Effect Of TSG101 To Breast Cancer Cell And The Relations With HIF-1α And MAPK/ERK Signal Pathway

The tumor susceptibility gene 101(TSG101)was originally found in murine NIH3T3 cells in 1996. TSG101 protein mediates a variety of biochemical and biological functions,such as the regulations of vesicle transport and cell cycle. TSG101 expressed abnormally in many kinds of human malignant tumors.But the relationship between TSG101 and tumorigenesis is not clear. Hypoxia-inducible factor-1α(HIF-1α) is a kind of transcription factors induced by hypoxic condition,play key roles in the pleiotropic response observed under hypoxic conditions. Extracellular signal-regulated protein kinase (ERK) is a classical signal transduction pathway of the mitogen-actived protein kinase(MAPK) family. ERK was thought to be associated with proliferation,differentiation and inhibiting apoptosis.A study showed that TSG101 overexpression in the mammary gland of transgenic mice led to abnormal activation of MAP kinases.Another study showed that many factors could induce the production of HIF-1αby activating MAPK/ERK signal transduction pathway.The regulations of these molecules are still unclear in human breast cancer so far. In the present study,we used small interfering RNA(siRNA) targeting TSG101 gene to transfect human breast cancer cell line MCF-7 cells,observed the effects of HIF-la and p-ERK expression and the biological traits of MCF-7 cells.Then we used immunohistochemical technique to detect the expressions of TSG101,HIF-la and p-ERK in human breast cancer tissues and to find the relationships among them.Materials and Methods1. The siRNA sequence targeted against human TSG101 gene was synthesized.The siRNA negetive control(siRNA NC),TSG101 siRNA were transfected into breast cancer MCF-7 cell line respectively via lipofection.MCF-7 cells that were transfected siRNA NC and MCF-7 cells were used as control groups.The proliferation abilities of MCF-7 cells transfected by TSG101 siRNA and control groups were tested by MTT.The cell cycle and apoptosis of MCF-7 cells before and after transfection were examined with flow cytometry. Changes of migratory capacities of MCF-7 cells before and after transfection were measured by Transwell. The expressions and locations of MCF-7 cells before and after transfection were detected with immunofluorescence.The protein expressions of TSG101,HIF-la and p-ERK in MCF-7 cells before and after transfection were assessed by Western blot.2. The expressions of TSG101,HIF-1αand p-ERK were detected with immunohistochemistry in 54 breast cancer samples.3. The SPSS 13.0 software was employed to analyze the data.P<0.05 was considered as statistical significance.Results1. The expression level of TSG101 protein in TSG101 siRNA transfected MCF-7 cells (0.1338±0.0086,0.0708±0.0091) was lower than those in the control groups (0.5460±0.0145,0.4782±0.6778) (P<0.05).2. TSG101 siRNA transfected group cells were significantly slower than the control groups in the rate of cell proliferation (P<0.05).The apoptosis rate of TSG101 siRNA transfected group cells was significantly higher(13.71±1.31%) than the control groups (4.37±0.87%,6.00±0.08%) (P<0.01).The percentage of G0/G1 stage cells in TSG101 siRNA transfected group cells was significantly higher(70.51±0.23%) than the control groups(59.15±0.77%,59.21±0.68%)(P<0.01); The percentage of S stage cells in TSG101 siRNA transfected group cells was significantly lower(23.65±0.78%) than the control groups (34.96±0.45%,34.89±0.30%) (P＜0.01)3. The migratory capacity of the TSG101 siRNA transfected group cells was lower than the control groups.The cell numbers of migratory capacity (4.60±0.80) were Significantly lower in the control groups (24.47±1.90,23.80±2.20) (P＜0.01)4. The expression of HIF-la protein was reduced significantly after TSG101 siRNA transfection (0.1235±0.0057) than before (0.6762±0.0080) (P＜0.01).The expression of p-ERK protein was reduced significantly after TSG101 siRNA transfection (0.1582±0.0093) than before (0.5078±0.0036) (P＜0.01)5. The positive expressions of TSG101, HIF-la and p-ERK were 74.07% (40/54),77.78%(42/54) and 81.48%(44/54) separately in invasive ductal carcinoma of breast.The correlations between the expression of TSG101and HIF-la and between the expression of TSG101and p-ERK in breast cancer were both significant (P<0.01)Conclusions1. Breast cancer cells were decreased proliferation and induced apoptosis with transfected TSG101 siRNA into them. The cell cycle of TSG101 siRNA transfected breast cancer cells was arrested at G1/S period.Inhibition of TSG101 protein expression by TSG101 siRNA could decrease migratory capacity of breast cancer cells.2. Maybe TSG101 is an important regulator of HIF-1αgene expression and transcriptional activation in breast cancer.3. TSG101 may play its biology effect through MAPK/ERK signal pathway in breast cancer.