RNAi Inhibit The Expression Of S100A4 Gene And Its Effect On Invasion And Metastasis Ability Of Osteosarcoma Cell

IntroductionOsteosarcoma is a common clinical osteogenic malignant tumor, accounting for 20% bone tumors. It is characterized by high risk of malignancy, recurrence, metastasis and poor prognosis. With the development of molecular biology, especially tumor molecular biology, theoretically and technically, the mechanism of the osteosarcoma at the molecular level has a new understanding. In recent years, studies have shown that the abnormal expression of S100A4 gene and protein is closely related to the biological behavior of malignant tumors, and plays a very important role in invasion and metastasis of tumors. The studies on biological characteristics of S100A4 protein and a variety of tumors have been carried out.S100A4 protein is a metastasis-related gene, which was cloned from highly metastatic mouse breast cancer cells by Sbralidze in 1989. The human S100A4 gene was cloned in 1992. This gene is located on chromosome 1q21, with the length of the gene is 2837bp, and encodes a protein of 101 amino acids. This area is easy to have the changes of chromosomal deletion, translocation and duplication, which reveals that S100A4 protein may play an important role in the tumorigenesis and development of osteosarcoma. S100A4 protein binding with Calcium may lead to the changes of the configuration of S100A4 protein. The S100A4 protein exposes the new binding points and makes it to combine with a range of target proteins. S100A4 protein promote the invasion and metastasis of malignant tumor by reducing tumor cell adhesion, increasing exercise capacity of tumor cells, remodeling extracellular matrix, inhibiting apoptosis, promoting cell dysplasia and angiogenesis. The research of S100A4 protein structure and function, as well as the gene encoding, remains to be further in-depth. S100A4 protein is not only expected to be diagnostic indicators of malignant tumors and assessment of prognosis of patients with tumor marker, but also to be the new target of curing malignant tumors by decreasing the expression of S100A4 gene.RNA interference (RNAi) is a new technology discovered in recent years, which is used to gene expression, regulation and function of organism. It utilizes specific silent phenomenon of homologous genes of biological cells caused by the small interfering RNA (siRNA). Its nature is siRNA specifically binding and degradation with the corresponding mRNA, thereby preventing mRNA translation. RNAi is the result of biological evolution, and the protective response of the organisms to be invasion by foreign source of nucleic acid such as the viral genes. It exists in a variety of organisms, performing the biological functions, such as anti-virus, stabilizing transposon and monitoring abnormal mRNA expression. RNA interference is not only able to provide an economical, fast, and efficient inhibition of gene expression in technical means, but also may build a new sight in the determination of gene function, gene therapy and so on.This study first verified the S100A4 gene in different human osteosarcoma cell lines and osteosarcoma tissues, and then applied the RNA interference technology, to restrain the S100A4 gene expression of human osteosarcoma MG-63 cells, and observed the impact on cell proliferation and in vitro invasion capacity, to study its mechanism. This article is divided into three parts. PartⅠ:Study the expression of S100A4 in osteosarcoma cell lines MG-63 and U-2OS. PartⅡ:Investigate the expression and relationship research of S100A4 and CD44V6 in osteosarcoma tissues. PartⅢ:RNAi inhibit the expression of S100A4 gene and its effect on cell biological behavior and invasion and metastasis ability of human osteosarcoma MG-63 cell, and its mechanism.ObjectivePartⅠ:Detect the expression of S100A4 gene in human osteosarcoma MG-63 and U-2OS cell line. PartⅡ:Study the expression of S100A4 and CD44V6 in osteosarcoma tissues, and analyze its clinical significance. PartⅢ:RNAi inhibit the expression of S100A4 gene in human osteosarcoma MG-63 cell, observe its commitment to cell proliferation and in vitro invasion capacity, and explore its mechanism. MethodsPart I:Human osteosarcoma MG-63 and U-2OS cell purchased from Shanghai Cell Institute, Chinese Academy of Sciences. The expression of S100A4 gene in human osteosarcoma MG-63 and U-2OS cell line was detected by Real-time PCR method. Part II:Thirty-three samples of osteosarcoma patients administrated from 2004 to 2008 and thirteen samples of osteochondroma patients administrated in 2008 were collected from First Affiliated Hospital of China Medical University. The expression of S100A4 and CD44V6 protein was detected by Streptavidin-Peroxidase method. The relationship between the expression of S100A4, CD44V6 in osteosarcoma tissues and the clinicopathological factors was discussed. Part III:Basing on the online designing principles provided by the Ambion Company, the corresponding siRNAs were designed according to the sequence of S100A4 gene in GenBank, and the siRNA vector of S100A4 is composed. The situation of transfection was observed by fluorescence inverted microscope. The changes of S100A4 gene expression both before and after transfection were detected by Real-time PCR method. The changes of S100A4 protein expression both before and after transfection were detected by Western-Blot method. The specific siRNA with high transfection efficiency was selected as the transfection experiment group for the follow-up tests. The changes of cell proliferation were determined by MTT method and flat colony-forming experiments. The changes of cell invasion and metastasis ability were determined by Transwell chamber assay. And the changes of CD44 and MMP2 gene expression both before and after transfection were detected by Real-time PCR method. The possible mechanisms were preliminary studied about RNA interference suppress S100A4 gene expression of human osteosarcoma cells to affect the cell invasion and metastasis.ResultsPartⅠ:The result of Real-time PCR reaction showed that human osteosarcoma cell line MG-63 and U-2OS both has the expression of S100A4 gene, the extent of two cell lines express identical. PartⅡ:The positive rate of S100A4 protein in osteosarcoma tissues is 69.7%, the positive expression locates in osteosarcoma cell cytoplasm. The control group could not find S100A4 protein expression. The positive expression of CD44V6 locates in cell membrane and cytoplasm near the cell membrane of osteosarcoma and osteochondroma cell. The positive rate of CD44V6 protein in osteosarcoma tissues is 60.6%, but only 7.7% in osteochondroma. The expression of S100A4 and CD44V6 in osteosarcoma tissues detected from this study is relevant (r= 0.413, P<0.05). The expression level of S100A4 and CD44V6 in osteosarcoma tissues is compared by the sex, age, primary tumor site, pathological type of tumor cells of patients, and no significant difference was found from the statistic point of view (P>0.05). Part III:Green fluorescence can be seen in non-specific siRNA transfection group (C2 group), specific siRNA transfection group (S1, S2, S3 group) by fluorescence inverted microscope. The positive expression locates in cell cytoplasm. The expression levels of S100A4 mRNA in each specific siRNA transfection group have varying degrees of reduction. SiRNA3 group is the most significant group. The expression levels of S100A4 protein of Western-blot detection are similar to the conclusions. MTT method and flat colony-forming experiments have shown that specific siRNA transfection group can inhibit cell proliferation. There is significant difference compared with the control group and non-specific group (P<0.05) Trans well chamber experiments showed that:The invasive ability of tumor cells significantly decreased after RNA interference S100A4 gene expression, the cell number through artificial basement membrane decreased significantly, there is significant difference compared with the blank control group and negative group (P<0.05). Comparing the changes of CD44 and MMP2 expression both before and after S100A4 siRNA-specific transfection with double standard curve method, the expression of S100A4 inhibition also affects the expression of CD44 and MMP2, CD44 expression decreased to 42.33%±0.0322, MMP2 expression decreased to 71.67%±0.0551, the difference was statistically significant (P<0.05)ConclusionPartⅠ:S100A4 gene expresses in Human osteosarcoma cell line MG-63 and U-2OS similarly. PartⅡ:S100A4 in osteosarcoma tissues has a higher level of expression in this study, and is related to the expression of S100A4 and CD44V6 in osteosarcoma tissues. PartⅢ:S100A4 siRNA-specific vector in vitro synthesis can effectively inhibit the expression of S100A4 in osteosarcoma cell line MG-63, cell proliferation and cell invasive ability in vitro. The decrease of S100A4 gene expression can inhibit the expression of CD44 and MMP2 gene, and reduce osteosarcoma cell invasion and metastasis capability.RNA interference technology provides a new thought for the treatment of osteosarcoma. S100A4 gene is closely related to the invasion and metastasis of osteosarcoma. Inhibiting S100A4 gene expression in osteosarcoma may become an important adjunctive method of surgery, chemotherapy, radiotherapy and other traditional therapy. In this study, the application of RNAi to the tumor provides experimental basis for gene therapy.