Dissertation
Dissertation > Medicine, health > Oncology > Genitourinary tumors > Breast tumor

Roles of Sphk1in the Malignant Transformation of Breast Cancer: a Preliminary Experimental Study

Author ZhangYan
Tutor JiangJun
School Third Military Medical University
Course Surgery
Keywords breast cancer Sphk1 malignant transformation proliferation apoptosis migration
CLC R737.9
Type Master's thesis
Year 2012
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In the process of malignant transformation of normal cells to tumor cells, a series ofcellular changes will occur, including morphological changes, loss of contact inhibition,excessive proliferation and migration ability, as well as enhanced expression of malignantgenes. Uncontrolled cell proliferation is considered to be the nature of malignanttransformation. Therefore, studies on regulation of cell proliferation and apoptosis inmalignant transformation are expected to help reveal the mechanism of tumor development.Thus, searching for new oncogenes or further revealing the functions of existing oncogeneor tumor suppressor gene is important to demonstrate the development mechanism ofmalignancy and to investigate the potential therapeutic targets.In1998, the Sphingosine kinase-1(sphingosine kinase-1, Sphk1) was first extractedfrom renal cells of rat. And until present, two isomers (Sphk1and Sphk2) have been foundin human and mouse tissues. In human cells, the Sphk1and Sphk2genes are located onchromosome17and19, respectively. They have different biological functions andregulation mechanisms. Sphk1molecules mainly distribute in the brain, heart, lungs, liver,spleen, and hematopoietic immune system, and are related to the excessive proliferation of avariety of tumor cells. Overexpression of Sphk1can improve the conversion from G1to Sphase in the cell cycle and reduce cell doubling time, which plays a role in cell cycleregulation. Suppressed expression of Sphk1can lead to cell cycle stagnation, accumulationof pro-apoptotic protein in mitochondrial membrane, release and activation of cytochrome.Therefore, it can promote cellular apoptosis by activating the intracellular pathway. In ourprevious study on breast cancer, we found that no Sphk1expressed in normal breast tissues,and it increased gradually in atypical hyperplasia tissues. However, there was a strongexpression of Sphk1in breast cancer tissues. The results showed that Sphk1might play animportant role in the progression of breast cancer. However, the exact role of Sphk1in theprogression of breast cancer and its mechanism remains unknown. In view of the above problems, we used chemical carcinogen (DMBA) induced normalmammary epithelial cells (MCF-10A). The expression levels of Sphk1mRNA weredetermined by real-time fluorescent quantitative PCR and then their relationship withapoptosis and proliferation was assessed. The siRNA was used to down-regulate theexpression of Sphk1, in order to investigate the effects of Sphk1on proliferation, apoptosisand migration of human breast cancer MCF-7cell line. This preliminary study investigatedthe role of Sphk1in the progression of breast cancer, in order to provide experimentalevidence of Sphk1in biological behavior of malignant tumors, and provide new ideas forthe research and development of molecular target of malignant tumors.Research methods and main results:1. Sphk1role in the process of malignant transformation of normal humanmammary epithelial cells.Methods: Using chemical carcinogen (DMBA)induced normal mammary epithelialcells (MCF-10A).The expression levels of Sphk1mRNA was determined by real-timefluorescent quantitative PCR. Cell proliferation and cell apoptosis was assessed by CCK-8assay and flow cytometry respectively. The ability of anchorage-independent growth of theMCF-10A cells transfected with DMBA was assessed by Soft agar colony formation.Results: The30thgeneration Cell of experiment group showed malignanttransformation phenotype:different sizes;most of the changes for the class circle shape;cytoplasm increased;nuclear atypia;compound layer growth. The DMBA significantlyincreased the mRNA expressions of sphk1in MCF-10A cells((P<0.05). The MCF-10Acells induced with DMBA showed a significant increase of cell proliferation compared tothe control cells. Moreover, significant decrease of proportion of apoptotic cells weredetected in the transformaed cells(P<0.05).The malignant transformed cells of MCF-10Ainduced by DMBA were formed small Cell colonies in soft agar in30thgeneration. thecontrol cells can were not formed small Cell colonies in soft agar.2. Effect of Sphk1gene on proliferation,apoptosis and migration in humanMCF-7breast cancer cells.Method: The siRNA against Sphk1was chemically synthesized and transfected intothe breast cancer MCF-7cell line, to silence the expression of Sphk1. Transfectionefficiency was examined by flow cytometry(FCM)and the expression levels of Sphk1 mRNA and protein were determined by real-time fluorescent quantitative PCR and Westernblotting. Cell proliferation and cell apoptosis was assessed by CCK-8assay and flowcytometry respectively. Cell migration was measured by transwell assay.Results: The efficiency of siRNA transfection into human MCF-7cells were89%. ThesiRNA against sphk1gene significantly reduced the mRNA and protein expressions ofsphk1in MCF-7cells(P<0.05). Among them the siRNA-2showed most significant effect(P<0.05).The MCF-7cells transfected with sphk1siRNA-2showed a significantdecrease of cell proliferation and migration compared to the control cells. Moreover,significant increase of proportion of apoptotic cells were detected in the transfected cells(P<0.05).ConclusionsSphk1may play an oncogenic-like role in the development of breast cancer, promotethe malignant transformation of normal epithelial cells to cancer cells, and enhance theability of proliferation and reduced apoptosis, leading to the increase of malignancy ofbreast cancer, which are based on the following evidences:(1) After using carcinogenic agents to induce cell malignant transformation, theexpression level of Sphk1was significantly increased. Sphk1may be an independentprognostic factor of breast cancer patients;(2) After malignant transformation, the ability of proliferation increased and theapoptosis reduced.(3) The transformed cells can form colonies in soft agar, which verified its malignantgrowth capacity.(4) Knockdown the Sphk1expression by siRNA in MCF-7breast cancer cell, theproliferation and migration were significantly reduced, and apoptosis was increased.

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