Effect Of MicroRNA-155-5p-PTEN Regulatory Axis On Burkitt's Lymphoma And Its Mechanism

Objection: To investigate PTEN methylation and microRNA-in Burkitt Lymphomna(BL)cell line Raji and human normal B lymphocyte cell line(BCELL)by methylation-specific PCR(MSP)and RT-PCR.155-5P(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 PTEN genes and proteins in lentivirus knockdown Raji cells were investigated.The relationship between MiR-155-5p and PTEN gene and its mechanism were studied.To explore whether the MiR-155-5p/PTEN regulatory axis can be a candidate for BL therapy.Method: This topic is divided into three parts.The first part: 1.qRT-PCR to investigate the difference of MiR-155-5p mRNA expression in Raji cells and BCELL cells,and the Raji cells were infected with lentivirus(anti-mir-55-5p).2.qRT-PCR confirmed the expression of miR-155-5P mRNA in lentivirus before and after Raji cells.The effects of lentivirus on Raji cell proliferation and cell migration were detected by CCK8,colony formation assay and cell migration assay.The apoptosis and cycle of Raji were detected by flow cytometry.The second part: 1.qRT-PCR explored the expression levels of phosphatase and tension homolog(PTEN)in Raji and BCELL cells.2.Methylation PCR(MSP)and pyrosequencing(BSP)were used to investigate the methylation level of PTEN gene in Raji cells of Burkitt's lymphoma cell line.qq-PCR was used to investigate the differential expression of DNMT1,3A and 3B mRNAs in the Raji 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 Raji cells before and after Raji cells.3.qRT-PCR and Western-Blot investigated the effect of azacitidine on the mRNA and protein of PTEN gene before and after Raji 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 PTEN gene before and after MiR-155-5P by lentivirus.3.To construct wild-type and mutant plasmids of PTEN 3-UTR region and co-transfect the two plasmids with miR-155-5P mimic into 293 T cells,and investigate the mRNA of wild-type and mutant plasmid PTEN mRNA by qRT-PCR.Express differences.4.WB explores the protein kinase kinase B(AKT)and Phosphatidylinositide Protein kinase B(P-AKT)proteins involved in the PTEN/PI3K/AKT pathway before and after lentivirus action on Raji cells.The level changes.Results: 1.qRT-PCR results showed that Raji cells expressed higher miR-155-5P than BCELL cells.2.qRT-PCR results of lentivirus interference in miR-155-5P in Raji cells showed that the expression of miR-155-5p mRNA in Raji cell experimental group(SI-KD)was significantly lower than that in the control group(SI-NC).The CCK8 results showed that the Raji 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 Raji cells in the experimental group was significantly lower than that of the control group(p<0.05).The results of flow cytometry detection of Raji 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 Raji cells showed no obvious apoptosis in both groups(p>0.05).The results of qRT-PCR showed that the expression level of PTEN in Raji cells was lower than that of BCELL,and the results of MSP and BSP showed that the PTEN gene was highly methylated.4.qRT-PCR results showed that azacitidine could reduce the methylation level of Raji cells after Raji cells.Apoptosis results showed that azacitidine could promote Raji cell apoptosis after Raji cells,and CCK8 results showed that azacitidine could inhibit Raji cell growth after Raji cells.At the same time,qRT-PCR results showed that azacitidine could increase PTEN mRNA and protein levels and decrease Mir-155-5p expression.At the same time,it can promote the inhibition of apoptosis and proliferation of Raji cells.The qRT-PCR results of lentivirus infection showed that the PTEN mRNA in the Raji cell experimental group was higher than that in the control group(p<0.05).6.Luciferase reporter gene results showed that the relative activity of wild-type PTEN-miR-155-5P fluorescein was significantly lower than that of PTEN wild type,PTEN mutant and PTEN mutant-miR-155-5P(P<0.05=.There was no significant difference in PTEN wild type,PTEN mutant and PTEN mutant-miR-155-5P.5.WB results showed that the molecular weight of P-AKT protein in experimental group was lower than that in control group after lentivirus infection of Raji cells(p<0.05).However,there was no significant difference in the molecular weight of the two AKT proteins(p>0.05).Conclusion: 1.The PTEN gene of Raji cells is hypermethylated and highly expressed miR-155-5P.2.Lentivirus-infected BL cell line Raji cell miR-155-5P can inhibit cell proliferation,decrease cell migration ability,and block cell cycle progression,but has no effect on apoptosis,and promotes PTEN gene expression..3.The methyltransferase inhibitor azacitidine can reverse the PTEN methylation level of Raji cells,promote the inhibition of Raji cell growth and promote its apoptosis,and promote the increase of PTEN gene expression level.4.Azacitidine can improve the expression of miR-155-5P and miR-155-5p can positive regulate the expression of PTEN gene through PI3K/AKT pathway.