| BACKGROUNDBreast cancer is one of the most common malignant tumors in women worldwide. The present results show that breast cancer is a genetic disease, whose occurrence and development are often accompanied with activation and inactivation of series of oncogenes and tumor suppressor genes, which may cause signal transduction pathway upstream and downstream the genes abnormal or mis-regulation of protein networks. In the past decades tumor suppressor gene has become the focus of attention and hotspot of research for global medical scientists. So far multiple tumor suppressor genes such as BRCA1, P53etc. have been confirmed to suppress tumor growth, invasion and metastasis in breast cancer. Now there are two hot directions of tumor suppressor gene research, one is the functions of tumor suppressor gene and mechanism research. The other one is tumor suppressor gene expression regulation. Based on the existing achievements, further studies on tumor suppressor gene function and mechanism of how tumor suppressor gene influences the occurrence and development of breast cancer and to explore new breast cancer related tumor suppressor gene for clinical gene targeting therapy may be the direction and emphases of future research.In2000, Tatarelli firstly cloned complete cDNA sequence of TES gene from human normal ovarian tissue. The length of human TES gene open reading frame (ORF) is1263bp, encompassing7exons, encoding a protein with421amino acid residues and the molecular weight of the protein is48kDa. The TES protein contains a PET domain at the N-terminal and three tandem LIM domains at the C-terminal (LIM1, LIM2, LIM3). Each LIM domain can form two separate zinc finger structure. This special tandom LIM domain is closely related with localization of TES gene in cells and the function of TES in regulating cell biological behavior. TES protein is mainly distributed in focal adhesion and cell junction area in cell to combine with a series of cytoskeletal protein such as talin, zyxin, VASP, Mena, EVL, alphall-spectrin, actin and Arp7A. Multiple protein binding ability of TES gene depends mainly on LIM domains at carboxyl terminus. For example, the LIM1domain of TES is the direct binding site for zyxin, whereas the LIM3domain combines with the Mena and VSAP, which together with zyxin to form zyxin-TES-Mena-VSAP complex and constitute part of the cell-cell junction. In Hela cell, destruction of zyxin-TES-Mena-VSAP complex by silencing TES can lead to reduction of actin stress fibers enrichment, then decrease the activity of RhoA which binds with GTP to influence Rho-GTPase pathway activity and increase cell motility, decrease cell spreading. These cell biological changes will eventually lead to increased tumorigenicity of tumor cells. The above study has important significance to clarify the function of TES gene and its tumor suppressor mechanism. Privious research showed that the TES gene expression in ovarian, prostate, thyroid, breast, stomach, kidney, colon and head and neck malignant solid tumors and leukemia was down-regulated and responsible for cancer development. The main reasons for TES gene expressional and functional abnormalities include D7S486loci LOH, promoter region methylation, point mutation and gene polymorphism (SNP) of TES gene. Over-expression of TES gene in cancer cells can inhibit proliferation and induce cell apoptosis.Mutation and methylation have been found to be the main mechanism for TES gene inactivation in breast cancer cells. In ZR-75breast cancer cell line, there is an insertion of thymine caused frameshift mutation at1186bp of TES gene coding region. The premature termination of translation in the378th amino acid (Full-lengthTES gene amino acid chain is421) may produce a truncated TES protein lack of LIM2and LIM3domain, affecting its normal function. The promoter region methylation of TES gene in the T47D breast cancer cell line causes gene silencing, whereas over-expression of TES gene cDNA can inhibit cell proliferation and colony formation, implanted the TES over-expressed T47D cell in nude mice in vivo was showed lower tumorigenicity and more cellular apoptosis compared with the control group. The mechanism may be that over-expression of TES can up-regulate the level of endogenous apoptosis related caspase-3and extrinsic apoptosis caspase-8protein and their corresponding apoptotic pathways. Delta9-tetrahydrocannabinol (THC) could activate the transcription factor JunD and make it shift into nucleus to inhibit the proliferation of breast cancer cells, at the same time the up-regulation of TES expression was observed; However in the JunD knockdown tumor cell the expression of TES did not significantly upregulated after the addition of THC, indicating that TES gene may be the downstream target of THC and JunD and can play an important role in anti-breast cancer by inhibiting cell proliferation. In summary, the tumor suppressor function of TES gene in breast cancer such as inducing apoptosis of tumor cells has been well demonstrated, for the mechanisms of TES gene silencing in breast cancer have also been reported in many studies. But whether TES gene has more anti-cancer functions in other aspects, what role TES gene plays in the occurrence and development of breast cancer, and the related molecular biological mechanism is still worthy of discussion and research in depth. OBJECT1. In this study we constructed over-expression and RNA interference vectors for TES gene and built TES over-expressing and silencing breast cancer cell line by using lipofectin transfection technology to investigate if TES gene influences biological behaviors including proliferation, invasion, angiogenesis, metastasis, tumorigenicity and motility in breast cancer cell and to further explore the molecular biological mechanism that TES affects the genesis and development of breast cancer.2. TES protein expression was detected by immunohistochemical and Western Blot technique in clinical specimens from breast cancer patients to explore the relationship between TES protein level and development and outcome of the breast cancer.METHODSRNA isolated from normal human ovarian tissue was used as template to reverse-transcripte genomic cDNA, then PCR to amplify TES gene cDNA sequence. Double-digest PCR product and pcDNA3.1eukaryotic expression vector to obtain double sticky ends. T4ligase was used to connect PCR product and linear vector to get TES gene over-expression vector named pcDNA3.1-TES; shRNA interference sequences targeting TES gene was designed according to the instruction of pGPU6-Neo-GFP interference vector and named pGPU6-Neo-GFP-shRNA-TES. Interference and over-expression vectors were successfully constructed due to sequencing results. The TES gene over-expression and RNA interference vector (pcDNA3.1-TES and pGPU6-Neo-GFP-shRNA-TES) and corresponding control vector (pcDNA3.1, pGPU6-Neo-GFP) were separately transfected into human breast cancer cell line MDA-MB-468and MCF-7, puromycin selection proceeded for about one month until all untransfected cells died. Stable TES gene over-expressing and silencing cell lines468-TES-OVE, MCF-7-TES-KD and the corresponding stable empty vector cell lines468-Control, MCF-7-Control were established. Over-expression or Interference effect on cellular mRNA levels of TES was detected by real-time quantitative PCR. Moreover, protein levels of TES influenced by over-expression and interference were determined by Western Blot.After stable cell line established, MTT method was used to detect cell proliferation efficiency by drawing growth curve in MDA-MB-468and MCF-7respectively. The cells were trypsined and seeded into culture dishes at very low density.2weeks later the condition of cell survive and clone formation were observed. In the cell invasion experiment,468-TES-OVE,468-Control and MCF-7-TES-KD, MCF-7-Control cells were seeded and grown in a matrix gel loaded Tranwell upper chambers, while in the migration tests directly seeded the cells into the upper chambers. The lower chamber was filled with TCM conditioned culture medium. The number of cells migrated through the chamber were counted through microscope to determine cell invasive ability and mobility. Growth factor stripped tumor cell conditioned medium was used to culture human umbilical vein endothelial cells (HUVEC) on matrix gel, after period of time the number of new formed capillaries was counted under microscope.468-TES-OVE、MCF-7-TES-KD and their related control cells were implanted in nude mice in vivo by subcutaneous injection or tail vein injection respectively. Tumor size was weekly measured and mice were euthanized and executed seven weeks after implantation. The number of pulmonary metastases was counted to assess tumorigenisis and metastatic ability of breast cancer cells in vivo.Due to existing literature confirms that miR-29b can regulate the expression of MMP-2protein to affect tumor cell invasion, metastasis and angiogenesis, we assume that the ability of TES gene influence above mentioned activities in breast cancer cells may be associated with the regulation of miR-29b. Using RT-PCR method respectively detected mRNA levels of miR-29b and MMP-2in468-TES-OVE/Control or MCF-7-TES-KD/ Control cells; then miR-29b inhibitors or mimics were respectively transfected into468-TES-OVE/Control or MCF-7-TES-KD/Control cells to measure mRNA level of miR-29b and MMP-2. Moreover, the cell invasion and migration assay and capillary tube formation assay were also repeated in the condition of transfecting miR-29b inhibitors or mimics to verify that TES regulates invasion, migration and angiogenesis in breast cancer cell through miR-29b and MMP-2.In order to identify the relationship between TES gene and development and prognosis of breast cancer, Western Blot was used to detect TES protein expression in the39cases of breast carcinoma tissue and adjacent normal tissue. We also used immunohistochemical technology to evaluate TES, MMP-2and CD34protein level in307different cases of paraffin embedded breast tissue including normal breast tissue, ductal hyperplasia without atypia (UDH), atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and invasive ductal carcinoma and adjacent normal breast tissue. In addition, we used the clinical pathological database from Yale University and the University of Helsinki with long-term follow-up results to analyze the connection between TES gene expression and prognosis of breast cancer patients as well as the expression of common clinical pathological indexes including ER, PR, HER2and P53.RESULTSSequencing results showed that TES over-expression and RNA interference vectors were successfully constructed and the vectors were transfected into human breast cancer cell MDA-MB-468and MCF-7. Real-time quantitative PCR and Western Blot showed that TES gene has been stably over-expressed or knocked down in very high efficiency.MTT results showed that over-express TES gene in MDA-MB-468breast cancer cell can decrease cell proliferation and clone formation capacity (P<0.01), whereas in MCF-7breast cancer cell knockdown of TES gene result in cell proliferation and clone formation increased (P<0.001). Tranwell results showed that over-expression of TES gene can reduce cell invasion (P<0.01) along with significantly decrease cell migration (P<0.001), on contrast knockdown of TES gene in MCF-7significantly increased cell invasion and migration (P<0.001). In the capillary tube formation assay, the number of capillary structure formed when HUVEC cells cultured with TCM conditioned medium from468-TES-OVE was far less than the control group (75.67±7.02vs37.33±5.51, P=0.0017), while capillary formation capacity of HUVEC significantly increased by culturing with the conditioned medium from MCF-7-TES-KD than MCF-7-Control (38.67±4.51vs66.33±7.02, P=0.0046).468-TES-OVE formed smaller tumor compared with468-Control after implanted in nude mice and fewer macrometastatic foci was found in lung tissue after injected468-TES-OVE than control group (42.23±7.02vs16±3.6, P=0.0045); The MCF-7-TES-KD cell formed earlier tumors and the tumors grew faster than MCF-7-Control group in nude mice and also knockdown TES could significantly promote lung metastasis (0.67±0.58vs14.34±3.51, P=0.0027).In468-TES-OVE and468-Control cell, the mRNA levels of miR-29b and MMP-2were detected by RT-PCR, the results showed that over-expression of TES could significantly increase miR-29b mRNA level (P<0.001) and was associated with decreased MMP-2mRNA levels (P<0.01); Inhibition effect of miR-29b on MMP-2is reversible by transfecting miR-29b inhibitor and makes MMP-2mRNA level increased significantly (P<0.001). Moreover, in cell invasion and angiogenesis assay transfecting the miR-29b inhibitor could also reverse the phenomenon that over-expression of TES decreased cell invasion and angiogenesis. In contrast, silencing TES gene in MCF-7breast cancer cell lead to significant reduction of miR-29b mRNA level (P<0.001) with MMP-2mRNA level dramatically increased (P<0.001). With treatment of miR-29b mimics, cell invasion and angiogenesis of MCF-7-TES-KD were no longer promoted. The above results showed that TES gene specifically regulates expression of miR-29b and MMP-2to influence the biological behavior of breast cancer cells.Western Blot results showed the expression of TES protein in31among39cases of carcinoma tissues decreased significantly compared with adjacent normal tissues. Immunohistochemical staining showed that TES gene was expressed in all normal breast tissues (50/50,100%) as well as in almost all cases of ductal hyperplasia without atypia (27/28,96.43%). Furthermore,70.59%(12/17) atypical ductal hyperplasia tissues expressed normal level of TES protein, and only about half of the ductal carcinoma in situ tissues showed positive staining of TES (13/25,52%), in invasive ductal carcinoma tissues TES protein expression rate was only29.95%(56/187). Statistical analysis showed that the breast relapse-free survival, cause-specific survival, distant metastasis-free survival and overall survival rates in breast cancer patients with high expression of TES were higher than that in patients with low TES protein level. In addition, the expression of TES was closely relevant to the protein level of ER in breast cancer patients.CONCLUSION1. Using breast cancer cell lines which were successfully transfected with TES over-express/knockdown vector In vitro proved that TES gene is closely related to a series of cellular biological behaviors including proliferation, invasion, metastasis and angiogenesis in breast cancer.2. Animal experiments in vivo proved that the TES gene funtions as a tumor suppressor on regulating growth and metastasis of breast cancer. Above results further supported the point of view that TES gene is a multi-functional tumor suppressor gene in breast cancer.3. TES regulates invasion, metastasis and angiogenesis in breast cancer cells through TES/miR-29b/MMP-2pathway.4. The relationship between TES protein level and survival rate of breast cancer patients suggest that TES gene can be used as an independent molecular marker to determine prognosis of breast cancer. SIGNIFICANCE1. Revealed the relationship between the TES gene and the occurrence and development of breast cancer and the signal transduction pathway related.2. Firstly studied and proved that TES can be used as a molecular assessment index of prognosis for breast cancer patients.3. To provide the basis and evidence for the TES gene as a new therapeutic target in breast cancer. |
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