Study On The Effect And Mechanism Of CRYAB On The Function Of Prostate Cancer Cells

prostate cancer(PCa)is one of the most common malignant tumors in men,and its incidence increases with age.According to the 2020 Cancer statistics [1],prostate cancer is currently the first cancer incidence in males,and the second cancer mortality,which is a major issue plaguing male genitourinary health.Compared with the developed countries in Europe and the United States,the incidence of prostate cancer in China has been relatively low in recent years.However,in recent years,with the aggravation of population aging,the change of dietary structure and the popularization of prostate specific antigen(PSA)testing in China,the incidence of prostate cancer is continuing to rise rapidly.Moreover,the proportion of advanced prostate cancer in patients initially diagnosed is relatively high,and prostate cancer has become one of the important diseases affecting the health of Chinese men [2].Due to the great advances in systematic and personalized prostate cancer therapy in the past decades,patients with localized disease have better prognostic outcomes.However,metastasis,which is the main cause of death in prostate cancer patients,remains a great challenge.Therefore,clarifying the causes and molecular mechanisms of metastasis is of great significance for early detection,diagnosis and individualized treatment.In order to further explore the mechanism of the occurrence and development of prostate cancer,we downloaded the datasets GSE46602,GSE103512 and GSE69223 from the GEO database,and found 2 common down-regulated genes and 4 common up-regulated genes through pooling analysis.Univariate and multivariate COX regression analysis of the six prostate cancer-related genes screened out showed that only CRYAB had certain diagnostic and clinical significance.CRYAB expression was also found to be significantly correlated with Gleason score.It indicates that CRYAB may play an important role in the occurrence and development of prostate cancer.As a protein-coding gene,CRYAB is mostly studied on the function of its molecular chaperone.CRYAB is also a gene-coding protein that has been studied relatively early and its function is very clear,but there are few studies related to prostate cancer.Therefore,we identified it as the research objective.First,we verified that the expression of CRYAB in prostate cancer tissues was lower than that in adjacent prostate cancer tissues through TCGA database.Then we performed real-time PCR and Western Blot experiments on prostate cancer-related cell lines,and the results showed that,The expression of CRYAB in cancer cells(including 22RV1,DU45 and PC3)was lower than that in prostate epithelial cells RWPE-1,which was consistent with the results of database screening.Then the characteristics of CRYAB detected by real-time fluorescence quantitative PCR experiment were used.Two cell lines PC3 and DU145,which express relatively low levels of CRYAB,were selected for cellular functional verification.The expression of CRYAB was up-regulated by overexpression plasmid transfection in two CRYAB low expression cell lines,and then CCK8 proliferation assay,cell scratch assay,Transwell invasion and migration assay and terminal deoxynucleotidyl transferase d UTP Nick end labeling assay were performed respectively.The results showed that CRYAB inhibited the proliferation,migration and invasion of prostate cancer cell lines,and promoted the apoptosis of prostate cancer cell lines.Further real-time PCR experiments showed that when CRYAB expression was up-regulated,the expression of E-cadherin gene was decreased,and the expression of N-cadherin gene was increased.The expression of E-cadherin and N-cadherin was verified by Western blot.Taken together,our results demonstrated that CRYAB inhibited prostate cancer migration and invasion by inhibiting EMT.We have preliminarily demonstrated the role of CRYAB in the development of prostate cancer and found that CRYAB may be a potential molecular biological target for the diagnosis and treatment of prostate cancer.Part 1Screening and validation of CRYAB and its expression in prostate cancer cell linesObjective To screen out prostate cancer-related genes and verify their expression levels in prostate cancer cell lines.Methods(1)Data collection,data collection,and data collection.(2)bioinformatics.(3)Cell resuscitation,culture,passage and freezing.(4)RNA extraction.(5)Reverse transcription of RNA.(6)Real-time fluorescence quantitative PCR.(7)Western Blot assay.(8)Statistical methods.Results(1)The genes related to prostate cancer were screened by downloading the GEO database GSE46602,GSE103512 and GSE69223 datasets,including 4 common down-regulated genes and 2 common up-regulated genes.(2)Univariate and multivariate COX regression analysis showed that only CRYAB had certain diagnostic and clinical significance in prostate cancer,and the expression of CRYAB was significantly correlated with Gleason score.(3)We verified that the expression of CRYAB in prostate cancer tissues was lower than that in adjacent tissues in 499 prostate cancer tissues and 52 adjacent tissues from TCGA database.(4)The expression of CRYAB in cancer cells(including 22RV1,DU45 and PC3)was lower than that in prostate epithelial cells RWPE-1,as verified by real-time PCR and Western Blot.Conclusions The expression of CRYAB in prostate cancer tissues is lower than that in adjacent prostate cancer tissues,and the expression of CRYAB in prostate cancer cell lines(including 22RV1,DU45 and PC3)is lower than that in prostate epithelial cell RWPE-1.CRYAB is closely related to the occurrence and development of prostate cancer.Part ⅡEffect of CRYAB on the biological function of prostate cancer cellsObjective To investigate the effect of CRYAB on the biological function of prostate cancer cells.Methods(1)Cells were resuscitated,cultured,passaged and cryopreserved.(2)Cell transfection;(3)RNA extraction.(4)Reverse transcription of RNA.(5)Construction of overexpression plasmid.(6)Real-time fluorescence quantitative PCR.(7)Western Blot assay.(8)CCK8 cell proliferation assay.(9)Cell scratch test.(10)transwell chamber migration and invasion assay.(11)Terminal deoxynucleotidyl transferase d UTP Nick end labeling assay.(12)Statistical methods.Results(1)The CRYAB overexpression plasmid cell line was successfully constructed,which was verified by inverted fluorescence microscope,real-time fluorescence quantitative PCR and Western Blot.(2)Overexpression of CRYAB inhibited the proliferation of PC3 and DU145 cells.(3)Overexpression of CRYAB inhibited the migration and invasion of PC3 and DU145 cells.(4)Overexpression of CRYAB increased the ability of apoptosis in PC3 and DU145 cells.Conclusion CRYAB can inhibit the proliferation,migration and invasion of prostate cancer cells,and promote the apoptosis of prostate cancer cells.Part ⅢExploration of the mechanism of CRYAB on the function of prostate cancer cell linesObjective To explore the mechanism of CRYAB on the function of prostate cancer cell lines.Methods(1)Cells were resuscitated,cultured,passaged and cryopreserved.(2)Cell transfection;(3)RNA extraction.(4)Reverse transcription of RNA.(5)Real-time fluorescence quantitative PCR.(6)Western Blot assay.(7)Statistical methods.Results(1)The expression of CDH1 decreased and CDH2 increased after CRYAB overexpression.(2)After overexpression,the expression of E-cadherin decreased,and the expression of N-cadherin increased.Conclusion CRYAB can inhibit the migration and invasion of prostate cancer cells by inhibiting EMT.