| Objective: Pancreatic cancer is a highly malignant cancer of the digestive system in humans and is the fifth most common cancer and second leading cause of cancer-related deaths worldwide.Pancreatic ductal adenocarcinoma(PDAC),derived from pancreatic ductal epithelial cells,is the main type of pancreatic cancer and accounts for ～80% of all pancreatic cancer cases.Currently,surgical resection is the most effective therapeutic approach for patients with PDAC;however,most cases are diagnosed at the stage of metastasis and cannot undergo surgery.Despite tremendous developments in diagnostic techniques and treatment strategies,the prognosis of patients with PDAC is still poor,with a 5-year survival rate of less than 5%.The mechanisms underlying the aggressive behavior and poor clinical outcomes of PDAC have not yet been elucidated.Therefore,understanding the mechanisms underlying the formation and progression of PDAC is crucial for the identification of effective therapeutic techniques.Long noncoding RNAs(lncRNAs)are a group of non–protein-coding transcripts with a length of >200 nucleotides.LncRNAs have been identified as novel gene regulators acting via multiple mechanisms,including microRNA(miRNA)competition,transcriptional modulation,chromatin remodeling,and histone modification.Existing evidence suggests that lncRNAs are implicated in the control of various biological behaviors,including cellular senescence,differentiation,metabolism,survival,apoptosis,and tumorigenesis.In the field of PDAC research,numerous lncRNAs are known to be aberrantly expressed,and their anomalous expression exerts a significant action on the malignant characteristics of PDAC because these lncRNAs function as cancer-promoting or tumor-suppressive molecules.MicroRNAs(miRNAs)are a family of endogenous noncoding short RNA molecules composed of 17–24 nucleotides.MiRNAs are the main regulators of gene expression,because they bind(through base-pairing)to the 3′-untranslated region(UTR)of their target mRNAs and cause translational inhibition and/or degradation of the target mRNAs.The altered expression of miRNAs was found to play oncogenic or tumor-suppressive roles in the progression of PDAC and crucial functions in a wide array of biological processes.The lncRNA-mediated tumor behaviors are dependent on the crosstalk between lncRNAs and mRNAs,which compete for shared response elements in miRNAs.This competing endogenous RNA(ceRNA)mechanism has aroused great interest in research on the genesis and progression of many types of human malignant tumors.Accordingly,investigation of the specific roles of PDAC-related lncRNAs may offer a novel and effective target for anticancer treatment.In recent years,LINC00657 has been reported to be dysregulated and contributes to tumor progression in various human cancers,such as non–small cell lung cancer,esophageal squamous cell carcinoma,glioblastoma,colon cancer,and hepatocellular carcinoma.However,to the best of our knowledge,studies on the LINC00657 expression profile or its participation in PDAC have not yet been conducted.Therefore,in the present study,we first measured LINC00657 expression in PDAC tumors and cell lines.Then,we carried out functional assays to determine the biological functions of LINC00657 in the malignant progression of PDAC as well as the associated downstream molecular mechanisms.Methods: A total of 56 pairs of PDAC tissue samples and tumor-adjacent tissues were collected from patients with PDAC who underwent surgical resection at The First Affiliated Hospital of China Medical University.None of the patients had received radiotherapy,chemotherapy,or other anticancer modalities before the surgical operation.First,real-time quantitative PCR(RT-qPCR)was used to detect the expression of LINC00657 in 56 pairs of PDAC tissues and adjacent tissues,four PDAC cell lines and human normal pancreatic cell line HPDE6c7.Using statistical analysis(including: Chi-square test and Kaplan-Meier method)the relationship between the expression level of LINC00657 and the clinicopathological factors of PDAC and the prognosis of patients.Later,the expression of LINC00657 was down-regulated in PDAC cell lines.The effects of LINC00657 on PDAC cell proliferation,migration,invasion,and apoptosis in vivo and in vitro were examined using the CCK-8,Transwell,flow cytometry,and subcutaneous xenograft experiments in nude mice.Next,we detected the localization of LINC00657 in PDAC cells by nuclear plasma separation and RT-qPCR experiments,and then applied bioinformatics to predict the target miRNA mi R-433 of LINC00657 and target mRNA PAK4 of mi R-433.The effects of LINC00657 on miR-433 expression levels,the binding of LINC00657 and miR-433,and the expression levels of miR-433 and PAK4 in PDAC cell lines,cancer tissues and adjacent tissues were detected by luciferase reporter genes,RIP experiments and RT-qPCR;Western blotting was used to detect PAK4 protein levels in PDAC cell lines.Spearman’s correlation was used to analyze the correlation between LINC00657 and miR-433 in PDAC,and the correlation between miR-433 and PAK4;Chi-square test and Kaplan-Meier method were used to analyze the relationship between miR-433 expression level and the clinicopathological factors of PDAC and the prognosis of patients.By down-regulating the expression of LINC00657 in the PDAC cell line,up-or down-regulating the expression of miR-433 or up-regulating the expression of PAK4,RT-qPCR,functional experiments(including: CCK-8 experiment,Transwell experiment,flow cytometry experiment)were used to detect the relationship between the competitive combination of LINC00657,miR-433 and PAK4 and their interactions on PDAC cell proliferation,Effects of migration,invasion,and apoptotic capacity.Then use the spss22.0 statistical analysis software to analyze the experimental data,P<0.05 indicates that the results are statistically significant.Results: 1.We found that the expression of LINC00657 was increased in the PDAC cell line(4/4),and the expression level of LINC00657 was significantly higher in 56 cases of paired PDAC tissues than in the adjacent tissues.And the high expression of LINC00657 is closely related to the pathological T stage of PDAC and lymph node metastasis.At the same time,Kaplan-Meier analysis showed that the overall survival of patients with high expression of LINC00657 was lower than that of low expression of LINC00657.2.We knocked out LINC00657 in the PDAC cell lines Panc-1 and Sw1990 with high expression of LINC00657.Functional experiments(CCK-8,Transwell,flow cytometry,and subcutaneous xenograft tumor experiments in nude mice)in vitro and in vivo have confirmed that knockout of LINC00657 inhibits the proliferation,migration,and invasion of PDAC cells,And promoted its apoptosis.3.In terms of the molecular mechanism of LINC00657 affecting the malignant phenotype of PDAC,we tested that miR-433 expression was significantly reduced in PDAC;The expression of LINC00657 and miR-433 are mainly localized in the cytoplasm,and the expression of miR-433 is negatively correlated with the expression of LINC00657.We have also found that LINC00657 has a binding site for miR-433,and directly binds to miR-433 through the binding site.After testing,we found that miR-433 in PDAC cells can also directly bind to the 3’-UTR of the target mRNA PAK4.Therefore,the three competitively combine and interact with each other to affect the malignant phenotype of PDAC.Finally,rescue experiments confirmed that LINC00657 competitively binds miR-433 as a competing endogenous RNA(ceRNA),resulting in increased PAK4 expression and affecting the malignancy of PDAC.Conclusion: 1.LINC00657 expression is up-regulated in PDAC tissues and cell lines.2.In vitro and in vivo experiments have demonstrated that knockdown of LINC00657 inhibits the proliferation,migration,and invasion of PDAC cells and promotes apoptosis.3.LINC00657 acts as a molecular sponge for miR-433 in PDAC cells.4.PAK4 is a target gene of miR-433 in PDAC cells.5.Inhibition of miR-433-PAK4 axis leads to increased PAK4 and thus weakens the effect on PDAC cells due to reduced LINC00657.These results indicate that a LINC00657–miR-433–PAK4-mediated targeted modality may be an effective approach to the prevention or treatment of patients with PDAC. |
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