Knockdown K-Ras Combined Drugs Delays The Malignant Behavior Of Castration-Resistant Prostate Cancer Through PLC?/pKC? Signaling Pathway

OBJECTIVE:As a common oncogene,K-Ras is often inseparable from the malignant behaviors of different types of tumors,but it is rarely studied in castration-resistant Prostate Cancer(CRPC).Our previous study found that the expression of H-ras and K-Ras increased in castration resistant prostate cancer,but the mechanism of the increase needs to be further explored.The purpose of this study is to explore the specific mechanism of K-Ras gene in CRPC,whether it is related to tumor progression,metastasis and drug resistance,and whether it can be used as a key molecule in the treatment of CRPC in clinical practice,and to provide a new scheme for the treatment of CRPC.Method:(1)Clinical data collection: tissue samples from 50 primary prostate cancer(PPC)patients and 41 CRPC patients were collected at the department of urology,the first affiliated hospital of chongqing medical university from January 2010 to July 2019.All subjects were informed of the purpose of the study and the procedures of the study.The CRPCpatients in our study were defined solely in accordance with the European urological association(EU)guidelines.This study retrospectively analyzed the patient's age,blood prostate-specific antigen(PSA)levels,metastasis,and drug resistance.Approved by the ethics committee of Chongqing medical university,and in accordance with the principles of the declaration of Helsinki.This topic by using immunohistochemical(IHC)and Western blot to detect prostate cancer tissues K-Ras,PLC epsilon,PKC epsilon protein expression levels of epsilon,statistical analysis of the change of protein levels of various genes and the correlation between each other,and analysis of K-Ras gene expression and the clinical characteristics,demographic characteristics and survival rate of statistical correlation analysis.(2)Cytological phenomena: culture of bicalutamide resistant cells(R-Bica cells)and bicalutamide + docetaxel dual resistant cells(R-B +D cells)based on LNCa P cells.The IC50 experiment was conducted to verify whether the two drug-resistant cell lines(R-Bica and R-B+D)were successfully constructed,and the drug concentration was obtained when the proliferation inhibition rate of different cell lines was at 50%.Meanwhile,the expression of K-Ras,PLC? and PKC? genes and proteins in LNCa P,R-Bica and R-B +D cells were detected by q PCR and Western blot.K-Ras expression in low-resistance cell lines(R-Bica,R-B+D)was knocked by three different small interfering rnas.The small interfering RNAs with thehighest knock efficiency were selected for follow-up study.IC50 was used to detect the sensitivity of cells to drugs in the cell lines with different treatments.Clone formation and cck-8 assay were used to detect cell proliferation in different treatment groups.BWB,invasion test and scratch test were used to detect the expression of metastasis and invasion related genes and the change of metastasis and invasion ability in K-Ras knockout group and control group,respectively.(3)Cytological mechanism study: sh-K-ras R-Bica,sh-K-Ras R-B+D cell lines with the highest k-ras inefficiencies and their corresponding NC(Negative Control)groups were selected,as well as untreated blank r-bica and r-b +D cell lines.(1)Western blot and q PCR were used to verify the changes in the levels of the downstream genes PLC?,PKC? and protein in the different treatment groups.(2)The expression of the downstream factor PLC? and PKC? was detected by Western blot and q PCR.The expression level of the membrane plasma protein of the downstream factor PLC? was used to locate the role of the downstream factor PLC? in the cells.(3)The co-precipitation assay(Co-Ip)confirmed the direct correlation between K-Ras,PLC? and PKC? at the protein level.Meanwhile,drug-resistant cells and untreated drug-resistant cell lines were selected for subsequent experiments.(4)The PKC? genes in drug-resistant cell lines R-bica and R-B +D were knocked down with three groups of PKC? small interfering RNA knocked down by Western blot and q PCR,and the group with thehighest efficiency was selected for subsequent experiments.(5)Gene and protein changes of transferrant-related factors such as VEGF,MMP2 and MMP9 were detected in drug-resistant cell lines with k-ras and PKC?knockdown.(6)Transwell and scratch test to detect its invasion and migration ability.(7)IC50 was used to test the drug sensitivity of various cell lines.(4)Drug study on inhibitor of K-Ras G12 C in vitro: Two groups of untreated drug-resistant cell lines(R-Bica and R-B+D)were selected.(1)Inhibitor9 and blank PBS were added into the medium.(2)Observe the changes in the gene levels and protein levels of the invasive-related factors MMP2,MMP9 and VEGF in the cell lines of each group.(3)CCK-8 was used to detect the inhibitory effect of inhibitor9 on the proliferation of drug-resistant cell lines and the most suitable concentration at the cytological level.(4)Through IC50 to observe the recovery of drug sensitivity after the use of inhibitors.(5)Xenograft experiment in nude mice: K-Ras inhibitor 9 was performed in vivo.Two drug-resistant cell lines(R-Bica and R-B+D)were selected for the experiment in male nude mice.Group of animals: male mice treated with inhibitor 9,Bicalutamide or Bicalutamide + Docetaxel were treated with PBS,and treated with placebo,bicalutamide or bicalutamide + docetaxel were treated with PBS:(1)The tail intravenous injection,build internal transfer model with flag fluorescent label tag twostrains resistant cells(R-Bica,R-B+D),will be grouped into mice: R-Bica(Inhibitor9),R-Bica(PBS),R-B+D(Inhibitor9),R-B+D(PBS),starting from 14 days to give Inhibitor9 tail intravenous injection,or PBS in 21 days after the administration through living imager,42 days to observe its internal transfer case.(2)Bone metastasis model: drug-resistant cell lines(R-Bica,R-B+D)were injected into the right tibial plateau of nude mice,which was consistent with the treatment method of the metastasis model.The invasion of tumor cells was observed by imaging examination at 42 days.The right tibia of nude mice was cut off,and the invasion of tissue specimens was observed after HE staining.(3)Subcutaneous tumorigenesis model: subcutaneous tumorigenesis was performed on the left side of the abdomen of nude mice respectively.The treatment method was consistent with the above model,and the size of subcutaneous tumorigenesis was observed in nude mice.RESULTS:(1)In CRPC tissues,the expression of K-Ras,PLC and PKC were upregulated to different degrees compared with that of PPC(P values were0.014,0.007,0.018,respectively).At the same time,there was a positive correlation between K-Ras and the expression levels of PLC and PKC proteins(PLC,r=0.4,P=0.0084),(PKC,r=0.42,P=0.0065).The expression of K-Ras,PLC and PKC in the tissues of 13 CRPC patients was detected by Western blot.The results were similar to those of immunohistochemistry.The expression levels of K-Ras and PLC and PKC were positively correlated(PLC?,r=0.58,P=0.04)and(PKC?,r=0.65,P=0.02).The demographic and clinical characteristics of the patients showed that the positive expression of k-ras was positively correlated with the metastasis of tumor patients(bone metastasis and visceral metastasis were respectively(P=0.01,P=0.018)in PPC patients and(P=0.009,P=0.046)in CRPC patients).Of 41 patients with CRPC survival analysis,through Kaplan Meier,median progression-free surial(recurrence-free survival,RFS)of K-Ras negative patients was 38 months,and K-Ras positive patients with median progression-free survival was 27 months,showed that K-Ras negative correlated to the RFS(P = 0.007).(2)By using IC50 of drug sensitivity of detecting various plant cells,according to the results than carew amine for drug-resistant cell line R-Bica cell inhibition rate of 50% was 105.9?M,more than carew amine + west he match on drug resistant cell lines R-B+D cell inhibition rate of 50% to111.5 ?M,its dosage is original LNCa P cell lines contrast carew amine(1.55?M)of 67.74 times,contrast carew amine+dorsey he(1.689 ?M)of68.45 times,drug resistant cell lines to build success.Western blot and PCR were used to detect the expression of K-Ras,PLC? and PKC? in LNCa P,R-Bica and R-B+D cells.The results showed that the expression of the three groups of genes and proteins in LNCa P cells was lower than that in R-Bica and R-B+D cells,and the results were statistically significant.TheIC50 results showed that the sensitivity of the two drug-resistant cell lines(sh K-Ras R-Bica and sh K-Ras R-B+D)that were knocked down by k-ras was significantly restored.The cell inhibitors of 50% were 36.09 ?M and45.49 ?M respectively,while the untreated drug-resistant cell lines were R-Bica(100.7?M)and R-B+D(113.3?M),respectively.At the same time,the proliferation of tumor cells that knock down K-Ras was inhibited by cloning and CCK-8 experiments.At the same time,the protein and gene expression of the transfer-related factors(MMP2,MMP9,VEGF)were all reduced.Meanwhile,Transwell and scratch experiments showed that the invasion and migration ability of the cells were decreased,and the results were statistically significant.(3)In the drug-resistant cell lines that knocked down K-Ras,the protein levels of the downstream target genes PLC? and PKC?,as well as the expression levels of the genes,were inhibited.We used K-Ras G12 C overexpression to verify whether this inhibition level could be reversed.(1)After the over-expression of K-Ras G12 C,RT-q PCR detection found that the expression level of K-Ras G12 C was increased;Western blot and RT-q PCR showed that the expression of PKC? was decreased in drug-resistant cells with PLC knocked down,and the expression of K-Ras G12 C in drug-resistant cells with PLC knocked down was reversed,suggesting that K-Ras may regulate the PKC? gene through PLC?.(2)The membrane localization of the PLC gene showed that the over-expression ofK-Ras G12 C caused the increase of the expression of the PLC gene located on the cell membrane,indicating that the activation of the PLC? gene was mainly realized by the translocation of the membrane.Co-immunoprecipitation(Co-Ip)results showed that k-ras directly bound to PLC? gene and PLC? gene and PKC? protein at the protein level,which proved that k-ras regulated PKC? through PLC on the cell membrane,and the three proteins were directly bound to each other at the protein level.(3)When PKC? gene was knocked down,the expression of factors related to invasion and metastasis of drug-resistant cell lines(VEGF,MMP2,MMP9)was down-regulated at the protein and gene levels.The results of the Transwell and wab-heal experiments suggested that the invasion and migration of CRPC cells decreased after PKC? was lowered.The results of IC50 indicated that the sensitivity of drug-resistant cell lines to drugs had been restored to a certain level.In the treatment group,sh-PKC r-bica(43.96 ?M)and sh-pkc R-B+D(51.25?M).Control group: R-Bica(103.1?M)and R-B+D(116.7 ?M).The above experiments confirmed that K-Ras can directly regulate the PLC? /PKC? pathway,and PKC? can regulate the malignant behavior of CRPC cells,suggesting that k-ras regulates the malignant behavior of CRPC cells through the PLC? /PKC? pathway.(4)Western blot and RT-q PCR showed that the expression of metastatic and invasive factors was down-regulated when R-Bica and R-B+D inhibitor K-Ras G12 C was added to the medium.CCK-8 detectionfound that the proliferation rate was inhibited with the increase of the drug concentration of the inhibitor.The test results of IC50 showed that the inhibitor could effectively increase the sensitivity of the drug(after adding the inhibitor in the experimental group,R-Bica(38.8 ?M)and R-B+D(50.10?M);Control group: R-Bica(103.6?M),R-B+D(111.5?M)).(5)In vivo transplantation of nude mice:(1)Visceral metastasis model:in both the inhibitor 9+ bicalutamide group and the inhibitor 9+bicalutamide + docetaxel group,the metastatic ability of tumor cells was significantly inhibited compared with that of drug-resistant cells-induced mice.In vivo imaging,metastasis was significantly reduced at 42 days compared with 21 days.(2)In the bone invasion group model,it can be seen on the X film that the invasive ability of tumor cells was significantly inhibited in both the inhibitor 9+ bicalutamide group and the inhibitor 9+bicalutamide+docetaxel group,and the periosteum of the experimental group was present,while the periosteum of the control group was unclear.HE staining showed that the tumor cells in the control group had penetrated through the periosteum,while the experimental group did not completely penetrate the penetrating cortex into the medullary cavity.(3)In the two experimental groups treated with inhibitor 9,the tumor size was smaller than that of the control group,and the results were statistically significant.Conclusion:This study investigated the role of K-Ras in the regulation of CRPCcell metastasis and drug resistance through the PLC /PKC signaling pathway.At the same time,it was revealed that the inhibitor of K-Ras G12 C could restore the sensitivity of CRPC cells to drugs,reduce the metastasis and invasiveness of drug-resistant cells,and reduce the degree of malignancy.An inhibitor of K-Ras G12 C has entered clinical trials and is expected to be a promising new treatment option for aggressive prostate cancer.