Effect Of MicroRNA-155-5p/TAOK1 Regulatory Axis On Acute Lymphoblastic Leukemia And Its Mechanism

Objection: To investigate TAOK1 methylation and micro RNA in Acute Lymphoblastic Leukemia(ALL)cell line Nalm6 and human normal B lymphocyte cell line(BCELL)by methylation-specific PCR(MSP)and RT-PCR.MiR-155-5p expression level.At the same time,the effects of MiR-155-5p and methyltransferase inhibitors on the biology of tumor cells and the TAOK1 genes and proteins in lentivirus knockdown Nalm6 cells were investigated.The relationship between MiR-155-5p and TAOK1 gene and its mechanism were studied.To explore whether the MiR-155-5p/TAOK1 regulatory axis can be a candidate for ALL therapy.Method: This topic is divided into three parts.The first part:1.qRT-PCR to investigate the difference of MiR-155-5p m RNA expression in Nalm6 cells and BCELL cells,and the Nalm6 cells were infected with lentivirus(anti-mir-55-5p).2.qRT-PCR confirmed the expression of mi R-155-5P m RNA in lentivirus before and after Nalm6 cells.The effects of lentivirus on Nalm6 cell proliferation and cell migration were detected by CCK8,colony formation assay and cell migration assay.The apoptosis and cycle of Nalm6 were detected by flow cytometry.The second part:1.qRT-PCR explored the expression levels of phosphatase and tension homolog(TAOK1)in Nalm6 and BCELL cells.2.Methylation PCR(MSP)and pyrosequencing(BSP)were used to investigate the methylation level of TAOK1 gene in Nalm6 cells of Acute Lymphoblastic cell line.qq-PCR was used to investigate the differential expression of DNMT1,3A and 3B m RNAs in the Nalm6 cells before and after treatment with the methyltransferase inhibitor azacitidine.Flow cytometry and CCK8 were used to investigate the effect of methylation enzyme inhibitor azacitidine on the apoptosis and proliferation of Nalm6 cells before and after Nalm6 cells.3.qRT-PCR and Western-Blot investigated the effect of azacitidine on the m RNA and protein of TAOK1 gene before and after Nalm6 cells.The third part: 1.qRT-PCR to explore the expression level of MiR-155-5P before and after azacitidine treatment.2.qRT-PCR explored the expression of TAOK1 gene before and after MiR-155-5P by lentivirus.3.Investigate the m RNA of wild-type and mutant plasmid TAOK1 m RNA by qRT-PCR.Express differences.4.WB explores the protein kinase(MST1/2)involved in the Hippo pathway before and after lentivirus action on Nalm6 cells.The level changes.Results: 1.qRT-PCR results showed that Nalm6 cells expressed higher mi R-155-5P than BCELL cells.2.qRT-PCR results of lentivirus interference in mi R-155-5P in Nalm6 cells showed that the expression of mi R-155-5p m RNA in Nalm6 cell experimental group(SI-KD)was significantly lower than that in the control group(SI-NC).The CCK8 results showed that the Nalm6 cell experimental group began to undergo proliferation inhibition at 48 h(p<0.05).The results of colony formation experiments showed that the number of Nalm6 cells in the experimental group was significantly lower than that of the control group(p<0.05).The results of flow cytometry detection of Nalm6 cell cycle showed that there was no significant change in cell ratio of G0/G1 phase in the two groups,but the ratio of G2/M phase cells in the experimental group was significantly higher than that in the control group(p<0.05).Flow cytometry showed that apoptosis of Nalm6 cells showed no obvious apoptosis in both groups(p>0.05).The results of qRT-PCR showed that the expression level of TAOK1 in Nalm6 cells was lower than that of BCELL,and the results of MSP and BSP showed that the TAOK1 gene was highly methylated.4.qRT-PCR results showed that azacitidine could reduce the methylation level of Nalm6 cells after Nalm6 cells.Apoptosis results showed that azacitidine could promote Nalm6 cell apoptosis after Nalm6 cells,and CCK8 results showed that azacitidine could inhibit Nalm6 cell growth after Nalm6 cells.At the same time,qRT-PCR results showed that azacitidine could increase TAOK1 m RNA and protein levels and decrease Mir-155-5p expression.At the same time,it can promote the inhibition of apoptosis and proliferation of Nalm6 cells.The qRT-PCR results of lentivirus infection showed that the TAOK1 m RNA in the Nalm6 cell experimental group was higher than that in the control group(p<0.05).5.WB results showed that the molecular weight of MST1 protein in experimental group was lower than that in control group after lentivirus infection of Nalm6 cells(p<0.05).However,there was no significant difference in the molecular weight of the two MST proteins(p>0.05).Conclusion: 1.The TAOK1 gene of Nalm6 cells is hypermethylated and highly expressed mi R-155-5P.2.Lentivirus-infected BL cell line Nalm6 cell mi R-155-5P can inhibit cell proliferation,decrease cell migration ability,and block cell cycle progression,but has no effect on apoptosis,and promotes TAOK1 gene expression.3.The methyltransferase inhibitor azacitidine can reverse the TAOK1 methylation level of Nalm6 cells,promote the inhibition of Nalm6 cell growth and promote its apoptosis,and promote the increase of TAOK1 gene expression level.4.Azacitidine can decrease the expression of mi R-155-5P and mi R-155-5p can negatively regulate the expression of TAOK1 gene through Hippo pathway.