MiR-100 Inhibits Cells Proliferation In Mantle Cell Lymphoma By Targeting MTOR

Objective: The purpose was to study the aberrant of miR-100 and mTOR in mantle cell lymphoma(MCL)and their possible biological function and pathogenesis.Method: 18 cases of MCL tissues and 3 cell lines(Jeko-1,Mino,Granta-519)were recruited in this research and 18 cases of proliferative lymphadenitis and normal lymphocyte cells were severed as the control.The expression of miR-100 and mTOR mRNA was detected by RT-PCR,and the expression of mTOR protein was detected by western blot in MCL specimens.Chi square test was used to analyze the correlation between the miR-100 and the clinical feature of MCL patient.The relationship between miR-100 and mTOR in MCL pathological tissues and MCL cells were studied by linear fitting.The LV-miR-up and LV-mTOR-RNAi were constructed with lentivirus and transfected into Jeko-1 and Mino cell lines to up-regulate miR-100 and down-regulate mTOR expression.RT-PCR and western blot were used to test the expression of miR-100 and mTOR.Cell proliferation was detected by CCK-8.Cell apoptosis and cell cycle were checked by flow cytometry.Bioinformatics prediction software was used to predict that miR-100 was targeted to mTOR gene and the complementary binding region between mTOR mRNA transcripts and miR-100.Then mTOR wild-type and mutant-type luciferase reporter vectors were transfected with the over-expressing plasmid of miR-100 respectively in double luciferase experiment to verify miR-100 targeting at mTOR-3’UTR.PV-mTOR-up and LV-miR-100-up co-transfected into Jeko-1 and Mino cell lines to certificate the correlation between miR-100 and mTOR and the effect on cell proliferation,apoptosis and cell cycle.Results:1.Compared to the control,the miR-100 mRNA in MCL tissues was lower expression(p<0.0001),while the mTOR protein was higher expression(p=0.003).The expression of mTOR protein was negatively correlated with miR-100 mRNA in MCL tissues(r=-0.9161,p<0.0001).The miR-100 expression was lower and the mTOR mRNA was higher in MCL cell lines(p<0.0001).There was a negative correlation between the miR-100 and mTOR in MCL cell lines(r=-0.9927,p<0.0001).2.LV-miR-100-up or LV-mTOR-RNAi transfected into Jeko-1 and Mino cells respectively and led to increasing miR-100 or reducing mTOR expression,inhibiting cell proliferation,inducing cell apoptosis and blocking cell cycle in G1 phase.3.Three bioinformatics softwires predicted that miR-100 was targeting the mTOR gene,and the complementary binding site at 295-301 region.The results of double luciferase reporter assay showed that the expression of luciferase in Luc-mTOR-3’UTR-wt was inhibited by miR-100,but it didn’t happen in Luc-mTOR-3’UTR-mut.It supported the prediction that mTOR was one of the target genes of miR-100.4.Upregulation of miR-100 could inhibit the expression of mTOR both mRNA and protein in Jeko-1 and Mino cells.PV-mTOR-up partially cancelled the effect of LV-miR-100-up on decreasing mTOR expression,inhibiting proliferation,inducing apoptosis and blocking cell cycle in G1 phase in Jeko-1 and Mino cells.Conclusions: There is an aberrant of miR-100 and mTOR in MCL,which includes downregulation in miR-100 and up-regulation in mTOR.The expression of mTOR is negatively correlated with miR-100.Up-regulating miR-100 or down-regulating mTOR could inhibit the cell proliferation,promote the cell apoptosis and block cell cycle in G1 phase.Aberrant of miR-100 and mTOR may play an important role in MCL pathogenesis.miR-100 targets at mTOR.It may be a potential gene for anti-mantle cell lymphoma.