| Background In current clinical gastrointestinal tumors, the incidence of gastric cancer is the highest and the mortality mortality rate ranks third in the world. Our country located in the region with high incidence of gastric cancer. Despite mortality rate of gastric cancer in our country have decline in recent years, but still in the top five. In the past few years, with advances in surgical techniques, chemical therapy, radiotherapy and molecular targeted therapy to cancers, the prognosis of GC was improved. But the long-term outcomes of GC patient remained dismal, especially for advanced GC with a 5-year overall survival rate of 40% or less.Lin28B protein is a homologue of Lin28, a highly conserved RNA-binding protein originally identified in Caenorhabditis elegans. Lin28B contains a cold shock domain and retroviral-type CCHC zinc fingers that confer RNA-binding ability. Lin28 is readily expressed in embryos and embryonic stem cells, yet its expression remains at low levels in normal adult tissues, suggesting that Lin28 may play a critical role in cell proliferation and differentiation during embryonic development. Furthermore, Lin28B promote malignant transformation, and their expression is associated with advanced stages of numerous types of tumors, including hepatocarcinoma, nephroblastoma, ovarian carcinoma and germ cell tumors. It is highly expressed in various tumors, such as hepatocellular carcinoma and colorectal cancer. Overexpression of Lin28 has been shown to promote cancer cell proliferation. High expression of Lin28 is a poor prognostic factor in hepatocellular cancer and it is significantly associated with lymph node metastasis in colorectal cancer.MicroRNAs (miRNAs) are small noncoding RNAs,22 nucleotides in length, that repress target messenger RNAs (mRNAs) through an antisense mechanism. Recently, increasing evidence has indicated that miRNAs are involved in the development and progression of cancers, acting as tumor suppressors or oncogenes. miRNA Let-7 which was first identified in C.elegans as a heterochronic gene, promotes the transition of larval stage 4-to-adult. Further research on Let-7 revealed its function in cell proliferation, differentiation, apoptosis, and metabolism. The Let-7 level were found to be low in different human tumors, and its loss or down-regulation was associated with increased tumors aggressiveness and poor clinical outcome. Ectopic expression of Let-7 reduced chemoresistance and invasiveness of cancer cells, suppressing tumor growth of human lung cancer in vivo.At the molecular level, Lin28 acts as a suppressor of let-7 microRNA biogenesis. Lin28 and its homolog, Lin28B have been known to regulate all let-7 family members through a maturation process and cellular differentiation. Lin28 was reported to act as a transacting regulator which binds let-7 pre-miRNA to block its maturation in embryonic stem cells. This particular function of let-7 was used in the process of establishing induced pluripotent stem cells(iPS cells) from human fibroblasts in order to enhance the efficiency of cellular formation. A number of studies have also suggested an association between Lin28 and let-7, with particular emphasis upon their involvement in cellular processes such as differentiation and proliferation.However, a correlation between Lin28 and let-7 and its effects on progress in gastric cancer has not yet been confirmed. Therefore, we intend to design the following experiments including in vivo and in vitro studies to clarify Lin28B and Let-7 regulatory relations and its effects on the progress in gastric cancer.Objective 1. Respectively in gastric cancer cell lines and gastric cancer, to detect the expression of Lin28B and Let-7 respectively and to analyze whether there is a correlation;2. In gastric cancer cells, to further confirm the presence of Lin28B-Let-7 regulation axis;3. In human gastric cancer cells with overexpression of Let-7, to observe the effect on cell proliferation and cell cycle;4. In human gastric cancer cells with overexpression of Let-7, to observe the changes of sensitivity to chemotherapeutic drug 5-fluorouracil (5-FU);5. To observe the effect of overexpression of Let-7 on growth of subcutaneous transplantation tumor in nude mice.Methods 1. The Western Blot assay and RT-PCR assay were performed to detect the Lin28 expression in gastric cancer tissue samples and normal tissues; the qRT-PCR assay was performed to detect the Let-7 expression in gastric cancer tissue samples and normal tissues; to detect expression of Lin28B and Let-7 in CD 133+/CD44+ cells sorted by flow cytometry (FCM) in gastric cancer cell line BGC-823.2. The lentivirus transfection method was performed to transfect the Let-7 into BGC-823 cells and then to detect Lin28B expression levels.3. CCK-8 assay was performed to detect the effect of overexpression of Let-7 on cell proliferation in BGC-823 cell.4. CCK-8 assay was performed to detect the sensitivity to chemotherapeutic drug5-fluorouracil (5-FU) in the BGC-823 cell and the BGC-823 cell transfected with Let-7.5. Sphere-forming assay was performed to evaluate the impact of cell differentiation in the BGC-823 cell line.6. To evaluate the impact of overexpression of Let-7 in a nude mouse xenograft model. Results 1. Lin28B and Let-7 in gastric carcinoma and adjacent normal tissue can be detected. The level of Let-7 expression in gastric tumor tissues was significantly lower than that in the adjacent normal tissues, the difference was statistically significant (P <0.05); the level of Lin28B expression level was significantly higher than those in adjacent normal tissue in gastric cancer tissues, the difference was statistically significant (P<0.05).2. Expression of Lin28B and Let-7 was found in gastric cancer cell line BGC-823,which was different between CD133+/CD44+cells and CD133-/CD44-cells.3. Expression of Lin28B and Let-7 in gastric cancer tissue was negatively correlated.4. In BGC-823 gastric cancer cell line transfected with Let-7, the expression of Lin28B significantly lower (P<0.05).5. BGC-823 gastric cancer cell line transfected with Let-7 suppressed BGC-823 cell proliferation by CCK-8 assay.6. CCK-8 assay adopted to detect cytotoxic showed that overexpression of Let-7 in gastric cancer cells to 5-fluorouracil improve chemotherapy sensitivity.7. Flow cytometry was used to detect cell cycle show overexpression of Let-7 can not change BGC-823 cell cycle.8. Sphere-forming assay with super adsorption cell culture plate showed overexpression of Let-7 reduce sphere-forming ability of BGC-823 cell.9. In nude mice experiments, overexpression of Let-7 significantly suppressed the tumorigenicity of BGC-823 cell.Conclusion 1. The study found Lin28B and Let-7 in gastric cancer cell lines were expressed and in gastric carcinoma also were expressed. Lin28B expression in gastric cancer tissues was significantly at a higher level than normal gastric mucosa; Let-7 expression in gastric cancer tissues was significantly at a lower level than normal gastric mucosa. Expression of Lin28B and Let-7 in gastric cancer tissue was negatively correlated.2. Furthermore, we demonstrated that Lin28B/Let-7 axis is in existence in the gastric cancer cell. Let-7 suppressed the expression of Lin28B.3.In vivo and in vitro studies we have found that Let-7 can suppress gastric cancer cell proliferation, Let-7 can increase the sensitivity of gastric cancer cells to 5-fluorouracil, Let-7 can not change gastric cancer cell cycle; Let-7 can suppress stem differentiation of gastric cancer cells, Let-7 can suppressed tumorigenicity of gastric cancer cells in vivo. |
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