Overexpressed Let-7a Inhibits Glioma Cell Malignancy By Directly Targeting K-ras, Independently Of PTEN Status

Research background：MicroRNAs (miRNAs) are a class of small non-coding RNAs and consist of20-25ribonucleotides. The mature miRNAs with full or partial complementarity tothe target mRNAs direct the cleavage of target mRNAs or act as repressors oftranslation, involved in modulating the development, differentiation, proliferation andapoptosis of cells. Recent studies have shown a distinct connection between miRNAsand the development of cancer, some of which are specially expressed inglioblastoma. We have collected different pathological diagnosis of malignant glioma,normal brain tissue as well as glioma cell lines, and then screen the glioma-specificexpression of miRNAs using miRNAs microarray methods to find some miRNAsshow low expression in human gliomas, let-7a is one of them. As an anti-oncomiRs,let-7a, has been reported to be downregulated in a variety of cancer. But less wasknown for the expression and function of let-7a in glioma up to now. As one of theRas oncogene family，K-ras had been reported to be correlated with many tumors viaits downstream signaling pathway. In our previous study, we found expression ofK-ras was positive correlated with giloma grades. Studies showed that expression ofPTEN was correlated with occurrence of glioma. PTEN could regulate downsteampathway of K-ras including PI3K/AKT and MAPK/ERK pathways. In this study, weverify K-ras is a target gene of let-7a in glioma via upregulation of let-7a aftertransfection of let-7a mimics and cotransfection with let-7a and K-raspcDNA(without3’UTR). We also test its affection and mechanism of action inglioma cells with different PTEN ststus. Taken together, these results suggest thatmodulation of the mechanism responsible for let-7a in GBM could be used as acritical therapeutic strategy for GBM intervention. Methods：1. Real-time RT-PCR Immunohistochemistry staining test expression of let-7a andK-ras in different grade glioma tissues.2. Selected human glioma cell line U87and U251, which were transfected withlet-7a mimics to upregulate let-7a status in cells, taking logarithmic phase for cellexperiments. MTT assay, Flow cytometric method, transwell assay andwound-healing assay test the maligant phenotype changes induced by let-7a inglioma cells.3. Bioinformatics, Luciferase reporter assay and western blot assay were used toanalysis whether K-ras is the target of let-7a. Immunohistochemistry staining testexpression of K-ras in grade glioma tissues and analysis correlation betweenlet-7a and K-ras.4. The effects of the proliferation, cell cycle, apoptosis, invasion and migrationwere evaluated by MTT assay, Flow cytometric method, transwell assay andwound-healing assay in U87and U251cells after transfected with let-7a orco-transfected with let-7a and K-ras(without3’UTR).5. Expression of PTEN was tested with western blot assay after transfected inLN229. PTEN siRNA was transfected into LN229to downregulate expression ofPTEN. And MTT assay, Flow cytometric method, transwell assay andwound-healing assay were used to test the effects of the proliferation, cell cycle,apoptosis, invasion and migration after transfection in LN229and LN229(PTENsiRNA) cells. The distinction of effect induced by let-7a and upregulation ofK-ras in different PTEN phenotype [U87（PTEN null）， U251（PTENmutant）,LN229(PTEN wild) and LN229(PTEN siRNA)] is analyzed togetherwith the result in step4.6. Western blot assay was used to study downstream signaling pathway of K-rasafter transfected with let-7a in different PTEN status.7. To test the growth inhibitory effects with let-7a, we employed a U87glioma cellsxenograft model, and evaluated the expression of K-ras and its downstream signaling pathway.Results:1. let-7a is upregulated in glioma tissues compared with in normal tissues.2. let-7a could suppress malignant phenotype in glioma cells.3. let-7a could inhibite expression of K-ras via target K-ras3’UTR, and expressionof let-7a and K-ras exsit correlation.4. K-ras is a functional target for let-7a to inhibit cell growth, induce apoptosis,suppress invasion and reduce migration in U87and U251. And these inhibitioneffect do not appear after cotransfection of K-ras (without3’UTR).5. PTEN is upregulated after transfected with let-7a in U87, U251and LN229cells.PTEN is downregulated after transfected with PTEN siRNA in LN229cells.There is no difference for let-7a to inhibit cell growth, induce apoptosis, suppressinvasion and reduce migration with different PTEN status analyzed together withthe result of step4.6. let-7a inhibites pAKT, pERK, pmTOR, pBAD and MMP9in glioma cellsindependent of PTEN status.7. let-7a suppresses glioma in vivo via inhibition of K-ras and its downstreamsignaling pathway.Conclusions:1. let-7a target suppressed K-ras and downstream signaling pathway in vitro andvivo and inhibited cell growth, induce apoptosis, suppress invasion and reducemigration.2. let-7a inhibited glioma malignancy via target K-ras independent of PTEN status.These results indicate for the first time that let-7a inhibits glioma malignancyindependent of PTEN status via targeting K-ras.