The Expression Of LMO2 And MiR-15a/16 In Prostate Stromal Cells And Its Preliminary Study In Development Of Prostate Cancer

Objective:Prostate cancer is one of the common malignancies in men.Clinically,70%to 75%of prostate cancers occur in the peripheral zone,and only 20%to 25%of prostate cancers occur in the transition zone.The reason for this difference is unclear,and stromal cells can play an important role in the progression of tumors.Therefore,this study intends to screen genes which are differentially expressed between stromal cells in the peripheral and transitional regions of the prostate by using gene chip and miRNA chip technology,and further study the regulatory relationship between the differential gene LMO2 and miR-15a/16,and identify the downstream target genes of miR-15a/16,so as to reveal the relevant mechanism of prostate cancer that inclines to occur in the peripheral zone,and provide a new theoretical and experimental basis for the intervention of the development of prostate cancer.Methods:Firstly,collect normal prostate tissue specimens from 5 male patients with radical cystectomy and surgical specimens from 45 patients with radical prostatectomy from March 2018 to September 2019 in Northern Jiangsu People's Hospital.Secondly,select peripheral and transitional zone tissues from normal prostate tissue samples obtained from patients with bladder cancer for cell culture.Thirdly,using gene chip and miRNA chip technology,screen differentially expressed genes between peripheral and transitional stromal cells.And verify differential expression of selected genes by real-time quantitative PCR and Western blot analysis.Fourthly,detect the expression of LMO2 protein in 45 prostate cancer specimens by immunohistochemical method,and analyze the relationship between the expression and the clinicopathological characteristics of the patient.Next,construct prostate peripheral stromal cells with over-expression and low-expression of LMO2,and detect the expression levels of miR-15a/16 in each group of cells by real-time quantitative PCR.Meanwhile,screen FGF2 and FGF9 as potential target genes of miR-15a/16 by the target gene prediction software,and construct the prostatic peripheral stromal cells with overexpression of miR-15a/16,and detect the expression of FGF2 and FGF9 proteins in the cells of the negative control group by Western blot.Then,use real-time quantitative PCR to detect the expression levels of FGF2 and FGF9 in prostatic peripheral stromal cells with overexpression and under-expression of LMO2.Finally,SPSS25.0 software is used for statistical analysis of the obtained data.Results:1.Gene chip results showed that the expression of LMO2 gene in peripheral and transitional stromal cells of prostate was different.Further real-time quantitative PCR results showed that the expression of LMO2 in peripheral stromal cells was higher than that in transitional stromal cells,and this difference was statistically significant(P<0.05).Western blot results also showed that the expression of LMO2 in peripheral stromal cells was higher than that in transitional stromal cells.2.MiRNA chip alignment revealed differential expression of miR-15a/16 in prostate stromal cells in different zones.Real-time quantitative PCR results showed that miR-15a/16 was highly expressed in transitional band stromal cells and low expressed in peripheral band stromal cells,and the difference between the two groups was statistically significant(P<0.05).3.From immunohistochemical results,it was showed that the positive rate of LMO2 protein expression in prostate cancer tissues of patients with Gleason score?8 was higher than that of patients with Gleason score<8,and the difference was statistically significant(P<0.05).At the same time,the positive rate of LMO2 protein expression in patients with regional lymph node metastasis was higher than that in patients without regional lymph node metastasis,and the difference was statistically significant(P<0.05).However,there was no statistically significant difference in LMO2 protein expression levels among patients with different ages,PSA scores and tumor stages(P>0.05).4.Real-time quantitative PCR results showed that compared with the negative control group,the expression of miR-15a/16 was down-regulated in the peripheral stromal cells transfected with the LMO2-expressing plasmid,and the difference was statistically significant(P<0.05).And after using siRNA to inhibit the LMO2 expression,the expression of miR-15a/16 was up-regulated,and the difference was statistically significant(P<0.05).5.The target gene database predicted the downstream target genes of miR-15a/16,and the results showed that FGF2 and FGF9 were potential target genes of miR-15a/16.Western blot results showed that,compared with the negative control group,the expression of FGF2 and FGF9 were down-regulated in the transfected miR-15a/16 mimic group.6.Real-time quantitative PCR results showed that,compared with the negative control group,the expression of FGF2 and FGF9 were up-regulated in the peripheral stromal cells transfected with LMO2-expressing plasmid,and the difference was statistically significant(P<0.05).And after using siRNA to inhibit the LMO2 expression,the expression of FGF2 and FGF9 were up-regulated,and the difference was statistically significant(P<0.05).Conclusion:1.The expression of LMO2 gene in stromal cells of peripheral prostate is higher than that of transitional stromal cells.2.The expression of miR-15a/16 gene in stromal cells of peripheral prostate is lower than that of transitional stromal cells.3.The difference in the expression of LMO2 and miR-15a/16 in the peripheral and transitional stromal cells of the prostate may be one of the reasons for that prostate cancer inclines to occur in the peripheral zone.4.The expression level of LMO2 protein in prostate cancer tissues is closely related to the Gleason score and regional lymph node metastasis.5.In prostate stromal cells,LMO2 can negatively regulate the expression of miR-15a/16,while miR-15a/16 can negatively regulate the expression of FGF2 and FGF9,and LMO2 can positively regulate the expression of FGF2 and FGF9.LMO2 may promote the expression of FGF2 and FGF9 by inhibiting the expression of miR-15a/16,and then promote the development of prostate cancer.