The Role Of LncRNA PVT1 In Colorectal Cancer

BACKGROUND:Colorectal cancer(CRC)is a malignant tumor that ranks third in morbidity and second in mortality in the world,and a serious threat to human health worldwide.In China,colorectal cancer is the fifth malignant tumor with both morbidity and mortality.The other four are lung cancer,gastric cancer,esophageal cancer,and liver cancer,which seriously threaten human's health and life.Surgical resection is the most effective treatment of the colorectal cancer,however,it is just suitable for patients with early mild colorectal cancer.For patients with advanced colorectal cancer,chemotherapy is still an important irreplaceable strategy in the treatment process.However,the cancer cells resistance to chemotherapy drugs limits the clinical efficacy.Therefore,there is an urgent need to identify new targets and improve clinical outcomes of CRC.The long noncoding RNA(lncRNA)is widely involved in the physiological and pathological processes of the body,plays an important role in tumor proliferation,apoptosis,drug resistance,and metastasis.and has a specific expression profile in a variety of tumors.And the lncRNA has a specific expression profile in a variety of tumors.Studies have confirmed that lncRNA PVT1 is highly expressed in CRC cells and tissues,and PVT1 upregulation is positively correlated with CRC cell proliferation,invasion,TNM stage,lymph node metastasis and metastasis.Therefore,PVT1 is potential to become a diagnostic marker for colorectal cancer.OBJECTIVE: To explore the diagnostic value of lncRNA PVT1 as a molecular marker for CRC,and its effect on the proliferation,migration and invasion of CRC cells;then further study of the miRNA molecules adsorbed by PVT1 and its possible mechanisms,such could provide a new idea for the early diagnosis of CRC.METHODS:In this study,we firstly analysis the PVT1 expression of 72 CRC patients' cancer and paracancer tissues with RT-PCR,and also the CRC cell lines.Then Kaplan–Meier analysis and ROC curve were used to analyze the clinical value of PVT1 as a diagnostic marker in CRC.Chi-square test was used to analyze the relationship between PVT1 and clinical characteristics of CRC.In order to further study the biological function of PVT1,three si RNAs of PVT1 were designed and screened by qPCR.The effect of proliferation of PVT1 in CRC cells was detected by CCK-8,and the cell cycle was detected using flow cytometry.And the cloning and scratch experiments were used to detect the effect of PVT1 on CRC cells' invasion and migration.On this basis,then Star Base,HMDD v2.0 and miRTar Base bioinformatics databases were used to analyze the possible downstream miRNA molecules of PVT1,which were further verified by luciferase reports and RNA pull down experiments.qPCR was used to verified the expression of target miRNA molecules in 72 pairs of CRC patients postoperative specimens.Pearson's correlation analysis confirmed the correlation between PVT1 and target miRNA expression.In vitro cell experiments verified the expression of miR-16-5p in HCT116 and SW480 cell lines after PVT1 knockdown.Consider that VEGFA-mediated AKT signaling pathway plays an crucial role in tumor pathogenesis,Western-blot was used to detect the expression of VAGFA,have VEGFR1 and phosphorylated AKT.At last,the model of human CRC xenografts in nude mouse was made to verify the effect of PVT1 and miR-16-5p on tumorigenesis and tumor growth in vivo.RESULTS: We first observed that expression levels of PVT1 were upregulated in most CRC specimens and cell linescompared with matched normal tissues and epithelial cell line FHC,respectively.Using a median PVT1 value as a cutoff,among the 72 CRC patients,47 cases were PVT-1 high and 25 cases were considered low in PVT1(PVT-1 low).Further statistical analysis shows that increased expression of PVT1 was significantly associated with lymph node metastasis(P < 0.01),distant metastasis(P < 0.05)and TNM stage(P < 0.05).Moreover,CRC patients with high levels of PVT1 reflected significantly shorter overall survival than those with the low expression of PVT1 by the analysis of Kaplan–Meier survival curve.Next,the receiver operating characteristic(ROC)curve was performed to assess the sensitivity and specificity of PVT1 expression in differentiating CRC from normal tissues(Cutoff value = 1.045,Youden index = 0.597,Sensitivity = 0.653,Specificity = 0.944).Notably,PVT1 showed remarkable predictive significance with an area under the ROC curve of 0.81(P < 0.01).Thus,elevated expression of PVT1 was significantly associated with poor prognosis for CRC patients.We designed three short hairpin RNAS(shRNAs)to silence PVT1 lncRNA expression in HCT116 and SW480 cell lines and these shRNAs decreased PVT1 expression at different levels,we then targeted PVT1 using shRNA-PVT1-2 for the subsequent experiments due to its highest inhibitory efficiency.CCK8 indicate silencing of PVT1 corresponds with the loss of cell proliferation in two cell lines.FACS showed that PVT1 knockdown in both the HCT116 and SW480 cell lines displayed significant increase in the percentage of cells in S-phase.The colony formation and scratch-wound healing assays showed that PVT1 knockdown significantly suppress invasion and migration in HCT116 and SW480 cell lines.Using Starbase,HMDDv2.0 and miRTar Base,we identify miR-15a-5p,miR-15b-5p,miR-16-5p,miR-17-5p and miR-195-5p as candidates that bind the PVT1 lncRNA and correspond with outcomes in human CRC.However,among these micro RNAs,miR-16-5p has been demonstrated closely related to the pathogenesis of CRC through both in vitro and in vivo experiments.Considering the relationship between miR-16-5p and PVT1 remains unclear we,therefore,focused on the specific mechanism of PVT1-miR-16-5p in the pathogenesis of CRC.Luciferase reporter assay demonstrated that overexpression of miR-16-5p significantly decreased the luciferase activity of the vector containing the complete PVT1 sequence,but did not affect the luciferase activity of the vector with mutant miR-16-5p-binding site.To further demonstrate the direct binding of miR-16-5p and PVT1,we utilized biotin labeled miR-16-5p and its mutant mimics to pull-down PVT1 in HCT116 and SW480 cells with PVT1 overexpression,and the results showed wild-type miR-16-5p captured more PVT1 compared with the mutant.We then evaluated the expression of miR-16-5p in 72 pairs CRC specimens.QRT-PCR result showed that miR-16-5p was significantly downregulated.Moreover,PVT1 expression was negatively correlated with the expression of miR-16-5p in CRC tissues(r =-0.321,p < 0.01).As expected,silencing of PVT1 increased the miR-16-5p expression in HCT116 and SW480 cell lines.Taken together,these data demonstrated that PVT1 acts as a miRNA sponge for miR-16-5p in CRC.Further more we found that targeted loss of miR-16-5p partially rescues the suppressive effect induced by PVT1 knockdown.Western-blot confirmed that vascular endothelial growth factor A(VEGFA),a direct downstream target of miR-16-5p was suppressed by PVT1 knockdown in CRC cells.Overexpression of VEGFA is known to modulate the AKT signaling cascade by activating the vascular endothelial growth factor receptor 1(VEGFR1).We,therefore,show that PVT1 loss combined with miR-16-5p overexpression reduces tumor volume maximally when propagated within a mouse xenograft model.CONCLUSIONS: We conclude that the PVT1-miR-16-5p/VEGFA/VEGFR1/AKT axis directly coordinates the response in CRC pathogenesis to suggest PVT1 as a novel target for potential CRC therapy.