| Part ? Screening and verification of differentially expressed miRNAs in pancreatic cancer tissuesObjective:Applying genechip technique to screen the abnormally expressed miRNAs in pancreatic cancer samples,and employing statistical softwares to analysis the experimental data.To establish the theoretical basis for pancreatic cancer treatment and element experience for further research.Methods:(1)39 cases of pancreatic cancer specimens and their matched adjacent normal tissues were collected,meantime all corresponding clinicopathological features of the patients were recorded.(2)The miRNA expression profiles in pancreatic cancer compared to corresponding normal tissues were analyzed with Affymetrix miRNA microarray.Then the most differentially expressed miRNAs were detected.(3)Among all of the microarray results,five dysregulated miRNAs were validated by performing qRT-PCR in the detection of another 36 specimens,including two miRNAs up-regulated(miR-422a and miR-628-5p)and three miRNAs down-regulated(miR-557?miR-563 and miR-658).(4)According to the expression levels of miR-557 in pancreatic cancer tissues,the relationship between miR-557 expression levels and pathological features such as gender,age,tumor size,lymph node metastasis,vascular invasion and TNM stage of pancreatic cancer patients was analyzed.Results:(1)Microarray results showed that compared with the normal tissues,558 miRNAs were differentially expressed in the pancreatic cancer tissues,including 213 miRNAs up-regulated and 345 miRNAs down-regulated.(2)Five candidate miRNAs,miR-422a?miR-628-5p?miR-557?miR-563 and miR-658,were validated by qRT-PCR,their expressions matched the data of microarray.Among these dysregulated miRNAs,miR-557 was chosen for further investigation.(3)The expression levels of miR-557 in pancreatic cancer tissues were found to be significantly more reduced than those of their matched adjacent tissues.The correlation analysis proved that there was an obvious correlation between miR-557 expression levels and differentiation status,lymph node metastasis,vascular invasion and TNM stage.However,there was no statistically significant association between the levels of miR-557 expression and the gender,age,tumor size or location.Conclusions:Differential miRNA expression profiles in pancreatic cancer tissues were screened by chip technology.miR-557 was significantly down-regulated in pancreatic cancer tissues and was closely associated with the malignant clinicopathological features of pancreatic cancer.Part II Effect of miR-557 on the malignant biological behavior of pancreatic cancer cellsObjective:To observe the effect of miR-557 overexpression on the malignant phenotype of pancreatic cancer cells and explore the potentially related mechanisms.Methods:(1)qRT-PCR was applied to detect the expression levels of miR-557 in three pancreatic cancer cell lines and normal pancreatic dutal epithelial cells.(2)The miR-557 gene was cloned to lentiviral vectors and was infected into PANC-1 cells to establish the stable miR-557 high expressing cell lines.(3)CCK-8 assays were used to evaluate the effect of miR-557 on the proliferative ability of PANC-1 cells in vitro.Annexin V-FITC/7-ADD double staining was performed to analyze the apoptosis of transfected cells.Transwell assays and wound-healing assays were used to observe the effect of miR-557 on invasion and migration ability of PANC-1 cells in vitro.(4)Immunofluorescence tests were applied to detect the effect of miR-557 on the expression of EMT markers and related pathway proteins.(5)Construct the tumor-bearing nude mouse model.Subcutaneous tumor formative study in nude mice was applied to examine the effect of miR-557 on tumorigenic ability of pancreatic cancer cells in vivo,while the lung metastasis model was performed to observe the influence of miR-557 on the distant metastasis of pancreatic cancer.Results:(1)The expression levels of miR-557 in three pancreatic cancer cells were significantly lower than that of pancreatic ductal epithelial cells(P<0.01).(2)qRT-PCR analyses demonstrated that the expression levels of miR-557 in PANC-1 cells were significantly increased after transfection,which proved that the construction and infection of lentivirus vectors were effective.(3)CCK-8 assays showed that 48 hours later,enforced expression of miR-557 resulted in significant restriction in cell growth of the PANC-1(P<0.01).The inhibitory effect was more obvious with time passing.Annexin V-FITC/7-ADD double staining showed that miR-557 had no significant effect on the apoptosis of PANC-1 cells(P>0.05).The Transwell assays showed that compared with control groups,the mean amount of cells penetrating Matrigel in miR-557 group was obviously reduced(P<0.01),indicating that miR-557 can significantly attenuate the invasion ability of PANC-1 cells in vitro.The results of wound-healing assays showed that the migration distance of PANC-1 cells was significantly shortened after miR-557 overexpression(P<0.01),suggesting that miR-557 can inhibit the migration activity of pancreatic cancer cells.(4)Immunofluorescence assays showed that the expression of epithelial marker E-cadherin was significantly increased after overexpression of miR-557 in PANC-1 cells,while the expression of the interstitial marker N-cadherin was obviously decreased,and the expression of regulatory molecule p-Smad2/3 was also significantly weakened,revealing that miR-557 may reverse the EMT progression of pancreatic cancer.(4)In vivo,compared with control groups,the time of subcutaneous tumor formation in miR-557 group was significantly prolonged,and the volume of tumor was powerfully decreased in miR-557 group.Furthermore,miR-557 also significantly inhibited the distant metastases of tumors in nude mice.Conclusion:The expression levels of miR-557 were significantly under-expressed in pancreatic cancer cells.Up-regulation of miR-557 can not only significantly inhibit proliferation,migration and invasion of pancreatic cancer cells in vitro,but also suppress tumor growth and distant metastasis in tumor-bearing nude mice.Part ? Mechanism of miR-557 regulating the malignant biological behavior of pancreatic cancerObjective:To predict the miR-557 target genes and conduct preliminary validation to reveal the potential molecular mechanisms of miR-557 regulating the progression of pancreatic cancer.Methods:(1)The potential target genes of miR-557 were screened by utilizing two bioinformatics prediction tools:miRanda and Target Scan.After comprehensive analyses,EGFR was chosen from these predicted target genes for further study.(2)Western blot was used to examine the protein expression of EGFR in PANC-1 cells which were transfected with miR-557 lentivirus vectors.(3)The dual luciferase gene reporter assays validated the targeted regulatory relationship between miR-557 and EGFR.(4)The EGFR plasmid vector was constructed.Rescue experimental strategy was designed to reveal the regulatory mechanisms of target gene,and the expression changes of EMT markers were analyzed.Results:(1)Applying bioinformatics analyses to speculate that EGFR is one of the target genes of miR-557.(2)Western blot analyses showed that overexpression of miR-557 could manifestly reduce the expression levels of EGFR protein.(3)Relative luciferase activity was significantly suppressed in PANC-1 cells after co-transfection miR-557 mimics and wild type psiCHECK-2-EGFR-UTR(P<0.05),which suggested miR-557 regulated EGFR negatively by binding 3'-UTR seed region.(4)CCK8 assays showed that compared with the miR-557 group,the proliferative ability of PANC-1 cells in miR-557/EGFR group was significantly enhanced(P<0.05),and the proliferation was even not significantly different from the control group(P>0.05),indicating EGFR can reverse the proliferation inhibition induced by miR-557 in PANC-1 cells.Transwell assays demonstrated that after transfection of EGFR plasmid,the number of transmembrane cells was significantly increased in miR-557/EGFR group compared with the miR-557 group,which was yet significantly reduced compared with the control group(P<0.05),suggesting that EGFR can only partially block the inhibition of invasion incited by miR-557 in PANC-1 cells.Wound-healing tests showed that the migration distance of the miR-557/EGFR group was significantly shorter than that of the control group(P<0.05),while the migration distance was significantly wider than that of the miR-557 group(P<0.05).This suggested that EGFR partly attenuates the ability of miR-557 to inhibit the migration activity of PANC-1 cells.(5)Immunofluorescence assays showed that the fluorescence intensity of E-cadherin in miR-557/EGFR group was weaker than that of miR-557 group,while the fluorescence intensity of N-cadherin and p-Smad2/3 was enhanced.Compared with the control group,the fluorescence intensity of E-cadherin in miR-557/EGFR group was increased,however,the fluorescence intensity of N-cadherin was decreased,and there was no significant change in the fluorescence intensity of p-Smad2/3(P>0.05).Conclusion:EGFR is one of the target genes of miR-557,and miR-557 can hinder the tumorigenesis and progression of pancreatic cancer by negatively regulating the expression of EGFR in pancreatic cancer cells.In addition,miR-557 may also influence EMT changes in pancreatic cancer through other target genes. |
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