| BackgroundProstate cancer (PCa) is a common malignant tumor in male urogenital system. In US the incidence of PCa is the highest among all malignant tumors in male. Due to the population aging in our country and the constant improvement in diagnosis, the detection rate of PCa has been raising at the rate of 10% each year, which imposes a serious threat to the health of middle-aged and elderly men. However, the pathogenesis of PCa is very complex. Many factors affecting the occurrence and development of PCa remain unclear, which limited its effective diagnosis and treatment. In recent years, the association of the prostate cancer 3 gene (differential display code 3 for prostate cancer, DD3) and PCa has received more and more attention.(1) Long non-coding RNA (lncRNA) and PCaLncRNA is a type of non-coding RNA that is longer than 200 nt and functions in regulating gene expression. Recent research found that lncRNAs play an anti-cancer or pro-cancer role in the tumorigenesis and development of tumors. They participate in biological processes including regulation of cell apoptosis, tumor infiltration and metastasis. Besides, they affect the growth of tumor cells through epigenetic regulation. LncRNAs could become novel tumor markers and therapeutic target and showed clinical application prospect in the diagnosis and treatment of tumors. DD3 is a type of lncRNA highly expressed and closely related to PCa. Comparing to traditional PSA, DD3 is superior in the diagnostic efficacy in terms of sensitivity and specificity, leading to its good prospect in clinical application(2) DD3 and PCaDD3 gene is a PCa-specific gene found by Bussemakers et al. in 1999. It locates in human 9q21-22 chromosome. DD3 does no code any protein. However it is very closely related to the occurrence of PCa. DD3 is specifically expressed in 95% of human PCa cells. Little or no expression is observed in normal prostate cells or benign prostatic hyperplasia cells. It has reported 100% accuracy for distinguishing PCa from benign prostate lesions using DD3. There is no DD3 expression either in benign or malignant lesions other than the prostate, making DD3 the most specific cancer marker for PCa. Different from PSA, DD3 is not affected by the age of patient, prostate size or other prostate diseases such as prostatitis. The expression of DD3 is significantly correlated to the PCa size, pathological stage and Gleason score. In patients with tumors>0.5mL and Gleason score≥7, DD3 showed elevated expression. In a multi-factor research on PCa prognosis, DD3 was identified as an independent predictor. Combing PSA and Gleason score, the accuracy for predicting the existence of extracapsular spread in PCa using DD3 reached 90%. Patients with low Gleason score, low PSA and DD3 expressions can only receive close monitoring and follow-up.DD3 is highly detected in the cells and cell debris in the urine of PCa patients. Using RT-PCR Liu et al. found that in 54 cases of PCa,48 cases were positive for DD3 mRNA in peripheral blood, there was no positive DD3 mRNA detection in the peripheral blood of 23 prostatic hyperplasia patients or 9 healthy men. It is likely that the phagocytosis of PCa cells by lymphatic cells in blood leads to its expression of DD3 mRNA in PCa patients. DD3 encodes lncRNA, which plays an important role in the growth and metastasis of multiple tumors.It is currently at clinical investigation stage regarding the specific function of lncRNA DD3 in PCa and the role it plays in the growth, infiltration and metastasis of PCa.ObjectiveSystematically investigate the mechanism of DD3 affecting the in the growth, infiltration and metastasis of PCa in vitro and in vivo through molecular, cellular and animal experiment techniques. This project will lay the theoretic foundation for further study of the role of DD3 in the development of PCa and provide novel insights to the clinical treatment of PCa.MethodsThis project was base on in vitro cultured PCa cell lines LNCaP and PC3, both of which are commonly used cell lines for studying PCa. The DD3 knock-down and overexpression vectors together with the respective control plasmids were constructed using siRNA interference and gene overexpression techniques. These vectors were packaged and transfected into cells by Lentivirus, resulting in the DD3 low-expression LNCaPDD3- cell line and the DD3 high-expression PC3DD+ cell line. The growth curve of LNCaPDD3-,LNCaPNC, PC3DD+ and PC3Ctrl cells were compared by cell count and MTT assay. After induction of apoptosis in LNCaPDD3-,LNCaPNC, PC3DD+ and PC3CtrL PCa cells using ABL-N, the early and late stage apoptotic events were monitored by Annexin V+PI double staining, suggesting the role of DD3 in apoptosis of PCa cells. Their invasion abilities were also tested by wound healing assay and transwell assay in order to investigate the role of DD3 in the suspended growth and invasion of PCa cells. At last the involvement of DD3 in the proliferation and apoptosis of PCa cells were studied in vivo. We measured the size and weight of the implanted tumors formed by different PCa cell lines in nude mouse transplantation tumor experiment. The resected transplantation tumors were paraffin-embedded and sliced. The Ki67 level was measured by immunohistochemistry staining and the tumor apoptosis level was measured by TUNEL assay.Results1. qPCR test result showed that specifically silencing the intracellular DD3 expression using RNAi caused significant reduction of DD3 in LNCaPDD3- cells comparing to the control LNCaPNC cells (P<0.01) while overexpressing DD3 in PC3DD3+ cells led to significant increase in DD3 expression comparing to PC3Ctrl cells (P<0.01). This result suggested the successful construction of LNCaPDD3- and PC3DD+ cells.2. Cell count method and MTT assay measured the proliferation rates and growth curves of LNCaPDD3- and PC3DD+ cells comparing to LNCaPNC and PC3Ctrl. Result showed that the growth rate of LNCaPDD3- cells was significantly slower than LNCaPNC cells (P<0.05), while the growth speech of PC3DD+ cells was significantly higher than PC3Ctrl cells (P<0.05).3. The Annexin V+PI double staining assay showed that the DD3-knockdown LNCaPDD3-cells were significantly less resistant to cell apoptosis than LNCaPNC cells (P<0.01) while the PC3DD+ cells were significantly more resistant to cell apoptosis than PC3Ctrl cells (P<0.01).4. Wound healing assay and transwell assay showed that the migration and invasion ability of LNCaPDD3-cells was significantly slower than LNCaPNC cells (P<0.05) while that of the PC3DD+ cells was significantly higher than PC3Ctrl cells (P<0.05).5. By measuring the size and weight of the transplantation tumors formed by LNCaPDD3-, LNCaPNC, PC3DD+ and PC3Ctr in nude mouse PCa subcutaneous transplantation model, we found that tumors formed by the DD3-knockdown LNCaPDD3- cells were significantly smaller and lighter than those formed by LNCaPNC cells (P<0.01), while tumors formed by PC3DD+ cells were significantly larger and heavier than those formed by PC3Ctrl cells (P<0.01). The Ki67 level was measured by immunohistochemistry staining and the tumor apoptosis level was measured by TUNEL assay. Results showed that the apoptosis level of tumors formed by LNCaPDD3- cells were significantly higher than those formed by LNCaPNC cells (P<0.05) and the former’s tissue cell growth ability was significantly lower than the latter (P<0.05). The apoptosis level of tumors formed by PC3DD+ cells were significantly lower than those formed by PC3Ctrl cells (P<0.05) and the former’s tissue cell growth ability was significantly higher than the latter (PO.05).Conclusions1. DD3 participates in the regulation of the growth of PCa cell, promotes the proliferation, migration and invasion, increase the resistance to apoptosis of PCa cells in vitro.2. DD3 is involved in the tumorigenesis of PCa cells, promotes the proliferation and increase the resistance to apoptosis of PCa cells in vivo.3. Further studying the mechanism of DD3 in PCa cells will facilitate the early diagnosis of PCa and the development of novel targeted drugs against PCa. |
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