Inhibitory Effect Of Valproic Acid On Proliferation Of Peripheral T Cell Lymphoma Cells Through MiR-3196 And Its Mechanisms

Background and objective:Peripheral T-cell lymphoma(PTCL)is an invasive disease,which is defined as a group of heterogeneous lymphoproliferative diseases caused by mature T cells or NK cells.At present,the survival time of the patients is short and the treatment progress is slow.Although the clinical efficacy of CD30 monoclonal antibody and PI3 K inhibitor has been achieved recently,the therapeutic effect is not satisfactory.Therefore,to deeply understand the molecular pathogenesis of PTCL,optimize its therapeutic means and develop therapeutic drugs are currently the key clinical topics to be solved urgently.Micro RNAs(miRNAs)are a class of small non-coding Rnas that can cause miRNA degradation or translation inhibition at the post-transcriptional level,thereby regulating the expression of downstream genes.It has been suggested that the abnormal expression of miRNAs is involved in the progression of PTCL,affects the survival of patients,and can be used as potential diagnostic and therapeutic targets for PTCL.miR-3196 acts as a tumor suppressor in a variety of tumors,inhibiting the progression of tumors,but the role of miR-3196 in PTCL remains unclear.Valproic acid(VPA)is a histone deacetylase inhibitor that has been proposed for the treatment of NHL.However,studies on the inhibitory effect of VPA in lymphoma and its mechanism are limited.This study aims to clarify the effect of valproic acid on PTCL cell proliferation and its relationship with miR-3196 expression.Part 1 The effect of VPA on the proliferation of peripheral T-cell lymphoma cells was mediated by miR-3196Objective: To investigate the effect of VPA on the proliferation of PTCL and its correlation with miR-3196 expression.Methods: 1.The effect of VPA on the proliferation of Hut 78 and A3 cells was detected by CCK8 assay.2.q RT-PCR was used to detect the expression of miR-3196 in Hut 78 and A3 cells after VPA intervention.3.miR-3196 mimics,miR-3196 inhibitor and miR-3196 NC were transfected into Hut 78 and A3 cells by liposome.after 24 hours of VPA intervention,cell proliferation was detected by CCK-8;Annexin V-FITC/PI double staining was used to detect cell apoptosis.The expressions of apoptosis-related proteins(Bad,Bcl-2,Cleaved Caspase-3)and PI3K/Akt pathway proteins were detected by Western blot.The changes of ATP,lactate and glucose uptake were detected by biochemical kits.ROS levels were measured by flow cytometry.Results:1.VPA treatment(0 m M,0.5 m M,1 m M,2 m M,4 m M,8 m M,16 m M,32 m M)significantly inhibited the proliferation rate of Hut 78 and A3 cells(P<0.01).2.VPA treatment(8 m M,16 m M,32 m M)significantly increased the expression of miR-3196 in Hut 78 and A3 cells,especially in 16 m M VPA treatment(P<0.01).Compared with the Control group,VPA and miR-3196 mimics significantly inhibited the proliferation of Hut 78 and A3 cells(P<0.01),Hut 78 and A3 apoptosis(P<0.01),and promoted the expression of Bad and Cleaved caspase-3 protein(P<0.01),inhibited the expression of Bcl-2 protein(P<0.01);However,miR-3196 inhibitor significantly promoted the proliferation of Hut 78 and A3 cells(P<0.01),inhibited cell apoptosis(P < 0.01),and significantly inhibited the expression of Bad and Cleaved caspase-3 protein(P<0.01),enhanced Bcl-2 protein expression(P<0.01);When combined with miR-3196 mimics,VPA could further promote cell apoptosis(P<0.01),and promoted the expression of Bad and Cleaved caspase-3 protein(P<0.01),inhibited the expression of Bcl-2 protein(P<0.01).4.Compared with the Control group,VPA and miR-3196 mimics significantly inhibited glucose uptake(P<0.01),ROS content(P<0.01),ATP content(P<0.01),increased lactic acid content(P<0.01);miR-3196 inhibitor significantly inhibited ROS and lactate(P<0.01),increased ATP content(P<0.01);Combined with miR-3196 mimics,VPA could further promote ROS content,lactate and glucose(P<0.01),inhibited ATP content(P<0.01).5.Compared with the Control group,the expression of p-PI3 K and p-AKT in Hut 78 and A3 cells was decreased in VPA group and miR-3196 mimics group(P<0.01).miR-3196 inhibitor significantly promoted the expression of p-PI3 K and p-AKT in Hut 78 and A3 cells(P<0.01);Combined with miR-3196 mimics,VPA further inhibited the expression of p-PI3 K and p-AKT in Hut 78 and A3 cells(P<0.01).Conclusions:1.VPA could significantly promote the expression of tumor suppressor miR-3196 in PTCL cells;2.VPA can promote the apoptosis of peripheral T cell lymphoma cells,inhibit cell proliferation and affect mitochondrial function through miR-3196.3.VPA inhibited the activation of PI3K/Akt pathway through miR-3196 in PTCL cells.Part 2 Study the inhibitory effect of VPA on the proliferation of peripheral T-cell lymphoma cells through the regulation of miR-3196 on KCNK3Objective: To investigate the correlation between the inhibition of PTCL cell proliferation by VPA via miR-3196 and the expression of KCNK3.Methods: 1.The data set in GEO(GSE132550)and online software such as miRTar Base and starbase2.0 were used to analyze the potential target genes of miR-3196,and the relationship between the target genes was verified by dual luciferase reporter gene vector.2.q RT-PCR and Western blot were used to detect the effect of VPA on KCNK3 expression in Hut 78 and A3 cells.3.Hut 78 and A3 cells were transfected with miR-3196 inhibitor and si-KCNK3 by lipofectamine.After 24 hours of VPA intervention,Ed U was used to detect the changes in cell proliferation.Annexin V-FITC/PI double staining was used to detect cell apoptosis.The expressions of apoptosis-related proteins(Bad,Bcl-2,Cleaved Caspase-3)and PI3K/Akt pathway proteins were detected by Western blot.Results:1.Through the analysis of GEO dataset(GSE132550)and online software such as miRTar Base and starbase2.0,it was found that KCNK3 and other genes were potential target genes of miR-3196.In GSE132550 dataset,KCNK3 log FC=7.19 and P<0.05.q RT-PCR and Western blot analysis showed that VPA treatment significantly inhibited the expression of KCNK3 in Hut 78 and A3 cells(P<0.01);miR-3196 inhibitor could significantly promote the expression of KCNK3 in Hut 78 and A3 cells(P < 0.01);VPA treatment significantly inhibited the expression of KCNK3 in Hut 78 and A3 cells(P<0.01).Luciferase assay showed that there was a target gene relationship between miR-3196 and KCNK3.2.Ed U assay showed that VPA treatment significantly inhibited the proliferation of Hut 78 and A3 cells(P < 0.01).Inhibition of miR-3196 expression significantly promoted Hut 78 and A3 cell proliferation(P<0.01);Compared with inhibitor group,the cell proliferation of VPA+inhibitor group was significantly decreased(P<0.01);Compared with the inhibitor group,si-KCNK3+inhibitor significantly inhibited cell proliferation(P < 0.01)compared with the si-KCNK3+inhibitor group,the cell proliferation of the VPA combined group was significantly decreased(P<0.01).3.Annexin V-FITC/PI double staining analysis showed that VPA treatment could significantly promote the apoptosis of Hut 78 and A3 cells(P<0.01).Inhibition of miR-3196 expression significantly inhibited cell apoptosis(P<0.01);Compared with inhibitor group,the level of apoptosis in VPA+inhibitor group was significantly increased(P<0.01);Compared with the inhibitor group,si-KCNK3+inhibitor could significantly promote cell apoptosis(P < 0.01);Compared with the siKCNK3+inhibitor group,the apoptosis of cells in the VPA combined group was significantly increased(P<0.01).4.VPA could significantly increase the expression of Bad and Cleaved caspase-3 protein in Hut 78 and A3 cells(P<0.01),inhibited the expression of Bcl-2 protein(P<0.01),while miR-3196 inhibitor significantly inhibited the expression of Bad and Cleaved caspase-3 proteins(P<0.01),enhanced Bcl-2 protein expression(P<0.01);Compared with inhibitor group,VPA+inhibitor significantly increased the expression of Bad and Cleaved caspase-3(P<0.01),inhibited the expression of Bcl-2 protein(P<0.01);Compared with the inhibitor group,the expression of Bad and Cleaved caspase-3 protein in the VPA combined group was significantly increased(P<0.01),and the expression of Bcl-2 protein was significantly decreased(P<0.01).5.VPA could significantly inhibit the expression of p-PI3 K and p-Akt proteins in Hut 78 and A3 cells(P<0.01),while miR-3196 inhibitor could significantly promote the expression of p-PI3 K and p-Akt proteins(P<0.01);Compared with inhibitor group,VPA+inhibitor significantly inhibited the protein expression of p-PI3 K and p-Akt(P<0.01);Compared with the inhibitor group,the si-KCNK3+inhibitor group could significantly inhibit the protein expression of p-PI3 K and p-Akt(P<0.01);Compared with si-KCNK3+inhibitor group,the expression of p-PI3 K and p-Akt protein was inhibited(P<0.01).Conclusions:1.KCNK3 is a target gene of miR-3196 in PTCL.2.VPA can inhibit the expression of KCNK3 through miR-3196,promote the apoptosis and inhibit the proliferation of peripheral T cell lymphoma cells.3.VPA inhibited the activation of PI3K/Akt pathway in PTCL cells after inhibiting the expression of KCNK3 through miR-3196.