The Research On Effect Of Prostate Peripheral Zones Stromal Cells On Prostate Cell Biological Behavior By Overexpression Of LMO2Gene

Objective：According to the prostate "zonal distinction" theory, we investigatedthe different gene expression between prostate peripheral zone stromal cells (PZs) andthe transitional zone stromal cells (TZs), and found overexpression of LMO2gene inPZs. Thus, the biological effects of LMO2on prostate cell proliferation, migration inthe stromal microenvironment were further explored, and the possible molecularmechanisms were explained.Methods：1. Primary culture different prostate stromal cells which were identifiedby immunohistochemistry. Coding genes and non-coding genes expressionaldistinction of different prostate stromal cells was screened by Affymetrix geneexpression arrays and miRNA expression arranys. We found LMO2gene wasup-regulated in PZs cells. To validate the microarray data, Q-PCR, Western-blottingand immunofluorescence analysis were supported the over expression of LMO2in PZscells.2. On one hand, Prostate stromal cell line which could stably express LMO2wasacquired by infection LMO2overexpression lentiviral vector（WPMY-1-LMO2）. Onthe other, shRNA-LMO2PZs were obtained by using LMO2specific shRNA plasmidwhich was transient-transfected to PZs（PZsshRNA-LMO2）. LMO2overexpression andinterference efficiency were observed by fluorescence microscopy and assessed byRT-PCR and Western-blotting afterwards.3. Use Transwell co-culture system tocompare the effect of stromal cells which overexpress/low-express LMO2gene on invitro proliferation and migration ability of co-cultured PC3and BPH-1cell lines. Protein factor chip and RT-PCR were used to screen expression of FGF-9and IL-11.After mixing Cytokines, FGF-9and IL-11, into medium of BPH-1and PC3cells, cellproliferation was measured by CCK-8and EdU assay; cell migration was observed bywound scratch assay. After co-cultured with LMO2overexpressing stromal cell line,the changes of BPH-1and PC3associated proteins were assessed by Western-blotting.4.The effect of WPMY-1-LMO2prostate stromal cells on proliferation and invasion ofPC3was verified by prostate in situ xenograft and subcutaneous xenograft model.Besides, immunohistochemistry was used to detect Ki-67expression in of xenografttumor.Results：1. After establishment the models of different prostate zonal stromal cellsand CAF which carried out for gene microarray profiling and miRNA microarrayprofiling. Results indicated512genes and27miRNA that expressed distinctly indifferent zone original stromal cells. Among these genes, we found that LMO2gene isoverexpression in PZs, additionally, it is confirmed by RT-PCR, western-blotting,immunofluorescence and immunohistochemistry.2. By using lentivirus transduction,LMO2stably expressed stromal cell line was successfully constructed. PZs wastransiently transfected of shRNA-LMO2plasmids and had the advantage of hightransfection efficiency which can effectively inhibit LMO2expression in PZs.3. Afterit co-cultured respectively with stromal cells which stably expressed LMO2, theproliferation and migration of both PC3and BPH-1were found increased, thoughshRNA-LMO2interference cell line had significantly effect on these of prostate cells.Protein factor chip found increased FGF-9and IL-11expression in the mediumsupernatant reserved from LMO2-overexpressed stromal cell line. RT-PCRrecapitulated and confirmed the results from protein factors chip. P-STAT3, AKTsignaling pathway may be involved in the mechanisms of these biological effectsabove mentioned. The expression of FGF-9and IL-11mRNA in WPMY-1-LMO2were upregulated higher than control group.4．Heterogeneous xenograft by mixing PC3and WPMY-1-LMO2stromal cells were inoculated into nude micesubcutaneously and prostate in situ to establish the animal model. The result showedthat the WPMY-1-LMO2stromal cells could promote tumor growth and metastasissignificantly compared with the control group.Conclusion：Distinct gene expression existed between different zonal originatedprostate stromal cells. Particularly, LMO2overexpresion was found in the peripheralzone stromal cells. The expression of LMO2in stromal cells could promote prostateepithelial cells growth and migration in vitro and vivo. Mechanisms of the abovephenomenons may be due to that LMO2overexpressed stromal cells should induceprostate epithelial cell growth and invasion via paracrine FGF-9, IL-11or othercytokines. Prostate epithelial-stromal interaction is the importance of the progress inprostate cancer, especially the key role of the stromal cells. The importance ofstromal–epithelial crosstalk in prostate cancer initiation and progression provides theimpetus for combinatorial microenvironment-targeting strategies. Specific targeting ofstromal can form the basis of personalized microenvironment-based anticancer therapyin the future.