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Objective:To observe the effects of microRNA-21 (miR-21) on cell invasion, migration and apoptosis of pancreatic cancer PANC1 cells, and explore the potential molecular mechanism.Methods:Recombinant plasmids carrying miR-21 or small interfering RNA targeting miR-21 were constructed. Using blank plasmid as the control, the recombinant plasmids were transfected with human pancreatic cancer PANC1 cells by liposome method, respectively to establish blank group, miR-21 overexpression group (overexpression group) and miR-21 silence group (silence group) PANC1 cell lines. Cell proliferation, apoptosis, invasion and metastasis were detected by MTT method, flow cytometry, transwell chamber and wound scratch test, respectively. ELISA and Western blot were used to measure the protein expression of programmed cell death factor 4(PDCD4), gene of phosphate and tension homology deleted on chromsome ten (PTEN), vascular endothelial growth factor (VEGF), survivin, matrix metalloproteinase 2 (MMP-2) and MMP-9.Results:After 24 h cell culture, the cell proliferation rate of blank group, overexpression group and silence group was (20.72±5.62)%, (28.46±6.12)% and (14.05±3.36 )%; cell apoptosis rate was (5.89±0.26)%, (4.62±0.19)% and (8.66±2.55)%; the number of transmembrane cells was (212.4±32.5), (508.8±50.7) and (50.9±10.6) per 200 times visual field; the area covered by migrated cells was (75.6±12.1), (118.8±20.2) and (48.8±9.5)mm 2 per 200 times visual field; the expression of PDCD4 was 0.85±0.22, 0.72±0.10 and 1.36±0.41; the expression of PTEN was 0.85±0.21, 0.28±0.09 and 1.36±0.45; the expression of VEGF was 0.79±0.24, 1.15±0.31 and 0.46±0.10; the expression of survivin was 1.02±0.33, 1.51±0.42 and 0.52±0.12; the expression of MMP-2 was 1.12±0.22, 1.86±0.52 and 0.56±0.18; the expression of MMP-9 was 1.06±0.15, 1.78±0.48 and 0.49±0.12. All the differences among the three groups were statistically significant (all P<0.05). Compared with blank group, the cell apoptosis rate, PDCD4 and PTEN expression were increased, while cell proliferation, invasion and migration, and the protein expression of VEGF, survivin, MMP-2 and MMP-9 were all decreased; the changes in silence group was totally contrary to those in overexpression group. All the differences among the three groups were statistically significant (all P<0.05). Conclusions:miR-21 silencing can inhibit the proliferation, migration and invasion of PANC1 cells and promote apoptosis, and the mechanism was possibly associated with the upregulation of PDCD4 and PTEN protein expression.
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OBJECTIVE To analyze the laboratory phenotype and FXII gene mutation in a consanguineous Chinese pedigree affected with factor XII (FXII) deficiency. METHODS Activated partial thromboplastin time (APTT), FXII activity (FXII:C) and FXII antigen (FXII:Ag) of the proband and her family members (10 individuals from 3 generations) were determined. Sanger sequencing was used to detect potential mutation within the 14 exons, their flanking regions and 5',3'-untranslated regions of the FXII gene. Suspected mutations were verified by backward sequencing. Conservation of the amino acids were analyzed with ClustalX-2.1-win. Four online bioinformatics software (PolyPhen-2, PROVEAN, SIFT and MutationTaster) were used to assess the impact of the mutations on the protein function. RESULTS The APTT of the proband and her elder brother have prolonged to 61.6 s and 68.6 s,and their FXII:C and FXII:Ag have decreased to 12%, 10% and 11%, 10%, respectively. The APTT of the paternal grandmother, maternal grandmother, father, mother, elder paternal aunt and elder maternal aunt were all normal, but their FXII:C and FXII:Ag have reduced to half of the normal value. Gene sequencing found that the proband and her elder brother have both carried a homozygous missense c.1078G>A (p.Gly341Arg) mutation in exon 10 of the FXII gene, for which the paternal grandmother, maternal grandmother, father, mother, elder paternal aunt and elder maternal aunt were heterozygous. Bioinformatic analysis suggested that the Gly341 is highly conserved, while p.Gly341Arg is a harmful mutation which may cause disease by affecting the function of FXII protein. CONCLUSION Homozygous p.Gly341Arg mutation, caused by consanguineous marriage, probably underlies the congenital FXII deficiency in this pedigree.