Curcumin Inhibits Proliferation And Migration Of Gastric Cancer Cells Via Upregulation Of MiR-133a-3p And Downregulation Of MMP15

Gastric cancer(GC)is the fourth common cancer and the second cause leading to cancer death in the world.There are about 989,600 new cases and 738,000 deaths worldwide each year.More than 70%of new cases and deaths occur in developing countries,half in East Asia and mainly in China.The pathogenesis of GC relates to genetic factors,diet,Helicobacter pylori infection and other factors.The survival rate of patients with gastric cancer is highly dependent on the stage of disease.Most patients with gastric cancer were diagnosed late for lack of early obvious symptoms and timely screening,leading to reduced 5-year survival after surgical resection.The accuracy of commonly tumor markers,such as carcinoembryonic antigen(CEA)and carbohydrate antigen CA19-9,is unsatisfactory in clinic.The endoscopic examination combined with gastric mucosal biopsy is still the most reliable diagnostic indicator.Therefore,the exploration of new biomarkers for early GC diagnosis is the main focus in the current study.MicroRNA(miRNA)is a long primitive non-coding small RNA that is transcribed by RNA polymerase Ⅱ.MiRNA can post-transcriptively silence the target gene by interacting with the untranslated region to regulate many important biological processes,such as cell period,cell differentiation,migration and apoptosis.Besides,miRNAs play an important regulatory role in expression of oncogene or tumor suppress genes to develop or inhibit tumors progress.MiR-133a is a muscle-specific miRNA that regulates development and differentiation of myoblast,and is correlative to many myogenic disorders and tumor development,including gastric cancer,colorectal cancer,ovarian cancer,lung cancer,breast cancer,bladder cancer and so on.However,the role and related mechanisms of miR-133a-3p,a functional mature form of miR-133a,in proliferation,migration,invasion,apoptosis of gastric cancer cell are rare reported.Matrix metalloproteinase(MMP)family is named as a cofactor for metal ions such as Ca2+,Zn2+,which consist of hydrophobic signal peptide sequences,propeptide regions,catalytic regions,hinge regions and carboxyl terminal regions.MMPs can degrade the protein composition of the extracellular matrix and destroy the barrier of tumor cell invasion to make for tumor invasion and metastasis.As a membrane-type MMP(MT-MMP),MMP 15 can induce the activation of Gelatinase A in a cell-mediated manner in breast cancer,ovarian cancer,cervical cancer,colorectal cancer,and other malignant tumors.According to bioinformatics software prediction,MMP15 and miR-133a-3p have a same conservative binding site.It is worthy of further study whether miR-133a-3p is able to regulate MMP15 and affect the progress of gastric cancer.Curcuma longa L belongs to the ginger family and is a perennial herb.Its dry rhizome as a medicine can "Xing Qi Po Yu,Tong jing zhi Tong".In recent years,curcumin from a major extract of turmeric,may contribute to resist tumor,inhibit platelet aggregation,reduce blood fat,inhibit bacteria,gallbladder,and blood pressure.The anti-cancer roles of curcumin is relative to accelerate the apoptosis of cancer cells,and regulate inflammation,angiogenesis and tumor microenvironment,as well as disturb many cancer signaling pathways,such as Ras,PI3K,p53,AKT,mTOR,Wnt-β catnin.In the present study,the expression of miR-133a-3p was checked by using real-time fluorescence quantitative PCR in gastric carcinoma,atrophic gastritis,gastric ulcer,superficial gastritis,normal gastric mucosa tissues and gastric juice.The expression of MMP15 was detected by immunohistochemical method in gastric carcinoma.In the current study we examined whether miR-133a-3p can infect proliferation,migration,invasion and apoptosis of gastric cancer cells,and whether curcumin can regulate proliferation,migration,invasion and apoptosis of gastric cancer by adjusting expression of MMP15 and miR-133a-3p.Part I The expression and clinical significance of miR-133a-3p and MMP15 in patients with gastric cancerOBJECTIVE:To study the expression of miR-133a-3p in gastric carcinoma,atrophic gastritis,gastric ulcer,superficial gastritis,normal gastric mucosa tissues and gastric juice,and to evaluate the possibility of miR-133a-3p screening for gastric cancer in gastric juice.To study the expression of MMP15 in gastric cancer tissue and analyze the correlation between MMP15 expression and miR-133a-3p and the relationship between the expression of miR-133a-3p and the mortality and survival rate and prognosis of gastric cancer patients.METHODS:Collected surgical biopsy specimens and 3ml gastric juice from subsidiary hospital of YangZhou University from June 2013 to June 2015,including 202 cases of primary gastric cancer,32 cases of atrophic gastritis,36 cases of gastric ulcer and 40 cases Superficial gastritis,normal mucosa 34 cases.Centrifuge the gastric juice,supernatant and tissue stored at-80℃.Extract the total RNA of gastric cancer,atrophic gastritis,gastric ulcer,superficial gastritis and normal gastric mucosa in tissue and gastric juice,and then reverse transcribe synthesis cDNA.The expression of miR-133a-3p was detected by real-time fluorescence quantitative PCR.The expression of miR-133a-3p was detected by real-time fluorescence quantitative PCR at 4 ± 1 0 C and tested at 0,3,6,9,12,15,18,21 and 24 hours to assess the stability of miR-133a-3p in gastric juice.The differences in miR-133a-3p expression were compared by one-way ANOVA,followed by Tukey multispectral examination.The correlation of miR-133a-3p expression in gastric juice and biopsy mucosa was analyzed by Pearson correlation test.To construct the ROC curve to evaluate the diagnostic value of miR-133a-3p in gastric juice.The expression of MMP15 in gastric cancer tissue was studied by immunohistochemical method,and analysis the correlation between MMP15 and miR-133a-3p expression.RESULTS:The gastric mucosa of patients with gastric cancer(F[4,204]= 56.5,p<0.001)and gastric juice(F[4,204]= 52.5,p<0.001)were significantly lower than those in other benign and normal controls.The Pearson correlation test showed that the level of miR-133a in gastric juice was significantly correlated with the expression level of miR-133a-3p in gastric juice,and the Pearson correlation coefficient was 0.972(p<0.0001).There was no significant difference in the expression of miR-133a-3p of gastric juice in different time(F[4,204]= 2.49,p = 0.32),it was stable.The area under the ROC curve of the miR-133a in gastric carcinoma,atrophic gastritis,gastric ulcer,superficial gastritis,gastric juice were 0.826.0.598,0.683,0.625(95%CI ＝0.744-0.912,p<0.001),with sensitivity and specificity of 82.4%and 71.8%respectively in GC.There was a significant difference of the positive MMP15 expression rate between miR-133a-3p overexpression and low expression(p<0.05),Low expression of miR-133a in gastric cancer was associated with a progression and poor prognosis of gastric cancer.CONCLUSIONS:The expression of miR-133a-3p is significantly reduced in gastric cancer patients,which may be involved in the development and progression of gastric cancer and may play an important role in regulating the expression of MMP15.It can be used as one of the high risk indexes of gastric cancer.The expression of miR-133a-3p was stable in gastric juice,high operability,high sensitivity.This discovery has a certain clinical value for the early diagnosis and treatment of gastric cancer and provides a new way for studying.Part Ⅱ MiR-133a-3p and MMP15 inhibit proliferation,migration and invasion of gastric cancer cells and involved mechanismOBJECTIVE:To study the effects of miR-133a-3p on the proliferation,migration,invasion and apoptosis in gastric cancer cells,and to explore whether its mechanism is related to targetting MMP15.METHODS:The biological information software Targetscan,MIRDB and RNA22-HSA were used to predict whether MMP15 and miR-133a-3p had conserved binding sites.Constructed MMP15 3’UTR-PGL3 and mut MMP15 3’UTR-PGL3 plasmids and then transfected into HEK293-T cells.The targeting effect of miR-133a-3p on MMP15 was detected by double luciferase reporter assay.Real-time fluorescence quantitative PCR was used to detect the expression of miR-133a-3p in normal human gastric epithelial cells GES1 and human gastric cancer cell lines SGC-7901,MGC-803,AGS,HGC-27,BGC-823 and MKN-45.Western blotting was used to detect the expression of miR-133a-3p in Epithelial cells GES1 and human gastric cancer cell lines SGC-7901,MGC-803,AGS,HGC-27,BGC-823,MKN-45.The MSCV-miR-133a-3p plasmid was constructed and transfected into SGC-7901 and HGC-27 cells and the cells were screened with puromycin.MTT assay was used to detect the effect of miR-133a-3p on the proliferation in gastric cancer cells SGC-7901 and HGC-27.Scratch test was used to detect the migration of SGC-7901 and HGC-27.The effection of miR-133a-3p on the invasion in gastric cancer cells SGC-7901 and HGC-27 was detected by Transwell invasion assay.Real-time quantitative PCR and Western blotting was used to detect the effect of miR-133a-3p on the expression of MMP15 mRNA and protein in gastric cancer cell SGC-7901 and HGC-27 to further determine the role of miR-133a-3p in the regulation of MMP15.RESULTS:The bioinformatics software predicted that MMP15 and miR-133a-3p had conserved binding sites.The double luciferase reporter assay showed miR-133a-3p could negatively regulate the expression of MMP15.The expression of miR-133a-3p in normal gastric mucosal cell GES1 was significantly higher than that in SGC-7901,MGC-803,AGS,HGC-27,BGC-823 and MKN-45(p<0.05)SGC-7901,HGC-27 cells,and we selected the lowest SGC-7901 and HGC-27 to do the following experiment.The levels of MMP15 protein in SGC-7901 and HGC-27 were higher than other cells.We observed green fluorescence in both SGC-7901 and HGC-27 cells transfected with miR-133a-3p and empty vector.The expression of miR-133a-3p was significantly increased in cells transfected with miR-133a-3p-MSCV-PIG(p<0.05).In the miR-133a-3p overexpressing SGC-7901 and HGC-27 cells,the proliferation ability of the cells was significantly inhibited(p<0.05),which indicated that exogenous miR-133a-3p could decrease the cell proliferation ability.In the miR-133a-3p overexpressing SGC-7901 and HGC-27 cells,the migration ability of the cells was significantly inhibited(p<0.05),which indicated that exogenous miR-133a-3p could decrease the cell migration ability.In the miR-133a-3p overexpressing SGC-7901 and HGC-27 cells,the invasion ability of the cells was significantly inhibited(p<0.05),which indicated that exogenous miR-133a-3p could decrease the cell invasion ability.In the miR-133a-3p overexpressing SGC-7901 and HGC-27 cells,the mRNA and protein levels of MMP15 were significantly decreased(p<0.05),suggesting that miR-133a-3p could negatively regulate MMP15.CONCLUSIONS:Overexpression of miR-133a-3p in gastric cancer cells inhibits the proliferation,migration,invasion.This mechanism may be related to miR-133a-3p target the MMP15 expression,the discovery provides a new direction for gastric cancer molecular biology research.Part Ⅲ Curcumin inhibits proliferation,migration,invasion of gastric cancer cells through upregulation of miR-133a-3p and downregulation of MMP15OBJECTIVE:To investigate the effect of curcumin on the proliferation,migration,invasion and apoptosis in gastric cancer cells SGC-7901 and HGC-27,and to explore whether this effect is related to the regulation of MMP15 by miR-133a-3p.METHODS:SGC-7901 and HGC-27 cells were grown to about 80%in logarithmic growth phase.Curcumin(0,5,10,20,30,40μg/ml)was added at different concentrations.MTT assay was used to detect the effect of curcumin on the proliferation of gastric cancer cells SGC-7901 and HGC-27.The effects of curcumin on the migration of SGC-7901 and HGC-27 in gastric cancer cells were compared by scratch test.Transwell invasion assay was used to detect the changes of gastric cancer cell line SGC-7901 and HGC-27 invasion after curcumin treatment.Flow cytometry was used to detect the changes of apoptosis rate of SGC-7901 and HGC-27 in gastric cancer cells treated with curcumin for 24 h.The effect of curcumin on mRNA expression of miR-133a-3p and MMP15 in SGC-7901 and HGC-27 cells was detected by real-time fluorescent quantitative PCR.Western blotting was used to detect the effect of curcumin on the expression of MMP15 protein in SGC-7901 and HGC-27 cells.RESULTS:Curcumin inhibited the proliferation of gastric cancer cells SGC-7901 and HGC-27,and had time and concentration dependence.Curcumin inhibited the migration and invasion of gastric cancer cells SGC-7901 and HGC-27 had concentration dependence.Curcumin induced apoptosis of SGC-7901 and HGC-27 cells in a dose-dependent manner,of which 30,40μg/ml of curcumin can significantly improve the early apoptosis rate,the apoptotic rate of HGC-27 was 8.75%and 13.83%,and the apoptotic rate of SGC-7901 was 16.56%and 19.94%,which was significantly higher than that of the control group(p<0.05).Real-time fluorescence quantitative PCR showed that curcumin could up-regulate the mRNA level of miR-133a-3p in SGC-7901 and HGC-27 cells in a dose-dependent manner and down-regulate the mRNA level of MMP15.Western blotting showed that curcumin in two cells can reduce the expression of MMP15 protein.CONCLUSIONS:Curcumin can significantly inhibit the proliferation,migration,invasion and induce apoptosis in SGC-7901 and HGC-27 cells,and the mechanism may be related to the curcumin-induced miR-133a-3P targeting MMP15.Our findings provide a theoretical basis and research direction for the development of new anti-gastric cancer drugs.In summary,the results of the present study proved the low expression of miR-133a-3p and overexpression of MMP15 in gastric cancer specimens and its association with development,invasion and metastasis of gastric cancer,suggesting that miR-133a-3p may be involved in against carcinogenesis and progression of gastric cancer.We further demonstrated that curcumin can significantly inhibit the proliferation,migration and invasion of gastric cancer cells,and induct apoptosis of the cells through downregulation of MMP15 and up-regulation of miR-133a-3p.These findings indicated that miR-133a was an independent diagnostic and prognostic factor of patients with gastric cancer,and even a potential therapeutic target in patients with gastric cancer.