MiR-221/222Regulate Tumorigenesis And Cellular Migration/Invasion In Basal-like Breast Cancer

Breast cancer is a malignant and complex disease that involved in the expression of abnormal of non-coding and coding gene,which is one of the most common malignancy of women. Clarifying the molecular mechanisms of breast cancer metastasis, and helping establish a reasonable prevention and treatment programs that can greatly improve long-term survival of patients and improve their live quality.MicroRNA (miRNA) are a class of19-23nucleotides in length endogenous small non-coding RNA. It can bind to the3’untranslated region (Untranslated region, UTR) of mRMA inhibit directly the translation or degradation of the target mRNA. Depending on the difference of target gene and target pathways, some miRNAs play tumor suppressor role, some miRNAs play tumor promotion role.In this study, we design the miRNA primers library by self and conduct quantitative real-time PCR to validate them one by one, developing a high specificity widely detect range and simple breast cancer miRNA microarray. Using our own design miRNA microarray to analyze abnormal miRNA expression in breast cancer patients, and with real time PCR method to verify the results of the chip. In addition, we analyzed miRNA expression pattern in101breast cancer patients to find abnormal miRNAs in human breast cancer. Again, we used bioinformatics software TargetScan and miRDB that selected miR-221and miR-222target genes with differential expression ofpredictive analysis, and transfected in vitro to breast cancer celllines MDA-MB-231, MCF-7, MDA-MB-453,T-47D and Hs578t such as indirect verification miR-221/miR-222mimics, and we do a potential target gene identification and validation. Finally, we have in-depth study the influence of miR-221and miR-222for the biological behavior of basal-like breast cancer cells MDA-MB-231,it include cell proliferation, migration, invasion andthe cell cycle.(1) Different expression of miRNAs in breast cancerThrough the results of miRNA microarray,the expression of miR-221and miR-222in basal-like breast cancer is higher than the luminal breast cancer. This high expression illustrate that miR-221and miR-222may play a role in the molecular typing of breast cancer.(2) Prediction and identification of potential target genes of miR-221and miR-222miRNAs interact with their target mRNAs and cause degradation of mRNA or inhibit the post-transcriptional gene expression. In this study, we use the biosoftware TargetScan and miRDB prediction analysis the expression with difference of the target genes in miR-221and miR-222. In the prediction of possible target genes, SOCS1and CDKN1B showed higher possibility.Meanwhile,101cases of microarray results of previous studieson breast cancer and normal tissues showed that only SOCS1and CDKN1B were predicted. Therefore, we may speculate CDKN1B and SOCS1that are target genes of miR-221and miR-222. Proved by Western blot in the MCF-7breast cancer cells over-expressing miR-221and miR-222, inhibition of protein expression of SOCS1. In the basal-like breast cancer cells MDA-MB-231cells, inhibiting the expressing of miR-221and miR-222,then,SOCS1occurs high expression.similarly, CDKN1B occurs high expression.(3) miR-221/222on cell proliferation, migration and invasion capacity and cell cycleThis study research the biology function of miR-221and miR-222in breast cancer. MTT assay show that the inhibitors of miRNA-221and miR-222can inhibit the growth of breast cancer cells, miRNA-221and miR-222could promote cell proliferation, which is the promoter of the miRNA. We further analyze the impact of miRNA-221and miR-222for inhibiting invasion and migration in the biological behavior of breast cancer cells.The invasion experimental results show that transfected miR-221/222mimics can significantly enhance the invasiveness of MDA-MB-231breast cancer cells. Furthermore,the migration experiments also show miRNA-221and miR-222mimics transfected into MDA-MB-231cells can significantly enhance their ability to migrate, the opposite result is that breast cancer cells transfected with the inhibitors of miRNA-221and miR-222can inhibit significantly the ability of migration in breast cancer cells MDA-MB-231, SUM159and Hs578t. These results suggest that the expression level of miR-221and miR-222related closely with the migration and invasion of breast cancer cells. In addition, we also analyse the impact of miR-221and miR-222on the cell cycle, in low serum (0.5%FBS) culture medium starvation conditions, miR-221and miR-222are synergistic cyclin D low expression for plus10%FBS medium containing cultured 12h, cyclin D expression of miR-221and miR-222and is induced obviously. A quantitative analysis showed both anti-miR-221and anti-miR-222are able to inhibit the G1/S transition, and arrest the cell cycle at G1phase.In summary,the miRNA microarray of breast cancer and quantitative real-time PCR confirmed the miR-221and miR-222expression was significantly high in breast cancer. SOCS1and CDKN1B might be target genes of miR-221and miR-222in breast cancer cells. In vitro studies confirmed that miR-221and miR-222over expression significantly increased proliferation,invasion and migration of breast cancer cell MDA-MB-231. Thus, suggesting that miR-221and miR-222are potential promote tumor miRNAs in in human basal-like breast cancer.