Construction Of Lentiviral Expression Vector With MicroRNA-100 Inhibitor And Its Effect On The Cellular Behavior Of BEAS-2B

[BACKGROUND]The occurrence of tumor is a complex process.We have been committed to the research of tumor pathogenesis and intervention.The role of mi RNAs in cancer is of great impor-tance.During our previous studies, we use the analysis of mi RNA hybridization to esta-blish the mi RNA(micro RNA,mi RNA,mi R)expression profiling in the SHP2+/ + wild type and SHP2D61G/+mutation of MEFs acute induced by As2O3.As a result,the expressio-n of mi R-100 is decreasing among dozens of differentially expressed mi RNAs.The mal-ignant cell behavior is more obvious after treated with As2O3 in SHP2D61G/+ mutated M-EFs. So we speculate that mi R-100 took part in the process of cell malignant transform-ation and we further study the role of mi R-100 in this process.In recent years, the research about the relationship between mi R-100 and tumor has made significant progresses.Study found that, in some tumors such as prostate cancer,its expression is elevated, and it promotes the metastasis of prostate cancer, while in oth-er cancers such as cervical, lung and bladder cancer, the expression is low, suggesting that it plays a role of tumor suppressor. Some investigators found that BEAS-2B cells chronically exposed to sodium arsenite increased proliferation and gained anchorage-in-dependent growth, having a certain degree of malignant transformation.Study also found that the most significant human health risk to arsenic exposure is the development of multiple cancers, including lung cancer.Therefore, both mi R-100 and arsenic isclosely related to lung cancer and we choose human bronchial epithelial cells BEAS-2B cells as the research object to research the effect of mi R-100 on the BEAS-2B celluar behavior. The lentiviral vector system can efficiently, stably import gene into cell lines with the advantage of high efficiency, wide infection spectrum and high safety.Thus we intend to study the effect on the cellular behavior of BEAS-2B after silencing the expression of mi R-100 by infecting BEAS-2B cells with mi R-100 inhibitor lentivirus expression vector. Lay the foundation for further study the role of mi R-100.[METHODS]1. Construct the mi R-100 inhibitor lentivirus expression vector and stable infect BEAS-2B cells with it.2. Detect the expression of mi R-100 after infecting with mi R-100 inhibitor lentivirus expression vector by RT-PCR.3. Detect the change of lentivirus-infecting BEAS-2B cell proliferation by MTT and plate colony assay; observe the non-anchored growth capacity of lentivirus-infecting BEAS-2B cells by soft agar assay.4. Test the ability of cell adhesion and migration of lentivirus-infecting BEAS-2B cells by adhesion and Transwell migration assay.[RESULTS]1. We have established BEAS-2B cell lines infecting with stable expression of mi R-100 inhibitor.2. The expression of mi R-100 in BEAS-2B cells infecting with mi R-100 inhibitor lentivirus expression vector was down-regulated obviously.3. The results of MTT and colony formation assay showed that the proliferation ability of BEAS-2B cells increased when the expression of mi R-100 was down-regulated.4. The ability of anchor-independent growth of BEAS-2B cells increased when the expression of mi R-100 was down-regulated,forming a larger colony.5. Adhesion and transwell migration assay showed that,the cell adhesion and migrationability of BEAS-2B cells increased in some extent when the expression of mi R-100 was down-regulated.[Conclusion]1.The expression of mi R-100 in BEAS-2B cells was down-regilated by infecting BEAS-2B cells with mi R-100 inhibitor lentivirus expression vector.2.It promoted the cell proliferation,growth and migration ability of BEAS-2B cells when the expression of mi R-100 was down-regulated.