The Mechanism Of M~6A RNA Methylation In GaMSCs Promoting Glioma Cells’ Proliferation And Chemoresistance To Temozolomide

Part Ⅰ The effects of gaMSCs on the proliferation,temozolomide resistance and total RNA m~6A levels of glioma cellsObjective:To explore the effects of glioma associated mesenchymal stem cells(gaMSCs)on the proliferation,temozolomide(TMZ)resistance and total RNA m~6A levels of glioma cells.Methods:Surgical specimens of gliomas(WHO grade Ⅲ and Ⅳ)were collected and used to isolate gaMSCs.The equal amounts of glioma cells(U87 or U251)were seeded in six-well plates and divided into co-cultured(CO)and mono-cultured(MO)groups.Glioma cell lines and gaMSCs co-cultivation was conducted using the non-contact coculture transwell system.After cultured with serum-free medium for 0 and 3 days,the two groups of glioma cells were counted by microscope after crystal violet staining.The colony formation experiments were also performed to compare the colony formation abilities of U87 and U251 cells under gaMSCs co-culture and mono-culture conditions.The sensitivities of glioma cells to TMZ in CO and MO groups were measured by CCK8 assay.Then the serum-free medium was used for culture of gaMSCs for 3 days and was collected as the gaMSCs-conditioned medium.U87 or U251 cells were seeded in 96-well plates and were treated with gaMSCs-conditioned medium(conditioned group)and serum-free medium(control group).Then the CCK8 assay was performed to detect cells’abilities of proliferation and TMZ resistance.The m~6A levels of the toal RNAs extracted from co-cultured and mono-cultured U87 and U251 cells were measured by Dot-blot assay and m~6A quantitative kit.Results:Numbers of cells and colonies in gaMSCs co-cultured groups of U87 and U251 cells were significantly larger than the mono-cultured groups.The U87 and U251 cells from co-cultured groups also exzerted stronger resistant abilities to TMZ.Moreover,U87 and U251 cells proliferated faster and were more resistant to TMZ under the stimuli of the gaMSCs-conditioned medium.However,the total m~6A levels of the co-cultured groups were obviously lower than the mono-cultured groups.Conclusions:GaMSCs promote glioma cells’proliferation and TMZ resistance,but reduce their total m~6A levels.Part Ⅱ CSF1 is an important downstream gene of gaMSCs promoting proliferation,TMZ resistance of glioma cells via m~6A RNA methylationObjective: To investigate the function of m~6A RNA methylation in gaMSCs’ promoting the malignant progression of gliomas.To identify the downstream genes of gaMSCs promoting glioma cells’ proliferation and TMZ resistance via m~6A RNA methylation.Methods: Me RIP sequencing and RNA sequencing of U87 cells from mono-cultured and co-cultured groups were performed to identify the differentially m~6A modified and differentially expressed genes(/log2FC/>0.585 and FDR<0.05),based on which the functional enrichment analysis was also conducted.Then the potential downstream gene was overexpressed or knockdown by transfection with plasmids in U87 and U251 cells and then colony formation experiments and CCK8 assay were conducted to explore its roles in glioma cells’ proliferation and TMZ resistance.Results: The hypo-methylated m~6A peaks,which were involved in many signaling pathways associated wth glioma’s malignant progression,dominated the differentially modified peaks(2590 of 2918)in U87 cells from the co-cultured groups.Colony stimulating factor 1(CSF1)was one of the differentially hypo-methylated and upregulated genes in the co-cultured groups.Functionally,overexpression of CSF1 enhanced U87 and U251 cells’ abilities of colony formation,proliferation and TMZ resistance,while knockdown of CSF1 had the opposite effects.Conclusions: GaMSCs may promote glioma progression by downregulating m~6A modification in glioma cells.CSF1 is an important downstream gene of gaMSCs promoting proliferation,TMZ resistance of glioma cells via m~6A RNA methylation.Part Ⅲ The mechanism of m~6A methylation by which gaMSCs upregulate CSF1 in glioma cellsObjective: To explore the mechanism of m~6A methylation through which gaMSCs increase the expression of CSF1 in glioma cells.Methods: The results of RNA sequencing were analyzed to identify the differentially expressed m~6A regulatory proteins between co-cultured and mono-cultured U87 cells.FTO protein levels of U87 and U251 cells from the co-cultured and mono-cultured groups were compared by western blot analysis.Then FTO was upregulated or downregulated by using the lentiviral system to explore its effects on the expression levels of CSF1 in U87 and U251 cells.Subsequently,the protein and mRNAs’ m~6A levels of CSF1 were measured by Western blot and me RIP-q PCR analysis,respectively.Moreover,colony formation experiments and CCK8 assay were performed to investigate the effects of FTO knockdown on U87 and U251 cells’ proliferation and TMZ resistance.The stable FTO-knockdown U87 and U251 cell lines were co-cultured with gaMSCs to check if FTO inhibition could reverse the CSF1’s upregulation by gaMSCs in glioma cells.YTHDF2 was knockdown by using si RNA and its effects on the expression of CSF1 were also analyzed by Western blot.Then the RIP-q PCR was performed to verify YTHDF2’s binding to CSF1 mRNA.The actinomycin D was used to inhibit the transcription and the mRNA stability assay was conducted to explore the effects of FTO and YTHDF2 on the decay of CSF1 mRNA.Results: The expression levels of FTO were increased in the co-cultured groups of U87 and U251 cells.The overexpression of FTO could elevate the expression of CSF1 in U87 and U251 cells.Moreover,knockdown of FTO could reverse the gaMSCs’ upregulation of CSF1 and inhibit the proliferation and TMZ resistance of U87 and U251 cells.Both of gaMSCs co-culture and FTO overexpression could reduce the m~6A levels of CSF1 mRNA in CDS region.Knockdown of YTHDF2 increased the protein levels of CSF1 in U87 and U251 cells.Furthermore,YTHDF2 could bind to CSF1 mRNA and knockdown of YTHDF2 inhibited the decay of CSF1 mRNA.Interestingly,the overexpression of FTO also slowed down the decay of CSF1 mRNA obviously.Conclusions: GaMSCs can reduce the m~6A levels of CSF1 mRNA in glioma cells through FTO-mediated demethylation,thereby inhibiting CSF1 mRNAs’ decay and increasing its expression.YTHDF2 can bind to CSF1 mRNA and promote its decay,and is its possible m6A reader.