| Gastric cancer is a serious threat to human health, whose incidence and mortality remain high in recent years. In 2012 the number of new gastric cancer cases reached 951,000 worldwide and ranked No. 5 in all tumors. China has a high incidence of gastric cancer. In 2012 the number of new gastric cancer cases ranked No.1 in the world, seriously affecting the health of residents and bringing a heavy burden to the national health resources. At present, there are various treatments for gastric cancer, including surgery, chemotherapy, radiation therapy, biological targeted therapy, immunotherapy and Chinese medicine treatment. A variety of treatment methods are constantly improved, but as a result of the insidious onset in some patients, weak health care awareness and the lack of simple and effective screening method, the overall detection rate of early gastric cancer is still low. Most patients have been in the advanced stage when diagnosed with gastric cancer. So despite the continuous improvement of medical treatment, the 5-year survival rate of patients with gastric cancer is still not ideal and there is no significant improvement in overall prognosis. It is very important to further explore the molecular mechanism of the biological behavior of gastric cancer and to provide new indicators and targets for the prognosis as well as the treatment of gastric cancer.X-box binding protein-1(XBP1) is an important transcription factor in eukaryotic cells and an essential component in the unfolded protein response(UPR) signaling pathway. When cells are in unfavorable microenvironment such as hypoxia, nutritional deficiencies or oxidative stress, endoplasmic reticulum protein folding disorders may occur during assembly, resulting in abnormal accumulation of a large number of unfolded or misfolded proteins in the endoplasmic reticulum and further triggering cascade of intracellular signaling stress response, which is called “unfolded protein response”. Its function is to activate cell signaling pathways to promote cell survival adaptation or apoptosis by inducing programmed cell death. Inositol-requiring enzyme 1α(IRE1α) / X-box binding protein-1(XBP1) pathway is one of the three main UPR pathways. In this process, IRE1α unconventionally splices XBP1 encoding m RNA with its endonuclease activity. The spliced XBP1 m RNA translates the active form of X-box binding protein 1(XBP1s), which enters the nucleus and regulates expression of target genes to facilitate re-folding or degradation of the unfolded or misfolded proteins in endoplasmic reticulum. This process eliminates the stress in the endoplasmic reticulum and promotes cell survival.The metabolism of tumor cells is vigorous. The blood supplies the oxygen and nutrients needed for the growth of tumor cells. As tumor grows, angiogenesis therein need some time. When blood vessel growth lags the tumor growth and the blood supply can not fully meet the demands of the growth and proliferation of tumor cells in oxygen and nutrients, cells will turn into a state of hypoxic, affecting the assembly and folding process of proteins in the endoplasmic reticulum, resulting in the endoplasmic reticulum stress and triggering the unfolded protein response. Previous studies have confirmed the existence of endoplasmic reticulum stress and UPR activation in tumor cells, which is related to the cancer cell survival and resistance to anti-cancer therapy. Over-expression of UPR components(e.g. 78 k Da glucose regulated protein(GRP78), 94 k Da glucose regulated protein(GRP94)) has been detected in breast cancer, liver cancer, stomach cancer and esophageal adenocarcinoma. Various studies have confirmed the close relationship between UPR component GRP78 and the prognosis in a variety of cancers. The expression of XBP1 is increased in breast cancer, which is associated to the rapid growth of breast cancer cells, resistance to endocrine therapy and lower survival rate. Some other animal experiments showed that the lack of XBP1 could significantly inhibit tumor formation and development. Therefore, it is of considerable significance to further explore the role of XBP1 in the development of tumors. As yet, there is no study regarding the XBP1 expression in gastric specimens or its role in the development of stomach cancers.The present study detected the expression of XBP1 in clinical specimens of gastric cancer, analyzed the relationship between XBP1 and clinic opathological features and its impact on the prognosis of patients with gastric cancer. Furthermore, gastric cancer cell lines were used to study the effects of XBP1 on gastric cancer cell proliferation, apoptosis, migration and invasion. The molecular mechanism was explored to provide a new therapeutic indictor and target for the prognosis of patients with gastric cancer.Part Ⅰ: the expression of of X-box binding protein 1 in gastric cancer and its impact on the prognosis of patientsObjective: To detect X-box binding protein 1 expression in gastric cancer and analyze its relationship with the clinical pathological features and prognosis of patients with gastric cancer.Methods: RT-PCR and Western blot were used to detect the RNA and protein expression of XBP1 in fresh cancer tissues and borderline tissues, normal gastric mucosa tissues. The expression differences between the three tissues were compared. Immunohistochemical assay was used to detect XBP1 expression in 30 cases of gastric cancer tissues, 30 cases of borderline tissues, 30 cases of gastric ulcer tissues and 30 cases of normal gastric mucosa tissues. Analysis of variance was used to compare the expression differences of XBP1 between different tissues; immunohistochemical assay was also used to detect XBP1 expression in 208 cases of gastric cancer specimens. We analyzed its relationship with clinicopathologic factors of patients with gastric cancer and its impact on their prognosis. Statistics analysis was performed using SPSS 19.0 statistical software; P <0.05 was considered statistically significant.Results: RT-PCR and Western blot analysis showed that both the RNA and protein levels of XBP1 in gastric cancer were higher than borderline tissues and normal gastric mucosa tissues. The difference was statistically significant(P<0.05). Immunohistochemical assay showed that in 30 specimens, XBP1 highly expressed in 22 cases of gastric cancer, in 10 cases of borderline tissues, in 12 cases of gastric ulcer and in 9 cases of normal gastric mucosa. In gastric cancer the XBP1 expression was higher than other groups. The difference was statistically significant in gastric cancer(P <0.05). However, the difference of XBP1 expression in borderline tissues, gastric ulcer tissues and normal gastric mucosa was not statistically significant. In 2008 cases of gastric cancer specimens, there were 129 cases of high XBP1 expression and 79 cases of low XBP1 expression. Chi-square test analysis found that XBP1 expression was associated with depth of invasion, lymph node metastasis, TNM stage(P <0.05), but not associated with the age of patient undergoing surgery, sex, blood type or tumor site(P> 0.05). Spearman rank correlation analysis showed that XBP1 expression was positively correlated with depth of invasion, lymph node metastasis and TNM stage(P <0.05). Kaplan-Meier survival analysis showed that in high XBP1 expressed patients the median survival time was 25 months, while in low XBP1 expressed group the median survival time was 60 months. Log-rank used to test 5-year survival rate between the two groups showed remarkable statistical significance(P> 0.05).Conclusion: The XBP1 expression in gastric cancer was higher than non-cancerous gastric tissue; The XBP1 expression in gastric was positively correlated with depth of invasion, lymph node metastasis and TNM stage. High XBP1 expressed gastric cancer patients had better prognosis than low XBP1 expressed patients; XBP1 is one of the independent factors affecting the prognosis of patients with gastric cancer.Part Ⅱ : the impacts of X-box binding protein 1 on gastric cancer cell proliferation, apoptosis, migration, invasion and the mechanismObjective: To study the impact of XBP1 on gastric cancer cell proliferation, apoptosis, migration and invasion and to explore the intrinsic molecular mechanisms.Methods: AGS gastric cancer cell line was used. The impact of XBP1 expression interference on gastric cancer cell proliferation, apoptosis, migration and invasion was studies by proliferation assays(with MTT assay), apoptosis study, chemotaxis assays and invasion assay; AGS cells were cultured under hypoxia condition(0.1% O2). Chromatin immunoprecipitation assay(CHIP) was employed to detect the association between cellular XBP1 expression and VEGFa, MMP9, COX2 and MMP2 gene; Co-Immunoprecipitation(Co-IP) was used to detect whether HIF1 interacted with XBP1.Results: Compared with the control group, the gastric cancer cell proliferation rate in si-XBP1 group was decreased but apoptosis was increased; migration and invasion were weakened. All the differences were statistically significant. After XBP1 expressing in cells was silenced, VEGFa, COX2 and MMP2 m RNA was dramatically reduced and MMP9 m RNA was reduced. XBP1 was combined with VEGFa, MMP9, COX2, MMP2 genes and there was interaction between XBP1 and HIF1.Conclusion: The expression of XBP1 can promote AGS gastric cancer cell proliferation, reduce the rate of apoptosis, migration and enhance the invasion capability of gastric cancer cell. Under hypoxic environment, XBP1 can regulate VEGFa, MMP9, COX2, MMP2 gene transcription through its interaction with HIF. |
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