The Role Of Histone Methyltransferase SMYD3 In The Development Of Pancreatic Cancer And Its Molecular Mechanism

Background Pancreatic cancer is one of the most common malignant tumors in the digestive system.It is characterized by difficulty in early diagnosis,limited treatment and poor clinical prognosis.The carcinogenic factors of pancreatic cancer are complex and the pathogenesis is still unclear.It has been a focus of clinical concern to explore the pathogenesis of pancreatic cancer and to find an effective therapeutic approach.Epigenetics,which is the genetic variation of genes in transcription and translation,has been shown to be a key mechanism for tumorigenesis.Epigenetics mainly includes DNA methylation,post-translational histone modification,and small ma-mediated silencing,especially histone modification.Histone modification includes histone methylation,histone phosphorylation,histone acetylation and histone ubiquitination.In recent years,the role of histone methylation in the pathogenesis of cancer has become a hot topic,among which histone methylated transferase SMYD3 has been found to be a key protein in the pathogenesis of many tumors,but there are few reports on its role in pancreatic cancer.Part One The expression of SMYD3 in pancreatic cancer tissues and cellsAim The expression of SMYD3 was abnormally expressed in many tumors and played an important role in the development of tumors,however its role in pancreatic cancer was still not known.In histological experimental section,the expression of SMYD3 in human pancreatic cancer tissues and normal pancreatic tissues was firstly detected by immunohistochemical method,and understand its relationship between clinical pathological characteristics and prognosis.Secondly in cytology test section,from the cellular level testing SMYD3 mRNA and protein expression in pancreatic cancer cell line.The aim was to study the expression of SMYD3 in pancreatic cancer,and to explore the role of SMYD3 in the pathogenesis of pancreatic cancer.Methods Immunohistochemistry was used to detect the expression of SMYD3 in 67 pancreatic cancer tissues and 20 non-tumor pancreatic tissues,and to analyze the relationship between their expression and the clinicopathological features and prognosis.mRNA and protein expression of SMYD3 in pancreatic cancer cell lines(SW1990,CFPAC-1,ASPC-1,PACA-2)was determined by RT-PCR and Western blot methods.Results The expression rates of the SMYD3 in 67 cases of PC and 20 non-tumor cases were 62.7%vs.35.0%(p<0.05).In further analysis,the expression of SMYD3 protein was associated with tumor size,T staging,TNM staging,lymph node metastasis and nerve infiltration.Kaplan-Meier survival analyses showed that patients with positive expression of SMYD3 had an obviously shorter survival period than those with negative expression.The mRNA and protein of SMYD3 were highly expressed in four pancreatic cancer cell lines including SW1990,CFPAC-1,ASPC-1,and PACA-2.Conclusions The SMYD3 protein was overexpressed in pancreatic cancer tissues and cells,and involved in the progression of pancreatic cancer.Part Two The effect of SMYD3 on proliferation,migration and invasion of pancreatic cancerAim Through our previous studies,we found that SMYD3 was significantly highly expressed in pancreatic cancer and was involved in the development of pancreatic cancer,but its specific role in the pathogenesis of pancreatic cancer was still unclear.In this study,the effect of SMYD3 on the proliferation,migration and invasion of pancreatic cancer cells SW1990 was firstly observed in the cytological experiment by up-regulating and down-regulating SMYD3 protein levels in pancreatic cancer cells SW1990.Secondly,the effect of SMYD3 on pancreatic cancer implants was studied by establishing an animal experimental model.To further explore the specific role of SMYD3 in the pathogenesis of pancreatic cancer.Methods The pancreatic cancer cell line SW1990 was transfected with the interference(shRNA-SMYD3)and overexpression(SMYD3-OE)lentivirus vectors,and the transfection was verified by fluorescence staining,RT-PCR and Western blot.The transfected pancreatic cancer cell lines were divided into shRNA-SMYD3 SW1990 group,SMYD3-OE SW1990 group and Ctrl SW1990 control group.Cell proliferation was detected by CCK-8 assay,and cell migration and invasion were detected by transwell method.A model of subcutaneous tumor implantation in nude mice with human pancreatic cancer was established,which was randomly divided into the control group SW1990 and the shRNA-SMYD3 SW1990 interference group.After 2 weeks,the volume and weight of the tumor implantation were observed.Results Successfully constructed pancreatic cancer cell lines with interference and overexpression of SMYD3.Interference with the expression of SMYD3 protein in SW1990 pancreatic cancer cell lines significantly inhibited the proliferation,migration and invasion of tumor cells,while overexpression of SMYD3 protein significantly enhanced the proliferation,migration and invasion of tumor cells.In animal experiments,we found that the tumor volume and weight of the mice in the shSMYD3 SW1990 interference group were significantly inhibited.Conclusions The SMYD3 protein significantly promoted the proliferation,migration and invasion of pancreatic cancer,and played an important role in the pathogenesis of pancreatic cancer.Part Three The molecular mechanism of histone methyltransferase SMYD3 in the pathogenesis of pancreatic cancerAim In previous studies,we found that SMYD3 protein plays an important role in the pathogenesis of pancreatic cancer,with its high expression significantly promoting the proliferation,migration and invasion of pancreatic cancer,and inhibiting the expression of SMYD3 significantly inhibiting the proliferation,migration and invasion of pancreatic cancer,but the specific mechanism of action is still unclear.Apoptosis and invasion and metastasis is an important process of tumor biological behavior,reported in a previous study cysteine aspartic acid proteinase 3(Caspase-3)and matrix metalloproteinase 2(MMP-2)as a target protein play an important role in the process,and extracellular regulating protein kinase(ERK)signaling pathways involved in the regulation of this process.Firstly,in the histological experiment,the expression of SMYD3,Caspase-3 and MMP-2 proteins in human pancreatic cancer tissues and normal pancreatic tissues was detected by immunohistochemistry,and the relationship between the three proteins was analyzed.Secondly,in the cytological experiment,the relationship between SMYD3 and ERK1,Caspase-3 and MMP-2 in pancreatic cancer cells was observed by interfering with the expression level of SMYD3 protein.Finally,the expression of ERK1,Caspase-3 and MMP-2 proteins in pancreatic cancer implant tissues was detected.To investigate the molecular mechanism of SMYD3 protein in the pathogenesis of pancreatic cancer.Methods Immunohistochemistry was used to detect the expression of caspase-3 and MMP-2 in 67 pancreatic cancer tissues and 20 non-tumor pancreatic tissues,and the correlation between SMYD3 and Caspase-3 and MMP-2 was analyzed.mRNA and protein expressions of ERK1,Caspase-3 and MMP-2 in t:he shRNA-SMYD3 SW1990 group and the control group were detected by RT-PCR and Western blot methods.A model of subcutaneous tumor implantation in nude mice with human pancreatic cancer was established,which was randomly divided into the control group SW1990 and the shRNA-SMYD3 SW1990 interference group.And the expression of ERK1,Caspase-3 and MMP-2 proteins in the tumor tissue was detected by Western blot.Results The expression rates of the Caspase-3 and MMP-2 in 67 cases of PC and 20 non-tumor cases were 65.7%vs.40.0%(p<0.05)and 59.7%vs.30.0%(p<0.05).In further correlation analysis showed that SMYD3 was positively correlated with the expression of Caspase-3 and MMP-2 proteins.RT-PCR and Western blot showed that the mRNA and protein expression of ERK1 and MMP-2 decreased after the intervention of SMYD3 protein expression in SW1990 pancreatic cancer cell lines,showing a significant correlation,but the mRNA and protein expression of Caspase-3 did not change significantly.In animal experiments,the expression of ERK1 and MMP-2 protein was significantly decreased in the interference group compared with the control group,while the expression of Caspase-3 protein was not significantly changed in the two groups.Conclusions The SMYD3 may regulate the expression of MMP-2 protein through ERK signaling pathway to regulate the development of pancreatic cancer.Summary SMYD3 protein was highly expressed in pancreatic cancer,which can promote the proliferation,migration and invasion of pancreatic cancer,and played an important role in the occurrence and development of pancreatic cancer.The mechanism may be that SMYD3 regulated the expression of MMP-2 protein through ERK signaling pathway to regulate the development of pancreatic cancer.