The Functions And Mechanism Of Interleukin-6 And Downstream Jak/stat3 Signaling Pathway In Prostatic Diseases

[Background]Interleukin-6 (IL-6) is a multi-functional cytokine. It is involved in the inflammatory process of chronic prostatitis. In addition, IL-6 may still play an important role on hormone refractory and neuroendocrine differentiation in prostate cancer. However, the mechanism of IL-6 in chronic prostatitis and prostate cancer is unclear. Molecular epidemiologic investigation of IL-6 in chronic prostatitis and the function of IL-6/STAT3 signal pathway on prostate cancer progression remain to be further explored.[Objectives]1. To investage IL-6 expressed level and gene polymorphism in patients with catⅢtype chronic prostatitis by molecular epidemiologic methods; 3. To analyse the influnce of IL-6/STAT3 signaling on biological behavior of prostate cancer cell line; 4. To target inhibite STAT3 signaling pathway with siRNA in prostate cancer cell line; 5. To study the influnce of IL-6/STAT3 signaling pathway on the malignant behavior of prostate cancer cell lines and its possible mechanism after targeted STAT3 silence.[Methods]1. For molecular epidemiologic analsis on IL-6 the baseline population was established by epidemiological investigation, according to the differences in environmental factors and symptoms the baseline population was further divided into two study cohorts; 2. Patients with catⅢtype chronic prostatitis type were included by NIH-CPSI questionnaire investation and further clinical screening and further divided intoⅢa andⅢb subtype groups. Levels of IL-6, IL-2, IL-8, IL-10, TNF-α, oxidative stress and the relationship between IL-6 and symptoms were systemic analysed; 3. Levels of IL-6 and other cytokines in EPS and serum were and detected by enzyme-linked immunosorbent assay and the relationship between IL-6 and other inflamatary factors was analysed; 4. ROS level in EPS was examined by DCFH2-DA; 5. TAOC level in EPS was detectd by ABTS; 6. -174G/C genotype of IL-6 promoter was analysed with tetramer ARMS-PCR; 7. PSMA specific single chain antibody fusion protein was constracted and identificated with molecular biology technology; 8. STAT3 specific siRNA were designed using molecular biology software. The targeted delivery ability of the fusion protein was identified by flow cytometry analysis and cell ELISA; 9. The prostate cancer cell line model by IL-6 treatment was established as the dual biological function on LNCaP cell line of IL-6; 10. LNCaP cells in which the STAT3 signaling was suppressed by STAT3 specific siRNA treatment were achieved and the expression of pSTAT3 was detected by Western Blot; 11. MTT method was used to examined the growth of prostate cells and the STAT3 signaling suppressed cells; 12. Cell cycle of LNCaP cells and the STAT3 signaling suppressed cells was examined by FCM analysis; Annexin V/PI dual-staining was used to detect cell apoptosis.[Result]1. Molecular Epidemiologic investation of IL-6 in cat III type chronic prostatitis1) The IL-6 level in EPS were significantly higher than that of control group in patients with both subtypes CP/CPPS, but not accompanying the changes of serum IL-6 level.2) IL-6, TNF-α, IL-2, IL-8 and IL-10 participated the pathological process of IIIa subtype CP/CPPS accompanying by the increased ROS level and decreased TAOC level.3) IL-6 was involved in the pathological process of subtype IIIb CP/CPPS. However, significant changes of the levels of TNF-α, IL-2, IL-8, IL-10 and oxidative stress were not found in the course of the symptoms aggravation.4) Although the type criterion of CP / CPPS is disputed we found the pathological mechanism of IL-6 in the two types CP / CPPS may be different based on the typing standard.5) CP/CPPS patients with IL-6 promoter -174 C / G genotype have a higher EPS IL-6 levels and the symptoms score was significantly higher than that of GG and CC homozygous patients.2. Exogenous IL-6 biological behavior of prostate cancer cell lines and its downstream signal transduction1) In initial phage of exogenous IL-6 treatment IL-6 can inhibite induce LNCaP proliferation by inducing cell cycle arrest and apoptosis.2) In inhibition phage of exogenous IL-6 treatment elevated PSA levels were deteced in LNCaP cells, indicating that IL-6 can induce the malignant phenotype of LNCaP cells.3) After targeting inhabite STAT3, with the reduction of phosphorylation STAT3, PSA secretion levels appeared significantly reduced. Therefore STAT3 plays an important role during the process which IL-6 induces the increased PSA levels in LNCaP cells.4) STAT3 is not involved in proliferation inhibition of IL-6 treatment as the MTT growth curve of IL-6-treatment-LNCaP cell is not influnced after siRNA targeting STAT3 silence.[Conclusions]1. IL-6 is involved in the pathological process of bothⅢa andⅢb subtype CP/CPPS, but the pathological mechanism of IL-6 may be different in the two subtypes CP/CPPS; 2. CP/CPPS patients with -174G/C genotype in IL-6 promoter have high expression levels of IL-6 in their EPS; 3. Exogenous IL-6 can induce the malignant phenotype of LNCaP cells with the increased secretion of PSA and JAK/STAT3 signaling pathway is involved in the malignant progression.