| INHIBITION
OF THE MALIGNANT PROGRESSION OF PROSTATE CANCER CELLS
BY SUPPRESSING THE EXPRESSION OF THE OSTEOPONTIN GENE
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Professor Youqiang Ke University of Liverpool |
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This project was mainly supported by a grant from the North West Cancer Research Fund. It is also supported by a small grant (G2005/05) from Prostate UK. These grants have jointly been used as a PhD studentship to provide maintenance, fees and consumables for Miss Yu Zhang to pursue her PhD degree. The project started in July 2006, and it is now about half way through. The original research plan has been adhered to and about 65% of the originally proposed work is now finished. Generally speaking, it has progressed very well.
Osteopontin (OPN) is a gene involved in promoting the growth and malignant dissemination of cancer cells in several different organs, but it was not previously clear whether OPN plays any role in the malignant progression of human prostate cancer. Our recent study showed that the levels of OPN expression in both prostate cancer cell lines and tissues are greatly increased when compared with those detected in the benign prostate cell lines and the benign prostatic hyperplasia (BPH) tissues. The increment of OPN expression is positively-related to the increased degree of malignancy and reversely-related to the length of patient survival time. Therefore, we concluded that the level of OPN expression can independently predict the potential of aggressiveness of prostate cancer.
One aim of this study is to investigate the possible role of OPN in prostate cancer through suppressing OPN expression. The other aim is to make therapeutic exploration of the highly malignant prostate cancer cells by suppressing the expression of the OPN gene so that the malignant progression can be effectively reversed in the animal model. In other words, this project is aimed at further investigation of the biological significance of the elevated expression of OPN in the malignant progression of human prostate cancer. According to our original plan, we designed several sets of experimental work to assess the effect of the inhibited expression of OPN in prostate cancer cells. We have used a new advanced technique named RNA interference (RNAi) to block the OPN expression in the highly metastatic prostate cancer cell line PC-3M. The effect of the suppressed expression of the OPN gene on the invasiveness, tumourigenicity of the cancer cells, and their ability of spreading to the secondary sites is now being systematically assessed.
As described in the original proposal, the entire project consists of
the following three sets of experiments:
A). Using the RNAi technique to block the OPN expression in the highly
malignant PC-3M cells so as to establish clones expressing undetectable
level of OPN.
B). Using the invasion assay and the soft agar assay or collagen gel penetration
assay to test the invasiveness and tumourigenicity of the transfectants
so as to ascertain whether or not suppressing OPN can reduce the malignant
characteristics of the cancer cells in vitro.
C). Inoculating the transfectant cells into the prostate gland of the
nude mice by a surgical technique called orthortopic implantation to
test the tumourigenicity and metastatic ability of the transfectant
cells so as to establish whether or not suppressing OPN expression can
reduce the abilities of the cancer cells to form tumours and to spread
to the secondary sites.
To suppress the level of OPN expression in the highly malignant PC-3M cells, Miss Yu Zhang has selected 5 short interference RNA sequences based on the nucleotide sequence of the OPN gene. She has cloned all these 5 molecules respectively into a pSilencer vector respectively and established 5 constructs that can express 5 different short interference RNAs. She then performed transient transfection experiments to transfect these 5 constructs into PC-3M cells respectively. To identify the most effective suppressor, she used Real-Time PCR and Western Blot to analyse ONP expressed in the transfectant cells at RNA and protein levels respectively.
Miss Yu Zhang has found that the ability to suppress OPN expression is different amongst the 5 RNAi molecules. The most effective suppressor, which generated a clone expressing the lowest level of OPN, was able to produce a 30-fold reduction of the OPN at the protein level. Therefore, this construct containing the most effective RNAi suppresser was stably transfected into the PC-3M cells. A “ring cloning” method was used to isolate 5 clones and the control clone was generated separately with the scrabble RNA. Through Western Blot analysis, Miss Yu Zhang has produced a clone which expressed a very low level of OPN (more than 30-fold reduction), a clone expressing moderately-reduced level of OPN (about 10-fold reduction) and the control which expressed similar level of OPN to the parental PC-3M Cells.
This program of work, including the design and synthesis of the RNAi sequences, cloning of these short molecules into the vector, performing the transient transfection experiments, assessment of the results with Real-Time PCR and Western Blot, establishing the stable transfectant clones, took Miss Yu Zhang about 14 months altogether and is a good achievement for a first year PhD student.
In the past few months, Miss Yu Zhang has started to perform the second set of experiments to assess the consequences of suppressing OPN expression in prostate cancer cell line PC-3M. She has completed the proliferation assay and the bio-assay for invasiveness. Both assays showed that suppression of the OPN expression greatly inhibited the cell proliferation rate and the invasiveness. Miss Yu Zhang now is preparing for the soft agar assay to test the nodule-producing ability and nude mouse assay for tumourigenicity and metastasis. We are confident that the project will be completed in the originally planned time.
Research interim report dated 05 May 2007
Project 2005/05