MiRNA17Family Regulates Cisplatin-Resistant And Metastasis By Targeting TGFbetar2in NSCLC

[BACKGROUND]For nearly30years, the mortality of lung cancer are more and more higher, lung cancer havereplace liver cancer to be the first cause of death of malignant tumor in the world. At presentsurgical is the still the most effective treatment for lung cancer, patients who failed to acceptsurgical only drug chemotherapy or radiation therapy can be chosen, but even if it is sensitive tochemotherapy in the beginning, patient swill eventually turn out acquired drug resistance and thentumours metastasis, these are the main reason of high mortality of lung cancer.MicroRNAs (miRNAs) have been proven to play crucial roles in cancer, including tumorchemotherapy resistance and metastasis of non-small-cell lung cancer (NSCLC). TGF signalpathway abnormality is widely found in cancer and correlates with tumor proliferation, apoptosisand metastasis. Here, miR-17,20a,20b were detected down-regulated in A549/DDP cells(cisplatin resistance) compared with A549cells (cisplatin sensitive). Over-expression of miR-17,20a,20b can not only decrease cisplatin-resistant but also reduce migration by inhibitingepithelial-to-mesenchymal transition (EMT) in A549/DDP cells. These functions of miR-17,20a,20b may be caused at least in part via inhibition of TGF signal pathway, as miR-17,20a,20b areshown to directly target and repress TGF-beta receptor2(TGF R2) which is an importantcomponent of TGF signal pathway. Consequently, our study suggests that miRNA17family(including miR-17,20a,20b) can act as TGF R2suppressor for reversing cisplatin-resistant andsuppressing metastasis in NSCLC.[OBJECTIVE]Seeking for the miRNAs can be a marker of the tumor sensitivity to cisplatin or a marker ofability of metastasis,then research the molecular mechanisms of them.Our results suggest thatmiRNA17family (miR-17,20a,20b) play important roles in the regulation of cisplatin-resistantand migratory capability by targeting TGF R2of TGF signal pathway. miRNA17family has thepotential as key regulatory factors for the chemotherapy resistance and metastasis of NSCLC.miRNA17family may be the sensitive markers for predicting cisplatin treatment and the ability ofmetastasis.[METHOD]1. Cell lines and in vitro comprising A549and his cisplatin-resistant model A549/DDP.2. Microarray detection of miRNA expression and their results were testified by qRT-PCR.3. Cytotoxicity assay: The cell-counting kit-8colorimetric assay was used to measure the cellviability after transfection of miRNA.4. Bioinformatics software to predict the miRNA target genes.5. qRT-PCR and Western boltting detection the change of targeted mRNA and protein afterincreased expression of miRNA.6. Dual luciferase reporter gene assay detects whether the miRNA and downstream targets fordirect control.7. transwell assay to test the ability of migration.8. The small interfering RNA (siRNA) si-TGF R2was used to silence TGF R2in A549/DDPcell line.9. Immunohistochemical staining to detect the E-cadherin and Vimentin’s protein of humantissues.[RESULT]1. miR-17,20a,20b expression level significance lower in A549/DDP than A549byqRT-PCR,these results conform to microchip.2. CCK-8assay shows after transfect with miR-17,20a,20b mimics or inhibitors, A549/DDPcell line resensitive to cisplatin and A549cell line much more resistant to cispaltin.3.Cellular morphology, A549cells exhibited the epithelial phenotype and A549/DDP cellsshowed the mesenchymal phenotype,A549/DDP cells transfected with miR-17,20a,20b mimics could change to epithelial phenotype,and A549cells transfected with miR-17,20a,20b inhibitorscouldchange to mesenchymal phenotype.4. Detection at mRNA and protein expression level by RT-PCR and Western Blotting.A549/DDP cells transfected with miR-17,20a,20b mimics decreased Vimentin expression andincreased E-cadherin expression, and A549cells transfected with miR-17,20a,20b inhibitorsshowed an increase in Vimentin expression and a decrease in E-cadherin expression.5. By transwell we find migratory capability of the A549/DDP cells exceed to A549cells.Results showed that over-expression of miR-17,20a,20b could increase migration in A549/DDPcells and inhibition of them could reduce migration in A549cells These results indicated thatmiR-17,20a,20b could regulate the metastasis efficiently in NSCLC.6. With bioinformatics software, we found TGF R2could be the downstream target of miR-17,20a,20b.7. We found that both the mRNA and protein expression levels of TGF R2were up-regulated inA549/DDP cells compared with A549cells by Western Blotting and qRT-PCR. The expressionlevels of TGF R2decreased in A549/DDP cells transfected with miR-17,20a,20b mimics andincreased in A549cells transfected with miR-17,20a,20b inhibitors. Thus, the expression ofTGF R2was enhanced when cisplatin-resistant occurs and regulated by miR-17,20a,20b.8. We generated luciferase reporter constructs containing specific mutations at putativemiR-17,20a,20b binding site.When A549cells were transfected with the wild type TGF R23’UTR, co-transfection of miR-17,20a,20b mimics inhibited luciferase activity. In contrast, theeffects of miR-17,20a,20b mimics were eliminated in A549cells transfected with the mutant typeTGF R23’UTR. These results suggested that miR-17,20a,20b binds directly to putative TGF R23’UTR regions, as predicted by the in silico model.9. TGF R2silented resulting in the inhibition of EMT: decreasing of migratory capability.Moreover, A549/DDP si-TGF R2cells were more sensitive to cisplatin-induced growth inhibitionthan A549/DDP cells.10. Immunohistochemical staining found that the expression of E-cadherin in primary cancerexceed to metastatic cancer, but the expression of Vimentin has the negative result.[CONCLUSION]1. miR-17,20a,20b expression level associated with the sensitivity to cisplatin.2. miR-17,20a,20b regulate EMT and migration.3. miR-17,20a,20b directly target and suppress TGF R2.4.Suppression of TGF R2expression reduces cisplatin-resistant and migration.