Mechanism Of YWHAG Gene Promoting Cell Proliferation And Autophagy Induction In Prostate Cancer

YWHAG is one of our screens out new high copy number and expresses significant differences genes based on the prostate sequencing dataset in the TCGA database and ONCOMINE database.that located at chromosome 7q11.23.The NCK1-AS1 gene was found to be transcribed according to ENCODE chromosome modification data and ChromHMM chromosome state segmentation data.We founded that YWHAG was over-expressed in prostate cancer(PCa)tissue compared with Benign prostatic hyperplasia tissue,indicate YWHAG can promote the development of PCa,and however,The underlying mechanism of action remains unclear.in this study,we investigate the expression of YWHAG,the function and its molecular mechanism in PCa.Part 1: The expression of YWAHG and clinical significance analysis in PCaObjectives: To detect the expression of YWHAG in PCa tissues and BPH tissues.The relationship between YWHAG expression level and clinicopathologic features was analyzed.Methods: Using the ONCOMINE database and the TCGA database to find genes related to prostate cancer proliferation,using gene expression profiling for differential analysis.Detecting the expression level of YWHAG in tissues and in prostate cancer cells by qRT-PCR,WB.Cellular immunotechnology is used to detect YWHAG protein localization.statistical analysis of the relationship between YWHAG and patient age,pathological stage,tumor size,and lymph node status.Results: A total of 20 genes related to prostate cancer proliferation and clinical stage were obtained by cross-alignment analysis of the TCGA database and the GSE6099 dataset.The results of qRT-PCR and WB showed that YWHAG was highly expressed in prostate cancer tissues(*P <0.05);IHC results showed that YWHAG was mainly located in the cytoplasm and basement membrane of prostate gland epithelial cells;YWHAG expression level and clinical index analysis The results showed that the expression level of YWHAG in prostate cancer was significantly correlated with clinical stage(*P <0.05)and lymph node metastasis(**P <0.01).Conclusion: The expression level of YWHAG in prostate cancer was significantly higher than that in benign prostatic hyperplasia and normal prostate tissue In multiple databases and clinical samples,and its expression level was correlated with clinical detection of prostate cancer patients.It is our newly identified biomarker for prognostic evaluation of prostate cancer.Part 2: YWHAG promotes cell proliferation in prostate cancerObjectives: The upstream promoter region of YWHAG gene and its regulatory mechanism were clarified,and the effects of YWHAG on the cycle,proliferation,colony formation and invasion of prostate cancer PC3 and DU145 cells were detected.YWHAG normal group and low expression stable transformed group were injected into nude mice.Detection of the difference in tumorigenic ability of prostate cancer cells.Methods: The luciferase recombinant plasmids with different truncated lengths in the 1500 bp region upstream of YWHAG were constructed,and the luciferase activity in different regions was obtained.The position of the YWHAG core promoter region was analyzed.qRT-PCR and WB detect the expression of YWHAG in prostate cancer cells PC3,DU145 and LNcaP;after silencing and overexpressing YWHAG,FCM,CCK-8,Edu and transwell detect cell cycle,cell proliferation,DNA synthesis and cells,respectively Invasion ability;plate cloning assay to detect the effect of YWHAG on cell colony formation ability,tumor formation assay in nude mice to detect the effect of YWHAG on cell tumor formation ability.Results: qRT-PCR and WB detected the expression of YWHAG in PC3,DU145,LNCaP and RWPE-1 cells,and found that YWHAG was highly expressed in both PC3 and DU145 cells.ChIP and qRT-PCR experiments confirmed that the transcription factor SP1 binds to the YWHAG promoter region,and SP1 directly regulates YWHAG expression.We used siRNA technology to silence YWHAG in PC3 and DU145 cells.The results of CCK8 assay showed that silencing YWHAG inhibited the proliferation of prostate cancer cells.The results of FCM showed that the number of cells in S phase was significantly decreased after silencing YWHAG,and the number of cells in G1 phase was significantly increased.Edu detection of silencing YWHAG inhibited DNA synthesis in prostate cancer cells and delayed cell in G1/S conversion.Process.Nude mice were injected subcutaneously into the normal control group and PC3 cells stably expressing YWHAG.Compared with the control group,the tumorigenic ability of the low expression group was significantly weakened.Conclusion: The transcription factor SP1 binds directly to the YWHAG promoter region and regulates its expression.YWHAG is abnormally highly expressed in prostate cancer and drives the cell cycle and promotes cell proliferation.Part 3: YWHAG induces autophagy in prostate cancer cellsObjectives: The expression of autophagy marker proteins LC3,P62,ATG5 and Beclin 1 in YWHAG control group,silencing group and overexpression group were examined to investigate the role of YWHAG in autophagy of prostate cancer cells and its molecular mechanism.Methods: The effects of silencing or over-expressing YWHAG on autophagy of prostate cancer cells were detected by WB and CY3-LC3;statistical analysis of the expression of YWHAG and LC3-I(LC3A)and LC3-II(LC3B)in the PRAD dataset of TCGA database;electron microscopy The autophagic vesicles were observed in different treatment groups.The autophagic flow was detected by confocal microscopy after transfecting prostate cancer cells with mRFP-GFP-LC3 double-labeled plasmid.The YWHAG treatment group was treated with chloroquine.The effect of LC3 protein expression;Si RNA interfered with the autophagy-associated gene ATG5,and the plate clone assay was used to detect the colony forming ability of prostate cancer cells;WB detected the change of the marker protein of autophagy pathway in prostate cancer cells after silencing or over-expressing YWHAG.Results: The WB results showed that the proportion of LC3 B in the YWHAG overexpression group was increased to LC3 B,the number of LC3-II accumulation in the silencing group was significantly decreased,and the expression levels of Beclin 1 and ATG5 protein were consistent with LC3 B,while the expression of P62 in each group was opposite to that of LC3 B.Immunofluorescence assay showed that LC3 protein in cells showed dot-like aggregation in the cytoplasm.Compared with the silencing group,LC3 aggregation in the normal group and over-expressed group was more obvious.TCGA data correlation analysis YWHAG was negatively correlated with LC3A(R=-0.16,P=0.00054),YWHAG was positively correlated with LC3B(R=0.33,P=8.5e-14);electron microscopy showed that the number of autophagic vacuoles in the silent group was significantly lower than that in the control and overexpression groups(p<0.05).Confocal microscopy showed that the intensity of autophagic flow in the overexpressed group was significantly higher than that in the control group,while the autophagy was blocked in the silencing group,and the occurrence of autophagy was not smooth;YWHAG overexpression group,overexpression combination The expression of LC3 in the green quinone co-incubation group was significantly higher than that in the DMSO-treated group(**p<0.01),and the expression level of LC3 in the YWAHG-silencing group was lower than that in the control group(*p<0.05);cell clones after silencing YWHAG and ATG5 Force decreased significantly(* p <0.05);YWHAG by the PI3K-AKT-mTOR signaling pathway in prostate cancer cells induced autophagy.Conclusion: This study found that silencing YWHAG promoted the expression of PI3 K type III phosphorylation and induced phosphorylation of AKT and mTOR,while the expression level of ATG5 was up-regulated in YWHAG overexpression group.Upregulation of ATG5 increased LC3 A to LC3 B cleavage and induced protection.The occurrence of autophagy eventually promotes the survival of prostate cancer cells.Part 4: Molecular Mechanism of miRNA 144 /YWHAG/CDK2Regulating Cycle and induced Autophagy in Prostate CancerObjectives: To determine the molecular mechanism of YWHAG in regulating prostate cancer cell cycle and proliferation.Methods: Gene expression profiling chip was used to detect and analyze the differentially expressed genes after silencing YWHAG,GO and pathway analysis of the biological functions and signaling pathways involved in differential genes;WB,qRT-PCR and dual luciferase experiments verified YWHAG,miR-144 and CDK2 interaction between the three.RESULTS: We used VAHTSTM mRNA-seq V2 Library Prep KIT for Illumina to detect differential gene expression after silencing YWHAG,and analyzed gene expression differences using Transcriptome Analysis Console and DEGseq software.The screening conditions were p<0.05 difference fold>2.0.A total of 2111 significant differential genes were detected;the biological functions involved in differential genes were analyzed by GO and pathway;the differential genes were mainly involved in the PI3K-AKT signaling pathway(PI3K-Akt signaling pathway)and the cancer signaling pathway(Pathways in cancer).Biological processes such as the FoxO signaling pathway,the Sphingolipid signaling pathway,the ErbB signaling pathway,and the cell cycle.Among them,PI3 K,AKT and CDK2 are involved in several major biological processes and signaling pathways as significant differentially expressed genes.Bioinformatics predicted the binding site of YWHAG to miRNA-144.The activity of wild-type YWHAG luciferase was significantly attenuated after PC3 cells transfected with miR-144 mimics,suggesting that miR-144 can affect the cycle of prostate cancer cells by regulating YWHAG expression.,proliferation and invasion ability.Conclusion: Gene expression profiling results indicate that PI3K/AKT/CDK2 is involved in multiple biological processes including autophagy and cancer after silencing YWHAG;miR-144 positive feedback regulates YWHAG expression,and low expression of miR-144 promotes prostate The cycle and proliferation of cancer cells.