MiR-9-5p Induces Apoptosis Of Pancreatic Cancer Cells Through SIRT1/NF-κB Pathway

Objective: By verifying the targeting relationship between micro RNA-9-5p(miR-9-5p)and silent mating type information regulation 2 homolog-1(SIRT1),to investigate the effect of miR-9-5p on the apoptosis of pancreatic cancer cells and its molecular mechanism,the expression of nuclear factor-κB(NF-κB)pathway marker protein and apoptosis-related gene cysteine-dependent aspartate-specifc proteases(Caspase-3)were detected.Methods:(1)Using the Target Scan database and dual luciferase reporter test,the interaction targets between miR-9-5p and SIRT1 were predicted and the targeting relationship between them was determined;(2)Human pancreatic carcinoma cell line PANC-1 was transfected with miR-9-5p mimic lentivirus as mimic group,empty vector as negative control group(NC Group)and untreated Panc-1 as blank group(3)CCK-8 assay was used to detect cell viability,(4)RT-q PCR was used to detect the m RNA expression levels of miR-9-5p,SIRT1 and Caspase-3 in PANC-1;(5)The expressions of miR-9-5p,SIRT1 and Caspase-3m RNA were determined by Pearson correlation analysis,(6)The expressions of SIRT1,NF-κ b p65,p-NF-κ b p65 and Caspase-3 protein were detected by Western Blot(7)The expression of SIRT1 and Caspase-3 was detected by Elisa,and(8)The apoptosis was detected by Hoechest staining.Results:(1)Target Scan Human database data show that there is a targeting relationship between miR-9-5p and SIRT1,and previous literature indicates that miR-9-5p is low expressed in pancreatic cancer cells;Dual luciferase reporter gene assay showed that miR-9-5p could inhibit the fluorescence activity of wild-type SIRT1 cells,indicating that miR-9-5p could target the expression of SIRT1;(2)RT-q RCR assay showed that miR-9-5p could inhibit the expression of SIRT1 in wild-type SIRT1 cells,the expression of miR-9-5p m RNA was significantly increased in mimic group(P<0.01);(3)The survival rate of mimic group was significantly lower than that of NC Group(P < 0.05);(4)Compared with the control group,SIRT1 m RNA expression was significantly down-regulated in mimic group(P<0.01),Caspase-3 m RNA expression was significantly up-regulated in mimic group(P<0.01),and SIRT1 m RNA expression was significantly down-regulated in mimic group(P<0.01),caspase-3 m RNA expression was significantly up-regulated(P<0.01);(5)Pearson correlation analysis found that the expression levels of miR-9-5p and SIRT1 were negatively correlated(r =-0.849;P < 0.05);the expression levels of miR-9-5p and Caspase-3 were positively correlated(r = 0.796;P < 0.05);(6)Western Blot showed that SIRT1 protein expression was significantly down-regulated in mimic group(P < 0.01),and the ratio of p-NF-κB p65 to NF-κB p65 protein expression was significantly up-regulated in mimic group(P<0.01),the expression level of Caspase-3 protein was significantly up-regulated(P< 0.01);(7)The expression of SIRT1 and Caspase-3 in mimic group was significantly down-regulated(P < 0.05)and up-regulated(P<0.01)compared with the control group and NC Group by ELISA;(8)Hoechest staining showed that the cells in mimic group had hyperchromatic nuclei and increased apoptosis.Conclusion:(1)Mir-9-5p can be targeted to inhibit the expression of SIRT1 in PANC-1cells;(2)overexpression of miR-9-5p can promote apoptosis of PANC-1 cells,the mechanism of which may be related to targeting SIRT1 followed by activation of NF-κB pathway.