| Objective: Colorectal cancer (CRC) is one of the most common malignant tumors, which is the most common cause of cancer-related death and has a high rate of morbidity in 40 to 60. In our country, incidence and mortality of colorectal cancer listed on the fourth place in all kinds of malignant tumors, just below the common malignant tumors in stomach, lung, esophageal. In recent years, local data shows that incidence of the disease is on rising ,with the improvement of the living standard and the changing of diet structure. Genesis and development of Colorectal cancer are complicated processes with multi-procedure. It is correlated with expression of many genes and proteins closely, including proto-oncogene activation, anti-oncogene mutation and loss and so on. Anomalies of signaling network penetrate into Genesis of cancer each stage. With the development of research, it found that signaling pathways is playing an important role in genesis and development of cancer. It is discovered recently, FLNa is an important suppressor factor in breast cancer cells'growth and metastasis. There is a lower or undetectable level in invasive Colorectal cancer cell lines. Reduction or loss of FLNa expression is involved with malignant tendency of human Colorectal tissue, but its specific mechanism in inhibiting invasive growth of tumor cells is not clear. So it is very important for understanding the mechanisms of invasion and metastasis and searching effective approaches to prevent the recurrence and metastasis of HCC is of great importance.In this study, FLNa gene was transfected into SW480. It provides a foundation for study on the mechanism of FLNa to inhibit tumor cells'invasion and metastasis in vitro.Methods: 1 Expressing vector pcDNA3.1/V5-His-TOPO/FLNa was tran- sfected into SW480 cells by lipofectamine protocols. After G418 selection,the cells SW480/FLNa expressing FLNa stably were obtained. Expression of FL Na in tumor cells was confirmed by reverse transcription polymerase chain reaction(RT-PCR) and Western blot.2 SW480 cells were transfected stably with either FLNa or vector plas- mid.SW480 cells or vector transfected SW480 cells were stimulated with EGF and U0126.Cytosolic and nuclear fractions were immunoblotted for phospho- ERK(p-ERK),total ERK,and MMP-9 expression,respecttively.3 The cells are classified into three categories (SW480, SW480 incuba- ted with U0126 for 20 min and SW480/FLNa).Cytosolic and nuclear fractions were immunoblotted for total ERK,p-ERK,and MMP-9 expression by Western blot,respectively.There are SW480/FLNa,SW480/FLNa incubated with EGF for 20 min and SW480.Cytosolic and nuclear fractions were immunoblotted for total ERK,p-ERK,and MMP-9 expression by Western blot,respectively.4 Using migration and invasion experiment to detect the SW480 cells'migratory and invasive abilities.Results: 1 FLNa gene was stably expressed in SW480 cells.Take GAP DH as the internal reference,and make quantitative measure according to FL Na/GAPDH.Expression of FLNa mRNA in different SW480 cells detected by RT-PCR show that FLNa mRNA were higher in SW480/ FLNa group than that in SW480/pcDNA3.1 and SW480 group, respect- tively [(1.27±0.11 vs. 0.12±0.02,P<0.05) and (1.27±0.11 vs. 0.14±0.03,P<0.05)].(Fig.1)2 FLNa protein was stably expressed in SW480 cells.Take GAPDH as the internal reference,and make quantitative measure according to FLNa/GA PDH.Expression of FLNa protein in different SW480 cells detected by West- ern blot show that FLNa protein were higher in SW480/FLNa group than that in SW480/pcDNA3.1 and SW480 group,respectively [(0.78±0.03vs. 0.01±0.00, P<0.05) and (0.78±0.03 vs. 0.01±0.00, P< 0.05)].(Fig.2)3 SW480 cells were stimulated with U0126 for 5,10,20 and 30 min. Expression of total ERK in different SW480 cells detected by Western blot show that SW480 incubated with U0126 for 20 min were lower than that the other group,respectively.(Fig.3)4 SW480/FLNa cells were stimulated with EGF for 5,10,20 and 30 min. Expression of total ERK in different SW480/FLNa cells detected by Western blot show that SW480/FLNa incubated with EGF for 20 min were higher than that the other group,respectively.(Fig.4)5 The cells are classified into three categories (SW480,SW480 incubat- ed with U0126 for 20 min and SW480/FLNa).Expression of total ERK, p- ERK in different cells detected by Western blot show that SW480 incubated with U0126 for 20 min were lower than that the other group,respectively.(Fig.5)6 The cells are classified into three categories (SW480/FLNa,SW480/ FLNa incubated with EGF for 20 min and SW480).Expression of total ERK, p-ERK in different cells detected by Western blot show that SW480/ FLNa incubated with EGF for 20 min were higher than that the other group,respecti- vely.(Fig.6)7 The cells are classified into three categories (SW480, SW480 incubated with U0126 for 20 min and SW480/FLNa).Using migration and invasion exp- eriment to detect the different cells'migratory and invasive abilities,the results of number in different cells detected show that SW480 were higher than that in SW480 incubated with U0126 for 20 min and SW480/FLNa group, respect- tively [(213±15vs. 82±13, P<0.05) and (213±15 vs. 75±11, P<0.05)](.Table1)8 The cells are classified into three categories (SW480/FLNa,SW480/ FLNa incubated with EGF for 20 min and SW480).Using migration and inva- sion experiment to detect the different cells'migratory and invasive ability- ies,the results of number in different cells detected show that SW480/ FLNa were lower than that in SW480/FLNa incubated with EGF for 20 min and SW 480 group,respectively [(54±12vs. 125±15, P< 0.05) and (54±12vs.130±14,P <0.05)].(Table2)Colusion:1 The cells are classified into two categories (SW480/FLNa and SW480). Expression of total ERK,p-ERK in SW480 cells detected by Western blot sh- ow that the inhibitory effects on SW480 were proved.2 Using migration and invasion experiment to detect the different cells'migratory and invasive abilities,the results show that inhibition effects medi- ated by filaminA(FLNa) on invasion and metastasis of human colon carcino- ma cell line SW480 in vitro.3 SW480 cells were stimulated with U0126 and it indicated that the inhibitory effects on proliferative and invasive potential of a human Colorectal carcinoma cell line SW480 were able to in the expression of ERK.SW480 /FLNa cells were stimulated with EGF and it indicated that the potentiation effects on proliferative and invasive potential of SW480/FLNa were able to in the expression of ERK.
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