Metformin Potentiates Anti-tumor Effect On Pancreatic Cancer Cells By Down-regulating MiR-23b-5p Which Is Targeted Activating STK4

Pancreatic Ductal Adenocarcinoma(PDAC)is one of the most aggressive malignancies worldwide,with a 5-year survival of less than 5%.It is the fourth-leading cause of cancer-related mortality on the global scale.Surgical resection may offer curative opportunities to select patients.However,the majority of PDAC patients are diagnosed at an advanced stage and have lost the opportunity for surgical treatment.For patients with locally advanced or metastatic inoperative pancreatic cancer,chemotherapy and radiotherapy are the standard treatments.Nevertheless,because of the inherent ability of pancreatic cancer to become chemoresistant and radioresistant,combined modality therapy has failed to universally improve outcomes.Therefore,it is urgent to identify novel pharmacological strategies for PDAC management.Recent studies have shown that components of metabolic syndrome,including diabetes mellitus(DM),obesity and hyperlipidemia,are significant risk factors for PDAC.Up to 80 % of pancreatic cancer patients present with either new-onset type 2 diabetes or impaired glucose tolerance at the time of diagnosis.Metformin is a widely used oral anti-hyperglycemic agent for type 2 diabetes mellitus(T2DM).Systemically,it reduces glycemia by inhibiting hepatic gluconeogenesis and increasing insulin sensitivity in peripheral tissues.Retrospective studies have shown a survival benefit in diabetic patients with many solid tumors including pancreatic cancer that have been treated with metformin compared with patients treated with insulin or sulfonylureas.Additionally,emerging evidences suggested that metformin may be useful in preventing and treating cancer,even in nondiabetic individuals,indicating the anti-tumor properties of metformin are independent of its hypoglycemic effect.Furthermore,metformin acts synergistically with 5-fluorouracil and oxaliplatin to induce cell death in cancers.However,the role and the underlying mechanism of metformin's effects on cancer-related chemotherapeutic agents' sensitivity remain unclear.Micro RNAs(mi RNAs)consist of a family of short,noncoding RNA molecules?They are evolutionarily conserved and stable in plasma.Accumulating evidence suggests that deregulation of mi RNAs may contribute to many human diseases.Through regulating different pro-oncogenic pathways,mi RNAs can contribute to uncontrolled and rapid cancer cell growth,resistance to anticancer therapy,and tumor invasiveness,metastasis,and aggression.mi RNA plays an essential role in tumorigenesis and tumor progression.Both breast cancer and pancreatic cancer have poor prognoses,and altered expression of several different mi RNAs have been reported to be associated with both cancers.Plasma mi RNAs could potentially be used as a diagnostic biomarker in several diseases,particularly early cancer detection.Clinical studies have reported that mi RNAs are used as novel biomarkers to detect early colorectal cancer and to predict the prognosis of some human cancers.The underlying mechanisms of the anticancer effects of metformin remain unclear.Modulation of mi RNA expression may be an important mechanism.Part? Screening anti-cancer effective mi RNA of metformin and predicting its target genes in pancreatic cancer Objective:The aim of the present study was to screen anticancer effective mi RNA of metformin in the PANC-1 human pancreatic cell line by Affymetrix mi RNA3.0 and Affymetrix HTA2.0 microarray techniques and to further explore the regulation of signaling pathways.Further study on the regulation of mi RNAs by metformin could result in both novel therapeutic strategies for recurrent or drug resistant cancer,and combination approaches with conventional anticancer therapies.Methods:Pancreatic cancer PANC-1 cells were cultured in vitro and treated with 20mmol/L metformin for 48 h.The PANC-1 cells treated with no metformin were set as the control cells.Then total RNAs were extracted by mir Vana RNA Isolation Kit and quantified by the Nano Drop ND-2100 and the RNA integrity was assessed using Agilent 2100.Affymetrix mi RNA3.0 and Affymetrix HTA2.0 microarray techniques are used to detect total RNA extracted from pancreatic cancer cells.Differentially expressed mi RNAs were then identified through fold change as well as P value calculated using t-test.The threshold set for up-and down-regulated genes was a fold change>= 2.0 and a P value<= 0.05.Target genes of differentially expressed mi RNAs were the intersection predicted with Targetscan,PITA,micro RNAorg.To obtain the effective mi RNAs and its target gene according to the results of HTA2.0 microarray test and previous study.Results:1.The quality of RNA samples of pancreatic cancer cells are all qualified.Chip quality of Affymetrix mi RNA3.0 and HTA2.0 chip are qualified and hybridization conditions of that are reliable.2.According to the screening criteria,we preliminarily obtained twenty four differentially expressed mi RNAs,of which seventeen up-regulated mi RNAs and seven down-regulated mi RNAs.3.Target genes of differentially expressed mi RNAs were the intersection predicted with 3databases(Targetscan,PITA,micro RNAorg)and 374 target genes were received totally.4.We acquire effective mi RNAs of metformin based on comprehensive analysis of the results of microarray test and previous study.It is has-mir-23b-5p and its target gene is STK4.Conclusion:With the use of microarray techniques,the effective mi RNA of metformin and its target gene were obtained quickly,efficiently and accurately,which is pointing out the direction for our study and laying a solid foundation for our follow-up work.Part? Effect of mi RNA-23b-5p on PANC-1cells biological behavior and its targeted regulation on STK4 Objective:The aim of the present study was to test the effect of mi R-23b-5p on pancreatic cancer cells biological behavior and further to verify its promotion on pancreatic cancer.Real-time fluorescent quantitive PCR and Western bloting were used to detect the effect of mi R-23b-5p on STK4 expression level in PANC-1 cells.We aim at proving the targeted combination of mi R-23b-5p with STK4 and further confirming the anti-cancer effect of metformin is achieved by down-regulating mi R23b-5p and activating targeted gene STK4.Methods:Pancreatic cancer PANC-1 cells were cultured in vitro.The experimental groups are PANC-1 cells transfected with mi R-23b-5p mimics or inhibitor,and the control groups are transfected with negative control.Total RNA and protein were extracted after the corresponding time,then relative expression of mi R23b-5p was detected to verify whether transfection was successful.CCK-8 method and Transwell invasion assay were respectively used to examine the proliferation and invasion abilities of PANC-1 cells transfected.The PANC-1 cells transfected with mi R-23b-5p mimics were simultaneously treated with 20mmol/L metformin for 48 h,then CCK-8 method and Transwell invasion assay were conduct.Primers of target gene STK4 and internal reference were designed and synthesized.We used Real-time fluorescent quantitive PCR and Western bloting to detect the expression level of STK4 in PANC-1 cells transfected with mi R-23b-5p mimics or inhibitor.Luciferase assays were carried out with PANC-1 pancreatic cancer cells transfected with STK4 fluorescence report plasmid vector and mi R-23b-5p mimics(or mi R-23b-5p inhibitor)as the experimental group and PANC-1 cells transfected with negative control as the control group.Results:1.Cell transfection was conducted successfully and quality of mi R-23b-5p mimics and inhibitor are reliable for subsequent experiments.2.CCK 8 assay showed that proliferative activity of PANC-1 pancreatic cancer cells transfected with mi R-23b-5p mimics was significantly elevated compared with negative control.Meanwhile,the proliferation of cells transfected with mi R-23b-5p inhibitor was restrained.The differences between experimental groups and control groups are statistically significant(P <0.01).3.In Transwell invasion assay,we found that cell number of mi R-23b-5p mimics group was markedly more than control group when mi R-23b-5p inhibitor group was much less.This discovery suggest that mi R-23b-5p could enhance invasion abilities of PANC-1 cells in vitro.4.The proliferation ability of PANC-1 cells treated with mi R-23b-5p mimics and metformin was obviously weaker than control group cells,but much better than cells treated with metformin only.5.After transfected with mi R-23b-5p mimics,the relative expression of STK4 m RNA in PANC-1 cells was reduced while significantly increasd in cells transfected with mi R-23b-5p inhibitor.Western bloting test showed a corresponding trend.The differences between experimental groups and control groups are statistically significant(P<0.01).6.Luciferase activity of PANC-1 cells transfected with wild STK4 sequence fluorescence report plasmid vector and mi R-23b-5p mimics was markedly raised compared with control cells,the differences are statistically significant(P <0.01).However,there are no statistically significant differences(P> 0.05)of luciferase activity of cells between mutant type and control groups.Conclusion:From the above results,we can initially come to the conclusion that mi R23b-5p significantly enhanced PANC-1 pancreatic cancer cells proliferation and migration ability.Moreover,mi R23b-5p could significantly suppress the inhibition of metformin on PANC-1 cells proliferation,which is achieved by targeted regulation on STK4.