LMO4,as A Nuclear Transcription Factor,Regulates Proliferation Of Prostate Cancer Cells

BackgroundProstate cancer is one of the most common malignancies in men,particularly in western countries.But the incidence and mortality of prostate cancer markedly increased among Chinese people compared with western individuals.For the last several years,advanced disease has been managed with androgen deprivation therapy,an approach that leads to disease regression,but rarely cure.Castration-resistant prostate cancer generally results in death over 30–40 months depending on disease extent at diagnosis.Cancer stem cell is a kind of malignant cancer cell with capacity for self-renewal and differentiation.which maintain cancer cell proliferation and differentiate into different cancer cells.At present,the source and marker molecules of prostate cancer stem cells are still unclear.CD133 was originally identified as a cell surface marker of human haematopoietic and mouse neuroepithelial cells.It was subsequently reported to function as cancer stem cells marker in solid tumors,such as neuroglioma,melanoma,etc.A subpopulation of prostate cancer cells,CD133⁺prostate cancer cells,show strong clonal formation ability,anti-radiotherapy and anti-chemotherapy abilities,but it is indefinite for the roles of CD133 involved in PCSCs.LMO4,a member of the LMO family,is a nuclear transcription factor.LMO4 play an important role in embryonic development,including nervous development,skin development and cochlear development,lmo4^-/-mice showed lethality owing to unclosed perinatal and neural tube.In adults,LMO4 usually expresses in different tissues,such as stomach,intestine,kidney,while less expresses in prostate,seminal vesicle.LMO4 expression is dramatically increased in many malignant cancers,such as breast cancer,pancreatic cancer,non-small cell lung cancer,squamous cell carcinoma,etc.LMO4 overexpression may be involved in migration and invasion of cancer cells.Previous studies proved that LMO4 combines LIM domain binding protein LDB-1,LIM family protein LMO2 and cycling-dependent kinase CDK9 to form a large complex and maintain stem cell proliferation.A small complex with LMO4 and LMO2generates and promotes cell differentiation once CDK9 leaving from the large complex after induced by stimulus.Till now,it is less known about roles of LMO4 in prostate cancer.Comparing with CD133^-cells,CD133⁺cells show highly resistant to?-radiation and chemotherapeutic drug.It is necessary to investigate if LMO4 is involved in CD133 expression and associated with the percentage of CD133⁺cells.This study will providing new insights for prostate cancer therapy.MethodsIn histological studies,H&E staining were used to observed the histological structure in the sections of prostate cancer tissues and prostatic hyperplasia tissues.Immunohistochemistry was used to detect the expression of LMO4 and CD133 in prostate cancer tissues and prostatic hyperplasia tissues.In cellular level study,RNA interference was performed to knockdown Lmo4 expression in prostate cancer cells PC-3.Western blotting and flow cytometric analysis were used to detect the expression of CD133 protein and the number of CD133⁺cells,respectively.EDU cell proliferation assay and soft agarose assay were used to determine proliferation of PC-3 cells.Co-Immunoprecipitation was used to detect the interaction between LMO4 and cycling-dependent kinase?CDK9?.ResultsHistological studies show that compared to prostatic hyperplasia tissues,CD133 and LMO4 expression dramatically increased in prostate cancer tissues.1 In prostate cancer tissues,the glandular structure disappear and CD133 is highly expressed on the surface of malignant epithelial cells.In 10 prostate cancer tissues,8cases of CD133 expression increased,while in prostatic hyperplasia,the gland structure was completely and CD133 expression was negative.?2?The expression of LMO4 significantly increased in prostate cancer tissues while negative In the prostatic hyperplasia tissues.In 10 cases of prostate cancer tissue,8cases of LMO4 expression increased,while in 7 cases of prostatic hyperplasia,only 2cases of LMO4 expression increased,LMO4 and CD133 were positive in 7 cases of prostate cancer tissue,LMO4 is consistent with the CD133 expression in prostate cancer.?2?Western blot results showed that both CD133 expression and numbers of CD133⁺cells significantly decreased in LMO4 knockdown PC-3 cells.The expression of LMO4is consistant with CD133.?3?Immunofluorescence experiments showed that interference with Lmo4 in the human prostate cancer cells,the fluorescence intensity of CD133 was reduced,and the number of CD133⁺cells was decreased.?4?Flow cytometry analysis showed that CD133 expression and the number of CD133⁺cells are decreased after Lmo4 knockdown in prostate cancer cells PC-3.?5?EDU cells proliferation experiment showed that cell proliferation ability of PC-3/SiLmo4 was markedly decreased comparing with both PC-3 cells and CD133⁺cells.?6?Cloning formation experiments showed that the number of CD133^-cells clone formation was lower than that of CD133⁺cells.The clone formation rate of CD133⁺cells was higher than that of CD133^-cells.However,cloning formation rate was significantly decreased when Lmo4 knockdown in PC-3 cells.?7?Co-Immunoprecipitation analysis showed that LMO4 and CDK9 could form a LMO4/CDK9 complex in prostate cells.However,CDK9 may leave from the complex owing to LMO4 overexpression in prostate cells.Interestingly,the LMO4/CDK9complex was not detected in PC-3/SiLmo4 cells.Conclusion?1?LMO4 expression in prostate cancer is consistent with expression of CD133.?2?LMO4 is involved in regulating proliferation of prostate cancer cells.?2?The proliferation of prostate cancer is independent of LMO4/CDK9 complex.