The Research Of The Expression And The Relationship Between Interleukin-6 And Prostate Cancer

Prostate cancer (PCa) is one of the commonest male malignancies worldwide and its mortality and morbidity vary greatly according to countries and regions. With increase of aging population, the incidence of PCa has been on the rise over years. Recent studies showed that interleukin-6 (IL-6) plays a critical role in the development and progression of PCa and the polymorphisms of the single nucleotide at its promoter region are intimately correlated with the development and progression of PCa. This study examined the expressions of IL-6 gene and protein in PCa cancer and their cell lines. A specific adenovirus expression vector with IL-6-shRNA and was constructed and introduced into the PC-3 cell line of human PCa in order to observe the effect of IL-6-shRNA on the growth and apoptosis of PC-3 cells. The polymorphisms of single nucleotide located at the promoter region of IL-6 gene in the Han People with PCa living in Hubei region were analyzed with an attempt to examine the relationship between the single nucleotide polymorphisms (SNPs) and development of PCa in Han People in Hubei region. The study falls into four parts.Partâ… . The expression of interleukin-6 in prostate cancer and its clinical significanceObjective:To study clinical significance and the relationship between the interleukin-6(IL-6) and disease progression in prostate cancer.Methods:â‘ Immunohistochemistry and RT-PCR were used to detect the expression of IL-6 protein and mRNA in frozen prostatic adenocarcinoma,adjacent benign prostatic tissue and prostate cancer cell lines such as PC-3 and LNCaP.â‘¡The serum levels of IL-6 in patients with prostate cancer and healthy control and the supernatants of prostate cancer cell cultures were measured by using ELISA.Results:â‘ The IL-6 protein levels in prostate cancer tissue and PC-3 cell were significantly higher than those in adjacent benign prostatic tissue and LNCaP cell.â‘¡The serum IL-6 levels in the patients with prostate cancer were markedly higher than those in the healthy control.The IL-6 levels in supernatants in PC-3 cell were notably higher than those in the LNCaP cell.Conclusions:The IL-6 gene may act as an important regulator in prostate cancer progression and may be one of the causes of prostate carcer conversion from an initially androgen-dependent state into an androgen-independent state.Partâ…¡. The construction and identification of Adenovirus expression vector targeting IL-6-shRNA geneObjective:To construct and identify adenovirus vector carrying shRNA targeting IL-6(rAd-IL-6-shRNA) and to establish a monoclone PC-3 cell line transfected with plasmid IL-6-shRNA, and detect its influence on expression of IL-6.Methods:To design 3 pairs of shRNA sequence targeting IL-6 and a pair of shRNA sequence making negative control(HK) respectively, and clone them into pGensil-1 vector by using technology of gene recombination. The recombinants were named pGensil-1_IL-6-shRNA1-,_ IL-6-shRNA2 and_ IL-6-shRNA3. They were transfected into Bacterium coli DH5a, and extracted with nucleic acid purification Kits. The sequence of recombinant plasmids was evaluated by electrophoresis and DNA sequencing. The adenovirus plasmid rAd-IL-6-shRNAl-3 was constructed by adeno-X expression system, and the control adenovirus plasmid rAd-HK was constructed by the same system. After verification by enzymolysis and PCR, the rAd-IL-6-shRNA1-3 vector was cotransfected into 293 cells where they were packed as the replication-deficient adenovirus rAd-IL-6-shRNAl-3. rAd-IL-6-shRNA1-3 was abundantly amplified and then virus titer was evaluated. RT-PCR and Western Blot were used to detect the IL-6 mRNA and protein expression in PC-3 cell lines.Results:rAd-IL-6-shRNA was successfully constructed and verified by enzymolysis and sequencing.RT-PCR and W-B analysis showed that the silencing effect of pGensil-1-IL-6-shRNA3 was more evident than other recombinant vectors.Conclusions:Successful construction of the recombinant adenovirus vector containing the IL-6-shRNA gene was achieved, pGensil-1_IL-6-shRNA3 was selected for the further study as the most effective plasmid in silencing expression of IL-6. A PC-3 cell line transfected with rAd-IL-6-shRNA was obtained.Partâ…¢. The Effects of IL-6-shRNA Gene on the Biological Behaviors of Prostate Cancer Cell (PC-3) in VitroObjective:To investigate whether the expression of gene IL-6-shRNA can effect the growth, proliferation and apoptosis of prostate cancer PC-3 cells in vitro. and research their inter-relation between IL-6 and tumorigenesis of prostate.Methods:The whole experiment was divided into 3 groups. The rAd-IL-6-shRNA was used to infect the human prostate cancer PC-3 cells. For control experiments, the vector rAd-HK was also transfected into PC-3 cells and nontransfected PC-3 cells. The effect of IL-6-shRNA on the cellular proliferation capacity of PC-3 cells was assayed by the growth curve. The cell apoptosis was detected by Hoechst33258 staining, and flow cytometry analysis. The cell migration assay was used to detect the difference of invasion and metastases between transfected and non-transfected cells.Results:MTT showed the cell proliferation was markedly inhibited compared with the control cells. Partial cancer cells presented morphological changes of apoptosis by Hoechst33258 staining and flow cytometry analysis. Contrast to control cells and rAd-HK transfected PC-3, the invasion ability of rAd-IL-6-shRNA transfected cells decreased obviously.Conclusion:IL-6-shRNA gene can suppress PC-3 cell growth and proliferation, in addition to acceleration of its apoptosis in vitro. IL-6 gene might play a crucial role in the development and progression of prostatic cancer and suppression of IL-6 gene expression might be a promising strategy for the treatment of human prostate cancer.Partâ…£. Relationship between Single Nucleotide Polymorphisms in Interleukin-6 and Prostate CancerObjective:To examine the association between the single nucleotide polymorphisms (SNPs) in-174G/C and-634C/G of interleukin-6(IL-6) promoter region and prostate cancer in the population of Han People in Hubei region.Methods:TaqMan PCR was employed for the gene-typing of-174G/C and-634C/G in promoter region of IL-6 gene to compare the prostate cancer patients and normal controls in terms of genetype frequency, allele frequency and risk of prostate cancer.Results:In all the subjects, the genetype of genetic locus-174G/C was found to be GG and no CG and CC were found. There were a significant difference in gene frequency of GG, CG and CC of-634C/G and allele frequency of G and C between prostate cancer patients and normal controls (P<0.05) and the gene frequency of GG+CG increased with the clinical stages and pathological grades of prostate cancer.Conclusion:No SNP in-174G/C IL-6 promoter region was found in the population of Han People in Hubei region. The SNP in-634C/G was, to some extent, associated with the development and progression of prostatic cancer. The population with GG+CG genetype have higher risk of prostate cancer.