MiRNA-491-5p Inhibits Cell Proliferation,Invasion And Migration Via Targeting JMJD2B And Serves As A Potential Biomarker In Gastric Cancer

BackgroundGastric cancer(GC)with high morbidity and mortality ranks one of the world’s most common gastrointestinal malignancies.From the worldwide perspective,the incidence of gastric cancer is the highest in Asia,it takes the second place in all the malignancies in China.Due to lack of sensitive and reliable GC diagnostic biomarkers for early GC diagnosis and untypical early symptoms of GC,GC patients are usually diagnosed at advanced stages.In addition,the five-year survival rate of GC patients is extremely low.As a consequence,it is imperative to find new biomarkers with high sensitivity and specificity for diagnosis.In our previous work,we have found that histone demethylase JMJD2B(also named KDM4B)was overexpressed in GC tissues compared to adjacent normal tissues and high level of JMJD2B induced GC cell proliferation,survival,invasion and metastasis.Nevertheless,the regulatory mechanism of JMJD2B has not been fully elucidated in the development and progression of gastric cancer.Studies have shown that miRNAs are single-stranded,non-coding RNA molecules,which can regulate the majority of cellular functions at the post-transcriptional level,like cell proliferation,apoptosis,differentiation and cell metabolism,contributing to cancer development and progression.Considering the function of miRNA on gene expression,we determined to explore whether JMJD2B expression was regulated by miRNA in GC development.Therefore,we predicted the miRNAs that could regulate the expression of JMJD2B by bioinformatics analysis softwares.And we found that miR-491-5p could regulate the expression of JMJD2B and participate in the development and progression of gastric cancer through a series of molecular biology experiments.Then we studied the regulatory mechanisim of miR-491-5p on the expression of JMJD2B and the significance of miR-491-5p as a potential biomarker for gastric cancer diagnosis.ObjectiveTo investigate the molecular mechanism of how miR-491-5p regulates JMJD2B in the development of gastric cancer and the potential value of miR-491-5p as a diagnostic marker of gastric cancer.Methods1、We searched for the potential miRNAs targeting JMJD2B via four databases:miRDB,miRanda/microRNA.org,Microcosm Targets and DIANA-microT v3.0.QRT-PCR was performed to reveal the different levels of miR-491-5p in GC cell lines HGC27,MGC803,SGC7901 and BGC823 compared with GES-1 cells(immortalized epithelial cell line).MiR-491-5p mimics or miR-negative control were transfected in GC cell lines MGC803 and SGC7901 using Lipofectamine 2000 following the manufacturer’s protocols.The expression of miR-491-5p was tested at 24h after transfection by QRT-PCR.Western blot analysis was performed after transfection with miR-491-5p mimics or miR-negative control to ascertain the protein level of JMJD2B.2、Dual luciferase report activity assay was performed by transfecting pmiR-RB-REPORT_h-KDM4B WT or pmiR-RB-REPORT_h-KDM4B MUT vectors,with miR-491-5p mimics or miR-negative control into GC cell line MGC803.After transfecion,the activity of luciferase was detected with the Dual-Luciferase assay kit according to manufacturer’s instructions.3、MiR-491-5p mimics or miR-negative control were transfected to GC cell lines MGC803 and SGC7901 using Lipofectamine 2000 following the manufacturer’s protocols.Colony formation assay was used to test the clony ability of GC cells after transfection of miR-491-5p.Transwell experiments were done to observe the invasion ability of the GC cells after transfection of miR-491-5p.Scratch wound-healing assay was carried out to detect the motility of GC cells after transfection of miR-491-5p.4、We detected the expression of miR-491-5p in GC tissues and GC patients’serum to speculate the possibility of miR-491-5p as a candidate biomarker of GC diagnosis.Results1、The evaluation from four public bioinformatic databases(miRDB、miRanda/microRNA.org、Microcosm Targets and DIANA-microT v3.0)revealed that miR-491-5p could target JMJD2B 3’ UTR.QRT-PCR was done and revealed that miR-491-5p was lower expressed in GC cell lines HGC27,MGC803,SGC7901 and BGC823 compared with GES-1 cells(immortalized epithelial cell line)(P<0.001).There was an evidently increase in miR-491-5p expression after transfection with miR-491-5p mimics compared with miR-negative control in two GC cell lines MGC803 and SGC7901(P<0.0001).Transfection of miR-491-5p mimics decreased the expression of JMJD2B in protein level in GC cell lines（P<0.01).2、According to the evaluation of the bioinformatic analysis by TargetScan,there are three binding sites of miR-491-5p on target JMJD2B 3 ’ UTR.We constructed five luciferase reporter vectors including wild type vector containing 3 binding sites,all-mutant vector in which 3 binding sites were mutated and 3 single mutant vectors with corresponding mutation separately.Our results showed that the luciferase activity was significantly amplified after co-transfection of pmiR-RB-REPORT_h-KDM4B WT and miR-491-5p mimics compared with miR-negative control(p<0.05).Whereas,the reduction of luciferase activity was abolished by transfecting the mutant vector of all mutations.Meanwhile,our result indicated that the luciferase activity was not significantly inhibited with individual mutation vectors compared with negative control.The above results indicated that miR-491-5p can directly bind to JMJD2B 3’ UTR.3、The colony formation assay showed that the colony numbers were significantly declined by ectopic expression of miR-491-5p in contrast to negative control(P<0.05).The transwell test displayed that miR-491-5p suppressed cell invasion in MGC803 and SGC7901 GC cell lines(P<0.05).Wound healing experiment revealed that over expression of miR-491-5p hindered the motility of MGC803 and SGC7901 cells.4、QRT-PCR was choosen to detect the level of JMJD2B expression.It was raised in GC tissues compared with that in adjacent normal tissues,while miR-491-5p expression was reduced in GC tissues in contrast with adjoining normal tissues.The expression of miR-491-5p detected by QRT-PCR was significantly decreased in serum of GC patients compared with that in the healthy cohorts.ROC curve analysis showed the sensitivity was 94.2%and the specificity was 76.2%with an AUC of 0.919(95%CI=0.874-0.963).Conclusion1、MiR-491-5p suppresses JMJD2B expression by directly targeting the three binding sites of of JMJD2B 3’ UTR and acts as a tumor suppressor through inhibiting cell proliferation,invasion and migration in gastric cancer.2、MiR-491-5p is lower expressed in the tissue and serum of GC patients compared with control,indicating that it might serve as a potential biomarker for the diagnosis of GC.