| Objective: T-cell acute lymphoblastic leukemia(T-ALL)is a malignancy of the blood system characterised by abnormal proliferation of malignant primary Tlymphocytes in the bone marrow and peripheral blood.Compared to B-cell acute lymphoblastic leukemia(B-ALL),T-ALL has a worse prognosis,is more likely to fail chemotherapy and has a higher relapse rate.T-ALL relapse followed by chemotherapy is poorly treated,with less than 7% of relapsed patients surviving long-term.Therefore,there is an urgent need to investigate the pathogenesis of T-ALL and to explore new treatments.For T-ALL which occurs as a result of the gradual accumulation of a series of genetic aberrations.Recent studies have shown that non-coding RNAs(non-coding RNAs,nc RNAs)play an important role in the development of T-ALL,with lnc RNAs(long non-coding RNAs,nc RNAs)and micro RNAs playing a significant role.Lnc RNA PCAT18 is highly expressed in a variety of cancers and promotes tumour progression,while its expression is down-regulated in gastric cancer and triple-negative breast cancer,acting as a tumour suppressor.Previous experiments have confirmed that PCAT18 and BCL2 are significantly highly expressed in T-ALL,and their detailed mechanism of action remains unclear.One study reported that mi R-107 expression was reduced in the peripheral blood of T-ALL patients compared to normal people,and the mechanism was unknown.We investigated the mechanism by which PCAT18 affects the development of T-ALL by verifying the relationship between mi R-107 and PCAT18 and BCL2 expression.Methods:(1)The expression of PCAT18 in the bone marrow of T-ALL patients and normal subjects was measured by real-time fluorescent quantitative PCR.Correlation analysis between PCAT18 gene expression and clinical tests in T-ALL patients.(2)Prognostic analysis between PCAT18 gene expression and patient survival.(3)The effect of transfection with sh PCAT18 on cell function was investigated by apoptosis and cell proliferation assays.m RNA and protein expression levels of apoptosis-related genes were detected by q RT-PCR and WB.(4)Application of star Base,Targetscan database predicted that mi R107 may have binding sites with PCAT18 and BCL2.The binding sites of mi R-107 and PCAT18 and BCL2 were verified by dual luciferase reporter assay;After silencing PCAT18 and overexpressing mi R-107,the regulatory relationship was verified by q RT-PCR and WB experiments.(5)The expression of mi R-107,BCL2 and endogenous and exogenous apoptotic pathway-related indicators in T-ALL patients were detected by q RT-PCR and WB.(6)Firstly,PCAT18 was knocked down in the cell lines,and the expression of mi R-107,BCL2,endogenous and exogenous apoptotic pathway-related indicators and apoptosis levels were detected.Then the cell lines were transfected with mi R-107 inhibitor,and the expression of mi R-107,BCL2,endogenous and exogenous apoptotic pathway-related indicators and apoptosis levels were detected after silencing mi R-107.Results:(1)In bone marrow specimens,PCAT18 expression was significantly higher in T-ALL patients(p <0.05).Peripheral blood leukocyte count,and bone marrow primitive cell ratio in T-ALL patients were positively correlated with PCAT18expression(p <0.05).(2)T-ALL patients with high levels of PCAT18 expression had a worse prognosis(p <0.05).(3)Transfection of the PCAT18 silencing plasmid decreased PCAT18 expression compared to the transfected empty vector group(p <0.05),and knockdown of PCAT18 increased apoptosis levels in both Jurkat E6-1,CCRF-CEM cell lines(p <0.05).(4)The prediction results showed that mi R-107 had a complementary binding sequence to PCAT18 and mi R-107 had a complementary binding sequence to BCL2.PCAT18-WT luciferase reporter gene vector and PCAT18-Mut luciferase reporter gene vector were cotransfected with mi R-107 mimics and luciferase activity was measured by luciferase assay after 48 h incubation.When transfection with mi R-107 mimics resulted in high expression of mi R-107,the luciferase activity of the PCAT18-WT reporter gene was significantly reduced(p <0.05).BCL2-WT luciferase reporter gene vector and BCL2-Mut luciferase reporter gene vector were cotransfected with mi R-107 mimics and luciferase activity was measured by luciferase assay after 48 h incubation.When transfection with mi R-107 mimics resulted in high expression of mi R-107,the luciferase activity of the BCL2-WT reporter gene was significantly reduced(p <0.05).When PCAT18 was silenced,mi R-107 expression levels in cells increased(p <0.05),When mi R-107 was overexpressed,both BCL2 gene and protein expression levels were increased(p <0.05).The presence of a binding site between mi R-107 PCAT18,BCL2 was demonstrated.(5)In T-ALL patient specimens,mi R-107 expression levels were significantly lower(p<0.05),while BCL2 expression levels were significantly higher(p <0.05)and the BCL2/BAX ratio was increased(p <0.05)compared to normal subjects.Endogenous apoptotic protein BCL2 was significantly highly expressed(p <0.05),BAX expression levels did not change significantly,and exogenous apoptotic pathway proteins Fas L and caspase8 expression did not change significantly.(6)Compared with the control group,knockdown of PCAT18 decreased PCAT18 expression levels(p <0.05),increased mi R107 expression levels(p <0.05),decreased m RNA and protein levels of BCL2,and increased apoptosis levels(p <0.05)in both Jurkat E6-1,CCRF-CEM cell lines,while BAX,Fas L and caspase8 expression levels were not significantly changed;compared to PCAT18 knockdown alone,PCAT18 knockdown with mi R107 inhibitor resulted in no change in PCAT18 levels,decreased mi R107 expression(p <0.05),increased m RNA and protein levels in BCL2(p <0.05),and decreased cell apoptosis levels decreased(p <0.05),and m RNA and protein expression levels of BAX,Fas L and caspase8 did not change significantly.Conclusions: PCAT18 reduced the binding of mi R-107 to BCL through targeted competitive binding of mi R-107,alleviated the inhibitory effect of mi R-107 on BCL2,indirectly increased the expression level of BCL2 and inhibited apoptosis in T-ALL cells. |
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