| Colorectal cancer(CRC) is one of the most common malignant tumors of human digestive tract, in the world wide, CRC morbidity is located in third of the common malignant tumors. In the world wide, morbidity of CRC is located in a low level in our country, but in the recent years, with the development of economic, the improvement of people's living standards and the change of lifestyles and living conditions, the morbidity,the prevalence rate and mortality rates of colorectal cancer are increasing.It is generally thought that the occurrence of CRC is an multifactor and multistep process, it's also the result of the internal factor interact with many external factors (such as biological, physical, chemical and immune factors). But which is the most important factor is unclear. The relation between cell apoptosis and tumor become the focus of Scientific research Since Kerr, Currie and Wyllie proposed the concept of apoptosis in 1972. It is thought that tumor occurrence is the result of the process of losing balance of cell proliferation and apoptosis. Twist is a highly conserved transcription factor that belongs to the family of basic helix-loop-helix proteins, and it plays an important role in the embryogenesis。Twist gene broadly take part in the process of tumor occurrence and cell proliferation and it also can alter tumor cell apoptosis, inhibit cellular differentiation, induce EMT process, take part in the process of tumor drug resistance and tumor angiogenesis. EMT plays an important role in embryogenesis, chronic inflammation and the process of tumor development ,the main character of EMT is the process of epithelial cells lose polarity and gain more ability of migration.Tumor cells gain more higher infiltrating and migration ability through EMT, so in the recent years research we discover that EMT have correlation with tumor infiltration and migration, if tumor cell take place EMT we may define it as tumor infiltration and migration. Twist gene plays a key role in adjusting EMT process.It is reported that in vitro and vivo experiment of mice breast cancer cell stub Twist gene was overexpressed in the tumor migrant step. The recent years research discover that Twist gene have the characteristic of cancer gene, it can code apoptosis inhibition protein and play an important role in the progress of tumor occurrence and development. It was shown that twist was over-expressed in various kinds of solid tumors, like breast cancers, prostate cancers, melanoma, osteosarcoma and so on. But the report about how the Twist gene worked in the colorectal cancer and it's mechanism is unclear in the domestic and international study.We research the function of Twist gene in colorectal cancer occurrence and development progress through three parts:1.we used RT-PCR and Western-blot methods to examine the expression of Twist mRNA and Twist protein in different transfer capabilities colon cancer cell lines;2.We up-regulated the Twist gene expression in human colon cancer SW480 cell line, and observed the effect of Twist gene on proliferation,apoptosis and invasive capabilities of colon cancer SW480 cells.3.We used siRNA to down-regulated Twist gene expression in human colon cancer HCT116 cell line and observe the effect of Twist gene on proliferation,apoptosis and invasive capabilities of human colon cancer HCT116 cell.Part 1 The research of the different expression of Twist gene in different transfer capabilities colon cancer cell linesObjective: We detected the expression level of Twist gene in different transfer capabilities colon cancer cell to separate the high and low Twist gene expression level cell lines, which will facilitate for the further study of the effect of twist gene on colon cancer cell biological character.Methods:1 We used RT-PCR to detected the expression level of Twist mRNA in SW480,SW620 and HCT116 colon cancer cell lines.2 We used Western-blot method to detected the expression level of Twist protein in SW480,SW620 and HCT116 cell lines . 3. We used SPSS 13.0 software to make Statistical analysis. The datas were presented as mean±standard deviation( (X|-)±S),P<0.05 means statistical significance.Results:1 The expression of Twist mRNA in different transfer capabilities cell linesRT-PCR results showed that the expression level of Twist mRNA in HCT116, SW620,SW480were 0.985±0.015,0.506±0.020 and 0.445±0.025 correspondingly. The expression of Twist mRNA in high transfer capability HCT116 cell line is higher than the other two cell lines and the difference has statistical significance (P<0.05).The Twist mRNA expression in SW480 is less than in SW620 cell line, well the difference has no statistical significance (P﹥0.05).2 The expression of Twist protein in different transfer capabilities cell lines.Western-blot results demonstrated that: the expression level of Twist protein in HCT116,SW620, SW480 were 1.488±0.060,0.751±0.022 and 0.651±0.030 correspondingly. The epprssion of Twist protein in high transfer capability HCT116 cell line has relatively more Twist protein expression than the other two cell lines and the difference has statistical significance(P<0.05). The Twist protein expression in SW480is less than in SW620 cell line, well the difference has no statistical significance(P﹥0.05).Conclusions: The gene of Twist has significant expression differences in three cell lines. In HCT116, which is colon cancer cell line with high transfer capability, the Twist gene has especially high expression level. That means the stronger invasion the tumor cells have, the higher expression level the Twist gene be.Part 2 Effects of up-regulated Twist gene expression on the proliferation,apoptosis and invasive capability of human colon cancer SW480 cellObjective: To choose the human colon cancer cell line which expressed Twist gene in lower level, and transfect the cell with high-expressed Twist plasmid, to evaluate the effect of Twist gene on the proliferation,apoptosis and invasive capability of human colon cancer through vitro and vivo experiments.Methods: We used RT-PCR and western-blot to examine the expression of Twist mRNA and protein in HCT116,SW480,SW620 three colon cancer cells, chose the cell which expressed Twist gene in lower level。We transfected the cell with high-expressed Twist plasmid and used RT-PCR and western-blot to examine the transfection result ,G418 experiment to sieve the cells which expressed Twist geng stably. We used MTT method to observe cell proliferation; used Wound Healing assay to observe cell motility rate; used Matrigel invasion assay to observe cell invasiveness; used FCM to observe the effect of Twist gene on cell cycle and apoptosis. We injected the cell to the nude mice and observed the tumor forming condition .Results:1 We used RT-PCR and Western blot method to detect transfer resultThe results showed that the Twist gene mRNA and protein expression in SW480 cells which transfected stably with Twist plasmid significantly higher than in control group (P<0.05).2 The effect of up-regulated Twist gene on vitro proliferation of SW480 cellMTT exprement result show:compared with control group, the growth rate of stably transfected group was significantly higher than control group since the 4th day.(P<0.05).3 The effect of up-regulated Twist gene on cell cycle of SW480 cellThe result of cell cycle detected by FCM show: the proliferation index(PI) of transfected group was(PI=61.279±1.709%)and the cell ratio in Sstage was (33.171±3.154%).The two number were significantly higher than control group(PI=26.142±1.518%,14.112±2.137%,P<0.05).The result show that: transfected with the Twist gene can intensify the vitro proliferation of SW480cell.4 The effect of up-regulated Twist gene on vitro migration of SW480 cellWound Healing assay results show: The transfected group cells migrated rapidly and closed the wound by (40.06±5.56)% after 24h and (25.24±2.65)% after 48h. In contrast,control group cells only closed the wound by (75.77±8.06)% and (35.37±6.79)% in the same time interval. The wound closed rate in transfected group cells was significantly higher than in control groups (P<0.05).The result show that: transfected with the Twist gene can intensify the vitro migration of SW480 cell.5 The effect of up-regulated Twist gene on vitro invasion of SW480 cellThe number of invasive cells in transfected groups were(154±12)and were(73±14) in control group. The invasive cells number in transfected groups was significantly higher than in control group(P<0.05). The result show that: transfected with the Twist gene can intensify the vitro invasion of SW480 cell.6 The effect of up-regulated Twist gene on apoptosis of SW480 cellFCM result show that : The apoptosis rate of SW480 cell in transfected group was 6.831±1.624%,the apoptosisi rate of cell in control group was 12.233±1.733%,the apoptosis rate in transfected group was significantly lower than in control group(P<0.05). The result show that: transfected with the Twist gene can lower the apoptosis rate of SW480 cell.7 The effect of Twist gene on the ability of tumorigenesis of human colon cancer of SW480cell in nude mice skin down implanted tumorwe can observed the tumor after injected transfected and control group cells into nude mice 16 days. The volume of tumors in transfected and control group were 1.804±0.419cm~3 and 0.636±0.228 cm~3 respectively, the volume of tumor in transfected group was significantly bigger than in control group(P<0.05). The weight of tumor in transfected and control group were 6.435±0.421g and 3.357±0.325g respectively, the weight of tumor in transfected group was significantly heavier than in control group(P<0.05).Conclusions:1 In this experiment Twist gene was transfected successfully in the SW480 cells and can be stably expressed.2 After we up-regulated the expression level of Twist gene it can be improve the vitro proliferation of the SW480 cell .3 After we up-regulated the expression level of Twist gene it can be improve the vitro migration and invasion capability of the SW480 cell. 4 After up-regulated the expression level of Twist gene it can be lower the apoptosis rate of SW480 cell.5 The vivo exprement result show: After up-regulated the Twist gene, the tumorigenesis of SW480 had no apparent change, but the tumor weight and volumn of the transfected group were significantly higher than control group.That result proved that Twist gene can intensify the proliferation of SW480 cell in vivo.Part 3 Effects of down-regulated Twist gene expression on the proliferation,apoptosis and invasive capability of human colon cancer HCT116 cellObjective: To observe effect of down-regulated the expression of Twist gene on the proliferation, apoptosis and invasive capability of human colon cancer HCT116 cell.Methods: we used siRNA technique to down-regulated the Twist gene expression,RT-PCR and Western blot to examine the transfection result,G418 method to choose the cells. We used MTT method to draw the Cell growth curve, used wound healing assay to observe the cell motility rate, used Matrigel invasion method to observe cell invasiveness in vitro. We injected the cell to the nude mice and observed the tumor forming condition .Results:1 We used RT-PCR and Western blot method to detect transfer resultThe results show: Twist mRNA and protein is lower in the Twist-si group cells than in control group cells(P<0.05).2 The effect of down-regulated Twist gene on vitro proliferation of HCT116 cellMTT method result show: compared with control group, the growth rate of Twist-si group was significantly lower than control group(P<0.05) scine the fourth day.3 The effect of down-regulated Twist gene on cell cycle of HCT116 cellThe result of cell cycle detected by FCM show:the proliferation index(PI) of Twist-si group was(PI=28.989±1.795%)and the cell ratio in Sstage was (15.818±2.115%).The two number were significantly lowerr than control group(PI=57.333±1.627%,30.318±3.137%,P<0.05).The result show that: down-regulated Twist gene can inhibit the vitro proliferation of HCT116cell.4 The effect of down-regulated Twist gene on vitro migration of HCT116 cellWound Healing assay results show: the Twist-si group cells migrated slowly and closed the wound by (15.06±2.56)% after 24h and (25.77±4.06)% after 48h. In contrast , control group cells only closed the wound by (25.24±1.65)%and (45.37±6.79)% in the same time interval. The wound closed rate in Twist-si group cells was significantly lower than in control groups (P<0.05).The result means that: down-regulated Twist gene can inhibit the vitro migration of HCT116cell.5 The effect of down-regulated Twist gene on vitro invasion of HCT116 cellThe number of invasive cells in Twist-si groups were(68±18)and were(123±20) in control group. The invasive cells number in Twist-si group was significantly lower than in control group (P<0.05). The result means that: down-regulated Twist gene can inhibit the vitro invaive capability of HCT116cell.6 The effect of down-regulated Twist gene on apoptosis of HCT116 cellFCM result show that : The apoptosis rate of HCT116 cell in Twist-si group was 30.625±3.644%,the apoptosisi rate of cell in control group was 10.423±3.506%,the apoptosis rate in Twist-si group was significantly higher than in control group(P<0.05). The result show that: down-regulated the Twist gene can raise the apoptosis rate of HCT116 cell.7 The effect of Twist gene on the ability of forming tumor of human colon cancer cell HCT116 in nude mice skin down implanted tumorwe can observed the tumorgenisis after we injected cell in nude mice 12 days. The volume of tumor in Twist-si and control group were 0.836±0.218 cm~3 and 1.814±0.257 cm~3 respectively, the volume of tumor in Twist-si group was significantly smaller than in control group(P<0.05). The weight of tumor in transfected and control group were 3.637±0.225g and 6.530±0.421g respectively, the weight of tumor in Twist-si group was significantly lighter than in control group(P<0.05).Conclusions:1 In this experiment siRNA can was successfully down-regulate the expression of Twist gene in the HCT116 cell.2 After we down-regulated the expression level of Twist gene it can be lower the vitro proliferation of the HCT116 cell .3 After we down-regulated the expression level of Twist gene it can be lower the vitro migration and invasion capability of the HCT116 cell.4 After down-regulated the expresion level of Twist gene it can be improve the apoptosis rate of HCT116 cell.5 The vivo exprement result show: After down-regulated the Twist gene, the tumorigenesis of HCT116 had no apparent change,but the tumor weight and volumn of the Twist-si group were significantly lighter than control group.That result proved that Twist gene can inhibit the proliferation of HCT116 cell in vivo. |
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