SiRNA inhibition of PRKC-σ-vb expression abrogates the aggressive phenotype of human prostate cancer cells

• Main Author: Yao, Sheng
• Published: University of Liverpool 2009
• Subjects: 616.994
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.510949

The work performed in this Thesis was aimed to elucidate a possible function of PKC~ in human prostate cancer cell-lines. In particular, the role of gene P RKC-(-vb, the predominat isoform expressed in these cells, playing in human prostate tumorigenesis and metastasis, was investigated. The Introduction gIves general information about prostate cancer, PKCs families, particularly PKC-~ and its role in prostate cancer. The Thesis includes six main chapters, presenting data as follows: The gene expression of PRKC-( (PRKC-(-vb, PRKC-(-vn) was determined in prostate malignant and non-malignant cell-lines. Overexpression of both variants of PRKC-(was detected in prostate cancer when compared to non-malignant cell-lines, indicating P RKC-( to be a potential biomarker of prostate cancer. Overexpression of P RKC-l was also detetected in prostate cancer in comparison to non-malignant cell-lines. This result indicated these two PKCs may play similar functions in prostate cancer through phosphorylation of different substrates. To further determine the function of PRKC-(-vb in prostate cancer, gene knockdown cell-lines PRKC-(-vb-Tl-6 and PRKC-(-vb-Tl-2 were established using vector-based RNAi. Reduced gene expression of PRKC-(-vb was confirmed in these two cell-lines when compared to PC3-M parental and scrambles control cell-lines. The transfected cell-lines PRKC-(-vb-Tl-6 and PRKC-(-vb-Tl-2 were further investigated by in-vitro and in-vivo assays. Altered morphology indicated the changed phenotype of the transfectant cells. Reduced proliferation, invasion and anchor-independent growth in transfectant cells indicated reduced malignancy and then suggested the role of PRKC-(-vb in promoting prostate cancer tumorigenesis and metastasis. Reduced tumorigenesis in gene knockdown cells was confirmed by in-vivo assays. Thereafter, potential oncogenic signalling pathways in the gene knockdown cells were investigated by oligonucleotide based micro array. A down-regulated canonical NF-Kl3 pathway was identified in the gene knockdown cell-lines. Moreover, several molecules involved in prostate cancer were detected, indicating that crosstalk of these molecules may exist in prostate cancer. In previously work, a novel sequence was detected to be expressed as extention of 5' primer of "vn". Based on expressed sequences, up-and down-stream of "vn" were determined using RT-PCR. An expressed sequence was detected as an extention of 3' down stream of "vn". However, the method need to be revised to detect the 5' up-stream sequence because Alu blocks were identified at 5' up stream "vn" within 1500bp disturbing further sequencing. Based on the original sequence confirmed previously, a new monoclonal antibody was generated according to the epitope that is different to that of PKC-s. A novel protein specific to prostate cancer was also detected, indicating that a new potential splice variant of PKC-s may be present in prostate cancer. In conclusion, the current study suggests the original hypothesis that expression of P RKC-( is not only a biomarker of prostate cancer but that it plays an important role in promoting prostate cancer tumorigenesis and enhancing tumour metastasis