Silencing Of FGFR4 Could Influence The Biological Function Of Gastric Cancer Cells And Its Therapeutic Value

Gastric cancer is one of the most common malignant tumor of the world, in Asia the incidence is higher than other continents. In China, the diagnosis rate of early gastric cancer is about 20%, which is far less than other Asian countries such as South Korea and Japan, especially the terminal cancer or the metastatic gastric cancer. The advanced gastric cancer in China, with poor outcomes, often needed to be given priority with surgery, radiation, chemotherapy and other auxiliary treatment. At present, there are many adjuvant chemotherapy and palliative chemotherapy for gastric carcinoma, many of which platinum and fluorouracil for first-line drugs, such as cisplatin and fluorouracil, fluorouracil plus oxaliplatin into, DCF, ECF, etc..Because of the complexity of the pathogenesis and uncertainty to gastric cancer, the survival time is not ideal. As the NCCN guidelines in America and the European society of medical oncology(ESMO) are not standard solution for the treatment of gastric cancer. In recent years, although targeted drugs in the treatment of cancer of the stomach have became a hot spot, and the occurrence of gastric cancer development are multiple factors and multiple stages and multiple genes involved in the process of its pathogenesis is not clear. Therefore, in addition to Hector plug butyl, most of the drugs did not got a breakthrough in the study of gastric cancer. Looking for new target genes and illuminating the mechanism of gastric cancer have important effects in stomach cancer therapy.As reported in research of targeted drugs in recent years, EGFR、VGFR etc. However, these studies are not effective for the treatment of gastric cancer. So looking for new target genes has important effect in treating stomach cancer.In recent years, there are many research on FGFR, FGFRs is one member of immunoglobulin superfamily, locating at a variety of cell membrane, and the FGFR4 Gly388Arg polymorphism is the study hotspot. Protein encoding by FGFR4 is a transmembrane protein distributing a variety of cells, which is a receptor tyrosine kinase (RTK). FGFR4 plays an important role in physiological responses, including cell proliferation and differentiation, trauma repairing, angiogenesis, muscle formation, and development of an individual. Some studies have shown that the expression of FGFR4 is abnormal in many cancers such as pancreatic cancer and breast cancer, so FGFR4-targeted therapeutics has a wide application prospect. But as so far, FGFR4 research rarely and not thorough in gastric cancer.Some reports suggest BGJ398 and FGFR family have very good affinity and selectivity, and BGJ398 can inhibit liver cancer cell proliferation. Some studies suggest that BGJ398 can reverse FGF19 specificity stimulation of AFP production increases, thus speculated that BGJ398 can inhibit the activity of FGFR4.20 ever found in a variety of fibroblast growth factor (FGF) in 10 can combine with FGFR4, only FGF19 combined with FGFR4 specificity.FGFR4 activated can make FGF19 promote liver cell proliferation, induction of liver cancer formation. Some studies suggest that BGJ398 can reverse FGF19 specificity stimulation of AFP production increases, thus speculated that BGJ398 can inhibit the activity of FGFR4.The current FGFR4 less research in gastric cancer, so this topic discusses FGFR4 in its role in the development of cancer of the stomach, for the early diagnosis and prognosis of gastric cancer, providing strong theoretical basis for gene therapy.Through the body function in vitro study FGFR4 detection in different sources of biological characteristics of tumor cell lines express the similarities and differences, discuss fibroblast growth factor receptor 4 (FGFR4) in gastric cancer (GC) function, and explore the agent for FGFR4 therapeutic value. The first two parts of this topic major on the genetic level and mRNA level and protein level To study the correlation of FGFR4 molecules with cancer of the stomach, And through the experiment in vitro,to research the influence of FGFR4 on biological characteristics of gastric cancer cell lines. By slow virus building FGFR4 RNAi, gastric carcinoma model used in nude mice, and in vivo in vitro correlation experiment, detect gastric cancer biology related protein expression and the changes. Using 5-Fu and BGJ398 single-agent or joint, intervention slow virus and cell lines, the in vivo and in vitro experiments, discusses the joint after the two drugs on gastric cancer biological behavior and mechanisms of the influence of a more comprehensive study FGFR4 in its role in the development of cancer of the stomach, provide the basis for BGJ398 applied in clinical treatment of gastric cancer, and provide a new evaluation index for the prognosis of gastric cancer.Part One The silencing of FGFR4 could influence the features of gastric cancer cellsChapter One The silencing of FGFR4 could influence the features of gastric cancer cellsBackground and projectiveGastric cancer (GC) is the fourth most common malignancies in the world. In China, the diagnosing rate of early GC is only 20%, and most patients are often diagnosed at advanced stage, so the prognosis is poor. Patients with GC often need combined therapy including operation, radiotherapy and/or chemotherapy. There are many chemotherapy regimens in which platinum and 5-fluorouacil are used as first-line drugs, such as cisplatin plus 5-fluorouacil, oxaliplatin plus 5-fluorouacil, DCF, ECF and so on, but improved survival time is dissatisfactory. Recently, study of targeting drugs for GC becomes a hot research point, but no breakthrough progress has been made in many drugs except trastuzumab. Therefore, it is critical for GC treatment to search new target genome and elucidate its pathogenesis. FGFR is one member of immunoglobulin superfamily, locating at a variety of cell membrane, and the FGFR4 Gly388Arg polymorphism is the study hotspot. Protein encoding by FGFR4 is a transmembrane protein distributing a variety of cells, which is a receptor tyrosine kinase (RTK). FGFR4 plays an important role in physiological responses, including cell proliferation and differentiation, trauma repairing, angiogenesis, muscle formation, and development of an individual. Some studies have shown that the expression of FGFR4 is abnormal in many cancers such as pancreatic cancer and breast cancer, so FGFR4-targeted therapeutics has a wide application prospect. However, the characteristics of FGFR4 in GC are rarely studied and remain unknown. This study constructed a FGFR4 silencing cell line mediated by Lentiviral Vector, to explore its influence on the capability of proliferation, invasion and apoptosis of gastric cell line. Moreover, we explored the possible mechanisms how FGFR4 silencing influence the biological behavior of gastric cancer cell line, via by comparing the expression of proteins among different treated-groups.MethodUsing the Real-time PCR detection FGFR4 in gastric cancer cell line HGC27, MKN45, MKN28, MGC803, BGC823, SGC7901 and 293T, select high expression cell. Construct FGFR4-silencing cell line, and identified the changes of proliferation between FGFR4-silencing cell lines and normal cell line via by CCK8. Flow cytometry sorting test FGFR4-silencing influence on the apoptosis of gastric cancer cell lines, and the changes of its invasion capability were tested by Transwell. Western Blot tested the changes of proteins expression among different treated-groups.Result:This study successfully constructed FGFR4-silencing cell line, and set up three groups including normal cell line, negative plasmid group and FGFR4-silencing group. The rate of proliferation and invasion capability was lower in FGFR4-silencing cell line than in normal cell line and negative plasmid group, the rate of apoptosis was enhanced, and the expression of PCNA and Bcl-xl decreased.ConclusionThis study screened out two FGFR4-overexpression strains from gastric cancer cell lines with different malignant degree, and constructed FGFR4-silencing gastric cancer cell line by lentiviral transfection. Then by studying its proliferation, invasion capability, apoptosis and related proteins, we found the ability of proliferation and invasion decreased in FGFR4-silencing cell line, the rate of apoptosis increased. It indicated that FGFR4 could promote the proliferation and invasion ability of gastric cancer cell, and increased its malignant degree.Chapter Two The silencing of FGFR4 could influence the features of Nude mice into gastric cancer tumorBackground and aims:FGFR4 was highly expressed in common malignant cancers such as pancreatic cancer, rhabdomyosarcoma, breast cancer and renal cancer. So far, only 10 of more than 20 fibroblast growth factors (FGF) can combine with FGFR4, and only FGF19 can combined with FGFR4 specifically. Currently, the characteristics of FGFR4 in gastric cancer haven’t been studied. Therefore, this study aims to study the role of FGFR4 in the development of gastric cancer, to provide theoretical evince for its early diagnosis, prognosis and gene therapy.Methods:The FGFR4-silencing cell line and the corresponding untreated-gastric cancer cell lines were injected into the back of nude mice subcutaneously, to construct mice model with gastric cancer. After 6-8 weeks of inoculation, the volume and size of tumor was recorded. The proteins related to proliferation and apoptosis were tested by western blotting, to analyze the effect of FGFR4-silencing on the growth of gastric cancer tissue.Results:The volume of tumor was smaller in nude mice injected HGC27 and MKN45 cell lines with FGFR4-silencing, than in negative control group and blank control group (P< 0.05). The difference was remarkable in 10th,13rd, and 16th day after inoculation. The apoptosis of cell lines was tested by TUNEL, and we found the rate of apoptosis increased in FGFR4-silencing group. In immunohistochemical staining, the expression of PCNA and Bcl-xl decreased and the expression of Caspase3 increased.Conclusion:FGFR4-silencing could inhibit the proliferation and enhance apoptosis of gastric cancer in nude mice model. Therefore, we deduce that low expression of FGFR4 should slow down the development of gastric cancer.Part Two The FGFR4 silencing could influence the features of gastric cancer cells and inhibit the efficacy of BGJ398 and 5-Fluorouracil towards gastric cancerBackground and projectiveBGJ398 and FGFR family have very good affinity and selectivity, and it can inhibit liver cancer cell proliferation. Although we can’t rule out BGJ398 may inhibit FGFR1, FGFR2 or FGFR3 to reach the purpose of inhibiting cell proliferation, but BGJ398 can reverse FGF19 specificity stimulation of AFP production increases, prompting BGJ398 inhibit cell proliferation mainly by inhibiting FGFR4. In addition, the BGJ398 for FGFR4 high expression of liver cancer cell lines HuH7d antiproliferative effect significantly better than FGFR4 lower expression of liver cancer cell lines and the cancer cells, thus further illustrate FGFR4, compared to the other FGFRs subtype of BGJ398 efficiency play a more important role. Little FGFR4 research in gastric cancer, so we will focus on FGFR4 in its role in the development of gastric cancer.MethodUsing BGJ398 and 5-Fu treat FGFR4 silencing cell line by single-agent or a combination of two drugs. respectively use no processing cell lines as a control, making a series of function test in vitro to evaluate the influence of the biological behavior of the gastric cancer cell lines with lower expression of FGFR4 by BGJ398 and 5-Fu. At the same time, we detect the changes of cell proliferation, invasion, apoptosis and cell cycle related protein expression after different treatment factors processing at the molecular level.Result:The gastric cancer cell line MKN45 and HGC27 cell proliferation was inhibited with 5-Fu or BGJ398 and combination of the two medicines increasing the rate of cell apoptosis. After constructed FGFR4 over expression cell lines by transfection technology, cell sensitivity of 5-Fu or BGJ398 and the combination of the two drugs iecreased, cell proliferation inhibition of increase, apoptosis rate increase comparing not the silencing of cell lines.ConclusionThe gastric cancer cell line MKN45 and HGC27 cell proliferation was inhibited with 5-Fu or BGJ398 and combination of the two medicines increasing the rate of cell apoptosis. FGFR4 silencing may be able to increase the effect of inhibition of gastric cancer cell line by 5-Fu and BGJ398.