| China is one of the countries with a high incidence of gastric cancer.The morbidity and mortality in Chinese patients with gastric cancer accounted for 42.6% and 45.0% of the global gastric cancer patients,respectively.According to the China Cancer Register Annual Report,there were approximately 679,000 new cases of gastric cancer,and approximately 468,000 deaths from gastric cancer in 2015.Moreover,gastric cancer had the characteristics of strong heterogeneity,poor efficacy,recurrence and poor prognosis,which seriously threaten people's health.Surgery is the main treatment for eradicating early gastric cancer,and traditional chemotherapy and radiotherapy are important treatments for advanced and recurrent metastatic gastric cancer.In recent years,precise treatment of gastric cancer had achieved initial efficacy.Although Individualized therapies targeting HER-2,VEGFR-2,and PD-1/PD-L1 had achieved breakthroughs in the treatment of advanced gastric cancer,the overall prognosis of gastric cancer remained to be improved.Therefore,it had become an academic focus to further research the molecular mechanism of gastric cancer development and to seek new and more effective therapeutic targets and methods.MicroRNAs(miRNAs)are single-stranded,non-coding RNAs of about 21-25 nucleotides in length that bind to the 3' untranslated regions(UTRs)of the target gene and can degrade or inhibit the expression of the target gene mRNA and exert post-transcriptional regulation.Numerous studies had confirmed that miRNAs can regulate about 30% of mRNAs in humans and have powerful biological functions.There was a causal relationship between dysregulation of miRNAs and many kinds of tumors,which may play an important role in tumorigenesis and development of tumors as oncogenes or tumor suppressor genes.Recently,miRNA mimics and molecules targeted at miRNAs(antimiRs)had shown promise in preclinical development.Several miRNA-targeted therapeuticses had reached clinical trial,and showed a good development prospect.MiRNA-33(miR-33)is located in the intron sequence of the sterol regulatory element-binding protein(SREBP)gene and includes both a and b members.They target genes involved in regulating cholesterol homeostasis and fatty acid metabolism(Abca1,Abcg1,Npc1,Ampk,Cpt1 a,Crot and Sirt6).The most classical pathway is the inhibition of ABCA1 by miR-33,resulting in reduced cholesterol output and reduced HDL synthesis.Recently,it had been reported that miR-33 may play a role in the development of malignant tumors.Most studies suggested that the main function of miR-33 a was tumor suppressor genes,but miR-33 a was also reported to be an oncogene.The signaling pathways regulated in different tumors were not the same,and the roles of miR-33 a in the different tumors were also varies.Thus,the roles and mechanisms of miR-33 a in malignancies need to be further studied.Moreover,the expression,function,and regulatory mechanisms of miR-33 in gastric cancer had not been extensively studied.In this study,two members of the miR-33 family,miR-33a/b,were detected by quantitative real-time reverse transcription-polymerase chain reaction(qRT-PCR)in gastric cancer and matched para-cancer tissue specimens.The relationships between the expression of miR-33 a and the clinicopathological features,tumor markers and prognosis of gastric cancer patients were analyzed.The expression of miR-33 a in one normal gastric epithelial cell line GES-1 and four SGC-7901,MKN-28,HGC-27 and BGC-823 gastric cancer cell lines was also detected by qRT-PCR.MiR-33 a was over-expressed or silenced by miRNA transfection technique.Flow cytometry,CCK-8(Cell-Counting Kit-8 assay),wound healing assay,and Transwell assay were used to investigate the biological functions of miR-33 a in SGC-7901 gastric cancer cell line,such as proliferation,cell cycle and invasion and migration.Bioinformatics software was used to predict miR-33 a downstream target genes,and its predicted target genes CDK-6,CCND-1,PIM1 and P53 were determined by dual luciferase reporter gene assay.The expression of target genes was detected by qRT-PCR and western-blot testing after transfection of miR-33 a mimic and inhibitor,and the regulation mechanisms of miR-33 a on target genes were clarified.The expression of CDK-6,CCND-1,and PIM1 in gastric cancer tissues and their paired paracancer tissues was detected to verify the relationship between miR-33 a and the target genes.The main research contents and results were shown as follows: Part one Expression of miR-33a/b in gastric carcinoma and its relationships with clinicopathological features and prognosisObjective: To detect the expression of miR-33 family in gastric cancer tissues and matched paraneoplastic tissues,and to explore the relationship between miR-33 family expression and clinicopathological features and prognosis of gastric cancer patients.Methods: The qRT-PCR technique was used to detect the differential expression of miR-200 family in gastric cancer tissues and paired noncancerous tissues.The relationships between miR-33 a expression and the clinicopathological features,tumor markers,and disease-free survival(DFS)of patients with gastric cancer were analyzed.Results:1.The expression of miRNA-33 a/b in gastric carcinoma and matched paracancer tissues.The down-regulation rates of miR-33 a and miR-33 b expression were 75.44%(43/57)and 56.14%(32/57)in cancer specimens and paired paraneoplastic tissue after operation in 57 patients with gastric cancer.MiR-33 a expression in gastric cancer tissues(Median: 0.04516;Interquartile Range:0.74817)was significantly lower than miR-33 a expression in paired paracancer tissues(Median: 0.07197;Interquartile Range: 1.39304,P<0.001).The expression of miR-33 b in gastric cancer tissues(Median: 0.00372;Interquartile Range: 0.00521)was not significantly different from that in adjacent tissues(Median: 0.00371;Interquartile Range: 0.00681)(P=0.113).2.The relationship between the expression of miR-33 a in gastric cancer and the clinicopathological features of gastric cancer.In 57 patients with gastric cancer,miR-33 a expression was significantly associated with tumor differentiation,tumor TNM stage,T stage,and M stage(P=0.000,P=0.002,P=0.005,and P=0.000),but miR-33 a expression was not significantly associated with gender,age,N stage,and blood vessel invasion(BVI)(P=0.211,P=0.127,P=0.113,P=0.643),respectively.Using multiple linear regression analysis,there was a significant negative correlation between tumor differentiation(Beta=-0.391,t=-3.164,P=0.003),tumor M staging(Beta=-0.325,t=-2.016,P=0.049)and the expression of miR-33 a,while there was no significant correlation between the expression of miR-33 a and tumor T stage(Beta=-0.029,t=-0.169,P=0.867),N stage(Beta=0.265,t=1.745,P=0.087)and BVI(Beta=0.038,t=0.297,P=0.768),respectively.3.The relationship between expression of miR-33 a and tumor markers in gastric cancer.The expression of miR-33 a was negatively correlated with the level of CA-199 in peripheral blood of gastric cancer patients(r=-0.407,P=0.002),but there were no significant correlations between the expression of miR-33 a and the expression of CEA,CA-50 and CA-724 in peripheral blood of gastric cancer patients(r=0.067,P=0.632;r=-0.214,P=0.132;r=-0.100,P=0.477).4.The relationship between expression of miR-33 a and DFS in postoperative patients with gastric cancer.In 57 patients with gastric cancer,4 patients were lost to follow-up,and 6 patients with stage IV were excluded.After eliminating these 10 patients,47 patients underwent postoperative DFS analysis.The median DFS for all patients was 13.6 months(range from 6.0 months to 28.9 months).Patients with miR-33a-overexpressing gastric cancer had significantly longer DFS than patients with low miR-33 a expression [median DFS(95% CI): 16.6 months(8.5 months-24.7 months)vs.9.8 months(4.6 months-15.0 months),P=0.04].Conclusions:1.The expression of miR-33 a was significantly lower in gastric cancer tissues than in paracancer tissues,but there was no significant difference in miR-33 b expression between gastric cancer tissues and paracancer tissues,suggesting that miR-33 a may play a role in the development of gastric cancer.2.The expression of miR-33 a was negatively correlated with tumor tissue differentiation,distant metastasis and CA-199 in peripheral blood of gastric cancer patients,respectively.These data suggested miR-33 a may be involved in differentiation,proliferation and metastasis of gastric cancer.3.The expression level of miR-33 a was significantly associated with DFS in patients with postoperative gastric cancer.Compared with patients with low expression of miR-33 a,patients with high expression of miR-33 a have significantly longer DFS.It suggested miR-33 a may be a potential prognostic factor for gastric cancer.Part two The effect of miR-33 a on biological function of gastric cancer cell and the regulation mechanismObjective: To investigate the biological function and the regulation mechanism of miR-33 a in gastric cancer cell.Methods: The qRT-PCR technique was used to detect the different expression of miR-33 a between one normal gastric epithelial cell lines GES-1 and 4 gastric cancer cells including SGC-7901,MKN-28,HGC-27 and BGC-823.MiR-33 a mimics and inhibitors were transfected into gastric cancer cell line SGC-7901 to achieve silence or overexpression of miR-33 a.The effects of miR-33 a on the cell cycle,cell proliferation,invasion and metastasis of gastric cancer cell line SGC-7901 were investigated by flow cytometry,CCK-8 assay,wound healing assay and Transwell assay.Results:1.The expression of miR-33 a in four gastric cancer cell lines and normal gastric epithelial cell line(Mean±SD).Compared with the expression of miR-33 a in normal gastric epithelial cell line GES-1(1.00±0.112),the expression of miR-33 a in gastric cancer cell line MKN-28(0.341±0.031,P=0.000),SGC-7901(0.581±0.042,P=0.000),BGC-823(0.538±0.029,P=0.000)and HGC-27(0.701±0.049,P=0.002)was significantly lower,respectively.This result was consistent with the difference in expression between gastric cancer tissues and paracancer tissues.2.The expression of miR-33 a after transfection of miR-33 a mimic and Inhibitor in human gastric cancer cell line SGC-7901.In this study,SGC-7901 cells were used to design an in vitro cell model,and miR-33 a mimic transfection group,miR-33 a Inhibitor transfection group and AllStars Negative Control siRNA transfection group(negative control group)and untreated group were detected by qRT-PCR after transfected 48 hours.The expression of miR-33 a showed no significant difference between the negative control group and the blank control group(0.96±0.005 vs.1±0.020,P=0.859).The expression of miR-33 a in mi R-33 a mimic group was significantly higher than that of blank control group(434.83±9.58 vs.1±0.020,P<0.001),while the expression of miR-33 a in miR-33 a Inhibitor group was significantly lower than that in the blank control group(0.36±0.03 vs.1±0.020,P<0.001).The above results suggested that the transfection experiments were successful and follow-up experiments could be conducted.3.The effect of miR-33 a on proliferation of human gastric cancer cell SGC-7901.CCK-8 cell proliferation assay showed that,after transfected 48 hours of SGC-7901 cells,the OD values(Mean±SD)of miR-33 a mimic transfection group,miR-33 a Inhibitor transfection group,blank control group,AllStars Negative Control siRNA transfection group(negative control group)were 0.549±0.03,1.54±0.12,1±0.09,and 0.953±0.06,respectively.Compared with the blank control group,the proliferation of SGC-7901 cells in the miR-33 a mimic transfection group was significantly decreased(P<0.001),and the proliferation of the SGC-7901 cells in the miR-33 a Inhibitor transfection group was significantly increased(P<0.001).4.Effect of expression of miR-33 a on cell cycle of human gastric cancer cell line SGC-7901.After transfected 48 hours,flow cytometry results showed that the ratio(Mean±SD)of G0/G1 phase,S phase,and G2/M phase of miR-33 a mimics transfected group was 68.30±0.85%,23.94±0.46%,and 7.76±0.89%,while the proportion of three phases of miR-33 a Inhibitor transfection group was 56.30±0.95%,39.43±0.55%,and 4.27±0.74%,respectively.The proportion of the three phases was 60.94±0.88%,35.74±0.75%,and 3.32±0.56%,respectively.There was a significant difference between the two groups in G0/G1 and S phases(P<0.05),respectively.SGC-7901 gastric cancer cells transfected with miR-33 a were arrested in G1 phase,suggesting an important role of miR-33 in regulating the G1/S transition.5.The effects of miR-33 a on migration and invasion of human gastric cancer cell SGC-7901.The wound healing scores of miR-33 a mimic transfection group,miR-33 a Inhibitor transfection group and the negative control group was 103.41±6.21%,95.376±6.55%,and 100.00±10.06%,respectively.Neither miR-33 a mimic group nor inhibitor transfection group had significant difference compared with the negative control group(P=0.253,P=0.146).Transwell assay results showed that the number of cells passing through each field of miR-33 a mimic transfection group,miR-33 a Inhibitor transfection group,and the negative control group was 87±4.9,83±7.2,76± 5.3,respectively.Neither the miR-33 a mimic transfection group nor the miR-33 a Inhibitor transfection group had significant difference compared with the negative control group(P=0.341,P=0.663).Conclusions:1.The expression of mi R-33 a in gastric cancer cell lines was significantly lower than that in normal gastric mucosal epithelial cells,which was consistent with the difference in expression between gastric cancer tissues and paracancerous tissues.2.miR-33 a inhibited the proliferation of SGC-7901 gastric cancer cells.3.SGC-7901 gastric cancer cells transfected with miR-33 a were arrested in G1 phase,suggesting an important role of miR-33 in regulating the G1/S transition.4.miR-33 a had no significant effect on the invasion and migration of SGC-7901 gastric cancer cells.Part three The regulation mechanism of miR-33 a on the Target Gene PIM1/CCND1/CDK6/P53 in gastric cancer.Objective: To study the regulation effect of miR-33 a on the predicted target gene PIM1/CCND1/CDK6/P53,and to clarify the regulatory pathway and mechanism of miR-33 a on proliferation of gastric cancer cells.Methods: Using the MicroRNA bioinformatics website and software,combined with the effect of miR-33 a on proliferation of gastric cancer cells,the potential target sequences were predicted and screened.Dual-luciferase reporter assays were used to detect the targeting action of mi R-33 a to its target genes.The qRT-PCR and Western-blot were used to detect the expression changes of target genes CDK6,CCND and PIM1 in the gastric cancer cell line SGC-7901 transfected with miR-33 a mimic and Inhibitor.The relationships between miR-33 a and target genes were further verified by detecting the expression of target genes in gastric cancer tissues and paired paracancer tissues.Results:1.Bioinformatics prediction of target genes of Hsa-miR-33 a.Through the online database of bioinformatics website FindTar 3.0,combined with the search results of miRBase,miRanda,PicTar and other databases,this study speculated that CCND1,CDK6,PIM1 and P53 were selected as potential predictive target genes for miR-33a-regulated cell proliferation.2.Verification of the miR-33 a predicted target genes CCND1,CDK6,PIM1,and P53 by dual-luciferase reporting system.Dual-luciferase reporter gene analysis showed that compared with AllStars Negative Control siRNA transfection group(negative control group),the relative fluorescence intensity of PIM1(0.78±0.03 vs.0.97±0.33,P<0.05),CCND1(0.56±0.01 vs.0.98±0.05,P<0.01)and CDK6(0.45±0.01 vs.1.05±0.03,P<0.01)in miR-33 a mimics transfection group were significantly reduced,but there was no significant difference in the relative fluorescence intensity of P53(0.87±0.06 vs.1.04±0.03,P=0.138).It is suggested that the PIM1,CCND1,and CDK6 genes are miR-33 a target genes in gastric cancer cells,whereas P53 is not a target gene of miR-33 a.3.Regulation of expression of miR-33 a in gastric cancer cell line SGC-7901 affects the expression of target genes CCND1,CDK6 and PIM1.After miR-33 a mimic was transfected into SGC-7901 gastric cancer cells,the mRNA expression levels of CDK6,CCND1,and PIM1 were significantly lower than those in the negative control group(0.18±0.01 vs.1.10±0.03,P<0.01;0.21±0.02 vs.0.94±0.09,P<0.01;0.43±0.09 vs.0.95±0.08,P<0.01),respectively.Conversely,the expression of CDK6,CCND1,and PIM1 mRNA in SGC-7901 gastric cancer cells transfected with miR-33 a inhibitor was significantly higher than those in the negative control group(3.24±0.17 vs.1.10±0.03,P<0.01;1.97±0.14 vs.0.94±0.09,P<0.01;2.13±0.11 vs.0.95±0.08,P<0.01),respectively.Similarly,after transfection of miR-33 a mimic into SGC-7901 gastric cancer cells,the protein expression levels of CDK6,CCND1,and PIM1 were significantly lower than those of the negative control group.On the contrary,the protein expression levels of CDK6,CCND1,and PIM1 of SGC-7901 gastric cancer cells transfected with miR-33 a inhibitors were significantly higher than those of the negative control group.4.The expression of CDK6,CCND1,PIM1 and P53 in gastric carcinoma tissues and their correlations with miR-33 a expression.Compared with adjacent tissues,49.1%(28/57),54.4%(31/57),68.4%(39/57)and 68.4%(39/57)of gastric cancer tissues had up-regulation of CDK-6,CCND-1,PIM1 and P53 mRNA expression.The mRNA expression of PIM and P53 in gastric cancer tissues was significantly higher than that in paracancerous tissues(P=0.004;P=0.039),and there was no significant difference in the mRNA expression levels of CDK6 and CCND1(P=0.429;P= 0.467).The expression of miR-33 a was significantly negatively correlated with the expression of CDK6,CCND1 and PIM1(r=-0.282,P=0.033;r=-0.353,P=0.010;r=-0.323,P=0.014),respectively,whereas miR-33 a expression has no significant correlation with P53 expression(r=-0.095,P=0.482)Conclusions:1.CDK6,CCND1 and PIM1 were Target Genes Regulated by miR-33 a in Gastric Cancer.2.miR-33 a could inhibit the proliferation of gastric cancer cells by inhibiting the expression of CDK6,CCND1 and PIM1,regulating cell cycle.Conclusions:1.The expression of miR-33 a in gastric cancer tissues was significantly lower than that in adjacent tissues,while the expression of miR-33 b in gastric cancer tissues was not significantly different from that in adjacent tissues.miR-33 a expression was significantly negatively correlated with tumor differentiation,distant metastasis,and CA-199 levels in peripheral blood of patients with gastric cancer,respectively.Compared with postoperative gastric cancer patients with low expression of miR-33 a,patients with high expression of miR-33 a had significantly longer DFS.2.The expression of miR-33 a in gastric cancer cell lines was significantly lower than that in normal gastric mucosa epithelial cells.miR-33 a had the effect of inhibiting the proliferation of gastric cancer cells.miR-33 a could regulate the proliferation cycle of SGC-7901 gastric cancer cells,arrest cell cycle at G1 phase,and inhibit the proliferation of gastric cancer cells.The miR-33 a expression had no significant effect on the invasion and migration ability of SGC-7901 gastric cancer cells.3.CDK6,CCND1 and PIM1 were target genes of miR-33a;miR-33 a could regulate cell cycle and inhibit gastric cancer cell proliferation by inhibiting the expression of CDK6,CCND1 and PIM1. |
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