Circular RNA CircPEX6 Inhibits Gastric Cancer Cell Proliferation,Migration And Invasion Through Regulating MiR-501/eIF4E3 Axis

Research background: Gastric cancer is the common malignant tumor of digestive tract worldwide.China has a high incidence of gastric cancer.According to the latest data in 2018,the deaths of gastric cancer in China is second only to that of lung cancer,accounting for about half of the global gastric cancer deaths.Due to the low detection rate of early gastric cancer in China,most cases are in the advanced stage when they are diagnosed,and the total five-year survival rate is less than 30%.Therefore,it is very necessary to help improve the prognosis of gastric cancer patients through investigating the molecular mechanism of the development and progression of gastric cancer and finding out the early diagnostic markers and effective therapeutic targets.Circular RNAs(circ RNAs)represent a new class of single-stranded,non-coding RNAs characterized by a covalently closed loop without 5? cap and 3? polyadenylated tails.Due to this particular structure,circ RNAs are resistant to the digestion of exonucleases.Most of the circ RNAs are located in the cytoplasm,and they are abundant in human cells with the characteristics of stability,conservation and tissue specificity.With the progress of high-throughput sequencing and novel bioinformatics algorithms,numerous circ RNAs have been identified.Majority of circ RNAs contain micro RNA(miRNA)response elements,through which competitively bind to miRNA via complementary base pairing,and subsequently inhibit the negative regulation ofmiRNA on downstream target gene expression.In recent years,circ RNA has been found to be involved in the development and progression of a variety of malignant tumors including gastric cancer.circPEX6 was a novel circular RNA molecule we screened from a high throughput sequencing database GEO(Gene Expression Omnibus).It originated from the backsplicing of exons 2 and 3 of PEX6(peroxisomal biogenesis factor 6)gene and was downregulated in the gastric cancer tissues compared to the paired normal gastric tissues.However,the role and underlying molecular mechanism of circPEX6 in gastric cancer are still not identified and elucidated.Micro RNA-501-5p(miR-501)has been reported to function as an oncogene in the liver cancer,gastric cancer,lung adenocarcinoma and cervical cancer.A binding site was predicted between circPEX6 and miR-501 by the miRanda,RNAhybrid and Target Scan bioinformatics analysis tools,and eIF4E3(eukaryotic translation initiation factor 4e family member 3)which is one of the member of eukaryotic translation initiation factor 4e family,was predicted to be the target gene of miR-501.Therefore,we will investigate the molecular mechanism of circPEX6 in suppressing the gastric cancer proliferation,migration and invasion as miR-501 sponge in order to provide a new potential target for the diagnosis and therapy of gastric cancer.Objectives:(1)To investigate the effects of circPEX6 on gastric cancer cell proliferation,migration and invasion.(2)To investigate the molecular mechanism of circPEX6 in regulating the proliferation,migration and invasion of gastric cancer cell through the miR-501/eIF4E3 axis.Methods:(1)We analyzed the differentially expressed circ RNAs in paired gastric cancer tissues and normal gastric tissues from Gene Expression Omnibus(GEO)database,and screened the downregulated circ RNAs in gastric cancer tissues.We used circ Base database to analyze the origin and location of circ RNAs.Actinomycin D assay and RNase R treatment assay were to verify the stability of circPEX6.The Fluorescent in Situ Hybridization(FISH)assay was used to determine the cellularlocation under a confocal microscope.(2)The overexpression plasmid of circPEX6(circPEX6)and empty plasmid(Vector),miR-501 mimic(miR-501)and its negative control(NC),co-transfection of circPEX6 plasmid and miR-501 mimic(circPEX6+miR-501),and si RNA against eIF4E3(si-eIF4E3)and its negative control(NCi)were transfected by Lipofectamine2000.(3)SYBR Green qRT-PCR was used to detect the expression levels of circPEX6,PEX6 m RNA and eIF4E3 m RNA in gastric cancer cells,and Taqman probe qRT-PCR was used to detect miR-501 expression level,and Western blot was used to detect eIF4E3 protein expression level.(4)The CCK-8 assay and cell colony formation assay were used to detect the gastric cancer cell proliferation,and the Transwell chamber assays with or without Matrigel were used to detect the migration and invasion abilities of each group of gastric cancer cells.(5)The wild-type circPEX6 sequence(circPEX6-wt)containing miR-501 binding site and its mutant type(circPEX6-mut)were synthesized and inserted into the Xba I site of the GV272 vector.The Dual-Luciferase Reporter Assay was used to evaluate the direct binding between circPEX6 and miR-501.Similarly,to evaluate the direct binding between miR-501 and eIF4E3,the wild-type eIF4E3 m RNA 3?UTR sequence(eIF4E3-wt)containing miR-501 binding site and a mutant type(eIF4E3-mut)were synthesized and inserted into the Xba I restriction site of GP-miRGLO vector.Results:(1)circPEX6,which was shown to be decreased in GC tissues from GEO database,also named as hsa?circ?0005038(chr6: 42941740-42942776),was derived from the exons 2 and 3 of PEX6 gene located at chromosome 6p21.1,whose genomic sequence length was 1036 bp and finally formed a sense-overlapping circular transcript of 248?nt.The qRT-PCR analysis showed that circPEX6 expression was significantly downregulated in SGC-7901,BGC-823,MGC-803 and AGS gastric cancer cells,compared to the normal human gastric epithelial cell line GES-1,especially inBGC-823 and MGC-803 cells.(2)The half-life of circPEX6 transcript measured by the qRT-PCR method exceeded 24?h in BGC-823 and MGC-803 cells after treatment with Actinomycin D which is a transcriptional inhibitor,whereas that of linear PEX6 m RNA transcript was about 12 h and 8 h respectively.As a result of qRT-PCR analysis,circPEX6 was significantly resistant to RNase R treatment,however,the levels of linear PEX6 m RNA were significantly reduced by 96.57% ± 0.23%(P?0.01)and 92.73% ± 0.50%(P?0.01)respectively.The confocal microscope showed that circPEX6 was predominately distributed in the cytoplasm of BGC-823 cells by the FISH assay.(3)The CCK-8 assay showed that the absorbance at 450 nm was reduced by27.26% ± 4.32% in the circPEX6 transfected BGC-823 cells(P?0.01)and by 31.3%± 3.72% in the circPEX6 transfected MGC-803 cells(P?0.01)when compared to the empty vector transfected groups.The cell colony formation assay revealed that the numbers of colonies in the circPEX6 transfected BGC-823 cells and MGC-803 cells were 384 ± 36 and 104 ± 14 respectively,and the significance was achieved(P?0.01)when compared to the Vector group(681 ± 25 and 228 ± 30 respectively).(4)The transwell chamber assay(without Matrigel)showed that the cell number penetrating through the membrane in the circPEX6 transfected BGC-823 cells was 303± 28,and there was a significant difference compared to the number of 577 ± 36 in the Vector group(P?0.01).In the MGC-803 cells,the cell number penetrating through the membrane in the circPEX6 group was 288 ± 33 and in the Vector group was 552 ± 62(P?0.01).The transwell chamber assay with Matrigel showed a similar trend with the transwell chamber assay without Matrigel,and circPEX6 group decreased the penetrating cell numbers relative to the Vector group(P?0.05).(5)The Dual-Luciferase Reportor Assay showed that co-transfection of the miR-501 mimic and circPEX6-wt vector into 293 T cells significantly decreased the luciferase activity by 34.52% ± 6.39% compared with co-transfection of negative control miRNA and circPEX6-wt vector(P?0.05),and there was no significant difference when co-transfection of miR-501 mimic and circPEX6-mut.The levels ofmiR-501 in SGC-7901,BGC-823,MGC-803 and AGS cells were significantly higher than that in GES-1 cells by qRT-PCR(P?0.05 or P?0.01).Cell proliferation,migration and invasion abilities were significantly promoted in BGC-823 and MGC-803 cells transfected with miR-501 mimic compared to the NC group(P?0.05 or P?0.01).(6)The luciferase activity was obviously reduced by 44.86% ± 4.82% in 293 T cells co-transfected with miR-501 mimic and eIF4E3-wt vector(P?0.01),however,co-transfection with miR-501 mimic and eIF4E3-mut failed to suppress luciferase activity.The ectopic expression of miR-501 led to a significant decrease in eIF4E3 m RNA compared with the NC group(P?0.05),whereas it was similar to the eIF4E3-si group.The Western blot analysis indicated that the expression of eIF4E3 protein was dramatically decreased by the transfection of miR-501 mimic.The silencing of eIF4E3 promoted the proliferation,migration and invasion capacities of BGC-823 and MGC-803 cells(P?0.05 or P?0.01).(7)The co-transfection of circPEX6 and miR-501 could rescue the enhanced proliferation,migration and invasion abilities caused by the ectopic expression of miR-501 in BGC-823 and MGC-803 cells.The Western blot results showed that circPEX6+miR-501 group and NC group had similar levels of eIF4E3 protein.Conclusions:(1)circ RNA overexpression can suppress the proliferation,migration and invasion abilities of gastric cancer cells.(2)eIF4E3 is a direct target of miR-501 in the gastric cancer cells and miR-501 can negatively regulates expression of eIF4E3 protein at a post-transcriptional level.Silenced eIF4E3 can enhance the proliferation,migration and invasion abilities of gastric cancer cells.(3)circPEX6 can competitively bind to miR-501 as a sponge to suppress the malignant behaviors of gastric cancer cells through relieving the negative regulation of eIF4E3 expression.circPEX6 might become the novel target of gastric cancer therapy.