Baicalin,the Major Component Of Traditional Chinese Medicine Scutellaria Baicalensis Induces Colon Cancer Cell Apoptosis Through Inhibition Of MicroRNAs

Objective:To explore the molecular mechanisms underlying the contribution of baicalin to the treatment of colorectal cancer.Methods:Cell culture:The human colon cancer cell line HT-29 were maintained in RPMI 1640 with 10%foetal bovine serum(GIBCO,CA,USA)and incubated in 5%CO2 at 37? in a water-saturated atmosphere.Cell Viability Assay:The cell viability tests were analyzed by the standard cell counting kit-8(CCK-8)assay method.HT-29 cells were seeded into a 96-well plate(1 ×104 cells per well)in cell culture medium.After 12 h,the medium was replaced with 100 ?L of fresh medium containing different concentrations of baicalin(0-600 ?M)and incubated for a further 24 h,or incubated with baicalin at 150?M for 0-48 h.Cells were then washed twice with PBS and incubated with 110 ?L fresh medium containing 10 ?L CCK-8 solutions for a further 3 h.Finally,the medium was removed and the absorbance at 460 nm was measured using a microplate reader(TECAN M200 infinite Pro).The cell viability(%)was calculated by the following formula,where[A]is the average absorbance:Cell Viability(%)=([A]460(sample)-[A]460(blank))/([A]460(control)-[A]460(blank))*100.Cell Transfection:To achieve miR-10a,miR-23a,miR-30c,miR-31,miR-151a and miR-205 overexpression,cells were transfected with miRNA mimics,while knockdown of these miRNAs was achieved by transfecting cells with miRNA antisense.Synthetic miRNA mimic,antisense and scrambled negative control RNAs(pre-miR-control and anti-miR-control)were purchased from GenePharma(Shanghai,China).A scrambled siRNA was included as a negative control.The cells were seeded in 6-well plates and transfected using Lipofectamine 3000(Invitrogen)according to the manufacturer's instructions.Baicalin treatment:Baicalin was dissolved in dimethyl sulfoxide(DMSO)in a 100?M stock solution,stored at-20?,and diluted to different concentrations with culture medium right before experimental use.A same volume of DMSO with a final concentration of 0.08%was added to the controls.Flow cytometry analysis of apoptosis:HT-29 cells were treated with baicalin at different concentrations(0,50,100,150 and 200?M)or c-myc siRNA or c-myc vectors for 48 h.Pan-caspase inhibitor Z-VAD-FMK(Biovision,USA)was applied following manufacturer's procedure.Cells were harvested and washed twice with PBS,and then re-suspended in binding buffer followed by staining with Annexin V-FITC/PI at room temperature for 15 min in the dark.Apoptotic cells were evaluated afterwards by gating PI-and Annexin V-positive cells on a fluorescence-activated cell-sorting(FACS)flow cytometer,and total apoptotic cells were counted as the sum of early apoptotic(PI-AV+)and late apoptotic(PI+ AV+)cells.All experiments were performed in triplicate.RNA isolation and qRT-PCR:Total RNA was extracted from cells and tissues with TRIzol.RNA was reverse-transcribed to cDNA with AMV reverse transcriptase and a stem-loop RT primer.TaqMan miRNA probes were used to quantify miRNAs.Real-time PCR was performed using a TaqMan PCR kit on an Applied Biosystems Roche Sequence Detection System.Relative expression of miRNA relative to the internal control U6 was determined using the 2-?? CT method:?? CT?(CTmiRNA-CTU6)target-(CTmiRNA-CTU6)control.Protein isolation and western blotting:All cells were washed with PBS,tissue samples were frozen solid with liquid nitrogen,and were subsequently grounded into powder.Protein extraction was achieved using RIPA Lysis buffer supplemented with a Protease and Phosphatase Inhibitor Cocktail.Extracted protein concentration was calculated using a Pierce BCA protein assay kit.The protein levels were analysed via western blotting using the corresponding antibodies,and were normalized to GAPDH expression.Profiling of miRNA expression:MiRCURY LNA Array system was used to detect the expression profiles of miRNAs in HT-29 cells treated with 150 ?M baicalin.The median normalization method was used to obtain normalized data.The threshold value for significance,with a fold change>2 and FDR<0.05,was used to define miRNA upregulation or downregulation.Establishment of tumour xenografts in mice:Mice were were maintained under specific pathogen-free conditions in accordance with institutional policies at Nanjing University.HT-29 cells were injected subcutaneously into six-week-old male nude mice(2 × 106 cells per mouse)to induce tumor formation.After inoculation for one week,the mice were treated with baicalin(50 mg/kg or 100 mg/kg)or vehicle(10%DMSO and 90%PBS)by intraperitoneally injection once every day for 3 weeks,with 5 mice in each group.Tumor sizes were measured every 3 days,and the ellipsoid volume was calculated as follows:Volume=(length)×(width)×(width)/2.After treatment for three weeks,mice were sacrificed and anatomized.The tumour xenografts were collected and weighed.A portion of the tissues were used for protein and total RNA extraction,the others were fixed in 4%paraformaldehyde and then processed for H&E staining or immunohistochemical staining to detect Ki-67 and cleaved-Caspase3.Pathological examinations and immunohistochemistry:Pathological examinations were carried out by Servicebio.For tissue morphology evaluation,H&E staining was performed on sections from embedded samples.Ki67 and cleaved-Caspase3 expression was assessed at a hot spot under×200 magnification using Image-Pro Plus software.The number of positive cells per 500 cancer cells was counted in each tumour.Results:Baicalin inhibits cell growth and induces apoptosis in HT-29 colon cancer cells:Baicalin has significant inhibition on growth in HT-29 cells with half-maximal inhibitory constants(IC50)of 165.5?M,and a time-dependent loss of cell viability after exposure to baicalin was observed.Baicalin induced the apoptosis of HT-29 cells in a dose-dependent manner,and it also induced apoptosis in colon cancer cell lines SW-480 and CAC02.Cleaved-caspase3 was significantly increased in baicalin-treated HT-29 cells compared with untreated cells,while apoptotic inhibitor Z-VAD-FMK significantly recovered the viability inhibition induced by baicalin.Baicalin suppresses the expression of a large number of oncomiRs in colon cancer cells:The miRNA expression in HT-29 cells treated with baicalin using an Exiqon miRCURY LNA microRNA Array showed that the level of miRNAs differed significantly between control samples and baicalin-treated samples.Of the 2000 miRNAs detected on the microarray,37 miRNAs were found to be significantly downregulated,while only 8 miRNAs were upregulated after baicalin treatment(fold change>2).Among the downregulated miRNAs,at least 20 miRNAs are reported as oncomiRs,including miR-92a,miR-222,miR-664b,miR-23a,miR-10a,let-7g,miR-93,miR-192,miR-205,let-7b,miR-191,miR-210,miR-31,miR-30c,miR-205,miR-151a,miR-106b,miR-200c,miR-34a and miR-32.Among the upregulated miRNAs,only two miRNAs(miR-204 and miR-638)are reported to have an anticancer effect.Therefore,we focused our study on the downregulated miRNAs.MiRNAs mimics the pro-apoptotic effects of baicalin in colon cancer cells:Levels of miR-10a,miR-23a,miR-30c,miR-31,miR-151a and miR-205 in baicalin treated HT-29 cells were remarkably decreased and miR-204 was increased in baicalin-treated cells compared with the control cells,while the expression of two control miRNAs(miR-1 and miR-16)had no significant changes between baicalin-treated cells and control cells.Compared with the baicalin only group,the cellular levels of miR-10a,miR-23a,miR-30c,miR-31,miR-151a and miR-205 were dramatically increased in HT-29 cells after transfection with corresponding mimics and dropped significantly after treatment with corresponding antisense.Transfection of miRNA inhibitors separately could significantly induce apoptosis in baicalin treated HT-29 cells,whereas transfection of their mimics markedly attenuated baicalin induced apoptosis.C-myc was suppressed in a dose-dependent manner in HT-29 cells treated with baicalin at various concentrations.The expression levels of miR-10a,miR-23a,miR-30c,miR-31,miR-151a and miR-205 were significantly decreased along with c-Myc,while the expression levels of miR-16 and miR-1 did not significantly change,and miR-204 was significantly increased in c-Myc siRNA-treated cells compared with the untreated control cells.C-myc siRNA induced cell apoptosis as well as baicalin did,while c-myc overexpression attenuated the inhibitory effect of baicalin on cell apoptosis.Baicalin suppresses tumour growth in xenografted nude mice:Compared with the control group,baicalin had a significant inhibitory effect on tumour growth.Haematoxylin and eosin(H&E)staining of xenograft tissues showed fewer mitotic cells in the group treated with baicalin compared with the control group,along with a smaller number of inflammatory cells and less necrocytosis.The cell proliferation rate evaluated by immunocytochemistry with the mouse monoclonal antibody Ki-67 revealed that the percentage of Ki-67-positive tumour cells was significantly declined in the baicalin treated group.Considerably stronger cleaved-caspase3 staining intensities were detected in baicalin-treated tumor tissues relative to control tissues.Consistent with the in vitro findings,miR-10a,miR-23a,miR-30c,miR-31,miR-151a and miR-205 were significantly downregulated in baicalin-treated mice,while miR-16 and miR-1 exhibited no significant change,and miR-204 was significantly upregulated.Conclution:Baicalin inhibits tumour growth by repressing the expression of c-myc and oncomiRs to induce apoptosis.