| BackgroundGastric cancer is a major disease which seriously impacts on the health of Chinese people; Shandong Province is the region of high incidence about stomach cancer in our country. Gastric cancer is characterized by late diagnosis and the relatively poor prognosis. Due to lack of effective treatment, the5-year survival of advanced gastric cancer was less than10%. Therefore, revealing the mechanism of gastric cancer is one of fundamental and important scientific problems that intervention on gastric cancer in the early stage.Abnormal gene regulation in host cells induced by H.pylori infection is the inner motivation to promote malignant transformation of cells. However, infective inflammation-induced malignant transformation involves in regulationary network, including numerous cell proteins, non-coding RNA, and orther biological activity molecules. The whole process is so complex. Previously, we applied high-throughput screening technologies on genome-wide and miRNA expression profiling (Roche-NimbleGen and Exiqon LNATM microRNA, Shanghai Kangcheng Biological Engineering Co., Ltd.) and bioinformatics database (NCBI, KEGGNCBI, Uniprot ID, Swissprot ID, Ensenbl ID, TIGR, TAIR, EMBL, Miranda, Targetscan, etc.). We speculated that Programmed cell death factor4(PDCD4) was likely to be the regulation node molecular on malignant transformation in gastric cancer.PDCD4is a tumor suppressor gene related to cell cycle and apoptosis; it not only affects programmed cell death, but also suppresses tumorigenesis by inhibiting protein translation initiation. Results showed that PDCD4protein expression declined to varying degrees in tissue specimens of gastric cancer, colorectal cancer, pancreatic cancer and other digestive cancer, and decreasion on protein expression in tissues was closely related to the differentiation of tumors; studies on knockout mice dispalyed that PDCD4gene knockout mice was susceptible to malignant B-cell lymphoma, therefore, PDCD4is considered to be a tumor suppressor factor which closely related with tumors, but the mechanism that PDCD4expression is inhibited by Helicobacter pylori remains unclear.MicroRNA (miRNA),a class of endogenous nucleotide noncoding RNA, was found in eukaryotic cells in recent years. Single-strand of miRNA can complementarily bind to3’ untranslated region (3’-UTR) of target genes, and negatively regulate gene expression at the post-transcriptional level. Currently most be-known miRNA located at the genomic regions associated with tumor and played an important role in the pathological process. Research have showed that many miRNA expression in gastric epithelial cells was affected by H.pylori infection, including miR-21, miR-146, miR-155, miR-27a, miR-106-93-25, miR-221-222cluster and miR-200family. While miR-21was proved to be involved in targeting and regulating PDCD4in variety human cell biological function. Therefore, changes on gene expression caused by H.pylori infection via affecting miRNA would set up a new bridge between pathogenic microorganism infection and host intercellular variation.Currently, during the process of molecular changes about Helicobacter pylori promote gastric cancer occurrence, the exact role of PDCD4and miR-21played, and whether there is regulationary correlationship between them are all not clarified. Meanwhile, cell proliferation is an important cellular effect, whether it involved in the mailignant transformation process was still needed to be exploring. The first part of thesis expanded a study on these issues.Epithelial-mesenchymal transition (EMT) refers to a cell biological process that epithelial cells are converted to the mesenchymal phenotype by specific program. It appeared that the epithelial markers (such as E-cadherin) were downregulated and mesenchymal cell markers (such as vimentin) were upregulated. There are many transcription factors and signaling pathways involved in the process. For example, zinc finger protein Snail1, Slug (Snai12), Zebl/2, spiral-loop-helix protein Twistl/2and so on are all important molecular about regulatiing EMT, they could inhibit epithelial markers expression or promote mesenchymal markers. Epithelial-mesenchymal transition is characterized by loss of polarity and loss of epithelial phenotype that connected to the basement membrane in epithelial cells, and acquisition of mesenchymal phenotype that has the higher ability to migration or invasionEpithelial-mesenchymal transition confers malignant properties to epithelial cells, which gastric epithelial cells show EMT is an important effect on malignant transformation. There is so much factors affecting epithelial-mesenchymal transition of human stomach tumors, Helicobacter pylori infection, as a environmental factor closely related to gastric cancer, has also been thought that have a closely relationship to the above process. The regulatory networks which Helicobacter pylori and CagA promote epithelial-mesenchymal transition in gastric cancer cells are extremely complex, including genes, proteins, metabolic molecules and non-coding RNA and so on. The mechanism on the process needs to be further elucidated. PDCD4is a regulator that affectes transcription or translation of various genes, recently it was demonstrated its potential links with tumor invasion and metastasis.However, whether it could induce EMT and malignant transformation by regulating PDCD4in H.pylori-associated gastric cancer still need to be explored. The second part of thesis expanded a study on these issues.Aim(1) We explored that whether H.pylori infection could regulate PDCD4via has-mir-21, thereby affect on the cell proliferation and promote the malignant transformation.(2) We attempted to find out if H.pylori could affect the cell epithelial-mesenchymal transition by regulating PDCD4expression.Methods and Results (1)We disclosed that H.pylori infection could induce cell proliferation in gastric cancer by inhibiting PDCD4.1. Expression of PDCD4was down-regulated in primary gastric cancer associated with H.pylori infection.To further understand the relevance between PDCD4expression and gastric carcinogenesis, initially we investigated the expression of PDCD4in gastric cancer specimens by immunohistochemical staining, including34primary GC tissues, the corresponding normal ones and30gastritis tissues, which are consecutive histological sections of Warth-starry staining and Giemsa staining double-positive. The results exhibited that expression level of PDCD4was significantly inhibited in cancer tissues compared with non-cancerous gastric tissues; and this down-expression trend was also confirmed by qRT-PCR, which showed PDCD4was strongly inhibited in gastric cancer tissues in comparison to the non-cancerous gastric tissues at the transcriptional level; Interestingly, we found that PDCD4expression was down-regulated gradully during the diferent stages from gastritis to gastric cancer.2. Helicobacter pylori and its key virulence factor CagA regulated PDCD4expression in gastric epithelial cell lines. Next, we tested whether H.pylori infection could affect PDCD4expression in gastric epithelial cell lines, AGS, BGC-823and GES-1. The results showed that both mRNA and protein expression levels of PDCD4were markedly down-regulated after H.pylori infection with time-and dose-dependent manners. To evaluate that whether down-expression of PDCD4by H. pylori infection was strain specific, the cell line GES-1was also infected with H. pylori strains26695, NCTC11637and SSI (1:100) for12h, and results also displayed that expression levels of PDCD4were substantially decreased after infection by all the three strains compared with that of control. We founded that transfection of CagA over-expression plasmid obviously inhibited the expression of PDCD4at both mRNA and protein levels. Herein, our results suggested that down regulation of PDCD4could be induced by H. pylori infection through its component of CagA.3. PDCD4was involved in cell proliferation by regulating the expression of p27kipl.We attempted to find out that whether the expression of p27kipl was regulated by PDCD4. The results showed that both mRNA and protein expression of p27kipl changed significantly in the same direction of PDCD4in AGS, BGC-823and GES-1cell lines after overexpression of PDCD4. Furthermore, using the dual luciferase activity assay, we found that the expression of PDCD4apparently regulated the luciferase activity of p27kipl promoter, suggesting that inhibition of p27kipl in gastric cancer was partially mediated by direct suppression of PDCD4. Next, results displayed that overexpression of PDCD4markedly inhibited colony formation compared with control by colony formation assay.4.The miR-21-PDCD4pathway mediated H.pylori-induced cell proliferation. Expression levels of miR-21in AGS, BGC-823and GES-1cells was overexpressed after H. pylori infection(1:100for12h) and CagA over-expression, which suggested that overexpression of miR-21by H.pylori probably played an important function in gastric carcinogenesis. Next, we determined whether miR-21-PDCD4pathway was involved in H.pylori-induced gastric cancer cell proliferation. The results showed that H.pylori or CagA-induced down-regulation of PDCD4was reversed by the inhibitor of miR-21, implying the existence of miR-21-PDCD4pathway in gastric carcinogenesis.(2)We found that epithelial-mesenchymal transition induced by H.pylori inhibiting PDCD4.1.Analysis of twistl, PDCD4and E-cadherin expression in H. Pylori-associated gastric cancer tissues.The expression of twistl, PDCD4and E-cadherin in cancer patients’ specimens and adjacent normal tissue specimens by immunohistochemistry (IHC), and all patients were positively H. Pylori infected, then the correlation between above three molecular and the malignant degree of the disease was analyzed by statistical analysis software. Twistl expression in tumor tissues was significantly higher than the adjacent normal tissues, while E-cadherin and PDCD4expression were opposited to twist Land they gradually changed as the malignant degree of the disease.2.The effect of H. Pylori CagA on the invasion and metastasis ability of gastric cancer cell by regulating PDCD4in vitro.By real-time quantitative PCR and western blot, the various gastric epithelial-derived cell lines infected with H. Pylori CagA showed expression changes of epithelial-mesenchymal transition-related genes (twistl and E-cadherin). As the first part, PDCD4expression was significantly decreased due to H. Pylori CagA. Then, gastric cancer cell was transfected with PDCD4-high expression plasmid and PDCD4 expression was recovery. We found that the role of H. pylori CagA on epithelial mesenchymal transition associated genes was obviously inhibited along with the increased expression of PDCD4in gastric cell. By Transwell cell invasion assay, H. pylori infection could induce the invasion and metastasis ability of cancer cell, while PDCD4-high expression plasmid was transfected into cell, the effect can be inhibited. The results suggested that H. pylori CagA may promote cell invasion and metastasis by regulating PDCD4expression in vitro.Conclusion(1) Helicobacter pylori and virulence factors CagA can promote cell proliferation;induce malignant transformation of gastric epithelial cells by "mir-21-PDCD4-p27kipl axis" signal pathway. Details were seeing in Figure1. That is to say, inhibition PDCD4expression caused by Hclicobacter pylori CagA is directly targeted and mediated regulation through mir-21; while PDCD4directly combined to the promoter region of p27kipl and affect on its expression, then promote cell proliferation and malignant transformation of host cells. Conclusions of the part enrich the molecular mechanisms of H. pylori-induced gastric cancer; also provide a theoretical basis for PDCD4as a potential cancer biomarker. Additional figure1:model diagram on the process that Helicobacter pylori and CagA promote cell proliferation and induce malignant transformation of host cells through "mir-21-PDCD4-p27kipl axis" signal transduction pathway(2)We initially confirmed that Helicobacter pylori CagA promoted cell epithelial-mesenchymal transition by inhibited PDCD4. Detailes were seen in additional figure2, which is H.pylori CagA infected cells, inhibited PDCD4expression, increased expression of the transcription factor twist1, downregulated epithelial marker molecules E-cadherin and started epithelial-mesenchymal transition in epithelial cells. Additional figure2:model diagram on the epithelial-mesenchymal transition induced by H.pylori by regulating PDCD4... |
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