Expression Of MicroRNA-425-5p And The Effect On Proliferation, Invasion And Metastasis In Gastric Cancer

In 1993, two team led respectively by Victor Ambros and Gary Ruvkun discovered and confirmed a class of mysterious micromolecules almost at the same time, which open a new avenue of research about micro RNA(micro RNA;mi RNA). The mi RNAs, which are about 22 nucleotides in length, are small noncoding RNAs highly conserved in evolutionary, and play the major role in gene expression through the way of post-transcriptional regulation. At present, it is considered that mi RNAs are strictly regulated by cell type and differentiation stage, and are expressed in particular tissues and specific stages. The specificity and scheduling of tissues about the expressions of mi RNAs play a decisive role in specific function of tissues and cells, and participate in important regulatory function in the process of cell growth and development. Accumulating evidence indicates that most neoplasms exist dysregulaton of mi RNA, which are crucial to cancer initiation, proliferation, invasion and metastasis. Many mi RNAs have been confirmed as the regulatory factors of oncogene, tumor suppressor genes and the cell signaling pathways. The function of mi RNAs, acting as either oncogenes or tumor suppressors, depends on the effect of their target genes. If mi RNAs act as oncogenes, their activity will increase. They will suppress the activity of tumor suppressor gene, and at the same time, promote cell proliferation and tumor progress. Decreased activity of mi RNAs, acting as tumor suppressor genes, would increase oncogenes translation, and promote tumor formation.Around the world, the disease incidence of gastric cancer is higher in Asia. However, the disease incidence is relatively low in the developed countries in Europe and the Americas. Gastric cancer is the fourth most common cancer diseases, and is the second leading cause of cancer-related death in the world. There are about 1 million new patients diagnosed as gastric cancer every year in the global. The formation of gastric cancer is due to multivariate factors such as genetics, environment, diet and living habits. The exact mechanism is still need for more in-depth exploration. The discovery and progress of relevant study about the mi RNAs provide a new way to research the occurrence and development of gastric cancer. Expressions of mi RNAs, such as mi R-9, mi R-421, mi R-27 a, mi R-143, mi R-145, mi R-29 and mi R-101, have been demonstrated to play an important role in regulating function in gastric cancer. And mi RNAs involve in the process of tumorigenesis and the development of gastric cancer by regulating the cell proliferation, apoptosis, invasion and metastasis ability. Combined detection of their expression levels promise to be early diagnostic and prognostic markers, even new targets of treatment in gastric cancer.In terms of cell proliferation, apoptosis, invasion and metastasis, multiple mi RNAs have an effect in gastric cancer in several ways. For example,mi R-199 a affects the function of gastric cancer cell in proliferation, apoptosis, metastasis. Mi R-301 a and mi R-1303 involves in the process of tumorigenesis and the development of gastric cancer in proliferation, apoptosis, invasion and metastasis. A large clinical sample test has been conducted by Ueda, using the microarray technology. The result has demonstrated that expression of micro RNA-425-5p in gastric cancer tissues is significantly higher than that of normal gastric mucosa tissues.In previous studies in gastric cancer, micro RNA-425-5p exists dysregulation of expression, which shows the possibility of involving in tumorigenesis and the development of gastric cancer. Now researches related to micro RNA-425-5p are unseen in cancer especially gastric cancer. This research is designed to investigate the expression of micro RNA-425-5p in gastric cancer cells and its impact on tumorigenesis and the development of gastric cancer, in order to break a new field in the pathogenic mechanism of gastric cancer and provide a theoretical basis for practical applications of micro RNA-425-5p in prognosis and treatment of gastric cancer.Based on the above issues, we have done the following researches in gastric cancer about micro RNA-425-5p: The Taqman probe is applied to detect expression level of micro RNA-425-5p in human gastric cancer epithelial cell lines, including SGC-7901, MKN-28, MKN-45, BGC-823 and AGS, and the human immortalized gastric mucosa normal cell line(GES-1). The objective is to appraise the difference about the expression of micro RNA-425-5p between the human gastric cancer epithelial cell lines and GES-1. If the significant difference between the human gastric cancer epithelial cell lines and GES-1 is exist in expression of micro RNA-425-5p. We should select specific gastric cancer epithelial cell lines for further study. By up-regulating or down-regulating the expression level of micro RNA-425-5p in the human gastric cancer epithelial cell lines, we assessed the effect of micro RNA-425-5p on proliferation, cell cycle, invasion and metastasis. And then, on the basis of cytology, we establish transplanted tumor animal model, and confirm micro RNA-425-5p further mechanism in vivo. We aimed to reveal the relationship between the abnormal expression of micro RNA-425-5p and tumor tumorigenesis as well as the development of gastric cancer. At the same time, its clinical potential value also initially was recognized.The main contents of this article are as follows: Part one Micro RNA-425-5p expression in gastric cancer cell linesObjective: To evaluate the expression level difference of micro RNA-425-5p in human gastric cancer epithelial cell lines, including SGC-7901, MKN-28, MKN-45, BGC-823 and AGS, and the human immortalized gastric mucosa normal cell line(GES-1).Methods: Total RNA was extracted from the cell lines using TRIzol® reagent. QRT-PCR(Taq Man Micro RNA Assay and Taq Man Universal PCR Master Mix) was used to detect the expression level of micro RNA-425-5p in human gastric cancer epithelial cell lines, including SGC-7901, MKN-28, MKN-45, BGC-823 and AGS, and the human immortalized gastric mucosa normal cell line(GES-1).The expression difference about micro RNA-425-5p between the human gastric cancer epithelial cell lines and GES-1 was evaluated.Results: Expression level of micro RNA-425-5p in human gastric cancer epithelial cell lines is significantly higher than that in GES-1. The expression level in MKN-45, SGC-7901 and BGC-823 increase most significantly.Summary: Compared with the human immortalized gastric mucosa normal cell line(GES-1), expression level of micro RNA-425-5p in human gastric cancer epithelial cell lines(SGC-7901, MKN-28, MKN-45, BGC-823 and AGS) is significantly higher. The expression level in MKN-45, SGC-7901 and BGC-823 increase most significantly. The result is consistent with previous study about gastric cancer tissue. The result reveals the potential relevance between abnormal expression of micro RNA-425-5p and tumorigenesis as well as development of gastric cancer. It is worthy of further study at the cellular level. Part two Impact of micro RNA-425-5p on proliferation, invasion and metastasis in gastric cancer cell lineObjective: To observe the impact on proliferation, cell cycle, invasion and metastasis of BGC-823 cell line, when the expression level of micro RNA-425-5p is up-regulated or down-regulated.Methods: After transient transfection with micro RNA-425-5pmimicsor micro RNA-425-5p inhibitor, the expression level of micro RNA-425-5pin human gastric cancer epithelial cell line BGC-823 is up-regulatedor down-regulated. Cell proliferation was assessed by a water-soluble tetrazolium salt(WST) assay using Cell Counting Kit-8. Soft agar colony formation assay was used to evaluate cell anchorage-independent growth capability. Haptotactic migration and matrigel chemoinvasion assays were used to appraise the ability changes of BGC-823 invasion and metastasis.Results: 1 In comparison with inhibitor NC and WT cell groups, the cell proliferation rate of BGC-823 cell transfected with micro RNA-425-5p inhibitor decreased significantly, which had statistical significance. When BGC-823 cell was transfected with micro RNA-425-5p mimics for 24 hours, the cell proliferation rate increased. And compared with mimics NC group, cell proliferation rate of micro RNA-425-5p mimics group increased with statistical significance. Because of up-regulation of micro RNA-425-5p, BGC-823 cells anchorage-independent growth capability increased. The results are highly suggestive that micro RNA-425-5p promotes cell proliferation. 2 In comparison with inhibitor NC and WT cell groups, the cell migration ability decreased significantly after BGC-823 cell transfected with micro RNA-425-5p inhibitors. The results have a statistical difference. The BGC-823 cell, transfected with micro RNA-425-5p mimics, has higher migration ability than the mimics NC cell group. Experimental results show that micro RNA-425-5p has a strong ability to promote gastric cancer cell migration. 3 In matrigel chemoinvasion assays, we found that compared with inhibitor NC and WT cell groups, the BGC-823 cell transfected with micro RNA-425-5p inhibitor has lower invasion capacity. The results have a statistical difference. After BGC-823 cell transfected with micro RNA-425-5p mimics, the cell invasion capacity was significantly increased than mimics NC cell group. The results have a statistical difference. Experimental results show that micro RNA-425-5p has a strong ability to promote gastric cancer cell invasion.Summary: 1 Human gastric cancer epithelial cell line BGC-823 which was transfected with micro RNA-425-5p mimics can effectively increase the expression level of micro RNA-425-5p. Compared with mimics NC group, the cell proliferation rate increased. BGC-823 cell line, transfected with micro RNA-425-5p inhibitors, has lower expression level of micro RNA-425-5p. The cell proliferation rate declined. Experimental results show that the micro RNA-425-5p involves in tumorigenesis and the development of gastric cancer by promoting the gastric cancer cell proliferation. 2 In soft agar colony formation assay, we found that the number of colonies formed from BGC-823 cells transfected with the mi R-425-5p inhibitor was less than half of that formed from the inhibitor NC and WT cell groups. BGC-823 cells transfected with the mi R-425-5p mimics displayed more colonies than the mimics NC group. The results are highly suggestive thatmicro RNA-425-5p promotes cell proliferation. 3 Compared with other groups, the cell migration ability of BGC-823 cell, transfected with micro RNA-425-5p inhibitor, decreased significantly. Transfected with micro RNA-425-5p mimics, the BGC-823 cell migration ability is significantly higher than mimics NC groups. The experimental results show that micro RNA-425-5p has significant ability to promote gastric cancer cell migration. 4 In matrigel chemoinvasion assays, the BGC-823 cell transfected with micro RNA-425-5p inhibitor has lower invasion capacity. After BGC-823 cell transfected with micro RNA-425-5p mimics, the cell invasion capacity was significantly increased. Experimental results show that micro RNA-425-5p has an obvious ability to promote gastric cancer cell invasion. Part three Impact of micro RNA-425-5p on pulmonary metastasis of nude miceObjective: to further appraise the effect of micro RNA-425-5p on tumor invasion or metastasis by nude mice experiment.Methods: 15 female BALB/C nude mice, weighing 15 ~ 16 g and 4 ~ 5 weeks old, were randomly divided into three groups, and 5 in each group. BGC-823 cells(1×106cells/mouse) were stably transfected with mi R-425-5p inhibitor or the negative control and WT were injected into 4-5-week-old female BALB/C nude mice through the tail vein. The mice were housed for six weeks following the injection and were then euthanized by cervical dislocation. The lungs were dissected and examined histologically. Briefly, formalin-fixed, paraffin-embedded tissues were stained with hematoxylin and eosin. Following staining, the tissues were observed under a light microscope and the number of metastases was counted.Results: Compared with inhibitor NC and WT nude mice groups, the number of lung metastatic nodes per mouse was markedly lower and the nodes were smaller in the mice injected with BGC-823 cells that had been transfected with the mi R-425-5p inhibitor. The results show that micro RNA-425-5p inhibitor inhibit the invasion and metastasis of tumor, which is consistent with the result of cytological experiments.Summary: The number of lung metastatic nodes per mouse was markedly lower and the nodes were smaller in the mice injected with BGC-823 cells that had been transfected with the mi R-425-5p inhibitor. The results show that micro RNA-425-5p inhibitor can inhibit the invasion and metastasis of tumor. So, high level of micro RNA-425-5p can promote tumor invasion and metastasis. On the contrary, low level of micro RNA-425-5p can inhibit tumor invasion and metastasis.Conclusions:1 Expression level of micro RNA-425-5p in human gastric cancer epithelial cell lines(SGC-7901, MKN-28, MKN-45, BGC-823 and AGS) is significantly higher than the human immortalized gastric mucosa normal cell line(GES-1). The expression level in MKN-45, SGC-7901 and BGC-823 increase most significantly.2 Human gastric cancer epithelial cell line BGC-823 which was transfected with micro RNA-425-5p mimics can effectively increase the expression level of micro RNA-425-5p. Compared with mimics NC group, the cell proliferation rate increased. BGC-823 cell line, transfected with micro RNA-425-5p inhibitors, has lower expression level of micro RNA-425-5p. Experimental results show that the micro RNA-425-5p involves in tumorigenesis and the development of gastric cancer by promoting the gastric cancer cell proliferation.3 In comparison with inhibitor NC and WT cell groups, the cell migration ability decreased significantly after BGC-823 cell transfected with micro RNA-425-5p inhibitors. The BGC-823 cell, transfected with micro RNA-425-5p mimics, has higher migration ability than the mimics NC cell group. Experimental results show that micro RNA-425-5p has a strong ability to promote gastric cancer cell migration.4 In matrigel chemoinvasion assays, the BGC-823 cell transfected with micro RNA-425-5p inhibitor has lower invasion capacity than with inhibitor NC and WT cell groups. After BGC-823 cell transfected with micro RNA-425-5p mimics, the cell invasion capacity was significantly increased than mimics NC cell group. Experimental results show that micro RNA-425-5p has an obvious ability to promote gastric cancer cell invasion.5 In comparison with inhibitor NC and WT nude mice groups, the number of lung metastatic nodes per mouse was markedly lower and the nodes were smaller in the mice injected with BGC-823 cells that had been transfected with the mi R-425-5p inhibitor. The results show that micro RNA-425-5p inhibitor can inhibit the invasion and metastasis of tumor. High level of micro RNA-425-5p can promote tumor invasion and metastasis. On the contrary, low level of micro RNA-425-5p can inhibit tumor invasion and metastasis.