Inhibiting miR-29 on growth, invasion and metastasis of PANC1 cells / 中华胰腺病杂志

ABSTRACT

Objective To investigate the effects of inhibiting miR-29 on growth,invasion and metastasis of pancreatic cancer PANC1 cells,and explore the potential mechanism.Methods Oligonucleotides inhibiting miR-29 (anti miR-29) and control oligonucleotides (miR NC) were used to transfect PANC1 cells to establish anti miR-29 PANC1 cells and miR NC PANC1 cells.Transient transfection of PUMA siRNA,E-cadherin siRNA or NC siRNA was used to construct cotransfected anti miR29 + PUMA-siRNA-PANC1 cells and anti-miR-29 + E-cadherin-siRNA-PANC1 cells.Number of colony formations was observed,cell survival was detected by MTT,cell apoptosis was measured by flow cytometry,cell invasion was detected by transwell chamber assay,and cell migration was detected by wound healing assay.Subcutaneous injection of anti miR-29 PANC1 cells was used to establish xenograft nude mice model,and venous injection of anti miR-29 PANC1 cells was used to establish lung metastasis nude mice model,and the subcutaneous and venous injection of PANC1 cells served as control.The growth of xenograft and the number of lung metastatic nodules were observed.TUNEL method was used to detect cell apoptosis in xenograft and immunohistochemical analysis was used to detect PUMA and E-cadherin in xenograft.Results The survival rate of PANC1,miR-NC-PANC1 and anti-miR-29-PANC1 cells was 100％,(96.8 ± 2.8) ％ and (24.4 ± 3.2) ％.The number of colony formation was (213 ± 36),(196 ± 28) and (37 ± 6) per 100 high power field.The number of transmembrane cells was (56.3 ± 9.6),(49.8-± 7.3) and (11.2 ± 3.4) per 400 high power field.The distance of cell migration was (260 ± 48),(247 ± 46) and (53 ± 7) μm.Cell apoptosis rate was (1.5 +0.9) ％,(2.6 + 0.9) ％ and (22.4 + 2.8) ％.There was statistically significant difference between anti miR 29 PANC1 cells and other PANC1 cells (P ＜0.05).The survival rate,apoptosis rate,transmembrane cells and migration distance of anti-miR-29 + PUMA-siRNA-PANC1 cells was (84.7 ± 10.9) ％,(1.3 ± 0.8) ％,(49.7 ± 6.4) per 400 high power field and (182 ± 36) μm,indicating that the effects of miR 29 inhibition on PANC1 cells were abolished (all P ＜0.05).The volume of the xenograft of PANC1 and anti-miR-29-PANC1 cells was (3 800 ±270) and (1 890 ± 160)mm3,the cell apoptosis rate was 0.93 ±0.14 and 8.26 ± 1.15,the number of metastatic lung lesions was (26.4 ± 6.5) and (8.6 ± 2.7),the PUMA positivity was (7.2 ±1.6) ％ and (43.8 ± 7.6) ％,E-cadherin positivity was (8.3 ± 3.6) ％ and (47.4 ± 5.7) ％,respectively.The xenograft volume and the number of metastatic lung nodules of anti miR29 PANC1 cells was obviously decreased or decreased,but cell apoptosis rate,PUMA positivity and E cadherin positivity were obviously increased,and the differences were all statistically significant (P ＜ 0.05).Conclusions Inhibiting miR-29 expression can decrease cell proliferation,migration and metastasis of PANC1 cells,and the potential mechanism may be associated with the upregulation of PUMA and E-cadherin.