Analysis Of TRIM28 Expression And Study The Mechanism For Its Correlation With Tumors

① To construct pc DNA5-TRIM28 recombinant plasmid, and pc DNA5- TRIM28-HEK293 stable inducible cell lines. ② To investigate the role of TRIM28 gene regulation at the molecular level and its expression in tumors. ③ To analysize the tumor-associated mechanisms with TRIM28. Methods: First, Construction of pc DNA5-TRIM28 recombinant plasmid, and pc DNA5-TRIM28-HEK293 stable inducible cell lines were screened out successfully by PCR technology, digestion, sequencing verified the positive clones and Western bloting. Secondly, extracting protein of TRIM28-HEK293 cells and seting control, mass spectrometry for detection TRIM28 target genes were identified by two-dimensional electrophoresis.The gene–specific primers were designed. Select DOX optimal induction time(12h) and the optimal induction dose(0.1μg/ml) induced stably expressing TRIM28-HEK293 cell line, extracted RNA, under the action of the enzyme reverse transcribed into c DNA, reverse transcriptase product as a template, PCR amplification with specific gene primers were performed by real-time PCR to verify the results of two-dimensional electrophoresis. Futher more, the over expression of TRIM28 in He La cells after transfection of 24 h RNA extraction, reverse transcription into c DNA, using reverse transcription products as template, real-time fluorescence quantitative PCR amplification was performed by 11 pairs of gene specific primers to verify two-dimensional electrophoresis results. Again, extracted seven different tumor cell lines RNA, transcribed into c DNA, reverse transcription product as a template by TRIM28 and internal reference(18S RNA) specific primers, then real-time fluorescence quantitative PCR amplification was performed to test TRIM28 m RNA expression levels in different tumor cells. We collected eleven cases of adjacent normal breast tissues and thirty-three cases of breast cancer tissues, tissue samples were extracted RNA, transcripted into c DNA as a template, TRIM28 and internal reference(18S RNA) specific amplification primers were used for real-time PCR amplification for adjacent normal tissues and tumor tissues to check the levels of m RNA expression by quantitative PCR. To detect TRIM28 and TWIST1 correlation with tumor, the over expression of TRIM28 in He La and 4T1 cells, RNA and protein were extracted, fluorescence quantitative PCR and Western blot were used for detection of TRIM28 and TWIST1. Results: pc DNA5-TRIM28 recombinant plasmid and stable expression TRIM28-HEK293 cell lines have been successfully constructed. The pc DNA5-TRIM28 recombinant plasmid and p OG44 plasmids were co-transfected into HEK-293 cell line, with hygromycin B positive clones. Detected by Western Blot, in cells with empty vector control HEK-293 cells, both plus DOX-induced or not, were not detected TRIM28 expression. In the co-transfection of the recombinant plasmid pc DNA5-TRIM28 and p OG44 plasmid Flp-In-HEK-293 cell lines by adding DOX-induced experimental group, TRIM28 was expressed significantly, demonstrating stable expression of inducible pc DNA5-TRIM28-HEK293 cell line was successfully constructed. Extracted induce stable expression pc DNA5-TRIM28-HEK293 cell line protein, two-dimensional electrophoresis testing were performed, and mass spectrometry found 11 TRIM28 target genes, including 3 up-regulated and 8 down-regulated genes. According to two-dimensional electrophoresis detected 11 genes, primers were designed, induced the expression of TRIM28 in TRIM28-HEK293 stable cell lines and extracted m RNA, after real-time quantitative reverse transcription PCR, the analysis found that we verified the results of two-dimensional electrophoresis partially in inducible expression of TRIM28-HEK293 stable cell lines, TRIM28 has a role in the regulation can be down-regulated the expression of certain genes. The 11 TRIM28 target genes were further verified in He La cell by overexpressing TRIM28 partially. TRIM28 expression were performed by reverse transcription and quantitative PCR in 7 tumor cell lines, and found that in MDA-MB-231 cell line, T47 D cell line, MCF-7 cell line, MDA-MB-435 cell lines and HEK293 cell lines, the TRIM28 m RNA expression were significantly increased, but not in normal cell MCF10 A, indicating that TRIM28 is tumorgensis or metastasis-associated. Collected 11 cases of adjacent normal breast tissues and 33 cases of breast cancer tissue samples, the quantitative PCR fluorescence quantitative analysis found that, compared with adjacent normal tissues, TRIM28 m RNA expression levels in breast tumor tissues were significantly increased, which is nearly four times than in the normal tissues, suggesting that the TRIM28 expression has an important relationship with the development of tumors. Overexpress TRIM28 in 4T1 cells we found, compared with only transfected with empty vector controls, TWIST1 protein level was increased, about 1.71 times 1.28 times respectively in the two independent experiments. Over-expression of TRIM28 increased TWIST1 protein, and TWIST1 plays an important role in cancer metastasis, which indicates that TRIM28 has played an important role in tumorigenesis or metastasis may be through TWIST1. Conclusion: We successfully established stable inducible TRIM28-HEK293 cell line; discovery and detection of 11 TRIM28 target genes by two-dimensional electrophoresis, mass spectrometry and fluorescence quantitative PCR. By extracting RNA seven different cell tumors and 44 cases of tissue RNA, found that TRIM28 m RNA expression levels increased in tumors(cells or tissues). TRIM28 expression and TWIST1 expression have correlation by Western blot, further proof that TRIM28 correlation with cancer and TRIM28 play an important role in breast cancer. But whether TRIM28 control the occurrence of tumors by adjusting the TWIST1, and how to regulate the occurrence and development of tumors? Further studies are needed.