Clinical And Experimental Study On Correlation Between The Expression Of MicroRNA-21 And Clinicopathological Features As Well As The Prognosis Of Prostate Cancer

Study Background and SignificanceProstate cancer is the most commonly malignant cancer among men worldwide.There are false positive results and false negative results of PSA screening.Therefore,it is of great importance to find proper prostate cancer biomarkers to help diagnose this disease in early stage.RNA biomarker is a novel type because it can be detected conveniently in blood and reflect disease progression more accurately.MicroRNA-21(miR-21)plays a pivotal role in cancer cells migration,proliferation and invasion via regulating numerous down-stream target genes.It has been clear that miR-21 participates prostate cancer pathogenesis,however most studies focus on miR-21 expression in prostate tissues not in the circulating system.The disadvantage of detecting tissue miR-21 expression is that the tissue sample obtains from invasive operation,thus causing physical injury and making it difficult to get continuous data.Hereby,a reliable detection strategy is highly need,which is more sensitive,more accessible in a non-invasive manner,more consistent with real clinical progress,and more convenient to detect.Prostate cancer in early stage is occult,and progresses to distant metastasis,particularly bone metastasis in advanced stage.Therefore,to investigate the mechanism of prostate cancer metastasis,and find potential treatment target are of the great importance for reducing prostate cancer-related mortality.miRNA is one of the prostate cancer research focus,and various studies have suggested a link between prostate cancer and miRNAs,including miR-21.Although great progress has been made in prostate cancer study,it still remains further understanding of its complex metastasis mechanism.This study is consist of two parts.In the first part,we observed expression of peripheral blood mononuclear cell(PBMC)miRNA-21 in prostate cancer patients,analyzing the association between miR-21 level and clinical characteristics including TNM stage and risk of disease.We also discussed whether miR-21 level was associated with diagnosis and risk of biochemical recurrence after radical prostatectomy.In the second part,we investigated how miR-21 loss could affect metastasis of prostate cancer in order to further understanding the role of miR-21 in progression of prostate cancer.We detected expression of miR-21 in prostate cancer cells and normal prostatic epithelial cells.Then we investigated the effect of down-regulated miR-21 on proliferation,invasion and migration of prostate cancer in vitro,as well as related proteins expression.Part IStudy Objectives:To investigate the relationship among miR-21 expression in PBMC and clinicopathological features and biochemical recurrence of prostate cancer,which will provide compelling evidence to support the opinion that miR-21 is an effective noninvasive screening and prognostic biomarker in patients with prostate cancer.Methods:Initially,we detected PMBC miR-21 relative expression of 92 prostate cancer patients,85 benign prostatic hyperplasia patients and 97 health controls via quantitative real-time polymerase chain reaction(qRT-PCR).We analyzed the relationship between miR-21 relative expression in PBMC and clinicopathological characteristics of different groups.The relative operating characteristic(ROC)curves were drew to analyze the diagnosis value of prostate cancer.Kaplan-Meier method and Log-rank test were used to evaluate the biochemical recurrence-free survival rate.The Cox proportional hazard risk regression analysis was used to screen the independent factors affected the biochemical recurrenceResults:qRT-PCR results demonstrated that the expression levels of miR-21 in prostate cancer(2.61 ± 0.88)group were increased compared to BPH(1.34 ± 0.50)and control group(1.24±0.47)(P<0.001).In prostate cancer patients with Gleason score ≤7 and Gleason score>7,there was a significant difference between these two sub-groups(2.38±0.82 and 2.76±0.88,respectively,P<0.05).Patients’miR-21 expression increased with clinical stages of cancer(T1,2.28 ± 0.83;T2,2.44 ± 0.50;T3,2.74±1.12;T4,3.62±0.64;F=7.249,P<0.001).Moreover,patients with lymphatic metastasis(2.94±0.98)expressed higher miR-21 in PBMC than patients without lymphatic metastasis(2.41±0.75)(P<0.01).Patients with distant metastasis(4.30±0.83)also expressed higher miR-21 in PBMC than patients without distant metastasis(2.45±0.69)(P<0.001).And a significant difference of PBMC miR-21 expression was observed among patients at low risk,moderate risk and high risk(1.74±0.41,2.51±0.86 and 2.89±0.81,respectively;F=15.192,P<0.001).However,age stratification and serum PSA level did not significantly influence prostate cancer patients’ PBMC miR-21(P>0.05).ROC curves demonstrated that the area under the curve(AUC)of prostate cancer and BPH patients distinguished by the miR-21 were 0.917 and the sensitivity and specificity were 84.8%and 83.5%,respectively,with a cut-off value of 1.838.ROC curves demonstrated that the AUC of control group distinguished by the miR-21 were 0.938 and the sensitivity and specificity were 87.0%and 86.6%,respectively,with a cut-off value of 1.755.As for prostate cancer and BPH patients whose PSA level was not more than 10ng/ml,AUC of ROC curve distinguished by PBMC miR-21 was 0.862,with sensitivity and specificity of 76.9%and 74.7%,respectively,with a cut-off value of 1.728;AUC of ROC curve distinguished by PSA was 0.868,the sensitivity and specificity of them were 76.9%and 75.9%,respectively,with a cut-off value of 4.750;AUC of ROC curve distinguished by both miR-21 and PSA was 0.946,with sensitivity and specificity of 92.3%and 86.7%,respectively,with a cut-off value of 2.793.The Kaplan-Meier Method of survival analysis demonstrated that the time-dependent biochemical recurrence free rate in low expression of miR-21 group(1-year,100%;3-year,92.4%;5-year,65.7%)is greater than that in high expression group(1-year,92.9%;3-year,39.0%;5-year,20.8%)(P<0.001).Cox proportional hazard risk regression model showed that miR-21 expression level,Gleason score and clinical stage were independent factors to predict the post-operation biochemical recurrence of prostate cancer patients,and the Exp(B)is 3.940(P=0.006),5.645(P=0.008)and 3.618(P=0.016),respectively.Conclusions:We identified a diagnostic capacity of PBMC miR-21,which was related with Gleason score,TNM stage and cancer risk level.And patients with elevated miR-21 expression had higher biochemical recurrence rate,suggesting that miR-21 was a dependent predictive factor for prostate cancer recurrence.Part ⅡStudy Objectives:To find the effect of down-regulated miR-21 on proliferation,invasion and migration of prostate cancer in vitro,and how miR-21 interacts with non-receptor protein tyrosine phosphatase 14(PTPN14),phosphatase and tensin homolog(PTEN)and metastasis of matrix metalloproteinase 2(MMP2),matrix metalloproteinase 9(MMP9),which might be the potential therapeutic target.Study method:First of all,we detected the expression of miR-21 level in 3 types of prostate cancer cells(PC3,LNCap and DU145 cell lines)and normal prostatic epithelial cells(PrEC)by qRT-PCR.Then we knocked down expression of miR-21 in DU 145 cells by siRNA interference technique to observe the effects of miR-21 knockdown on the proliferation,invasion and migration by MTT analysis,Transwell chamber test and wound healing test.To further investigate the mechanism,we detected the expression of PTPN14,PTEN,MMP2 and MMP9 by Western Blot.Results:qRT-PCR results showed that expression of miR-21 was higher in DU145 cells than those in PrEC,PC-3 and LNCap cells,suggesting that miR-21 might be associated with metastasis of prostate cancer.In MTT analysis,growth rate of DU145 with low expression of miR-21 was slower than those of controls.Also,the number of cells in DU145-AMO group migrated across the membranes decreased dramatically than blank control DU145 and negative control DU145-SCR groups(P<0.01).Wound healing test showed that less DU145-AMO cells migrated into scratch center than controls.Further Western Blot demonstrated that DU 145-AMO expressed more PTPN14 and PTEN than DU145(78%and 98%,respectively,P<0.01),as well as DU145-SCR(79%and 57%,respectively,P<0.01).Moreover,DU 145-AMO expressed less MMP2 and MMP9 than DU 145(71%and 61%,respectively,P<0.001),as well as DU145-SCR(70%and 64%,respectively,P<0.001).Conclusions:We confirmed that miR-21 loss could inhibit the growth of prostate cancer cells DU 145.Moreover,we also demonstrated that miR-21 level is highly associated with invasion,proliferation and migration capacity of cancer cells.PTPN14、PTEN、MMP2 and MMP9 might be involved in the tumor-suppression mechanism of miR-21 loss.Conclusions for this ProjectPBMC miR-21 expression in prostate cancer patients was significantly elevated,and was associated with clinicopathological features and biochemical recurrence,indicating its diagnosis and prediction potency as a biomarker.PBMC miR-21 as a prostate cancer biomarker demonstrates high sensitivity and specificity,with even higher sensitivity and specificityfor patients whose PSA level was not higher than lOng/ml when combined with PSA detection.Decreased miR-21 expression could inhibit proliferation,invasion and migration of prostate cancer.In addition,miR-21 is involved in prostate cancer cells’ proliferation invasion and migration by regulating expression of PTPN14,PTEN,MMP2 and MMP9,suggesting a prominent target of prostate cancer.In conclusion,miR-21 is highly associated with pathological characteristics of prostate cancer,as well as cancer cells’ migration,invasion and proliferation capacity.In further study,larger sample size is needed to investigate the potential of miR-21 as a diagnosis and prognosis biomarker of prostate cancer,and a therapeutic target.