| Background: Pancreatic cancer,also known as the "king of cancers," is a type of digestive system tumor with an extremely high malignant degree and a poor prognosis.The morbidity and mortality rates are increasing globally.The onset of pancreatic cancer is insidious,and there is no sensitive early detection method.Most pancreatic cancer patients have already lost the opportunity for surgery at the time of diagnosis,and only about 20% of patients are candidates for radical surgery.However,the postoperative recurrence rate of patients undergoing radical surgery remains high,with a poor prognosis.In addition to surgery,radiotherapy and chemotherapy are frequently used to treat pancreatic cancer at different stages.However,due to acquired drug resistance and insensitivity to radiotherapy,the curative effect is quite limited even after standard radiotherapy and chemotherapy,and the overall survival time of patients has not been significantly improved.Immunotherapy,targeted therapy,and other new therapeutic methods are also not ideal in pancreatic cancer.As a result,indepth investigation of the molecular mechanisms underlying the occurrence and progression of pancreatic cancer,as well as the search for sensitive tumor markers,effective therapeutic targets,and drug-resistant solutions,are critical for achieving early diagnosis,early treatment,delayed recurrence,and improved prognosis of pancreatic cancer.Initially,CDK18 was thought to be a neuronal kinase that phosphorylates the microtubule-associated protein TAU.Now CDK18 has been discovered to affect the life activities of cells in a variety of ways as research has progressed.For example,CDK18 binds to cyclin A2/E,which acts as a cycle-dependent kinase.CDK18 can promote replication of stress signals and genomic stability.CDK18 also regulates cell migration and adhesion by negatively regulating the activity of local adhesion plaque kinase.Furthermore,studies have shown that CDK18 is abnormally expressed in some malignant tumors and influences tumor progression,implying that CDK18 may be a driving factor in cancer.But the role of CDK18 in pancreatic cancer remains unknown.We intend to investigate the effects of CDK18 on the malignant phenotype and gemcitabine chemotherapy sensitivity of pancreatic cancer cells,and further clarify the mechanism of CDK18 in pancreatic cancer to provide a theoretical foundation for the discovery of new oncogenes and new effective pancreatic cancer targets.Methods: 1 To detect the differentially expressed genes in gemcitabine resistance PDX and control PDX.High-throughput sequencing was used to examine the differentially expressed genes in two PDX models.The expression of CDK18 in two groups of PDX models was verified using q RT-PCR,WB,and immunohistochemistry.2 To detect the expression of CDK18 in pancreatic cancer tissues and normal tissues.q RT-PCR,WB,and immunohistochemistry were used to detect CDK18 expression in pancreatic cancer tissues and adjacent normal tissues.3 To detect the expression of CDK18 in pancreatic cancer cells and normal pancreatic ductal epithelial cells.q RT-PCR and WB were used to detect CDK18 m RNA and protein expression in pancreatic cancer cells(As PC-1,Capan-1,Bx PC-3,CFPAC-1,PANC-1,MIA Paca-2,Capan-2)and normal pancreatic ductal epithelial cells(HPDE6-C7).Pancreatic cancer cell lines were screened for experimental use.4 Correlation analysis of CDK18 expression with the clinicopathological parameters and the survival of pancreatic cancer patients.To evaluate the correlation between CDK18 expression level and 46 pancreatic cancer patients’ age,gender,drinking history,smoking history,CEA,CA19-9,tumor size,TNM stage,lymph node invasion and distant metastasis.The connection between the expression level of CDK18 and the survival of pancreatic cancer patients was examined using the Kaplan-Meier method.5 To investigate the effects of CDK18 on pancreatic cancer cell proliferation,stemness,and cell cycle.To investigate the effect of CDK18 differential expression on pancreatic cancer cell proliferation,stemness,and cell cycle in vitro using CCK-8,spheroid formation assay,and cell cycle assay.In addition,q RT-PCR and WB were used to detect the expression of stemness-related genes(OCT4,NANOG,Sox2,CD133)in pancreatic cancer cells that expressed CDK18 differentially.In vivo,the effect of differential expression of CDK18 on tumor growth was analyzed by tumor formation assay in nude mice,and the expression of proliferation marker(Ki-67)and stemness marker(CD133)in pancreatic cancer xenograft tumor with differential expression of CDK18 was further detected by immunohistochemistry.6 To evaluate the effect of CDK18 on chemosensitivity to gemcitabine in pancreatic cancer.In vitro,to investigate the effect of CDK18 differential expression on pancreatic cancer chemosensitivity to gemcitabine using the colony formation assay and the CCK-8 assay.In vivo,the effect of differential CDK18 expression on pancreatic cancer chemosensitivity to gemcitabine was studied in tumor formation assay in nude mice.7 To investigate the effect of ALKBH5 on CDK18 stability.Me RIP-q PCR was used to detect CDK18 m6 A enrichment in pancreatic cancer cells(As PC-1,Capan-2)and normal pancreatic ductal epithelial cells(HPDE6-C7),and q RT-PCR was used to investigate the relationship between ALKBH5 and CDK18 in 46 pancreatic cancer specimens.q RT-PCR,WB,and Me RIP-q PCR were used to investigate the effects of ALKBH5 on CDK18 expression and m6 A enrichment in pancreatic cancer cells.The effect of ALKBH5 on CDK18 stability was investigated using the actinomycin D assay.8 To detect the effect of CDK18 on signaling pathways in pancreatic cancer.High-throughput sequencing was used to investigate the downstream signaling pathway regulated by CDK18.The expression of target pathway-related proteins in CDK18 differentially expressed pancreatic cancer cells was detected by WB.Results: 1 CDK18 expression was significantly higher in the gemcitabine-treated group than in the saline-treated group in the PDX model.High-throughput sequencing revealed a difference in CDK18 expression between the gemcitabine group and the saline group in the PDX models.Then,the expression of CDK18 in the gemcitabine group and the saline group was detected by q RT-PCR,WB and immunohistochemistry,and it was found that the expression of CDK18 in the gemcitabine group was significantly higher than that in the saline group.2 CDK18 expression was significantly higher in pancreatic cancer tissues than in adjacent normal tissues.q RT-PCR,WB,and immunohistochemistry were used to detect CDK18 expression in pancreatic cancer tissues and normal tissues,and it was found that CDK18 expression in pancreatic cancer tissues was significantly higher than that in normal tissues.3 The expression of CDK18 in pancreatic cancer cells was significantly higher than that in normal pancreatic ductal epithelial cells.q RT-PCR and WB were used to detect CDK18 expression in pancreatic cancer cell lines and normal pancreatic ductal epithelial cell lines.CDK18 expression was significantly higher in pancreatic cancer cell lines(As PC-1,Capan-1,Bx PC-3,CFPAC-1,PANC-1,MIA Paca-2,Capan-2)than in normal pancreatic ductal epithelial cell line(HPDE6-C7).Among the seven pancreatic cancer cell lines studied,As PC-1 and CFPAC-1 cells had the lowest CDK18 expression,while Bx PC-3 and Capan-2 cells had the highest.These four cell lines were chosen as the experimental cell lines.4 CDK18 expression was positively correlated with tumor size,TNM stage,lymph node invasion and distant metastasis of patients with pancreatic cancer,and negatively correlated with the survival of patients with pancreatic cancer.The CDK18 expression was found to be positively correlated with tumor size,TNM stage,lymph node invasion,and distant metastasis in pancreatic cancer patients after analyzing the CDK18 expression and clinicopathological parameters of 46 pancreatic cancer patients.The Kaplan-Meier method was used to analyze the relationship between CDK18 expression and pancreatic cancer patient survival,and it was found that high CDK18 expression was associated with poor pancreatic cancer patient survival.5 CDK18 promoted pancreatic cancer cell proliferation,stemness,and cell cycle progression.The effects of differential CDK18 expression on pancreatic cancer cell stemness,proliferation,and cell cycle were studied in vitro using the CCK-8 assay,spheroid formation assay,and cell cycle assay,and it was found that CDK18 could promote pancreatic cancer cell proliferation,stemness,and cell cycle progression.Furthermore,q RT-PCR and WB were used to detect the effect of differentially expressed CDK18 on stemness gene expression in pancreatic cancer cells,and it was found that CDK18 could promote the expression of stemness-related genes(OCT4,NANOG,Sox2,CD133)in pancreatic cancer cells.The effect of differential CDK18 expression on tumor growth was studied in vivo using tumor formation assay in nude mice,and it was found that CDK18 could promote tumor growth.Furthermore,immunohistochemistry was used to detect the effect of CDK18 differential expression on the expression of proliferation and stemness markers in transplanted tumors.CDK18 was discovered to promote the expression of the proliferation marker(Ki-67)as well as the stemness marker(CD133).6 CDK18 reduced pancreatic cancer chemosensitivity to gemcitabine.The effects of CDK18 differential expression pancreatic cancer cells on chemosensitivity to gemcitabine were studied in vitro and in vivo using colony formation assay,CCK-8 assay,and tumor formation assay in nude mice,and it was found that CDK18 reduced pancreatic cancer chemosensitivity to gemcitabine.7 ALKBH5 reduced CDK18 stability.The m6 A enrichment of CDK18 in pancreatic cancer cells and normal pancreatic ductal epithelial cells were detected using Me RIP-q PCR.CDK18 m6 A enrichment was found to be significantly higher in pancreatic cancer cells(As PC-1,Capan-2)than in normal pancreatic ductal epithelial cells(HPDE6-C7).The correlation between ALKBH5 and CDK18 expression was studied using q RT-PCR in 46 pancreatic cancer specimens,and it was found that there was a significant negative correlation between ALKBH5 and CDK18 expression.The effects of ALKBH5 on CDK18 expression and m6 A enrichment in pancreatic cancer cells were studied using q RT-PCR,WB,and Me RIP-q PCR,and it was found that ALKBH5 could reduce CDK18 expression and m6 A enrichment.The effect of ALKBH5 on the stability of CDK18 was investigated using the actinomycin D assay,and it was found that ALKBH5 could reduce CDK18 stability.8 CDK18 affected the biological function of pancreatic cancer cells through the Hippo signaling pathway.The differentially expressed genes were clearly enriched in the Hippo pathway according to high-throughput sequencing analysis.To determine whether CDK18 was involved in the regulation of the Hippo signaling pathway,WB was used to detect Hippo signaling pathway-related protein expression in pancreatic cancer cells with differential expression of CDK18.Overexpression of CDK18 was found to significantly down-regulate the related protein p-LATS2 and p-YAP expression of the Hippo signaling pathway,whereas inhibition of CDK18 had the opposite effect.It was also demonstrated that CDK18 could inhibit the Hippo signaling pathway activity.Conclusions: 1 CDK18 is positively associated with tumor size,TNM stage,lymph node invasion and distant metastasis,while negatively associated with the survival of pancreatic cancer patients.2 CDK18 can promote cell proliferation,stemness,and cell cycle process,as well as reduce chemosensitivity to gemcitabine in pancreatic cancer.3 The high expression of CDK18 mediated by m6 A modification in pancreatic cancer is regulated by ALKBH5.4 CDK18 affects the biological function of pancreatic cancer cells through the Hippo signaling pathway. |
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