| BackgroundRecently,multiple lines of studies have shown that long-non-coding RNA linc00662 plays an oncogene in a variety of cancers,however,the exact oncogenesis mechanisms of linc00662 in colorectal cancer(CRC)remains unknown.In the GEO database,we found expression of linc00662 was significantly upregulated,further studies have found that in un-resectable CRC tissues,linc00662 expression is dramatically increased,in the TCGA database,we found that patients with linc00662 over-expression had significantly shorter overall survival(OS).Therefore,in this study,we aim to explore the biological role of linc00662 in the regulation of CRC progression.Objective1.To verify the expression of linc00662 in colorectal cancer and adjacent tissues;2.Linc00662 locates in the cytoplasm,accumulating evidence suggested that linc00662 could act as a competing endogenous RNA(ceRNA),MicroRNA and its downstream regulated target genes are identified by bioinformatics analysis and the dual-luciferase reporter assay;3.To clarify the specific molecular mechanism of linc00662 affecting the proliferation,apoptosis and migration of colorectal cancer cells;4.The regulation relationship among linc00662,downstream regulated microRNA and downstream regulated target genes was determined by the recovery experiment.Methods1.The expression of linc00662 in colorectal cancer and the relationship between linc00662 and survival prognosis were analyzed using GEO and TGCA database;2.qRT-PCR was used to detect the expression of linc00662 in cancer tissues and paired adjacent tissues of 56 CRC patients,and clinicopathological parameters were analyzed;3.Using normal colorectal mucosa epithelial FHC cell lines and colorectal cancer cell lines SW480,Caco-2,Lovo,HT-29,HCT116 in vitro experiments,first of all use qRT-PCR to detect the linc00662 expression in the FHC cell line and SW480,Caco-2,Lovo,HT-29,HCT116,Then,the two cell lines with the highest expression levels were selected as experimental subjects,and lentivirus transfection with small interfering RNA was used to construct the low-expression CELL model of linc00662.Then,cell count Kit 8(CCK-8)and in vivo experiments were used to verify the proliferation ability of SW480 and CACO-2 cell lines after linc00662 expression was knocked down.Cell apoptosis rate and cell cycle percentage were detected by flow cytometry,and cell migration and invasion ability were detected by Transwell method.4.Starbase2.0 predicted that miR-16-5p,miR-195-5p,miR424-5p,and miR-497-5p shared two joint sites with linc00662.Dual-luciferase reporter assay was conducted to further verify the four candidate miRNAs.Then the screened microRNA was screened out for its related target genes in three online bioinformation databases,miRtarscan,miRDB and Targetscan Human7.0,and the target genes predicted jointly by the three databases were extracted by the Venn diagram,and their functions were enriched for analysis,and finally the functions of the target genes were identified.Finally,the recovery experiment was used to verify the regulatory relationship among linc00662,downstream regulated microRNA and downstream regulated target genes.The results of this study are divided into the following three parts:Part Ⅰ:Linc00662 was highly expressed in CRC and was negatively correlated with clinical prognosis.To explore linc00662 expression in CRC and its correlation with clinical prognosis,we initially analyzed the data in the GEO database(GDS3141,GDS 4379,GDS4381,GDS4718,GDS4516,GDS4393,GDS3501),we found that linc00662 expression in CRC tissues is higher than normal mucous tissues,further studies have found that in un-resectable CRC tissues,linc00662 expression is dramatically increased(Figure 1A).Then,we downloaded data of 237 patients from the TCGA-COAD dataset,12 patients with incomplete registration in the data were deleted finally,225 patients’ data were available.Based on the median expression level of linc00662,we divided these patients into two groups,including a low expression group(n=135)and a high expression group(n=90).And the data showed that patients with linc00662 over-expression had significantly shorter overall survival(OS)(P=0.037).Besides,RT-qPCR was used to detect the expression level of linc00662 in the CRC tissues and paired adjacent normal tissues in our hospital.Consistently,the linc00662 expression is higher in CRC tissues than normal colorectal mucosa,and 77.8%(43 of 56 paired)of the CRC tissue samples showed linc00662 over-expression.Based on the median value of the linc00662 expression,we separated these patients into high expression group and low expression group,our data showed that the expression level of linc00662 was positively associated with the positive lymph node metastasis,TMN stage,and poor-moderate differentiation.Finally,in the prognosis assessment,the result indicated that the linc00662 low expression group had longer survival time than the high expression group(P=0.022).Part Ⅱ:Linc00662 was up-regulated in CRC cell lines.Linc00662 promotes the migration,invasion and EMT processes of CRC cells,thus promoting tumor metastasis.To further verify the biological role of linc00662 in CRC progression,RT-qPCR was performed to detect the expression level of linc00662 in five CRC cell lines and normal cell line.The results confirmed that the expression of linc00662 was dramatically increased in all CRC cell lines compared to the normal cell line,especially inSW480 and Caco-2 cell lines.Thus,we selected the Caco-2 and SW480 cell lines for the following studies.Then,we constructed two siRNAs and negative control(NC)siRNA targeting linc00662 were transfected into Caco-2 and SW480 cells,named sh-linc00662-1 and sh-linc00662-2 and si-Control,which was confirmed with the RT-qPCR assay,and the results revealed that the linc00662 expression was knocked-down in two cell lines.To further assess the potential effects of linc00662 on cell proliferation,CCK-8assay was carried out at 24,48,72and 96 hours after sh-linc00662 transfection,suggesting that knockdown of linc00662 observably decreased the cell viability relative to the control group.To date,sh-linc00662-1 showed higher depletion efficiency(70%)of linc00662,compared to sh-linc00662-2(60%)in Caco-2 cells.Consistently,sh-linc00662-1 showed higher depletion efficiency(70%)of linc00662,compared to sh-linc00662-2(55%)in Caco-2 cells.Meanwhile,sh-linc00662-1 could inhibit more cell proliferation,compared to sh-linc00662-2 in both SW480 and Caco-2 cells.Consequently,sh-linc00662-1 was chosen as positive group in the in vivo research,since it showed better anti-tumor effect.After 21 days,we discovered that the tumor volume of the sh-linc00662-1 groups were dramatically smaller than the siRNA-NC groups.Consistently,the tumor weights of the sh-linc00662-1 groups were also markedly decreased,compared to the siRNA-NC groups.To further verify our results,tumors isolated from mice were also collected for H-E,Ki-67 and TUNEL detection.From the IHC results,we found Ki-67 was inhibited in si-linc00662-1 group,while TUNEL was relatively higher expressed in si-linc00662-1 group,compared to control groups,which again confirm our conclusions.The results of flow cytometry assay indicated that knockdown of linc00662 increased the early apoptosis ratio of Caco-2 and SW480 cells compared with the control groups.Moreover,the following cell cycle assays indicated that knockdown of linc00662 could arrest more cells at the G2/M phase.The results of Transwell assays revealed that knockdown of linc00662 expression restrained the migration and invasion abilities of Caco-2 and SW480 cells.As we knew,the epithelial-mesenchymal transition(EMT)is an important way to promote tumor cell metastasis,then the data of western blotting showed that the silencing of linc00662 could significantly up-regulate the E-cadherin which was the epithelial marker,and down-regulate the mesenchymal markers N-cadherin,vimentin,and Snail.Therefore,we concluded that linc00662 promoted the migration,invasion and EMT process of CRC cells,and then promoted tumor metastasis.Part Ⅲ:Linc00662 promotes the occurrence and development of CRC by regulating miR-497-5p/AVL9 axisBased on the results of starbase2.0 software,we discovered that a series of microRNAs,including miR-16-5p,miR-195-5p,miR424-5p and miR-497-5p,had two common joint sites with linc00662.We further conducted the dual luciferase reporter assay to further test the four candidate miRNAs,while these results indicated that the luciferase activity could be suppressed by miR-497-5p mimics with highest efficiency,among the up 4 selected miRNAs.Our data displayed that the expression of miR-497-5p in CRC tissues was lower than that in normal tissues(P<0.05),and 69.6%(39 of 56 paired)of the CRC tissue samples possess the.down-regulation of miR-497-5p.According to the Pearson correlation coefficient analysis,the miR-497-5p expression level was negatively associated with linc00662 expression in CRC tissues(r=-0.5134,P<0.001),which was identified with the dual-luciferase reporter assay.Consistent with the previous results,the results of RT-qPCR revealed that miR-497-5p expression was markedly down-regulated in CRC cell lines relative to the normal colorectal cell.Next,RT-qPCR showed knockdown the expression of linc00662 significantly increased miR-497-5p expression in Caco-2 and SW480 cells.These data confirmed that linc00662 exerts its function at least partially,through sequestering miR-497-5p.In order to further investigate the ceRNA network mechanism in CRC,three online bioinformatics databases(including miRDB,targetscan Human 7.2 and miRtarbase)were applied to predict the potential target genes of miR-497-5p,then we generated Venn diagram by online webtool(http://bioinformatics.psb.ugent.be/webtools/Venn/)to visualize the intersecting genes between the results of three databases.From Venn results,17 potential miR-497 target genes were identified(KANK1 SALL1 CBX4 IPPK WNT7A ZNRF3 RECK ACVR2A CYP26B1 LURAP1L CCND2 CACUL1 SPRED1 CHAC1 AVL9 ZNF622 CDC25A).We further verified their expression levels in the GEPIA database(http://gepia.cancer-pku.cn/),as AVL9,CBX4 ZNRF3 and CHAC14 were shown to be up-regulated in CRC.Meanwhile,Gene ontology(GO)function enrichment analysis that the up 17 genes were functionally concentrated in tumor related cell motility,cellular process,intracellular organelle and membrane part and so on,while AVL9 was involved in all these functions.Finally,the potential binding sites of miR-497-5p and AVL9 were also predicted using bioinformatics databases(https://cm.jefferson.edu/ma22/Interactive/),while dual luciferase reporter assays showed that luciferase activity was weakened in AVL9 wild-type cells,after transfection of miR-497-5p.Therefore,we supposed AVL9 might be the downstream of miR-497-5p.To determine the ceRNA network between linc00662 and AVL9 in CRC,we detected the coloration.TCGA-portal data(http://tumorsurvival.org/)revealed that CRC patients with higher level of AVL9 were more likely to have poorer overall survival(P=0.0246).Surprisingly,TCGA-portal database also showed that linc00662 expression was positively correlated with the expression of AVL9(R=0.34,P<0.001).Furthermore,linc00662-downregualted SW480 and Caco-2 cells showed that down-regulating linc00662’s expression could significantly reduce the AVL9 mRNA levels in two cell line,while the miR-497-5p inhibitor could again increase AVL9’s expression in linc00662-downregualted CRC cells.Consequently,we supposed linc00662 might regulate suppresses proliferation and metastasis abilities in CRC,by sponging to miR-497-5p and regulating AVL9.To verify the relationship between linc00662,miR-497-5p and AVL9 in CRC,we explored the function of miR-497-5p and AVL9,we tried to dysregulate the expression of miR-497-5p and AVL9 in SW480 cells,as sh-linc00662-1 SW480 cells showed higher efficiency on regulating the expression of miR-497-5p and AVL9.In our research,the expression of AVL9 was again found to be significantly down-regulated in sh-linc00662-1 SW480 cells,but reduced by miR-497-5p inhibitor.Furthermore,Again,AVL9-specific siRNA could greatly induce overexpression of AVL9(siRNA-AVL9)in SW480 cells.Interestingly,the results of CCK-8 showed that the overexpression of miR-497-5p inhibitor increased the cell growth in sh-linc00662-1 SW480 cells,but again inhibited by siRNA-AVL9.Consistently,the anti-cell migration and invasion effect induced by sh-linc00662-1,was reversed by miR-497-5p inhibitor.However,siRNA-AVL9 could rescue this effect,as less migration and invasion cells were observed from Transwell assays.Consequently,these results indicate that AVL9 might be a target gene of linc00662/miR-497-5p axis during CRC,s development.Part Ⅳ:AVL9 is upregulated in colorectal cancer(CRC)and could be a predicated biomarker in CRCBase on the GEO,TCGA,GEPIA database,our study confirmed that AVL9 was highly expressed in CRC lesions,compared with the adjacent normal tissues(P<0.001).High expression of ALV9 was negatively associated with the survive outcome(P<0.05).GO analysis showed that AVL9 expression-related genes were enriched in single organismal cell-cell adhesion,posttranscriptional regulation of gene expression,negative regulation of vascular endothelial growth factor receptor signaling pathway(P<0.05).The KEGG pathway analysis showed that these genes were mainly involved in Progesterone-mediated oocyte maturation,axon guidance,insulin signaling pathway and ubiquitin mediated proteolysis signaling pathways(P<0.05).PPI analysis showed that KBTBD2,KIAA1147,EPDR1 and RNF216 genes interact with AVL9,and GEPIA predicts that their expression levels are all positively correlated with AVL9.Furthermore,clinic-pathological parameters analysis found that high AVL9 expression was positively correlated with differentiation,TNM stages.RT-qPCR detection in serum further showed that AVL9 expression was up-regulated in the plasma of CRC patients compared to that of healthy controls.Conclusions1.Long non-coding RNA Linc00662 was significantly up-regulated in CRC and was negatively correlated with clinical prognosis.2.In vitro and in vivo experiments showed that long non-coding RNA Linc00662 promoted the migration,invasion and EMT of CRC cells,thus promoting tumor metastasis.3.Linc00662 specifically regulates AVL9 expression and participates in CRC progression and metastasis by means of sponge adsorption of miR-497-5p.4.The up-regulated expression of AVL9 in colorectal cancer(CRC)promotes tumor progression as an oncogene and may serve as a non-invasive biomarker for diagnosis and treatment.Therefore,our results may shed light on CRC diagnosis and therapies. |
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