Study On Function And Mechanism Of The M6A Demethylase ALKBH5 In The Regulation Of Pancreatic Cancer Cell Proliferation And Migration/invasion

Objective: To explore the expression of ALKBH5 in pancreatic cancer and corresponded normal pancreas.To identify the correlation between ALKBH5 expression and clinical indicators in pancreatic cancer.Methods:(1)Immunohistochemical was performed to detect the ALKBH5 protein expressing level in a 180-site pancreatic cancer tissue chip,and analysis was carried out to detected the relationship between ALKBH5 protein expressing level and pathological grades,clinical stages and disease prognosis.(2)The data of 183 cases pancreatic cancer was downloaded from TCGA data bank.The analysis was carried out to detect the relationship between ALKBH5 expressing level and pathological grades,clinical stages and disease prognosis.(3)RT-PCR and WB were used to explore the m RNA/protein expression of ALKBH5 in fresh pancreatic cancer tissues/normal pancreas tissues.Results: The immunohistochemical results showed that about 62.2%(56/90)pancreatic cancer cases had lower ALKBH5 expression in tumor tissues,about 22.2%(20/90)cases had equivalent ALKBH5 level in tumor and normal pancreas tissues,and about only 15.6%(14/90)cases had higher ALKBH5 expression in tumor tissues.The statistical results showed that ALKBH5 expressing level was higher in normal tissues than in tumor tissue(p<0.001),and lower in cases with mutation of KRAS,CDKN2 A,TP53 or SMAD4 than in cases without mutation.RT-PCR and WB revealed that tumor tissues had higher ALKBH5 m RNA/protein expression.(2)Cases with worse pathological grades or clinical stages had lower ALKBH5 expression.In the column of tissue chip,the high ALKBH5 expressing group(N=46)possessed medium survival of 25.5 months,five-year survival rate of 40.5%,and the low ALKBH5 expressing group(N=44)possessed medium survivalof 11 mouths,five-year survival rate of 11.3%.The high ALKBH5 expressing group had longer survival than the low ALKBH5 expressing group(p<0.05).In the column of TCGA,the high ALKBH5 expressing group(N=95)possessed medium survival of 23 months,five-year survival rate of 42.5%,and the low ALKBH5 expressing group(N=87)possessed medium survival of 18 mouths,five-year survival rate of 15.7%,The high ALKBH5 expressing group had longer survival(p<0.01).Conclusions: ALKBH5 was downregulated in pancreatic cancer.The ALKBH5 level was negative correlated with the clinical outcomes.Additionally,pancreatic cancer tissues with the mutations of KRAS、CDKN2A、TP53 or SMAD4 had lower ALKBH5 expression than these without mutations,which indicated that downregulation of ALKBH5 play a great role in pancreatic cancer tumorigenesis.Objective:To investigate the effect of m6 A demethylase ALKBH5 on the proliferation and invasion/metastasis of pancreatic cancerMethods:(1)The si RNA interference technology,Crispr/cas9 technology and lentiviral delivery system were used to change the ALKBH5 expression in pancreatic cancer cells.(2)The m6 A level in tumor tissues/normal pancreas tissues and cell lines was detected via m6 A RNA Methylation Quantification Kit.(3)The effects of ALKBH5 on the proliferation,invasion/metastasis of pancreatic cancer cells were tested in vitro using CCK-8 test,colony formation assay,wound healing assay,and transwell assay.(4)Subcutaneous transplanted model and orthotopic transplanted model in balb/c nu mice were performed to investigate the effects of ALKBH5 on the proliferation,invasion/metastasis of pancreatic cancer cells.Results:(1)The m6 A level was higher in tumor tissues than in normal pancreas tissues.Compared with blank cells,the m6 A level was higher in ALKBH5 down-regulated cells,was lower in ALKBH5(wild)upregulated cells and did not change in ALKBH5(H204A)up-regulated cells.(2)The proliferation and migration/invasion increased in ALKBH5 down-regulated cells,reduced in ALKBH5(wild)upregulated cells,and kept the same level in ALKBH5(H204A)up-regulated cells.(3)ALKBH5 knockout cells gained upregulated proliferation and migration/invasion ability in vitro and in vivo.Conclusions:ALKBH5 was a tumor suppressor gene.ALKBH5 inhibited proliferation and migration/invasion,dependent on its demethylase capacity.Objective:To explore the upstream/downstream mechanism of ALKBH5Methods:(1)The correlation between TP53 and ALKBH5 was analyzed on websites of Oncomine,Gepia and c Bio Portal.After changing the TP53 expression with si RNA,wild/mutant overexpression vector or crispr/cas9 system,the ALKBH5 expression was tested by WB.Moreover,ALKBH5 promoter luciference reporter system was used to investigate the relationship between TP53 and ALKBH5 transcription.(2)After down-regulating ALKBH5 in Panc-1,the Me RIP-sequence and m RNA expression profiling sequence were performed.Than the RT-PCR test was carried to verify the results.(3)WB,RT-PCR,APC m RNA 3’-UTR dual luciferase reporter assay and Top Flash reporter system were used to detect the effect of ALKBH5 on APC/β-catenin/wnt pathway.After upregulating APC,the proliferation and migration/invasion were investigated in vitro by CCK-8 test,wound healing assay and transwell assay.Results:(1)ALKBH5 expression was positive corrected with wild type TP53 expression.It was wild type TP53,but not mutant type TP53,promoted ALKBH5 transcription,(2)The Me RIP-sequence identified 47647 peaks,associated with 8147 genes in NC group,and 49134 peaks,associated with 8434 genes in si ALKBH5 group.The NC group had 6702 unique peaks,associated 2631 genes.The si ALKBH5 group had 8192 unique peaks,associated 3127 genes.The m RNA expression profiling sequence showed that 1361mRNA changed significantly in si ALKBH5 group,associated with 535 m RNA increaseed and 826 m RNA reduced.Seven candidate genes,RHOBTB2,TAF1,KSR1,APC,CDKN1 A,WT1 and HSF4,were chose.APC were identified finally after RT-PCR assay.(3)The APC/β-catenin/wnt pathway changed significantly.Downregulation of ALKBH5 suppressed APC m RNA/protein expression,promoted β-catenin nuclear location and TCF4/LEF transcription ability.Upregulation of ALKBH5 promote APC m RNA/protein expression,inhibited β-catenin nuclear location and TCF4/LEF transcription ability.Upregulation of mutant ALKBH5 had no effect on APC/β-catenin/wnt pathway(4)Overexpression of APC could rescue the promotion on proliferation and migration/invasion caused by low ALKBH5 expression.Conclusions:ALKBH5 was down-regulated in pancreatic cancer,cause the general mutant type Tp53 loss the function of promoting ALKBH5 transcription.APC/β-catenin/wnt pathway disorder was the crucial mechanism of that low ALKBH5 expression promoted proliferation and migration/invasion of pancreatic cancer cells.Treatment targeting ALKBH5/APC/β-catenin/wnt pathway should be a new strategy for pancreatic cancer therapy...