MicroRNA-26b Inhibits Cell Proliferation And Invasion By Regulation CDC6 Expression In Gastric Cancer

Background: Gastric cancer(GC)is one of the most common malignant diseases in the world,with an increasing incidence rate year by year especially in China.To date,it has been became a serious threat to human health and life due to the high mortality rate.R0 curative resection and adequate lymphadenectomy is the only potentially curable treatment method for advanced GC patients.Due to vague clinical manifestations and signs,GC patients are usually diagnosed at the advanced stage of disease.On the other hand,a high proportion of these patients experienced tumor recurrence and metastasis even after curative resection.Therefore,the long-term survival outcome of GC patients was still unsatisfactory.Although serological tumor biomarkers such as carbohydrate antigen 19-9(CA19-9),carbohydrate antigen125(CA125)and carcinoembryonic antigen(CEA)has been widely used in clinical practice,these do not show a sufficient specificity and sensitivity for early diagnosis of gastric cancer.It is very important to identify novel effective biomarkers for early detection of gastric cancer,tumor recurrence surveillance and uncovering the molecular mechanisms involved in GC development and progression.Cell division cycle 6(CDC6),which is located at human chromosome 17q21.3,is a essential regulatory protein for initiation of DNA replication in eukaryotic cells.It has been proved that the major function of CDC6 is involved in the assembly of pre-replication complexs(pre-RC)at DNA replication origins during the G1 phase.In addition,CDC6 also play a important role in the activation and regulation of cell cycle checkpoint,which is required for transition from G1-phase to S-phase and subsequent mitotic entry.Therefore,the aberrant expression of CDC6 have severe consequences on DNA replication,genomic stability and cell proliferation,which could finally result in tumorigenesis and malignant progression.It has been reported that expression of CDC6 is significantly elevated in many malignant tumors such as epithelial ovarian cancer,breast cancer,prostate cancer and gallbladder cancer.However,the expression and clinical significance of CDC6 in gastric cancer has notyet reported.Further investigation need be performed to clarify whether the expression of CDC6 is associated with malignant properties in gastric cancer cell lines such as proliferation and invasion.In addition,genetic alteration and molecular mechanisms esulted in the aberrant expression of CDC6 in gastric cancer remains unclear.MicroRNAs(miRNAs)are a group of small non-coding RNAs with length of19-25 nucleotides in eukaryotic cells,which could negatively regulate gene expression at the post-transcriptional level.Previous studies indicated that miRNAs could bind to the specific sequences at the mRNA 3’UTR(untranslated regions)of target genes,and result in the degradation of the targeted mRNA or translation inhibition.miR-26 b,which is a member of human miR-26 gene family,has been shown to function as a tumor suppressor or tumor promoter by regulate the expression of various target genes in many malignant tumors.Some studies reported the overexpression of miR-26 b could significantly inhibit the cancer cell proliferation,migration and invasion ability.On the contrary,other studies demonstrated that the overexpression of miR-26 b could have a positive effect on these malignant biological behaviors of tumor cells.However,little is known regarding the role of miR-26 b in mediating the development and progression of gastric cancer.The identification of specific oncogenic driver and its regulation network is helpful for the early diagnosis and treatment of gastric cancer.Thus,we expect that the present study could provide experiment evidence for targeted therapy and facilitate the understanding on molecular mechanism of tumorigenesis and progression in gastric cancer.Objective: The aim of this study was to investigate the expression level of miR-26 b and CDC6 and their clinical significance in gastric cancer,and explore the impact of the expression of miR-26 b and CDC6 on biological function in gastric cancer cell lines.In addition,the relationship between miR-26 b and CDC6 expression was further evaluated to determine whether the regulatory effect of miR-26 b on cell growth and invasion was related to the expression of CDC6.Methods: Gastric cancer tissues and adjacent normal tissues were collected from 48 patients.All patients underwent curative resection at the Department of Surgical Oncology,the First Affiliated Hospital of China Medical University between January2016 and January 2016.Real-time PCR analysis was performed to examine the expression level of miR-26 b in 48 gastric cancer samples.The relationship between and the expression of miR-26 b and clinicopathologic characteristics of these patients was evaluated.A total of 50 paraffin-embedded gastric cancer tissue samples were collected from the patients who underwent curative resection in our institution during the same period.The expression level of CDC6 in gastric cancer tissue and adjacent normal tissues were detected by immunohistocheistry staining.The clinicopathologic information was collected and reviewed,and the relationship between expression level of CDC6 in gastric cancer tissue and clinicopathologic factors was evaluated.The relationship between miR-26 b and CDC6 expression in gastric cancer tissues was determined by Speraman’s correlation analysis.In addition,the expressing correlation of between miR-26 b and CDC6 in gastric cancer cell lines MGC-803,BCG823 and SGC7901 was assessed by using qRT-PCR analysis,and normal gastric epithelial cell line GES1 was used as a control.The loss-or-gain of function assay was performed to investigate the biological function of miR-26 b and CDC6 in gastric cancer.miR-26 b mimic was synthesized to elevate the expression level of miR-26 b,and the plasmid with specific small interfering RNA for CDC6 was construct to reduce the expression level of CDC6 in gastric cancer cell lines.After these cells were transfected with miR-26 b mimic or the plasmids expressing the specific CDC6 interfering RNA(CDC6-siRNA),qRT-PCR and western blot analysis were used to validate the efficacy of transfection.MTS assay was performed to evaluate the impact of miR-26 b overexpression and CDC6 silence on the growth ability of SGC7901 cell lines in vitro,respectively.Transwell assay was performed to evaluate the impact of miR-26 b and CDC6 on invasion ability of gastric cancer cells in vitro,respectively.In addition,flow cytometry was used to detect cell apoptosis via AnnexinV-PI staining after upregulation of miR-26 bexpression or downregulation of CDC6 expression.To identify the putative candidate genes of mir-26 b in gastric cancer,we searched the TargetScan,miRanda and miRBase database to predict the potential binding site.The luciferase reporter vectors containing wild type(pmiR-CDC6-3’UTR-wt)or mutant type binding sites(pmiR-CDC6-3’UTR-mut)for mir-26 b was constructed.We performed the luciferase reporter assay to determine whether CDC6 was a direct target of miR-26 b.In addition,western blot analysis was used to further evaluate the negative regulation relationship between miR-26 b and CDC6 expression.To address whether the regulatory effect of miR-26 b on biological functions was depended on CDC6,we performed MTS assay and Transwell assay again after ectopic restoration of CDC6 expression in those cells transfected with miR-26 b mimic.Results: The expression level of miR-26 b was significantly lower in gastric cancer tissues than in the adjacent normal tissues(P<0.05).Low expression of miR-26 b detected by qRT-PCR analysis was associated with increased seroal invasion(P<0.05)and more advanced pathological stage(P<0.05).On the contrary,CDC6 protein was primarily detected in cytoplasm and cell nuclei of gastric cancer tissues,and the positive rate of CDC6 expression was significantly in gastric cancer tissues higher than in adjacent normal tissues(62% vs 24%,P<0.05).The high expression of CDC6 in gastric cancer tissues was significantly associated with lymph nodes metastasis(P<0.05),tumor infiltration growth pattern(P<0.05)and lymphovascular invasion(P<0.05).According to the result of Speraman’s correlation analysis,the relative expression level of miR-26 b was inversely associated with CDC6 protein expression in gastric cancer tissues(r=-0.591,P<0.01).Similarly,there was a significant negative correlation between miR-26 b and CDC6 expression in gastric cancer cell lines.Compared with the normal gastric epithelial cell line GES1,the expression level of miR-26 b was markedly decreased(P<0.05)and the expression level of CDC6 was markedly increased in MGC-803,BCG823 and SGC7901cells(P<0.05).qRT-PCR analysis showed that transfection with miR-26 b mimic in SGC7901 cells could result in a significant increase in the expression level of miR-26b(P<0.05).The growth ability of SGC7901 cells transfected with miR-26 b mimic was significantly inhibited(P<0.05)and the number of induced cell apoptosis was significantly increased in vitro(2.8±0.2% vs 6.5±0.5%,P<0.05),compared with AgomiR-NC group.In the Transwell invasion assay,the ectopic upregulation of miR-26 b expression markedly reduced the number of invasion cells through the Matrigel membrane(AgomiR-NC group: 98.7±5.5 vs AgomiR-26 b group:68.3±7.1,P<0.01).On the other hand,the results of western blot analysis demonstrated that the expression of CDC6 in the protein level was reduced to certain extents in SGC7901 cells transfected with the plasmids expressing the specific small interfering RNA(siRNA).MTS assays and cell apoptosis assays showed that knockdown of CDC6 had a similar inhibition effect on the growth of SGC7901 cells(P<0.05)and significantly induced cell apoptosis in vitro(Si-CDC6 group: 8.0±0.4% vs Si-NC 组 group :3.3±0.2%,P<0.05),respectively.Transwell assay indicated that the number of invasion cells through the Matrigel membrane was 85.7±3.2 in SGC7901 cells with CDC6-siRNA,which was significantly lower than that of in the blank control group(115.3±8.4)and Si-NC group(109.3±7.6).There was a significant statistical difference between Si-CDC6 group groups and Si-NC group(P<0.05).These results revealed that the downregulation of CDC6 expression could significantly inhibit the growth and invasion ability of gastric cancer cells,which were in accordance with the effect of upregulation of miR-26 b expression on the biological behaviors.More importantly,our results indicated that ectopic upregulation of CDC6 expression could reverse the inhibitory effect of miR-26 b overexpression on cell growth and invasion.Bioinformatics analysis indicated that there was a conserved and specific binding site for miR-26 b in the 3’UTR of CDC6 mRNA(starting at positions 109 and 116).In addition,we found that the expression level of endogenous CDC6 protein in SGC7901 cells was significantly decreased after ectopic overexpression of miR-26 b using miR-26 b mimic,compared with the control group.The luciferase reporter assaydemonstrated that the luciferase activity of the reporter plasmid containing the wild type CDC6-3’UTR was significantly suppressed by miR-26 b mimic(P<0.05),but the upregulation of miR-26 b expression had no effect on the luciferase activity of the reporter plasmid containing the mutant type CDC6-3’UTR.Conclusion: There was a significantly negative correlation between the expression level of miR-26 b and CDC6 expression in gastric cancer tissues and cell lines.The overexpression of miR-26 b or silence of CDC6 expression had a significantly inhibitory effect on the growth and invasion ability of gastric cancer cells.miR-26 b and CDC6 played an important role in gastric cancer development and progression.More importantly,CDC6 was a direct target of miR-26 b,and its expression level was negatively regulated by miR-26 b.The impact of CDC6 on malignant biological behaviors was,at least in part,mediated by miR-26 b in gastric cancer.miR-26 b and CDC6 may be considered as new potential targets for the early detection and therapeutic intervention of gastric cancer.