The Molecular Mechanisms Of The MiR-1224-5p/ELF3 Axis Regulating The Malignant Behaviors Of Pancreatic Cancer

Objective: Pancreatic cancer is one of the most malignant tumors in the digestive tract.The most common pathological type is pancreatic ductal adenocarcinoma(PDAC)originating from the pancreatic ductal epithelial cells.According to the data from the American Cancer Society,pancreatic cancer is the tenth leading cause of new cancer cases in men and the ninth leading cause of new cancer cases in women in 2020.Pancreatic cancer remains the fourth leading cause of cancer-related deaths with a five-year survival rate of about 9%.In China,the five-year survival rate for pancreatic cancer patients is about 7.2% which ranks the lowest compared with the other cancer types.Previous epidemiological studies have shown that smoking,overweight,lack of exercise,poor diet,diabetes mellitus,chronic pancreatitis,Helicobacter pylori infection,and genetic or acquired gene mutations are important factors associated with the initiation and development of pancreatic cancer.Due to the characteristics of insidious onset,unobvious symptoms,and early metastasis of pancreatic cancer,the patients are often in the middle or advanced stage of the disease at the first visit to a doctor.Most of the patients can’t have the opportunity to accept radical surgical treatment.Therefore,increasing the early diagnosis rate is one of the keys to improve the survival and prognosis of pancreatic cancer patients.In the treatment of pancreatic cancer,surgical resection is currently considered as the only possible method to cure.Pancreatic cancer patients are not sensitive to conventional chemotherapy or radiation therapies.Recently,immunotherapies are expected to improve the therapeutic effect of pancreatic cancer,but further large-scale researches are required to confirm its function.In conclusion,pancreatic cancer is characterized by the low early diagnosis rate,unsatisfactory therapeutic effect,and poor prognosis due to its unique biological characteristics and anatomical location.To further improve the efficiency of diagnosis and treatment of pancreatic cancer,we need to investigate the molecular mechanisms behind the occurrence and development of pancreatic cancer and identify more sensitive biomarkers and targets.microRNA is a class of non-coding RNA with a length of 18-22 nucleotides.As an important epigenetic regulator,microRNA can regulate the expression of corresponding genes at the post-transcriptional level.Previous studies have shown that microRNAs are involved in a variety of malignant biological behaviors of tumor cells by regulating the expression of specific genes,such as tumor genesis,cell proliferation,cell migration,cell invasion,cell cycle,and cell apoptosis.Previous studies have shown that the expression of miR-1224-5p is down-regulated in lung cancer,glioma,and intestinal-type gastric cancer.miR-1224-5p can be used as a prognostic marker in patients with glioma and intestinal-type gastric cancer,and the patients with higher miR-1224-5p expression level in the above patients have a better prognosis.In the aspect of molecular mechanisms,the researchers confirm that miR-1224-5p can inhibit the metastasis of intestinal-type gastric cancer by directly targeting the FAK gene through cellular and animal experiments.As for glioma,miR-1224-5p can inhibit its progression by targeting the CREB1 gene.Under the stimulation of oxidative stress from acute liver failure,the expression level of miR-1224-5p in liver cells is significantly increased.miR-1224-5p can inhibit the proliferation and promote apoptosis of liver cells by targeting the anti-apoptotic gene Nfib.The microRNA expression profiles of five pairs of keloid and corresponding normal skin tissues are detected by using the miRNA microarray to identify the differentially expressed microRNAs.The study finds that the expression of miR-1224-5p is lower in the keloid tissues compared with the normal skin tissues.The cellular experiments show that overexpression of miR-1224-5p can inhibit the proliferation,migration,and invasion and promote apoptosis of keloid fibroblasts.These studies have shown that the miR-1224-5p plays different roles in a variety of benign and malignant diseases,but the biological role of miR-1224-5p in pancreatic diseases hasn’t been reported.Therefore,this study intends to further investigate the biological role of miR-1224-5p in pancreatic cancer.Methods: 1.Firstly,three miRNA sequencing datasets of pancreatic cancer were downloaded from the public database GEO to analyze the expression of miR-1224-5p between pancreatic cancer tissues and normal tissues including GSE119794,GSE32678,and GSE24279.The relative expression level of miR-1224-5p in 20 pairs of pancreatic cancer tissues and corresponding adjacent normal tissues was detected by qRT-PCR.Besides,the relative expression level of miR-1224-5p in four pancreatic cancer cell lines(As PC-1,Capan-2,PANC-1,and SW1990)and human pancreatic ductal cell lines(h TERT-HPNE)was also detected by qRT-PCR.The correlation analyses between the miR-1224-5p expression level and clinicopathological information and prognosis of pancreatic cancer patients were performed by using the data from the public database TCGA.Besides,cell transfection was used to overexpress or inhibit the expression of miR-1224-5p in the As PC-1 and PANC-1 pancreatic cancer cell lines for further cellular functional experiments.Subsequently,CCK8,colony formation,wound healing,and transwell assays were used to detect the changes of proliferation,migration,and invasion abilities of pancreatic cancer cells after overexpression or inhibition of miR-1224-5p.2.Our researches showed that miR-1224-5p was involved in the regulation of malignant behaviors of pancreatic cancer.To further investigate the specific mechanisms behind miR-1224-5p in pancreatic cancer,the potential target genes of miR-1224-5p were predicted by using three microRNA target prediction websites,such as Target Scan,miRanda,and miRWalk.A comprehensive analysis of the predicted results from the above websites suggested that ELF3 might be the target gene of miR-1224-5p.Subsequently,the dual-luciferase reporter assay was used to detect the binding site between miR-1224-5p and ELF3 gene.Meanwhile,after knockdown or overexpression of miR-1224-5p in As PC-1 and PANC-1 pancreatic cancer cell lines,the changes in the m RNA and protein levels of the target gene ELF3 were detected.To clarify the biological role of the ELF3 gene in pancreatic cancer,the GEPIA database was firstly used to analyze the differential expression of the ELF3 gene between pancreatic cancer tissues and normal tissues.Then,western blot and immunohistochemistry were used to detect the expression of the ELF3 gene in the clinically collected pancreatic cancer tissues and adjacent normal tissues.The expression of the ELF3 gene in four pancreatic cancer cell lines(As PC-1,Capan-2,PANC-1,and SW1990)and human pancreatic ductal cell line(h TERT-HPNE)was detected by western blot.The correlations between ELF3 gene expression and clinicopathological information and prognosis of pancreatic cancer patients were analyzed by using the data from the TCGA database.Furthermore,the lentivirus transfection technique was used to stably knockdown ELF3 gene expression in the As PC-1 and PANC-1 cell lines.Then,CCK8,colony formation,wound healing,and transwell assays were performed to detect the changes of proliferation,migration,and invasion abilities after knockdown ELF3 gene in the As PC-1 and PANC-1 cells.Rescue experiment was designed to confirm that miR-1224-5p regulates the malignant biological behaviors of pancreatic cancer by suppressing ELF3 gene expression.The rescue experiment contained two designs,one design was to make the comparison between miR-1224-5p and ELF3 gene overexpression at the same time and miR-1224-5p overexpression alone,and the other design was to make the comparison between knockdown of miR-1224-5p and ELF3 gene at the same time and knockdown of miR-1224-5p alone.Also,the subcutaneous tumor model of nude mice was used to further verify the effect of miR-1224-5p and ELF3 on pancreatic cancer progression in vivo.3.To further clarify the specific molecular mechanisms of the miR-1224-5p/ELF3 axis regulating the progression of pancreatic cancer,the GSEA enrichment analysis was performed based on the transcriptional sequencing data of pancreatic cancer patients from the TCGA database to identify the ELF3 gene-related signaling pathways.Besides,the STRING database was used to construct the protein interaction network related to the ELF3 gene,and the 20 core genes with the highest correlation with the ELF3 gene were screened for subsequent GO/KEGG enrichment analysis to identify the molecular signaling pathways related to the ELF3 gene.The correlation analyses between ELF3 gene expression and six kinds of immune cell infiltration were performed by using the TIMER database.Finally,the western blot assay was used to detect the changes of key molecules in EMT/PI3K/Akt /Notch signaling pathways in As PC-1 and PANC-1 cells after knockdown of the ELF3 gene.Results: 1.By analyzing three datasets(GSE119794,GSE32678,and GSE24279)from the GEO database,the expression level of miR-1224-5p in the pancreatic cancer tissues was significantly down-regulated compared with that in the pancreatic normal tissues.The PCR method was used to detect the expression of miR-1224-5p in pancreatic cancer tissues and cells,and results showed that the expression level of miR-1224-5p in pancreatic cancer tissues and cells was significantly lower compared with the normal pancreatic tissues and cells.Based on the data from the public database TCGA,analysis results showed that miR-1224-5p expression was significantly down-regulated in pancreatic cancer patients with advanced-stage and lymph node metastasis.The survival analysis showed that pancreatic cancer patients with low miR-1224-5p expression have a poor prognosis.The cellular experiments confirmed that overexpression of miR-1224-5p could significantly inhibit the proliferation,migration,and invasion of As PC-1 and PANC-1 cell lines.2.The microRNA target gene prediction software and dual luciferase assay confirmed that miR-1224-5p could directly inhibit the expression of the ELF3 gene.Preliminary analysis using the GEPIA database showed that the m RNA expression level of the ELF3 gene in pancreatic cancer tissues was significantly higher than that in normal tissues.The protein expression level of the ELF3 gene detected by the western blot and immunohistochemical assays was significantly up-regulated in pancreatic cancer tissues compared with that in the adjacent normal tissues.The expression level of the ELF3 gene in the four pancreatic cancer cell lines(As PC-1,Capan-2,PANC-1,and SW1990)was also higher than that in the human pancreatic ductal cell line(h TERT-HPNE).Subsequently,data from the public database TCGA were used for analysis,suggesting that ELF3 gene expression was significantly upregulated in pancreatic cancer patients with advanced stage.Survival analysis showed that ELF3 expression was significantly correlated with the prognosis of patients with pancreatic cancer,and patients with high ELF3 expression levels had a poor prognosis.Stable knockdown of ELF3 gene expression in As PC-1 and PANC-1 cell lines could significantly inhibit the proliferation,migration,and invasion of pancreatic cancer cells.The rescue experiments were designed and performed to confirm that miR-1224-5p could regulate the malignant behaviors of pancreatic cancer by directly inhibiting the ELF3 gene expression.Using the subcutaneous tumor model in nude mice,it was confirmed that overexpression of miR-1224-5p or knockdown of the ELF3 gene could significantly inhibit the progression of pancreatic cancer in vivo.By comparing the effects of overexpression of miR-1224-5p and ELF3 gene at the same time and overexpression of miR-1224-5p alone in vivo,it was further confirmed that miR-1224-5p could inhibit the progression of pancreatic cancer by regulating the expression of the ELF3 gene.3.Subsequently,the GSEA enrichment analysis was used to identify the ELF3gene-related signaling pathways.Besides,the STRING database was used to construct the ELF3 gene-related protein interaction network,and the GO/KEGG analysis was performed to identify the ELF3 gene-related signaling pathways.The results of western blot showed that knockdown of ELF3 in As PC-1 and PANC-1 could significantly increase the expression of E-cadherin and reduce the expression of N-cadherin,Vimentin,MMP-9,Snail,and Slug.The expression level of P-PI3 K and P-Akt was significantly inhibited after ELF3 knockdown in the As PC-1 and PANC-1 cells.Besides,the expression level of Notch-1,C-myc,Cyclin D1,Hes-1,and VEGF which serve as key factors in the Notch signaling pathway were significantly inhibited after knockdown of the ELF3 gene in pancreatic cancer cells.Taken together,these results suggested that the ELF3 gene could regulate the progression of pancreatic cancer by modulating the EMT/PI3K/AKT/Notch signaling pathway.Conclusion: 1.The expression level of miR-1224-5p in pancreatic cancer tissues was significantly down-regulated compared with normal tissues,and the expression level of miR-1224-5p was significantly down-regulated in pancreatic cancer patients with advanced-stage and lymph node metastasis.The expression level of miR-1224-5p was significantly correlated with the prognosis of pancreatic cancer patients,and patients with a low expression level of miR-1224-5p had a poor prognosis.The cellular assays showed that overexpression of miR-1224-5p could significantly inhibit the proliferation,migration,and invasion of pancreatic cancer cells.Conversely,inhibition of miR-1224-5p could significantly improve the proliferation,migration,and invasion of pancreatic cancer cells.2.The dual-luciferase report assay and cell transfection experiment confirmed that miR-1224-5p could inhibit the expression of the ELF3 gene in pancreatic cancer cells.The expression level of the ELF3 gene was higher in pancreatic cancer tissues and cells.The ELF3 gene expression was significantly correlated with the clinicopathological information and prognosis of pancreatic cancer patients.Stable knockdown of the ELF3 gene could significantly inhibit the proliferation,migration,and invasion of pancreatic cancer cells.The rescue and in vivo animal experiments proved that miR-1224-5p could inhibit pancreatic cancer progression by inhibiting the expression of the ELF3 gene.3.The miR-1224-5p/ELF3 axis regulated the malignant behaviors of pancreatic cancer via regulating the PI3K/AKT/Notch signaling pathways.The miR-1224-5p/ELF3 axis regulated the malignant behaviors of pancreatic cancer by modulating the epithelial-mesenchymal transition(EMT)process.The expression level of the ELF3 gene was negatively correlated with the infiltration of the immune cells,suggesting that the abnormal expression of the ELF3 gene was correlated with the abnormal immune microenvironment of pancreatic cancer.