The Effect And Mechanism Of Action Of Metformin On FaDu Cell Proliferation In Vitro

Background:Hypopharyngeal carcinoma is one of the most highly malignant tumors of the upper aerodigestive tract and is difficult to diagnose early due to occult lesion location.Despite constant progresses in medical and surgical therapy,the five-year survival rate of this cancer has not improved over the past decade.Indeed,the prognosis of hypopharyngeal carcinoma is very poor,and the 5-year overall survival rate is approximately 15%to 45%.Metformin,as an insulin sensitizer,is the first line drug for the treatment of type 2 diabetes.Several studies have demonstrated that metformin can reduce the incidence of a wide variety of tumors in diabetic patients.In addition,metformin has been shown to inhibit the growth and proliferation of many cancer cells in vitro and in vivo(e.g.,liver,prostate,cervical and ovarian cancer).However,the effect of metformin on hypopharyngeal carcinoma is unclear.Abnormal expressions of the microRNA,miR-21-5p,and its downstream target gene,programmed cell death 4(PDCD4)have been reported to occur in multiple tumors.Moreover,the expression of miR-21-5p in hypopharyngeal carcinoma is significantly upregulated.To date,there is little published information about the effects of metformin on the expression of miR-21-5p and PDCD4 in hypopharyngeal tumor cells.Purpose:We designed a study to investigate the effects of metformin on the proliferation of the human hypopharyngeal carcinoma cell line(FaDu)and the expression of miR-21-5p and PDCD4 in these cells.Methods:1.Cells culture:The human hypopharyngeal carcinoma cell line(FaDu)was purchased from the American Type Culture Collection(ATCC(?),Manassas,VA,USA)and stored in liquid nitrogen until use.Cells were cultured in Dulbecco's modified Eagle's medium(DMEM)with 10%fetal calf serum(FCS),100 IU/ml penicillin and 100 mg/ml streptomycin at 37 ? in 5%carbon dioxide in air.2.Cell dividing:After three consecutive passages,cells were divided into groups:0 mmol/L metformin(control),25 mmol/L metformin,50 mmol/L metformin,75 mmol/L metformin,100 mmol/L metformin,and 125 mmol/L metformin(Sigma,St Louis,MO,USA).3.Cell proliferation assay:Cells were plated at 2x104/ml/well in 96-well plates,cultured for 36 h,washed twice with phosphate buffered saline(PBS),then cultured as described(one plate/timepoint,three wells/group).Cells in each group were cultured for 12 h,24 h and 36 h.Cell proliferation was quantified via CCK-8 assay,according to the manufacturer's instructions(Dojindo,Kunamoto,Japan).Optical density(OD)was measured three times at 450 nm,and growth inhibiting rate was calculated as:(OD experimental group-OD blank)/(OD control group-OD blank)�100%.4.RNA extraction and Real time qRT-PCR:Cells were incubated with metformin(25 mmol/L,50 mmol/L,75 mmol/L and 100 mmol/L)for 24 h.Total RNA was extracted using Trizol reagent(Invitrogen,Grand Island,NY,USA)according to the manufacturer's instructions,and cDNA was synthesized using the poly-A tailing method.17 PCR was performed for miR-21-5p with U6 RNA as internal reference,using a microRNA qRT-PCR detection kit(FulenGen Corporation,Guangzhou,China).Cycling conditions were lOmin at 95 ? followed by 40 cycles of 95? for 10s,60 ? for 20s,and 72 ? for 10s.PCR for PDCD4 was performed using a SY-BR(?)Green I real time qPCR(Roche Corporation,Basel,Switzerland)with P-actin as internal reference.Cycling conditions were 95 ? for 20s,followed by 40 cycles of 95? for 3s,and 55 ? for 30s.All PCR experiments were performed in triplicate.Expression levels of PCR targets in experimental groups were determined relative to control cultures using the 2-??Ct method.5.Western blot:Cells were incubated with metformin(25 mmol/L,50 mmol/L,75 mmol/L and 100 mmol/L)for 24 h,washed twice with PBS,and lysed with RIPA buffer(Beyotime,Jiangsu,China)on ice for 30 min.Lysates were centrifuged at 12000 �g for 5 min at 4 ?,and the protein concentration of the supernatant was determined using a BCA assay kit(Beyotime).Total protein(40 mg)was separated by 10%sodium dodecyl sulphate-polyacrylamide gel electrophoresis,transferred to polyvinylidene difluoride membranes,and blocked with 5%skimmed milk at room temperature for 2 h.Membranes were incubated overnight at 4 ? with mouse monoclonal antihuman-b-actin antibody(1:1000 dilution;Santa Cruz Biotechnology,Santa Cruz,CA,USA)or rabbit polyclonal antihuman PDCD4 antibody(1:2000 dilution;Abcam Corporation,Cambridge,UK),washed three times with tris buffered saline�Tween 20,then incubated at room temperature for 1 h with horseradish peroxidase labelled goat antirabbit or goat antimouse secondary antibody(1:5000,Santa Cruz Biotechnology).Immunoreactive protein bands were detected using an enhanced chemiluminescence(ECL)kit,according to the manufacturer's instructions(Beyotime)and quantified via densitometric analysis using software Image J(Image Processing and Analysis in Java).All assays were performed in duplicate.6.Statistical analyses Data were presented as mean � SD.Between group differences were analysed using one-way analysis of variance(ANOVA)for multiple group comparisons and Student-Newman-Keuls q-test for paired comparison.Statistical analyses were performed using SPSSversion 17.0(SPSS Inc,Chicago,IL,USA)for Windows(?).And P-value<0.05 was considered statistically significant.Results:1.Metformin significantly inhibited cell proliferation in a dose-(P<0.05)and time-dependent manner(P<0.05).There was no significant difference in cell proliferation between 100 mmol/L and 125 mmol/L metformin at any timepoint.2.Incubation with metformin for 24 h significantly inhibited miR-21-5 p expression(each dose P<0.05 vs control;Table 2).Levels of PDCD4 mRNA(each dose P<0.01 vs control)and protein(each dose P<0.05 vs control)were significantly upregulated by incubation with metformin.Conclusions:1.Metformin can significantly inhibit the proliferation of FaDu cells in a dose-and time-dependent manner.2.The inhibition dues to the downregulation of miR-21-5p,and the upregulation of PDCD4.