TC-1 Mediates The TRE17 Oncogene Induced Migration Of MCF-7 Breast Cancer Cells

[Object]TRE17 (also named as TRE2, USP6) was originally found expressing in Ewing sarcoma, leiomyosarcoma, rhabdomyosarcoma, osteosarcoma and breast cancer, etc. Then TRE17 was proved to be a novel proto-oncogene, because overexpression of TRE17 is sufficient to induce malignant transformation of NIH 3T3 fibroblasts and cause tumor formation in nude mice. However, the tumorigenic mechanism and the effect on cells' invasion and metastasis biology behavior of TRE17 have not been clear. Thyroid cancer gene-1 (TC-1,also known as C8orf4), is highly expressed in papillary thyroid cancer, gastric cancer, lung cancer, breast cancer and colon cancer, and it can promote malignant transformation of the normal thyroid cells.TC-1 can combine with Chibby (Cby) to competitive?-catenin sites, and thereby enhances the Wnt/?-catenin signaling pathway by relieving the antagonistic function of Cby on the ?-catenin-mediated transcription, and then upregulates the expression of ?-catenin target genes such as MMP9?VEGF?TCF, etc, they are implicated in invasiveness, migration, proliferation and other aggressive behavior of cancers. Therefore, this study mainly discusses the migration molecular mechanism of TRE17 which upregulate Wnt/?-catenin signaling TC-1, to further explain the role of TRE17 oncogene in the tumor.[Method]1. In order to study the effect of TRE17 overexpression on MCF-7 cells migration, Flag-TRE17 and empty carrier Flag-pcDNA3.0 plasmid was transfected into MCF-7 and HEK293 cells, then transwell migration experiment and scratches assays were made to analyze the effect.2. Gene chip technology was perfomed to screen TRE17 high expression of differentially expressed genes to explore the mechanism of which TRE17 affect cells migration ability. Flag empty vector served as control, TRE17 oncogene was transfected into human breast cancer cells MCF7, each group consisted of 2 cases for gene chip detection. Gene chip results suggest that 404 species genes are differentially expressed in breast cancer cells after transfected TRE17. The genes which were raised higher were selected to identified. The genes include C8orf4 (TC-1, thyroid carcinoma genes 1) was detected in the mRNA and protein level with Real Time PCR and Western Blot methods. TRE17 is composed of USP32 and TBC1D3, in order to further clarify which structure of TRE17 regulates TC-1 expression in MCF-7 cells, kinds of mutant plasmid of Flag-TRE17, including TBC structure domain deletion mutant Flag-TRE17 (?59-116), and the Flag-TRE17 (?117-174), calmodulin (Camodulin, CaM) binding sites mutant Flag-TRE17 (F328E), USP active site mutant Flag-TRE17 (USP-), and Flag-TRE17 were extracted and transfected into MCF-7 cells and the cell lysates were detected the expression lever of TC-1 with Western blot.3. Overexpression of TC-1 can induce the transformation of normal mammary epithelial cells into tumor cells, but its influence on breast cancer cell migration is absent. TC-1 eukaryotic expression plasmid was constructed to detect the function of TC-1 in the migration of breast cancer MCF-7 cells. Total RNA was extracted from MCF-7 cells, full length open reading frame (ORF) of TC-1 cDNA was amplified with RT-PCR, and was inserted into the eukaryotic expression vector Flag-pcDNA3.0, and the eukaryotic expression plasmid Flag-TC-1/pcDNA3.0 was constructed. The recombinant plasmid was transfected into MCF-7 cells, then ecombinant plasmid fusion protein Flag -TC-1 expression was analyzed with Western blot. Empty vector and recombinant plasmid were transfected into MCF-7 and BT549 cells, transwell migration experiments and scratches assays were performed respectively to analyze the role of TC-1 in the migration of breast cancer cells.4. In order to define the role of TC-1 in where TRE17 affect the migration of MCF-7 cells, we build TC-1 shRNA lentivirus which target different position of human TC-1 gene:TC-1 (653) shRNA, TC-1(1512) shRNA, and TC-1 (1566) shRNA and control lentivirus NC shRNA. MCF-7 cells were infected by the lentivirus. TC-1 gene expression was detected by Western blot to determine the effectiveness of specific shRNA. To perform Transwell migration experiments, Flag empty vector served as control, Flag-TRE17 was transfected into MCF-7 cells infected NC shRNA and TC-1(1566) shRNA. Recent research suggest that TC-1 carcinoma protein can enhance the transcription of downstream target genes of wnt/?-catenin signaling pathways (including genes related cells'migration). In order to explore the role of Wnt/?-catenin signaling pathway in the TRE17-mediated migration of MCF-7 cells, MCF-7 cells were treated with Wnt/?-catenin signaling pathway inhibitor XAV-939, and used to transwell migration experiment.[Result]1. We conducted transwell to discuss the influence of TRE17 oncogene on cells' migration. Transwell results showed that TRE17 overexpression can enhance migration ability of MCF-7 cells. Migration experiment results suggested that migration of overexpressed TRE17 in HEK293 cells were significantly improved.2. In order to further study the molecular mechanism of TRE17 which promotes cells'migration, gene chip technology was performed to screen the differentially expressed genes in MCF-7 cells which overexpressed TRE17. The experment found the differentially expressed genes were as many as 404 in breast cancer cells which were transfected TRE17. MCF-7 cells were transfected with FLAG-pcDNA3.0 and FLAG-TRE17-pcDNA3.0 recombinant plasmid respectively, then total RNA was extracted. Real-Time RT-PCR was conducted to confirm some certain differential expressed genes. The results showed that TC-1 was raised about two times. Agarose gel electrophoresis results of total RNA revealed that the brightness ratio of 28 s band to 18 s band is about 2:1, and the decent quality of RNA (P< 0.01). Western blot analysis showed that TC-1 expression increased in MCF-7 cells transfected with TRE17, while endogenous protein level in MCF-7 cells which were transfected with Flag was low. All the results suggested that TC-1 gene and protein increased markedly in MCF-7 cells which overexpressed TRE17. Kinds of mutant plasmid of Flag-TRE17, including TBC structure domain deletion mutant Flag-TRE17?(?59-116), and the Flag-TRE17 (?117-174), calmodulin (Camodulin, CaM) binding sites mutant Flag-TRE17 (F328E), USP active site mutant Flag-TRE17 (USP-), Flag-TRE17and Flag-pcDNA3.0 were transfected into MCF-7 cells,and the results shows that compared with the empty vector control, overexpressed Flag-TRE17, the mutant plasmid Flag-TRE17 (F328E and Flag-TRE17(-USP) respectively could increase the protein lever of TC-1, however, the TBC structure domain deletion mutant Flag-TRE17 (?59-116) and Flag-TRE17 (?117-174) lost the role of promoting the expression of TC-1. These results suggest that TRE17 promotes the expression of TC-1 depends on the TBC domain structure, but does not depend on USP domain, and the CaM bind or not does not affect the role of which TRE17 stimulates expression of TC-1.3. In order to study the migration effect of TC-1 overexpression on MCF-7 cells, full length ORF sequence of TC-1 cDNAwas cloned by RT-PCR, and agarose gel electrophoresis experment showed that the DNA band lied in 337bp, which was accordance with the anticipated obdective strap size. The RT-PCR products of recycling were treated with the BamH I and Xho I double enzyme, agarose gel electrophoresis was conducted and were extracted and purified, and then were connected with eukaryotic expression vecto Flag-pcDNA3.0 which was treated by the same enzyme, then the recombinant plasmid Flag-TC-1-pcDNA3.0 generated.The plasmid showed 327 bp characteristic fragments after treating with BamH I and Xho I double enzyme; DNA sequencing confirmed that the sequence was correct. After MCF-7 cells were transfected with the recombinant plasmid Flag-TC-l-pcDNA3.0 and the vector Flag-pcDNA3.0, transwell results showed migration ability of MCF-7 which overexpressed TC-1 increased. Similarly, the results of scratch experiment of human BT549 breast cancer cells showed that 12 hours after the scratch, scratch size had no obvious change in BT549 cells which were transfected with empty vector,while scratch size had shrunk nearly 50% in BT549 cells which were transfected with TC-1; 24 hours after the scratch, scratch size had shrunk no more than 1/3 in BT549 cells which were transfected with empty vector, while scratch size disappeared nearly in BT549 cells which overexpressed TC-1. These results indicate that high expression of TC-1 can enhance the migration of human breast cancer cells.4. In order to define the role of TC-1 in the progress of which TRE17 induced MCF-7 cells'migration, empty vector Flag was carrier for comparison,Flag-TRE17 was respectively transfected into the MCF-7 cells which were infected with control NC shRNA and lentivirus shRNA-TC-1(1566) which specificly inhibit TC-1 expression, and transwell assay was conducted. Transwell migration experiment results showed that TRE17 could promote the migration of MCF-7 cells when TC-1 protein level was not knockdown. While after TC-1 gene was knockdown by treated lentivirus TC-1 (1566) shRNA, the migration in MCF-7 cells induced by overexpressed TRE17 or not were both inhibited (P<0.05). These results indicated that the TC-1 oncoprotein regulate the migration of human MCF-7 breast cancer cells, and promote partially MCF-7 cells'migration mediated by TRE17 oncoprotein at least. MCF-7 cells were treated with Wnt/beta-catenin signaling pathway inhibitor XAV939, and subsequent Transwell migration experiment results showed that the overexpression of oncoprotein TRE17 and TC-1 can promote migration MCF-7 cells, respectively. After adding Wnt/?-catenin signaling pathways inhibition XAV-939, not only the migration of MCF-7 cells transfected with empty vector decreased significantly, but also the migration ability promoted by TRE17 or TC-1 overexpression was inhibited (P< 0.05). These results suggested that TRE17 may promote migration of MCF-7 breast cancer cells by TC-1/Wnt/?-catenin signaling pathway.[Conclusion]1. Overexpression of TRE17 can promote migration of human MCF-7 breast cancer cells and HEK293 embryonic renal epithelial cells;2. TRE17 oncogene promotes increasing of TC-1 oncogene and protein level through TBC domain;3. TRE17 may promotes the MCF-7 cells' migration by expression regulation of TC-1 partly;4. TRE17 may promotes the migration of MCF-7 cells by regulating the Wnt/?-catenin signal pathway which is in the downstream of TC-1.