| Background:Prostate cancer is one of the most common malignant tumors in males,androgen deprivation therapy（ADT）is an important tool in the treatment of early prostate cancer,but 80%to 90%of patients progress to Castration-Resistant Prostate Cancer（CRPC）after ADT.Metastasis and drug resistance are the main causes of death in CRPC patients.Studies have shown that the main mechanism of malignant progression of prostate cancer is reactivation of the androgen receptor signaling pathway after ADT.The two currently recognized pathways are the acquisition of the ability of prostate cancer cells to synthesize androgens on their own and the production or increase in androgen splice variants.Both activate the androgen signaling pathway at the ligand and receptor levels,respectively,which promotes malignant progression of prostate cancer.Current studies have confirmed that aldo-keto reductase AKR1C3 is a key enzyme in the synthesis of androgens by prostate cancer cells via cholesterol,and that prostate cancer cells adaptively overexpress AKR1C3 and gain the ability to synthesize androgens after ADT.However,the existence of a mechanism by which AKR1C3 contributes to the malignant progression of prostate cancer by a non-enzymatic pathway is not yet clear.Androgen receptor splice variant 7（AR-V7）increases in positivity or expression abundance with the use of anti-androgen drugs,AR receptor antagonists.AR-V7 also has intrinsic activity to naturally activate the androgen receptor signaling pathway in the absence of androgens.Therefore,the overexpression of AKR1C3 and the generation/increase of the androgen receptor splice variant AR-V7 are two major drivers of prostate cancer progression.Recently,it was found that AKR1C3 and AR-V7 were positively expressed simultaneously in some patients with metastatic CRPC,and it was also reported that AKR1C3 had a stabilizing effect on AR-V7 protein,which suggests that the two of them may have a regulatory relationship.However,in addition to the above regulatory mechanisms in the protein degradation pathway,whether there is a loop regulatory mechanism between the two of them is still unclear.The aim of this study was to explore the regulatory role and mechanism between AKR1C3 and AR-V7,and to lay a theoretical foundation for seeking new mechanisms and targets to inhibit the malignant progression of prostate cancer.The therapeutic effects of double silencing of AKR1C3 and AR-V7 synergised with enzalutamide were explored in vivo and in vitro to provide a new strategy for the combination therapy of malignant prostate cancer.Methods:（1）Bioinformatics screening of differential genes in prostate cancer progression dataset and selection of intersecting genes.To assess the expression level of AKR1C3 in prostate cancer patients at different stages of progression and the relationship with prognosis.Cellular experiments were performed to verify the molecular function of AKR1C3.Proteomics was used to screen the differential proteins between LNCa P-AKR1C3 and parental cells.Construct PPI network to screen core proteins.One-way Cox analysis was performed on the core proteins using TCGA database as a training set to screen the risk proteins and construct a risk model.Two external datasets of GEO were applied to validate the reliability of the model and confirm the role of AKR1C3 in the malignant progression of prostate cancer.（2）To determine the regulatory mechanism of AKR1C3 on AR-V7,we used plasmid overexpression and siRNA silencing,cat RAPID prediction and rescue experiments in CRPC cells.JASPAR database prediction,luciferase reporter genes,and chromatin immunoprecipitation（Ch IP）experiments were performed to clarify the direct regulatory role of AR-V7 on AKR1C3.Protein expression of AKR1C3 and AR-V7 was detected in tissue samples from prostate cancer patients by IHC.（3）We constructed dual siRNA nanoparticles（siA-A@PP）and observed the serum,RNAase,and p H stability of siA-A@PP by agarose gel electrophoresis.The effects of siA-A@PP and synergistic enzalutamide were confirmed in vitro and in subcutaneous xenograft tumor model.Results:（1）Bioinformatics results showed that AKR1C3 was a common highly expressed differential gene in the dataset of three types of prostate cancers,namely,castration-resistant,metastatic,and drug-resistant.TCGA database searches showed that the expression level of AKR1C3 was strongly correlated with lymph node metastasis in patients,and with the Gleason score.Kaplan-Meier survival analysis showed that the patients with high expression of AKR1C3 had a significantly lower overall survival was significantly lower and was highly expressed in patients who died.Results showed that AKR1C3 promoted the proliferation of LNCa P cells and reduced sensitivity to enzalutamide,promoted the migration of LNCa P and 22RV1cells.Silencing of AKR1C3 restored the sensitivity of 22RV1 cells to enzalutamide and attenuated the migration ability in vitro.A PPI network was constructed for 484up-regulated differential proteins and topological analysis was performed to identify 18 hub proteins.One-way Cox analysis with risk ratio>1 and P<0.05 as the judgement indicator identified CDC20,SRSF3,UQCRH,INCENP,TIMM10,TIMM13,POLR2L and NDUFAB1 as the 8 risk genes associated with AKR1C3.In the training set with two validation sets,GSE116918 and GSE54460,the patient mortality and the expression of the 8 risk genes increased with the increase of the risk value.Kaplan-Meier survival analysis showed that patients in the high-risk group had a poor prognosis.Time-dependent ROC curves showed that the model was better at predicting patient prognosis in the long term than in the short term.Expression of SRSF3,CDC20,and INCENP among the 8 risk genes was positively correlated with the IC50of androgen receptor blockers.（2）AKR1C3 regulates AR-V7 production through SRSF3 at the splice level.After blocking androgen signaling with enzalutamide in LNCa P cells,m RNA expression of AR-V7 and AKR1C3 was simultaneously upregulated.The protein level expression of AR-V7 was upregulated after overexpression of AKR1C3 in22RV1 and VCa P cells.The protein level expression of AR-V7 was similarly upregulated after overexpression of AR-V7.These results suggest that AKR1C3and AR-V7 may regulate each other.In addition,protein expression of SRSF3 was increased after overexpression of AKR1C3 in 22RV1 and VCa P cells.The protein level expression of AR-V7 was also increased after overexpression of SRSF3.cat RAPID software predicts that SRSF3 has a binding site to the intron between pre-m RNA exon 3 and specific exon 3B of AR.Silencing of SRSF3 in 22RV1 cells did not affect the m RNA levels of AR-FL,AR-V1,AR-V3,and AR-V4,whereas the down-regulation of m RNA and protein levels of AR-V7.The above results suggest that AKR1C3 regulates AR-V7 generation at the splice level through SRSF3.AR-V7 transcriptionally regulates AKR1C3 expression.m RNA expression level of AKR1C3 was elevated after overexpression of AR-V7 in 22RV1 cells.JASPAR database predicted that AR（AR-V7）has a binding site with the promoter of AKR1C3.The results of luciferase reporter assay showed that AR-V7 could bind to the promoter region of AKR1C3 to promote the increase of luciferase activity.Ch IP results showed that AR-V7 mainly binds to the promoter region of AKR1C3-437 bp～-423 bp.This indicates that AR-V7 regulates AKR1C3 expression at the transcriptional level.（3）We successfully constructed dual gene silencing nanocomplex siA-A@PP and enhanced drug sensitivity to enzalutamide in drug-resistant prostate cancer cells to enzalutamide.PAMAM-PEG binded to siRNA with an optimal N/P of 40and was not cytotoxic.siA-A@PP was stable in serum,RNAase,and p H environments.In vitro,sensitivity of CRPC cells to enzalutamide could be further increased compared to single-target silencing.In vivo,the tumour inhibition rate was 23%for siA-A@PP and 55%for synergistic enzalutamide.There was no significant differences in mouse body weights were observed through the treatment course.HE results showed that siA-A@PP with enzalutamide led to a decrease in mitotic figures and an increase in necrotic area of tumour tissues.The IHC results showed that siA-A@PP was able to reduce the expression of AKR1C3 and AR-V7.Conclusion:（1）AKR1C3 promoted prostate cancer proliferation,metastasis and enzalutamide resistance.AKR1C3-associated 8-gene risk model could effectively predict the prognosis of prostate cancer patients.（2）AKR1C3/AR-V7 formed a positive feedback loop.AKR1C3 could specifically regulate AR-V7 expression through the splicing factor SRSF3.AR-V7functions as a transcription factor that facilitates the transcription of AKR1C3,thereby establishing a positive feedback loop that contributes to the malignant progression of prostate cancer.（3）siA-A@PP had better safety and stability.siA-A@PP could increase the sensitivity of drug-resistant cells to enzalutamide in vitro and in vivo. |
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