Studies On The Key Role Of HMGA2 In Maintaining Malignancy Of Osteosarcoma Cells And The Molecular Mechanism

High mobility group protein A2 (HMGA2) is a member of the HMGA family. HMGA2 was composed with three AT-hook binding domain and an acidic C-terminal domain. AT-hook can help HMGA2 recognize and bind to the AT-rich regions of DNA small groove specifically, resulting in DNA bending, stretching, looping or melting, regulating replication, transcription and DNA repair, also known as architectural transcription factor. HMGA2 showed high expression at the embryonic period, whereas in the normal mature tissues almost no expression, but in some malignant tumors showed high expression, such as breast cancer, non-small cell lung cancer, pancreatic cancer, retinoblastoma, squamous cell carcinoma, suggesting that HMGA2 play an important role in malignant transformation of tumors. More and more studies have shown that cancer stem cells (CSC) are not sensitive to the chemotherapy and radiotherapy, resulting in cancer recurrence, metastasis and hard cure. It has been reported, overexpression HMGA2 and H-Ras gene can inhibit the differentiation of breast cancer stem cells in order to facilitate the maintenance of their self-renewal, but the miRNA let-7 can promote the differentiation of breast cancer stem cells to treat breast cancer by inhibiting the expression of HMGA2 and H-Ras gene.Osteosarcoma incidence growths rapidly and tends to younger, can occur lung metastasis within a few months. It has high degree of malignancy and poor prognosis, and chemotherapy or radiotherapy is ineffective. Therefore, searching for effective treatment for osteosarcoma is the focus of osteosarcoma study, and targeted gene therapy is considered one of the best treatments, so to find effective target genes for treatment are extremely important. In view of differential expression of HMGA2 in tumor cells and normal cells, RNAi gene therapy has certain advantages. In this study, we culture human osteosarcoma U2OS cells, utilize RNAi to inhibit the expression of HMGA2, and to observe the biological behaviors of U2OS cells after silencing of HMGA2, and to investigate the postential mechanisms, and to explore the possible relationship between HMGA2 and cancer stem cells.Partâ… HMGA2-shRNA transfected into U2OS cells reduced the expression of HMGA2Objective:To construct an effective HMGA2-shRNA silencing plasmid, and detect its expression in human osteosarcoma cell U2OS. Methods: According to the full cDNA sequence of HMGA2 (N0. 003483) in GenBank, select the specific sequence 5'-CGCCAACGTTCGA TTTCAT-3 'for the target of the interference effect, and construct the sequence to the eukaryotic expression plasmid pGCsi-H1, containing the green fluorescent protein GFP. Select the scrambled plasmid which will not interfere with any human mRNA as negative control. HMGA2-shRNA and scrambled plasmids were transfected into U2OS cells, selected by G418 and obtained stable cell clone of the HMGA2-shRNA and scrambled respectively, RT-PCR detect silencing effect of HMGA2-shRNA. Results: Obtained stable transfection cell clones of HMGA2-shRNA and scrambled, RT-PCR results showed that HMGA2-shRNA plasmid reduced mRNA expression levels of HMGA2 significantly. Conclusion: Obtained effective stable cell clones of silencing HMGA2 gene successfull.Partâ…¡Effects on proliferation and apoptosis of HMGA2 in U2OS cellsObjective:To study the effects of silencing HMGA2 on cell morphology, growth curve, colony forming ability, cell apoptosis and cell cycle of human osteosarcoma U2OS cell, to detect mRNA levels of related gene, and to explore its possible mechanism. Methods: Utilize microscopy observe cell morphology; CCK-8 kit detect cell growth curve; soft agar colony forming experiment testing the ability of cell cloning;Hoechst33342 dyes detect cell apoptosis; PI staining with flow cytometry detect cell cycle changes. Utilized real-time RT-PCR detecting mRNA level of relative genes. Results: compared with untreated and scrambled group, HMGA2 gene silencing group cell proliferation activity was decreased significantly (P<0.01); capacity of clonal growth was decreased; apoptotic cells increased significantly (P <0.05); the cell number of S phase decreased, most of the cell were blocked in the G0/G1 phase. The same culture conditions, untreated and scrambled group cells were spindle-type, cell body were more prominent after adheren, the majority of HMGA2 silencing group cells in shape of irregular polygons, cell body relatively flat after adherent. Real-time RT-PCR results show that the expression of the apoptosis gene caspase-3 and caspase-9 are increased,and the expression of the apoptosis inhibitor gene Bcl-2 and suvivin are decreased, and the expression of CDKN1A is increased. Conlusion: HMGA2 through inducted the expression of CDKN1A, overcomed the G1/S block to promote the proliferation of U2OS cells. HMGA2 inhibited spontaneous apoptosis of U2OS cells by reducing the expression of caspase-3 and caspase-9 and increasing the expression of Bcl-2 and suvivin. Partâ…¢Effects on migration and invasion of HMGA2 in U2OS cellsObjective:To study the effects of HMGA2 gene on cell spreading, adhesion, migration, invasion in human osteosarcoma U2OS, to detect mRNA levels of related gene, and to explore its possible mechanism. Methods: Utilized microscope to observe cells spreading on FN; CCK-8 kit detect cells adhesion ability on FN and matrigel; scratch test detect cell migration; Boyden chamber observed cell invasion; Real-time RT-PCR detect related gene mRNA levels. Results: compared to untreated and scrambled group, HMGA2-shRNA group cells capacity of spreading on FN is diminished (P <0.05); and the capacity of adhesion on the FN (P <0.01) and Matrigel (P <0.05) were decreased significantly; migration ability of Cell injury repair and migrate to the chemokines were significantly reduced; Boyden chamber invasion assay results showed, the cell number of HMGA2-shRNA group reached the carbonate membrane is significantly lower than untreated and scrambled group(P <0.05). Real-time RT-PCR results showed that the expression of MMP-2 and MMP-9 were decreased significantly, while the expression of TIMP3 was increased. Conclusion: Reduced the expression of HMGA2 can decrease spreading and adhesion, migration and invasion of U2OS cell; real-time PCR results suggested that one important molecular mechanism of HMGA2 to maintain migration and invision of U2OS was increasing the expression of MMP-2 MMP-9 and reducing the expression of TIMP-3 expression.Partâ…£the role of HMGA2 in maintaining chemotherapy resistance of U2OS cellsObjective:To study the effects of HMGA2 gene on chemotherapy sensitivity of U2OS cells; to detecte the mRNA level of stem cells markers, and to explore the relationship between HMGA2 and cancer stem cells. Methods: CCK-8 detected the response of U2OS cells to chemotherapeutic drugs vincristine; Real-time RT-PCR detected related gene mRNA levele of stem cells. Results:compared with untreated group(IC50=136.39ng/ml) and scrambled group (IC50=148.26 ng/ml), HMGA2-shRNA group(IC50=71.73 ng/ml) cells increased the sensitivity to chemotherapy vincristine(P<0.05). Real-time RT-PCR results showed that the expression of PTEN was increased and the expression of SOX2 was reduced. Conclusion: HMGA2 played an important role in maintaining the resistant to chemotherapy vincristine in U2OS cells, the results provided an experimental basis for the treatment of osteosarcoma by RNA interference targeted to HMGA2. For the first time, we found that HMGA2 can induce the expression of stem cell marker SOX2, suggesting that in the osteosarcoma U2OS cell populations exist stem cell-like cells, and associated with the expression level of HMGA2. The tumor suppressor gene PTEN, further confirmed the important role of HMGA2 in maintaining malignant phenotype of osteosarcoma cells, suggesting that HMGA2 is a promsing target gene in the RNA interference treatment osteosarcoma.Partâ…¤Isolation and biological behaviour analysis of prostatic carcinoma PC3 cell subpopulationObjective:To identify the characteristic of cancer stem-like cells in prostatic carcinoma PC3 cell line, and analyze the morphous and clone growth, investigate the method of discriminating the different tumor cell subpopulation by Rhodamin123 dye. Methods:Flow cytometry analyze the PC3 cells stained with Rhodamin123 dye; using fluorescent microscope to observe cells morphous and clone growth stained with Rhodamin123 and DAPI. Results:Two different tumor cell subpopulation exist in PC3 cell line, and the morphous, reproductive activity and colon growth are significant different in the two cell subpopulation. Conclusion:Cancer stem-like cell subpopulation exist in prostatic carcinoma PC3 cell line, the character of some cells exclude Rhodamin123 dye can be used to discriminate tumor stem cells and other tumor cells.