The Impact Of KLF4 Iinhibition On Malignant Phynotype Of Breast Carcinoma MCF7 Cells

In the past 20 years, the incidence of breast cancer has nearly doubled over the world. Each year, about 120 million women worldwide was diagnosed to have breast cancer,50 million women died of breast cancer. WHO reported that in Western countries, breast cancer has surpassed other tumors, as to be the primary cause of death of women aged 20-59. Although the incidence in China is lower than in Western countries, but in the past 30 years, breast cancer is one of the fastest growing cancer, which is a serious threat to the health of women in our country!With the deepening of biomarkers research, the level of breast cancer diagnosis and individualized treatment gradually increase. The discovery and in-depth research of molecular markers such as ER, PR, HER-2 has now expanded breast cancer treatment to the field of endocrine therapy and molecular targeted therapy. So looking for more and better molecular orgene therapy target become a very important hot spots.Transcription factors are a group of specific protein which could bind in a sequence-specific way to 5'end of genes, ensuring the target gene express in the specific strength and in a particular time and space. The change of the transcription factors'level will lead to disorders in the expression of the target gene and the protein, and then may affect the tumor occurrence, growth, invasion and metastasis. Human Kruppel-like factor 4 (KLF4) as a newly discovered transcription factor, has been greatly valued because its important role of inducing the mice fibroblast cells to produce iPS cells (induced pluripotent stem cells), they regulate target genes expression in cells and tissues by activating or inhibiting the transcription and the cell cycle phase associated dynamic expression, so as to regulate many cellular activities, such as cell growth, proliferation, differentiation and apoptosis. Studys found that KLF4 expression increased in primary breast cancer, and the high level KLF4 expression in nuclear indicate high malignancy and poor prognosis of breast cancer. Thanks to the basic findings, researchs around KLF4 in the pathogenesis, growth, metastasis, treatment and prognosis of breast cancer has been gradual developing.Our study select KLF4 as the research object, the human breast cancer cell MCF7 which has natural high KLF4 expression level as a research model, and we use the artificial siRNA (small interfering RNA) transfection technique to suppress the expression of KLF4. So we can pry into the impact of inhibiting KLF4 on the MCF7 cells'malignant phenotype such as cell growth, colony formation ability. Details are as follows:1.Find the breast cancer cells with natural high KLF4 expression level:1.1 Culture the human breast cancer cell lines:MCF7, Bcap37, MDA231, T47D, SK-BR3; including the human lung cancer cell line H322 with natural low KLF4 expression level, and H322/KLF4 cell line with stable KLF4 plasmid transfection, they are negative and positive controls, respectively.1.2 Cell lines'KLF4 expression were detected both in protein levels and RNA levels, MCF7 cell line was selected with natural low KLF4 expression both in RNA and protein, and was to be the following experiment model.2.Using KLF4-siRNA to knockdown the KLF4 expression in MCF7 cells: Use Oligofectamine Reagent to transfect KLF4-siRNA into MCF7 cells, then MCF7 cells with KLF4 low expression was obtained; same way to transfect the control-siRNA into some other MCF7 cells. Collect both groups'RNA, detect the expression of KLF4 using RT-PCR.3.MTT assay to evaluate the change on the growth ability of MCF7 cells with KLF4 knocked down:The MCF7/KLF4-siRNA cells and the control group MCF7/control-siRNA calls were respectively seeded in 96 well plates, cultured for 1,3,5 days, each incubated with MTT, then the crystals was dissolved with DMSO, read the absorbance at 570 nm, finally draw a growth curve. The results suggested that, KLF4 knockdown cells showed a significantly slower growth trend compared with the control group(P<0.01).Colony formation observed the affect of KLF4 expression inhibition on MCF7 cell clone forming ability:the MCF7/KLF4-siRNA cells and MCF7/control-siRNA cells were inoculated in 6-well plates, cultured for 14 days, and then added crystal violet staining Solution, and finally we observed the number of clones, the result showed, MCF7 cell cloning ability has been declined significantly with KLF4 knockdown compared with control cells(p<0.01).Through our current research, we draw forth the following preliminary conclusions:1.human breast cancer MCF7 cell line has natural KLF4 high expression;2.KLF4 expression inhibition can significantly slow down the growth of MCF7 cells;4.KLF4 knockdown can significantly reduced the colony formation ability of MCF7 cells.