,the expression of miR-1297,miR-153-5p and miR-21-5p in prostate cancer DU145 and PC-3 cells was significantly different from that in normal prostate RWPE-1 cells,and PTEN protein was not expressed in PC-3 cells.This study supposed that PTEN might be the key factor of miRNA and PI3K/AKT signal pathway.In order to ensure the difference in the results of Qi Lan Decoction regulating PTEN protein in subsequent experiments,DU145 cell was finally selected as the target cell line for subsequent experiments.(4)Difference of miRNA expression after intervention of Qi Lan Decoction.Compared with the control,Qi Lan Decoction could significantly down-regulate the expression of miR-1297 on DU145 cells(P<0.01),and down-regulate the expression of miR-21-5p on DU145 cells(P<0.05).Although Qi Lan Decoction could up-regulate the expression of miR-153-5p on DU145 cells,it had no statistical significance(P>0.05).(5)Retrieving the miRNA combined with PTEN in the database.The miR-1297 and miR-153-5p can bind to PTEN,of which miR-1297 has the highest binding degree with PTEN,while miR-21-5p has no binding site with PTEN.(6)Screening results of miRNA.There is no binding site between miR-21-5p and PTEN,and the binding degree between miR-1297 and PTEN is the highest,and after intervention of Qi Lan Decoction,the expression of miR-1297 on DU145 cells can be significantly reduced(P<0.01),while the expression of miR-153-5p has little change(P>0.05),so miR-1297 is selected for subsequent experiments.2.In vitro and vivo study on the effect of Qi Lan Decoction on proliferation and apoptosis of DU145 cells:(1)When the concentration of Qi Lan Decoction is 100 μg/ml、200 μg/ml、400μg/ml,with the increasing concentration,the cell morphology gradually changed,especially after 48 h and 72 h of drug intervention,the cell growth,contour definition and adhesion ability were significantly inhibited or decreased.Compared with the cell morphology at 24 h and 48 h,400 μg/ml Qi Lan Decoction had the greatest effect on DU145 cells at 72 h.The cell growth was almost completely inhibited,and the cell morphology was seriously damaged.(2)In the CCK-8 experiment,Qi Lan Decoction was divided into 10 concentration groups(0,1.56μg/ml,3.125μg/ml,6.25μg/ml,12.5μg/ml,25μg/ml,50μg/ml,100μg/ml,200μg/ml,400μg/ml)to screen out high,medium and low concentrations for subsequent experiments.The results showed that when the concentration of Qi Lan Decoction was 0,1.56μg/ml,3.125μg/ml,6.25μg/ml,12.5μg/ml,25μg/ml,50μg/ml,100μg/ml,it had little effect on the survival rate of DU145 cells.When the concentration of Qi Lan Decoction was 200μg/ml,400μg/ml,the survival rate of DU145 cells decreased significantly,and 400μg/ml Qi Lan Decoction inhibited the proliferation of DU145 cells most significantly.Therefore,the high,medium and low concentrations of Qi Lan Decoction in subsequent experiments were selected as 400μg/ml,200μg/ml,100μg/ml.(3)After treatment of DU145 cells with Qi Lan Decoction,the number of cells in each concentration of drug group in G2 phase was significantly increased,while the number of cells in the Qi Lan Decoction group(200μg/ml and 400μg/ml)at S stage decreased significantly.Western blot found that compared with NC group(control group),the protein expression of Cyclin D1 in 400μg/ml Qi Lan Decoction significantly decreased(P<0.01),and the protein expression of Cyclin D1 in400μg/ml Qi Lan Decoction is lower than 100μg/ml and 200μg/ml Qi Lan Decoction(P<0.05).(4)The flow cytometry results showed that Qi Lan Decoction could significantly promote cell apoptosis,and with the increase of Qi Lan Decoction concentration,the cell apoptosis rate also increased significantly.When the concentration is 400μg/ml,the apoptosis rate reached the highest.Western blot results showed that compared with NC group(control group),the protein expression of Bcl-2in 200μg/ml and 400μg/ml Qi Lan Decoction decreased significantly(P<0.01).Compared with NC group(control group),the protein expression of Bcl-2 in400μg/ml Qi Lan Decoction was lower than 100μg/ml and 200μg/ml Qi Lan Decoction(P<0.01).Compared with NC group(control group),the protein expression of Bax,Cleaved-caspase 3 in 200μg/ml and 400μg/ml Qi Lan Decoction increased significantly(P<0.01).And the protein expression of Bax,Cleaved-caspase 3 in200μg/ml and 400μg/ml Qi Lan Decoction was higher than 100μg/ml Qi Lan Decoction(P<0.01).(5)Animal experiment:(1)HE staining results of tumor tissue.There was no obvious change in NC group(control group),while there were obvious tumor tissue growth in M group(control group),with a large number of tumor cell necrosis,large nuclear atypia,inflammatory cell infiltration(lymphocytes,neutrophils)and other pathological changes.Compared with M group(control group),a small amount of tumor cells were seen in QH group(high concentration group of Qi Lan Decoction);in QM group(middle concentration group of Qi Lan Decoction),with a large number of tumor cell necrosis,large nuclear atypia,and visible mitotic image;in QL group(low concentration group of Qi Lan Decoction),with a large number of tumor cell infiltration and necrosis,large nuclear atypia,and visible mitotic image.To sum up,the animal model in this study was successfully replicated,and QH group(high concentration group of Qi Lan Decoction)significantly alleviated the above pathological changes.(2)Western blot results showed that compared with NC group,the protein expression of Cyclin D1 and Bcl-2 in M group increased significantly(P<0.01),and the protein expression of Bax and Cleaved caspase-3 decreased significantly(P<0.01).Compared with M group,the expression level of Cyclin D1 and Bcl-2protein in each concentration group of Qi Lan Decoction decreased to varying degrees,while the expression of Bax and Cleared caspase-3 protein increased to varying degrees in a concentration-dependent manner,of which QH improved most significantly(P<0.01).3.To explore the mechanism of Qi Lan Decoction on regulating the proliferation and apoptosis of prostate cancer cells in vitro based on miR-1297/PTEN/PI3K/AKT Signaling Pathway:(1)The miR-1297 mimics was correlated with PTEN expression,and they were negatively correlated.In the six groups in this experiment,miR-1297 mimics+QLF referred to the intervention of Qi Lan Decoction when miR-1297 mimics is applied for 24 hours,and SC79+QLF referred to the observation of adding SC79 and Qi Lan Decoction at the same time,then changing the fresh drug-containing medium every24 hours after adding the medicine.(2)Expression of genes and proteins related to miR-1297/PTEN/PI3K/AKT signal pathway.Compared with NC mimics group,When miR-1297 is over-expressed by miR-1297 mimics,the expression of PI3 K and p-AKT protein increased significantly(P<0.01),while the expression of PTEN m RNA and protein decreased,indicating that the over-expression of miR-1297 decreased the expression of PTEN,and PTEN negatively regulated the PI3K/AKT pathway and increased its activity.Compared with the corresponding control group,Qi Lan Decoction could significantly increase the expression of PTEN m RNA and protein in DU145 cells(P<0.01),and significantly reduced the expression of PI3 K and p-AKT protein in DU145 cells(P<0.01),which might be related to Qi Lan Decoction regulating the expression of miR-1297 and inhibiting the activity of PI3K/AKT signaling pathway.(3)The results of CCK-8 experiment showed that compared with NC mimics group,the activity and increment rate of DU145 cells in NC mimics+QLF group decreased significantly at 48h(P<0.01);However,the activity and proliferation rate of DU145 cells in miR-1297 mimics group and SC79 group increased significantly at24 h and 48h(P<0.01).Compared with the miR-1297 mimics group,the activity of DU145 cells in the miR-1297 mimics+QLF group decreased significantly at 24 h and48h(P<0.01),and the proliferation rate of DU145 cells in the miR-1297 mimics+QLF group decreased significantly at 48h(P<0.01).Compared with SC79 group,the activity and proliferation rate of DU145 cells in SC79+QLF group decreased significantly at 48h(P<0.01).To sum up,Qi Lan Decoction could significantly inhibit the proliferation of prostate cancer DU145 cells after 48 h of intervention,and its effect on inhibiting the proliferation of DU145 cells might be related to regulating the over-expression of miR-1297 and inhibiting the activity of PI3K/AKT signaling pathway.(4)Ed U results showed that compared with NC mimics group,the rate of Ed U positive cells in NC mimics+QLF group decreased significantly(P<0.01).Compared with SC79 group,the rate of Ed U positive cells in SC79+QLF group was significantly reduced(P<0.01),indicating that the proliferation of DU145 cells in SC79 group could be significantly inhibited after adding Qi Lan Decoction.In conclusion,the inhibition of DU145 proliferation by Qi Lan Decoction might be related to the inhibition of PI3K/AKT signaling pathway activity.(5)The results of flow cytometry showed that compared with the corresponding control group,the proportion of G2 phase cells in DU145 cells treated by Qi Lan Decoction increased significantly(P<0.01),indicating that Qi Lan Decoction inhibited cell proliferation by regulating the cell cycle of DU145 cells.In addition,Qi Lan Decoction could significantly reduce the protein expression of Cyclin D1 in DU145 cells(P<0.01),suggesting that Qi Lan Decoction might affect the cell cycle of DU145 cells by regulating the protein expression of Cyclin D1,thereby inhibiting its proliferation.(6)The flow cytometry results showed that compared with the corresponding control group,the Q1-LR+Q1-UR increased significantly after the intervention of Qi Lan Decoction(P<0.01).In addition,compared with the corresponding control group,the protein expression of Bcl-2 was significantly decreased after the intervention of Qi Lan Decoction(P<0.01),and the protein expression of Bax and Cleaved caspase-3 was increased to different degrees(P<0.05 or P<0.01).Conclusion:1.Qi Lan Decoction could inhibit the proliferation of DU145 cells and promote the apoptosis of DU145 cells.2.Qi Lan Decoction could exert the anti-cancer effect through down-regulating the expression of miR-1297 and then inhibiting the activation of PTEN/PI3K/AKT signaling pathway.3.Qi Lan Decoction might inhibit the expression of miR-1297,increase the expression of PTEN,and reduce the phosphorylation of PI3K/AKT,then regulate the expression of Cyclin D1,Bcl-2,Bax and Cleaved-caspase 3,so as to achieve the purpose of inhibiting the proliferation of DU145 cells and promoting the apoptosis of DU145 cells. |
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