| ObjectiveProstatic Cancer is the most common malignancy in the genitourinary system.The latest epidemiological statistics show that in recent years,with the aging of China's population and changes in dietary habits,the incidence of prostate cancer and the number of lethal cases in China are on the rise,and the incidence of the population is younger and the situation is grim.The onset of prostate cancer is often hidden,the clinical manifestations are lack of specificity,and the incubation period is long.Most patients are in the middle and late stage when more severe hematuria,urinary tract obstruction,bone pain and pathological fractures are diagnosed.The prognosis of the focal lesions after radical surgery is good,but the treatment is limited in the middle and late stage,and the prognosis is poor.Therefore,early detection,early diagnosis and early treatment are especially important for prostate cancer.However,there is still a lack of reliable biochemical indicators for early screening of prostate cancer and effective targeted treatment sites;therefore,in-depth exploration of the genesis and development of prostate cancer Mechanisms,the search for new screening indicators and targeted treatment sites have been hot topics in clinical and related researchThe ATPase 26S subunit(ATPase 2,PSMC2)is a protein-coding gene and is an essential component of the 19S regulatory granule of the 26S proteasome.It binds to ATP and nucleotides and has hydrolase and The function of nucleoside triphosphatase plays a key role in the selective degradation of proteins in the cell,and is involved in many biological activities such as regulation of cell differentiation and proliferation,apoptosis,energy metabolism and signal transduction Studies have shown that PSMC2 and 26S proteasomes are closely related to the occurrence and development of a variety of tumors,including breast cancer,hepatocellular carcinoma,esophageal cancer and pancreatic cancer,but there are no reports on PSMC2 and prostate cancerThis study is divided into three parts.The first part is to detect the expression of PSMC2 and analyze its clinical significance.The expression of PSMC2 protein in tissue samples was examined,and the relationship between PSMC2 and clinicopathological features of prostate cancer was analyzed,and its clinical significance was analyzed.At the same time,the expression level of PSMC2 mRNA in each prostate cancer cell line was detected,and the relationship between PSMC2 and prostate cancer was analyzed at the cellular level.The second part is the functional study of PSMC2.The effects of PSMC2 expression changes on proliferation,growth,apoptosis,two-dimensional migration and invasion ability and tumorigenic ability of prostate cells in PC-3 and DU145 were observed.The third part is the mechanism of PSMC2 regulating the development of prostate cancer and exerting its biological function.The upstream and downstream related protein molecules of PSMC2 were screened and treated with Akt inhibitor MK2206.The effects of PSMC2 expression and PI3K/Akt pathway inhibition on the expression of key proteins in prostate cancer cells were observed.The main regulation of PSMC2 in prostate cancer cells and its dependent transduction signaling pathway were analyzed.As preliminaryly revealing the mechanism of PSMC2 regulating the occurrence and progression of prostate cancer,it can provide potential biomarkers and molecular therapeutic targets for prostate cancer,providing new ideas for early diagnosis and targeted molecular therapy,providing theoretical basis and experiment for related researchMethod1.Specimen collection,cell culture and PSMC2 mRNA and protein expression detection:152 cases of cancer tissue specimens from patients undergoing radical resection of prostate cancer admitted to our hospital from January 2016 to December 2018 and matched cancers were collected.50 cases of normal tissues were detected by immunohistochemistry.The expression of PSMC2 protein in tissue samples was detected by immunohistochemistry.The cancer tissue group and the adjacent tissues group were set up.The cancer tissue group was subgrouped according to the clinical stage and pathological grade of the tumor,and analyzed.The relationship between PSMC2 and clinicopathological features of prostate cancer was analyzed.At the same time,prostate cancer cell lines PC-3,DU145 and C4-2 were cultured in vitro,and the expression level of PSMC2 mRNA in each prostate cancer cell line was detected by real-time fluorescent quantitative PCR.The relationship between PSMC2 and prostate cancer was analyzed at cellular level2.Cell model and animal model construction;PSMC2 gene knockdown prostate cancer cell model PC-3 and DU145 were constructed by plasmid construction and lentiviral transfection technique,and negative control group and PSMC2 knockdown group were set up by qRT-PCR and Western blot was used to detect the expression of PSMC2 mRNA and protein in cells,and the knockdown efficiency of PSMC2 was verified.Nude mice were housed in SPF-level conditions,and mouse xenograft models were established by subcutaneous injection of PC-3 cells and PSMC2 knockdownPC-3 cells.Negative control group and PSMC2 knockdown group were set up,and vernier calipers and small animals were used.The imaging system was used to observe the tumor formation and tumor growth in the animal model.The nude mice were sacrificed and the weight was weighed,and the expression of Ki-67 protein was detected by immunohistochemistry 3.Based on the above-mentioned PSMC2 knockout prostate cancer cell model PC-3 and DU145,the negative control group and the PSMC2 knockdown group were set up,respectively,using Celigo cell counting cell proliferation assay,plate cloning experiment,flow apoptosis experiment,scratch healing experiment,Transwell invasion experiment and xenograft experiment in nude mice.The impact of expression of PSMC2 in the proliferation,growth,apoptosis,two-dimensional migration,invasion ability and in vivo tumorigenic capacity of prostate cells PC-3 and DU145 were observed4.Based on the PC-3 cell model with down-regulated PSMC2 gene expression,human apoptotic antibody array chip was used to detect apoptosis-related proteins,such as pre-apoptotic proteins BIM,P21,anti-apoptotic proteins HSP27,IGF-II,Survivin.,STNF-R1,STNF-R2,TNF-?,TRAILR-4 and XIAP.The negative control group and the PSMC2 knockdown group were set in to analyze the effect of PSMC2 expression on the expression of apoptosis-related proteins and the key elements of death and the network of interactions.At the same time,combined with literature research and biosignal analysis,the possible signal transduction pathways of PSMC2 in biological function of prostate cancer were screened,and the expression of each key protein was detected by Western blot5.Construct a model of prostate cancer cells overexpressing PSMC2,and treat cells with Akt inhibitor MK2206.Set the group:Control-group:transfected negative control sequence cells;PSMC2-group:PSMC2 gene overexpression model cells;Control+group:transfection Negative control sequence cells+MK2206 treatment;PSMC2+group:PSMC2 gene overexpression model cells+MK2206 treatment.Western blot was used to detect the expression of key proteins Akt,p-Akt and apoptosis star protein Cyclin D1 and CDK6 in PI3K/Akt signal transduction pathway,and to observe the effect of PI3K/Akt pathway inhibition and PSMC2 expression upregulation on prostate cancer cell apoptosisResult1.Immunohistochemistry results showed that PSMC2 protein was localized on the cytoplasm and cell membrane of the cell membrane,showing a brownish yellow color.The positive expression rate and expression intensity of PSMC2 protein in prostate cancer tissues were significantly higher than those in adjacent normal tissues,and the expression of PSMC2 was higher in tumor tissues with higher Gleason score,indicating that PSMC2 was positively correlated with Gleason grade of prostate cancer.Spearman rank correlation analysis showed a significant correlation between PSMC2 expression and Gleason scores and grades.The results of qRT-PCR showed that PSMC2 was expressed at a higher level in prostate cancer cell lines PC-3,DU145 and C4-2,and the background expression levels were 8.29±0.34,8.96±0.37,and 7.71±0.28,respectively2.The results of cell model construction showed that the expression of PSMC2 mRNA in PSMC2 knockdown group was significantly lower than that in the negative control group,and the knockdown rate was 73.3%.The difference was statistically significant(0.268±0.021 VS)1.003±0.096,P<0.001);PSMC2 protein expression in PSMC2 knockdown group was significantly lower than that in negative control group,the difference was statistically significant(P<0.05).In DU145,PSMC2 mRNA expression in PSMC2 knockdown group was significantly lower than that in negative control group,and the knockdown rate was 65.9%,which was statistically significant(0.342±0.054 VS 1.004±0.097,P<0.001).The expression of PSMC2 protein in PSMC2 knockdown group was significantly lower than that in the negative control group,and the difference was statistically significant(P<0.05)3.Cell proliferation assay results of Celigo cell counting showed that the cell proliferation fold of PSMC2 gene knockdown group was significantly lower than that of control cells,and the difference was statistically significant(P<0.001).The results of plate cloning experiments showed that the number of cell clones in the PSMC2 knockdown group(164±11)was significantly lower than that in the control group(360±28),and the difference was statistically significant(P=0.0028)In DU145 cells,the number of cell clones in the PSMC2 knockdown group(210±13)was significantly lower than that in the control group(527±6),and the difference was statistically significant(p<0.001).The scratch healing results showed that the two-dimensional migration distance of PSMC2 gene knockdown group was significantly shorter than that of the control group at the beginning of 24 to 48 hours,and the difference was statistically significant(P<0.001)The results of Transwell invasion assay showed that the number of invasive cells in the PSMC2 knockdown group(225±1.34)was significantly lower than that in the control group(659±2.22),and the difference was statistically significant(P<0.001);In DU145 cells,the number of invasive cells in the PSMC2 knockdown group(45±4.89)was significantly lower than that in the control group(382±3.46),and the difference was statistically significant(p<0.001).Flow cytometry apoptosis assay showed that the apoptosis rate of PSMC2 knockdown group(15.56±0.52)was significantly higher than that of the control group(4.22±0.26)in PC-3 cells,the difference was significant.Statistically significant(P<0.001);in DU145 cells,the apoptosis rate of PSMC2 knockdown group(8.52±0.27)was significantly higher than that of the control group(3.63±0.46),with significant difference and academic significance(p<0.001).This indicates that PSMC2 has an anti-apoptotic effect on prostate cancer cells4.The results of tumor formation in nude mice showed that the growth curve showed that no obvious prostate cancer cell formation was formed in the nude mice of PSMC2 knockdown group The tumor formation in the control nude mice was significant,and the difference was statistically significant(P<0.001).In vivo imaging system detected very few prostate cancer cell-forming tissues in nude mice of PSMC2 gene knockdown group,and obvious tumors were detected in the control nude mice,the difference was statistically significant(P<0.001).After the nude mice were sacrificed,the tumors were removed,and no obvious tumors were found in the nude mice of the PSMC2 knockout group.More large tumors were found in the control nude mice,and the difference was statistically significant(P<0.001).Immunohistochemical results showed that Ki-67 protein expression was positive in the tumor body of nude mice in the control group,indicating that tumor cells implanted in the control group had good proliferation and the tumor-bearing results in vivo were reliable.In vivo studies have further demonstrated that knockdown of PSMC2 can significantly inhibit the tumorigenicity of prostate cancer cells.Immunohistochemical results showed that Ki-67 protein expression was positive in the tumor body of nude mice in the control group,indicating that tumor cells implanted in the control group had good proliferation and the tumor-bearing results in vivo were reliable.In vivo studies have further demonstrated that knockdown of PSMC2 can significantly inhibit the tumorigenicity of prostate cancer cells5.The results of human apoptotic antibody array chip showed that the levels of pre-apoptotic proteins BIM and P21 in PSMC2 knockdown group were significantly up-regulated compared with control cells,and the difference was statistically significant(P=0.0415,P=0.0127).3a);anti-apoptotic proteins HSP27,IGF-II,Survivin,STNF-R1,STNF-R2,TNF-?,TRAILR-4 and XIAP were down-regulated compared with the control group,and the difference was statistically significant(P=0.0358,P=0.0374,P=0.0122,P=0.0335,P=0.0294,P=0.0338,P=0.0457,P=0.0070).The results of Western blot showed that the levels of Akt,p-Akt,Cyclin D1,CDK6 and PIK3CA in PSMC2 knockdown group were significantly lower than those in control group,and the difference was statistically significant(P<0.05)6.Western blot analysis showed that the levels of p-Akt,Cyclin D1 and CDK6 in PSMC2 overexpressing group were significantly higher than those in the control group.After treatment with Akt inhibitor MK2206,the levels of p-Akt,Cyclin D1 and CDK6 in cells were detected Significantly down-regulated;while the inhibitor treatment was given,the expression of PSMC2 gene was up-regulated,and the levels of p-Akt,Cyclin D1,and CDK6 in cells were significantly up-regulated compared with those treated with inhibitor alone,and the difference was statistically significant(P<0.05).It is indicated that PSMC2 is dependent on the PI3K/Akt signal transduction pathway and promotes the proliferation and apoptosis of prostate cancer cells by promoting the expression of Cyclin D1/CDK6,thereby promoting the occurrence and progression of prostate cancerConclusion1.PSMC2 is highly expressed in prostate cancer tissues and cell lines,and its expression level is closely related to the clinicopathological features of prostate cancer,and positively correlated with Gleason grade of prostate cancer.It is a potential cancer-promoting gene for prostate cancer;2.PSMC2 promotes prostate cancer cell proliferation,colony formation,two-dimensional migration,invasion and tumor formation in vivo,and inhibits apoptosis3.PSMC2 may down-regulate the expression of pre-apoptotic proteins BIM and P21 and up-regulate the expression of anti-apoptotic proteins HSP27,IGF-II,Survivin,STNF-R1,STNF-R2,TNF-?,TRAILR-4 and XIAP.Thereby exerting an anti-apoptotic effect on prostate cancer cells.4.After PSMC2 gene knockdown,the expression of Akt,p-Akt,Cyclin D1,CDK6 and PIK3CA protein was decreased.The expression of Cyclin D1 and CDK6 protein was decreased in cells treated with Akt inhibitor MK2206,and Cyclin D1 was promoted by overexpression of PSMC2.,CDK6 expression and reversal of the inhibition of Akt inhibitors;suggest that PSMC2 regulates the development and progression of prostate cancer through the PI3K/Akt/Cyclin D1/CDK6 transduction pathway. |
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