The Effect Of LncRNA NCK1-AS1 Abnormal Expression On Prostate Cancer And Its Molecular Mechanism

Prostate cancer(PCa)is one of the common malignant tumors in men.Its development is closely related to external environmental factors and internal cellular factors.With the development of gene sequencing research,the proportion of non-coding region RNA in transcriptional genes has been paid more and more attention by researchers.Long non-coding ribonucleic acid(LncRNA)is a kind of non-coding region RNA,which participates in various physiological and pathological processes including human cancer,metastasis,invasion,cell differentiation and apoptosis.Although it cannot be directly translated into protein,it can regulate the expression of genes in multiple layers such as transcription,post-transcriptional level,epigenetics,and cell differentiation.lncRNA NCK1-AS1 is one of the lncRNAs that are abnormally expressed in many cancers and plays an important role in biological functions such as tumor proliferation and metastasis.A variety of LncRNAs can interact with TGF-?1 signaling pathway proteins to affect tumor cell proliferation,migration,invasion,and even drug resistance.However,the clinical role and potential biological functions of lncRNA NCK1-AS1 in prostate cancer are still unknown.Therefore,the aim of this study was to investigate the role and molecular mechanism of abnormal expression of lncRNA NCK1-AS 1 in prostate cancer.The experiment consists of the following three parts:Part ?:NCK1-AS1 distinguishes PCa patients from BPH patientsObjective:To investigate the expression of NCK1-AS1 in plasma of PCa,BPH and healthy men.Methods:?The plasma of PCa patients(n=60),BPH patients(n=58)and healthy males(n=60)were collected in this study.The quantitative analysis of NCK1-AS1 in each group was performed by fluorescence quantitative qRT-PCR and analyzed expression data by performing one-way ANOVA and Tukey test.?ROC curve analysis was performed based on the mRNA level of NCK1-AS1 to evaluate the diagnostic value of plasma NCK1-AS1 for PCa.PCa patients were taked as true positive cases,BPH patients were true negative cases,and ROC curves were drawn.The PCa patients were considered as true positive cases,and the healthy males were true negative cases,and the ROC curve was drawn.?According to the patient's clinical data,analyze the relationship between the expression of NCK1-AS1 and the patient's Gleason score and clinical stage.Results:?The plasma level of NCK1-AS1 in PCa group was significantly higher than the other two groups,the difference was statistically significant(p<0.05).There was no significant difference between BPH patients and healthy men(p>0.05).(2)PCa patients were taked as true positive cases,BPH patients were true negative cases,the area under the ROC curve(AUC)was 0.95,the standard error was 0.017,and the 95%confidence interval was 0.92-0.99.PCa patients were considered as true positive cases,and healthy males were true negative cases.The area under the curve was AUC of 0.95,the standard error was 0.018,and the 95%confidence interval was 0.92-0.98.? With the increase of Gleason score,the level of NCK1-AS1 increased slightly,but it was not statistically significant(p>0.05).With the increase of clinical stage,the level of NCK1-AS1 increased slightly,and the change of stage ? was the most obvious,but the statistical analysis showed that the results were not statistically significant(p>0.05).Conclusions:The NCK1-AS1 in plasma can effectively distinguish prostate cancer patients from benign prostatic hyperplasia and healthy men,and has potential clinical application value as a diagnostic marker of prostate cancer.Because the level of NCK1-AS1 is not a good indicator of the clinical stage of prostate cancer,it needs further study as a prognostic indicator after prostate cancer surgery.Part ?:Effects of abnormal expression of NCK1-AS1 on PCa cellsObjective:To explore the effects of overexpression of NCK1-AS1 on the migration and invasion ability of DU145 and 22Rv1 cells.Methods:?Fluorescence real-time quantitative PCR was used to detect the expression level of NCK1-AS1 in DU145,22Rv1 and RWPE-1 cells.?To construct DU145 and 22Rv1 cells overexpressing NCK1-AS1,and to detect the migration ability of DU145 and 22Rv1 cells and DU 145 and 22Rv1 cells overexpressing NCK1-AS1(without Matrigel)and invasive ability(including Matrigel)by Transwell method.Results:?The expression level of NCK1-AS1 in DU145 cells was 2.7 times higher than that in RWPE-1 cells,and the expression level of NCK1-AS1 in 22Rv1 cells was 3.3 times higher than that in RWPE-1 cells(p<0.05).?The migration ability of DU145 and 22Rv1 cells overexpressing NCK1-AS1 was significantly higher than that of DU145 and 22Rv1 cells,and DU 145 and 22Rv1 cells(NC group)transfected with empty vector.The difference was statistically significant(p<0.05).The results of invasiveness test showed that the invasion of DU145 and 22Rv1 cells overexpressing NCK1-AS1 in DU145 and 22Rv1 cells(NC group)transfected with empty vector was significantly increased,and the difference was statistically significant(p<0.05).Conclusions:NCK1-AS1 was highly expressed in prostate cancer cells DU145 and 22Rv1,which were significantly higher than normal prostate cells.The migration and invasion ability of DU145 and 22Rv1 cells overexpressing NCK1-AS 1 were stronger than that of normal DU145 and 22Rv1 cells,and NCK1-AS1 could promote the invasion of prostate cancer cells.Part ?:NCK1-AS1 regulates PCa cells through TGF-?1Objective:To investigate the role of TGF-?1 in promoting PCa cell invasion by NCK1-AS1.Methods:?The expression of transforming growth factor-?1(TGF-?1)in plasma of PCa patients was detected by enzyme-linked immunosorbent ELISA.The correlation between TGF-?1 and NCK1-AS1 in plasma of PCa patients was analyzed by linear regression.?The diagnostic value of TGF-?1 for PCa was analyzed by ROC.PCa patients were regarded as true positive cases,BPH patients were true negative cases,and ROC curves were drawn;PCa patients were treated as true positive cases,and healthy males were used as true negative cases,and ROC curves were drawn.?The NCK1-AS1 and TGF-?1 expression vectors were constructed,and the protein levels and mRNA levels of TGF-?1 in DU145 and 22Rv1 cells overexpressing NCK1-AS1 were detected by Western blot and RT-PCR,respectively.The level of NCK1-AS1 in TGF-?1 overexpressing DU145 and 22Rv1 cells was detected by RT-PCR.?The migration(without Matrigel)and invasive ability(including Matrigel)of NCK1-AS1 and TGF-?1 cells were detected by Transwell method.Results:?TGF-?1 and NCK1-AS1 showed a significant positive correlation(p<0.05).?Patients with PCa were regarded as true positive cases,BPH patients were true negative cases,and ROC curves were drawn.The area under the statistical analysis curve was 0.96(standard error:0.017;95%confidence interval:0.92-0.99).The PCa patients were treated as true positive cases,and the healthy males were true negative cases.The ROC curve was plotted.The area under the statistical analysis curve was 0.93(standard error:0.019;95%confidence interval:0.91-0.97).?Compared with the control group or cells transfected with empty vector,TGF-?1 was significantly up-regulated in mRNA and protein levels in PCa cells with overexpression of NCK1-AS1,and the difference was statistically significant(p<0.05).There was no significant difference in NCK1-AS1 in PCa cells with overexpression of TGF-?1 compared to the control group or cells transfected with empty vector(p>0.05).?The results showed that the invasiveness and migration of DU145 and 22Rv1 cells overexpressing NCK1-AS1 and TGF-?1 were increased compared with the control group or cells transfected with empty vector,and the difference was statistically significant(p<0.05).After treatment with TGF-?1 inhibitor SB431542(10 nM,SB,Sigma-Aldrich)for 24 hours,the ability of cell migration and invasion with overexpression of NCK1-AS1 was weakened,and the difference was statistically significant(p<0.05).Conclusions:NCK1-AS1 is specifically up-regulated in PCa patients and PCa cells,and promotes migration and invasion of PCa cells by regulating TGF-?1.