| Background and ObjectivesProstate cancer (PCa) and Breast cancer (BCa) are one of the most common malignant tumors for men and women, respectively, and both have a very high fatality rate. According to the data from American Cancer Society (ACS), the new PCa patients achieve 220,800 cases in 2015 in USA, which account for 26% of all the new cancer patients in 2015. PCa has a quite high fatality rate. The five-year survival rate of four stage prostate cancer patients is only around 20% and the ten-year survival rate of four stage prostate cancer patients is below 5%. Although morbidity of PCa in China is lower than that in Europe and America, the morbidity of PCa in China keeps increasing recently. For example, the morbidity and fatality rate of PCa in China increases by 212.5% and 124.9% from 1998 to 2008, respectively. According to the data from American Cancer Society (ACS), the new BCa patients in USA reach 231,840 cases in 2015, and 40,290 patients died because of BCa. Also the morbidity of BCa in China increases continually. According to statistical data from Chinese Academy of Medical Sciences, the number of new of BCa patients in China reaches around 200,000 cases in 2010. The development of PCa and BCa involves multiple genes, however the molecular mechanisms of PCa and BCa are not completely known currently.Long non-coding RNAs (long ncRNAs, lncRNA) are non-protein coding transcript than 200 bases. lncRNA in mammals accounts for 4% to 9% of the entire RNA. lncRNA plays a very important role in a series of cell functions, including gene transcript regulation, mediated chromatin reconstruction, histone modification and so on. More and more research results demonstrate that the abnormal expression of lncRNA are highly correlated to the development of many diseases including PCa and BCa.In previous study, researchers found a lncRNA which appears very frequently in PCa patients. This lncRNA was named PlncRNA-1. PlncRNA-1 plays a quite important role in the occurrence and development of PCa. PlncRNA-1 can not only interact with androgen receptor (AR), but also the decrease of PlncRNA-1 can promote the apoptosis of cancer cells. As the first lncRNA found in PCa, the mechanism of PlncRNA-1 in the development of PCa is not completely understood yet currently.The earlier research found that the expression of Her-2 changes when PlncRNA-1 promotes the apoptosis of cells. Such result suggests that the mechanism of PlncRNA-1 promoting cell apoptosis probably acts via Her-2 signaling pathway. In this instance, this thesis aims to determine whether PincRNA-1 induces apoptosis in PCa cells through Her-2 signaling pathway and also preliminarily explores the mechanism of PincRNA-1 in the development of BCa. This thesis hopes to offer help for the theoretical study and intervention approaches for PCa and BCa.Materials and MethodsIn the study of PCa, Real-Time PCR is utilized to measure the expression of PlncRNA-1 and Her-2 in the tissues of PCa patients in 23 cases and adjacent normal tissues. Then the influence of the deregulation of PlncRNA-1 gene on the expression of Her-2 gene in LNCaP cells and the effect of up-regulation of PlncRNA-1 gene on the expression of Her-2 gene in RWPE-1 cells are investigated using Real-Time PCR technology. The effect of PlncRNA-1 on cell cycle is studied by flow cytometry technology. Animal experiments are performed to analyze the effect of PlncRNA-1 expression on PCa cells. Finally, Western blot is used to explore the influence of PlncRNA-1 on the expression of CyclinD, a cell cycle protein.In the research of BCa, Real-Time PCR is used to test the expression of PlncRNA-1 in tissues of BCa patients in 81 cases and adjacent normal tissues, and also the expression of PlncRNA-1 in different BCa cell lines. This thesis uses CCK8 methods to measure the effect of PlncRNA-1 on the proliferation ability of BCa cells, utilizes Transwell method to study the influence of PlncRNA-1 on the migration and invasion ability of BCa cells, refers to animal experiments to investigate the effect of PlncRNA-1 on the transplant,growth and pulmonary metastasis (PM), and uses Western bolt to test the relationship between PlncRNA-1 and BCa cell, EMT, related protein and the effect of the expression of PlncRNA-1 on PTEN/PI3K/AKT signaling pathway.Results1. The mechanism of PlncRNA-1 in prostate cancer1.1 The high expression of PlncRNA-1 and Her-2 in tissues of prostate cancerThis thesis tests the expression of PlncRNA-1 and Her-2 in 23 cases of prostate cancer and adjacent normal tissues. The test results suggest that the expression of PlncRNA-1 is higher, and PlncRNA-1 and Her-2 are highly correlated with each other.1.2 The effect of PlncRNA-1 expression on Her-2 expressionThe down-regulation of PlncRNA-1 expression in LNCaP cells can result in the decrease Her-2 expression. The up-regulation of PlncRNA-1 expression in RWPE-1 cells can lead to the increase in Her-2 expression.1.3 The effect of PlncRNA-1 expression on cell cycleThe down-regulation of PlncRNA-1 expression in LNCaP cells can result in the decrease in the expression of CyclinD1 and lead to the increase the ratio of G2/Mstage cells. The up-regulation of PlncRNA-1 expression in RWPE-1 cells can cause the increase in the expression of CyclinD1 and the decrease in the ratio of G2/Mstage cells.1.4 The effect of PlncRNA-1 expression on transplantable tumour of prostate cancer cellsThe results of animal experiments suggest that the weight of transplantable tumour is obviously lighter than that of the control group after down-regulation of PlncRNA-1. At the same time, Immunohistochemical detection and Western Blot results testify that the expression of Her-2 and Cyclin-D1 is much lower than that of the control group.1.5 The mechanism of PlncRNA-1 regulating the cell cycleThe antagonist of Her-2, AC480, can block the effect of PlncRNA-1 on cycle distribution of PCa cells. The influence of PlncRNA-1 on cycle distribution of PCa cells is achieved via Her-2.2. The expression and role of PIncRNA-1 in breast cancer2.1 The expression of PlncRNA-1 in tissues and cell lines of breast cancerThis thesis firstly tests the expression of PlncRNA-1 in 81 cases of tissues of breast cancer and adjacent normal tissues using quantitative Real-Time PCR method. The experimental results demonstrate that the expression of PlncRNA-1 in tissues of breast cancer is higher than that in adjacent normal tissues. The PlncRNA-1 expression of cells, including T47D, SK-BR3, AU565, MDA-MB-231 and MDA-MB-435s, with higher invasive ability, is obviously higher than that of cells, including MCF10A, BT20, Hs578T and MDA-MB-468.2.2 The function of PlncRNA-1 in the proliferation of tissues and cells of breast cancerThe thesis firstly uses shRNA to decrease the expression of PlncRNA-1 in MDA-MB-231 and SK-BR3 cell. After the down-regulation of PlncRNA-1 gene, the proliferation speed of MDA-MB-231 and SK-BR3 cell becomes obviously slow. Then the thesis increases the expression of PlncRNA-1 in Hs578T and MDA-MB-468 cell. After the up-regulation of PlncRNA-1 gene, the proliferation speed of Hs578T and MDA-MB-468 becomes obviously quick. The experimental results above demonstrate that the expression of PlncRNA-1 in breast cancer cells can promote the proliferation of cells.2.3 The effect of PlncRNA-1 expression on skin mesenchymal associated protein of breast cancerIn MDA-MB-23 land SK-BR3 cell lines, the expression of E-cadherin and a-catenin increases obviously, while the expression of N-cadherin and Vimentin decreases after down-regulation of PlncRNA-l.In Hs578T and MDA-MB-468 cell lines, the expression of E-cadherin and a-catenin decreases obviously, while the expression of N-cadherin and Vimentin increases obviously after up-regulation of PlncRNA-1. These experimental results demonstrate that the expression of PlncRNA-1 is closely related with EMT of breast cancer.2.4 The effect of PlncRNA-1 expression on migration and invasion of breast cancerThis study analyzes the change of migration and invasion ability of different cell lines after gene modification of PlncRNA-1 through Transwell method. In MDA-MB-231and SK-BR3 cell lines, the number of migration and invasion cells becomes fewer obviously after down-regulation of PlncRNA-1. In Hs578T and MDA-MB-468 cell lines, the number of migration and invasion cells becomes more obviously after up-regulation of PlncRNA-1. Such experimental results indicate that the expression of PlncRNA-1 can enhance the migration and invasion ability of breast cancer cells.2.5 The effect of PlncRNA-1 expression on the growth of breast cancer transplanted tumourThis thesis studies the effect of up-regulation/down-regulation of PlncRNA-1 on the growth of breast cancer transplanted tumour.The breast cancer cell lines are planted into nude mice. Nude mice are killed six weeks later. The transplanted tumour is taken out and weighed. The data is recorded and the growth curve is plotted, In MDA-MB-231 lines, the size of final tumour decreases and the growth speed of the transplanted tumour slows obviously after down-regulation of PlncRNA-1. On the contrary, in Hs578T cell lines, the size of final tumour increases and and the growth speed of the transplanted tumour rises obviously. The experimental results above indicate that the expression of PlncRNA-1 can promote the growth of breast tumour.2.6 The effect of PlncRNA-1 expression on the metastasis of breast cancer to the lung.The breast cancer cell lines are planted into nude mice. Nude mice are killed six weeks later and lung of nude mice is taken out. The metastasis condition is observed based slices. In MDA-MB-231 cell lines, lung metastasis decreases after down-regulation of PlncRNA-1. However, in Hs578T cell lines, lung metastasis increases after up-regulation of PlncRNA-1. The experimental results demonstrate that the expression of PlncRNA-1 can promote metastasis of breast cancer to the lung.2.7 The relationship between PlncRNA-1 and PTEN/PI3K/AKT signaling pathwayIn this study, gene chips is used to test the variation of other genes in Hs578T line after high expression of PlncRNA-1. The experimental results indicate that PTEN/PI3K/AKT signaling pathway is changed obviously after the high expression of PlncRNA-1. This thesis also analyzes the related proteins of PTEN/PI3K/AKT signaling pathway in MDA-MB-231 cell line after down-regulation of PlncRNA-1 expression, and in Hs578T cell line after up-regulation of PlncRNA-1 expression. The experimental results suggest that in MDA-MB-231 cell line, the expression of PTEN increases, the expression of PI3K and AKT doesn't change, the expression of p-PI3K and p-AKT decreases, and the activity of PI3K/AKT signaling pathway decreases. In Hs578T cell line, the expression of PTEN decreases, the expression of PI3K and AKT doesn't change, the expression of p-PI3K and p-AKT increases, and the activity of PI3K/AKT signaling pathway increases. In order to investigate whether PlncRNA-1 affects the proliferation and migration of breast cancer cells through PTEN, this study decreases the expression of PTEN in MDA-MB-231-ShPlncRNA-1 cell. After the down-regulation of PTEN, the expression, proliferation, invasion and migration all restore to the level before the down-regulation of PlncRNA-1. The experimental results above suggest that PlncRNA-1 promotes the proliferation, invasion and migration of breast cells via negative regulation of PTEN.Conclusions1. As a cancer gene in prostate cancer, PlncRNA-1 is able to regulate the cell cycle and cell cycle protein,Cyclin-D1, and affect the proliferation and apoptosis of prostate cells via in vitro cell experiments and in vivo animal experiments.2. PlncRNA-1 plays an important role in the development of breast cancer as a cancer gene. PlncRNA-1 is able to promote the proliferation, migration, invasion, the growth of breast cancer transplanted tumour and lung metastasis of breast cancer. Such function of PlncRNA-1 in the development of breast cancer is relative to the PlncRNA-1 promoting EMT of breast cancer cells and the activation of PTEN/PI3K/AKT signaling pathway. |
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