Research On MicroRNA-25Suppressing Colon Cancer Cell Proliferation And Invasive By Targeting ANGPTL2

Background:The colorectal cancer (CRC) is the third most common malignant tumors worldwide, with the incidence and mortality increasing each year as the change of the dietary habits and structure and the aging of the population. As the early symptoms of CRC are not obvious, the most clinical cases are the advanced cases of CRC after diagnosis and the five year survival rate is only about fifty percent. Surgery is the major therapeutic method, radiation therapy, chemotherapy and targeting therapy are adopted as adjunctive treatment. CRC is tightly associated with multiple gene abnormalities, and inhibition of tumor growth by regulating key genes to block malignant transformation will be one of the most effective methods for the treatment of CRC. Currently, the major targets of colorectal cancer include the epidermal growth factor receptor (EGFR) and the vascular endothelial growth factor (VEGF), and chemotherapy with cetuximab and bevacizumab that target respectively to EGFR and VEGF in advanced stage of CRC showed good clinical effect. Therefore, identification and development of more effective drugs that target to novel oncogenes and molecular targets will be the breakthrough points for therapy of CRC in the future.Angiogenesis-like protein2(ANGPTL2) is a newly identified secretory extracellular matrix glycoprotein which belongs to angiogenesis-like protein family. ANGPTL2is induced by chronic hypoxia and involves in the angiogenesis and endothelial cell migration. Besides, This protein is closely related to inflammation, obesity and insulin resistance. In addition, ANGPTL2plays an important role in multiple diseases including rheumatoid arthritis, type II diabetes, coronary artery disease and malignancy processes. However, up to now, the expression and the regulatory mechanism of ANGPTL2has rarely been reported.MiRNAs (microRNA) are a class of small non-coding RNAs with about22nucleotides in length that involve in the regulation of approximately30%of human genes at the posttranscriptional level by either degrading or inhibiting translation of mRNA. Therefore, miRNAs are capable of regulating a variety of cellular events including cell proliferation, differentiation, cell death and metabolism, etc. Dysfunction of miRNAs could lead to the initiation and progression of malignant tumors. miRNA-25is upregulated in a variety of human malignant solid tumors, including esophageal squamous cell carcinoma, hepatocellular carcinoma and pancreatic cancer. The function of miRNA-25is very complicated as it could act as oncogene or tumor suppressor gene in different cancers. However, whether miRNA-25is involved in the regulation of ANGPTL2or not in CRC needs to be further studied which provides a basis for potential molecular targets researching. Part1ANGPTL2expression in CRC and its clinical significanceObjective:To examine the expression of ANGPTL2protein in colon cancer tissue and cells, and explore the association of ANGPTL2expression with pathological features of CRC.Methods:(1). Immunohistochemistry was used for analyzing100specimens including50human normal colorectal tissues and50human colorectal cancer tissues and statistical analysis was performed for analyzing the associations between ANGPTL2expression and clinicopathological features of colon cancer.(2). Real time PCR and Western blot were used to detect the expression of ANGPTL2in one normal colorectal cell HCEpic cell and4colorectal cancer cells including HCT-116、HT-29、SW480and SW620cells.Results:(1). ANGPTL2protein level was significantly up-regulated in human colorectal cancer tissues comparing to normal tissues, and the level was closely related to the colon tumor grade, clinical stage and lymph node metastasis, the difference was statistically significant (P<0.05).(2). ANGPTL2mRNA and protein level were significantly increased in colorectal cancer cells comparing normal cells, and the expression of ANGPTL2in the high invasive colon cancer cell line HCT-116and SW620was significantly higher than the low ones HT-29and SW480.Conclusion:ANGPTL2was significantly up-regulated in human colorectal cancer tissues and cells comparing to the normal ones, and the expression level was closely related to the colon tumor grade, clinical stage and lymph node metastasis, suggested that ANGPTL2might have an important role in the development process in colon cancer. Part2The effect of AGNTPL2on proliferation and invasion in colorectal cancer cellsObjective:To investigate whether the biological behaviors such as proliferation and invasion were affected by regulating ANGNTPL2expression level in colorectal cancer cell lines.Method:(1). Lentiviral expression vector Lv-ANGPTL2and lentiviral shRNA vector Lv-ANGPTL2-shRNA and their control vector had been contructed and transfected into colorectal cancer cells HCT-116and SW620, and the stable overexpression ANGPLT2cells (HCT-116/Lv-ANGPTL2and SW620/Lv-ANGPTL2) and the stable knockdown ANGPTL2cells (HCT-116/Lv-ANGPTL2-shRNA and SW620/Lv-ANGPTL2-shRNA) and corresponding control cells (HCT-116/Lv-NC, SW620/Lv-NC and HCT-116/Lv-NC-shRNA, SW620/Lv-NC-shRNA) were established. Real-time PCR and western blot were used to detect the expression of ANGPTL2in model cells after upregulation and silencing.(2). MTT and colony formation assays were used for detecting the proliferation and colony forming ability of the cells above, respectively.(3) Transwell assay and scratch adhesion test were used for detecting the invasive ability of the cells above.Results:(1). ANGPLT2was successfully upregulated or silenced by Lentiviral-mediated overexpression and Lentiviral-mediated RNA interference, respectively. The stable overexpression ANGPLT2cells and the stable knockdown ANGPTL2cells were also successfully constructed(2). MTT results indicated that the proliferation rate of HCT-116and SW620which stably transfected with Lv-ANGPTL2vector was faster than the cells transfected with Lv-NC and blank control cells, at24h,48h and72h. The difference was statistically significant (P<0.05). In contrast, the proliferation rate of HCT-116and SW620which stably transfected with Lv-ANGPTL2-shRNA vector was lower than the cells transfected with Lv-NC-shRNA and blank control cells, at24h,48h and72h. The difference was statistically significant (P<0.05).(3). Colony formation assays showed that the clone formation rate of HCT-116/Lv-ANGPTL2, HCT-116/Lv-NC and HCT-116was78±2.65%,61.3±7.51%,62.3±6.03%, respectively; SW620/Lv-ANGPTL2, SW620/Lv-NC and SW620was84.3±3.06%,67.3±8.50%,68.3±5.51%, respectively. HCT-116/Lv-ANGPTL2-shRNA, HCT-116/Lv-NC-shRNA and HCT-116was38.3±3.79%,64.7±3.51%,63±4.58%, respecively; SW620/Lv-ANGPTL2-shRNA, SW620/Lv-NC-shRNA and SW620was42.7±4.04%,64.3±5.03%,69.7±5.57%, respectively. Differences between in the groups were all statistically significant (P <0.05).(4). Transwell experiments showed that the invasive ability of HCT-116or SW620that were stably transfected with Lv-ANGPLT2was significantly higher than the cells stably transfected with Lv-NC and control group. Differences between groups were statistically significant (P <0.05). In contrast, the invasive ability in the stable knockdown ANGPTL2colon cancer cells is lower than those in control colon cancer cells, which were statistically significant differences (P<0.05).(5). Cell scratch experiments showed that ANGPTL2overexpression cell models of HCT-116or SW620had much narrower gaps compared to the control groups in24h, and the gaps almost disappeared in48h. However, ANGPTL2knockdown cell models of HCT-116or SW620had much wider gaps compared to the control groups in24h and48h.Conclusion:(1). Lentiviral vector-mediated ANGPTL2shRNA interference sequence or ANGPTL2cDNA into HCT-116and SW620cells have high transfection efficiency, and capable of knockdown or upregulation of ANGPTL2in HCT-116and SW620cells, respectively.(2). MTT and Colony formation assays suggested that ANGPTL2promotes cell proliferation of human colon cancer cell line HCT-116and SW620.(3). Transwell experiments and Cell scratch experiments results indicated that ANGPTL2promotes cell invasive ability of HCT-116and SW620. Part Ⅲ. The effect of MicroRNA-25on suppressing colon cancer cell proliferation and invasion by Targeting ANGPTL2Objective:To investigate the molecular mechanism of miRNA-25in regulation of ANGPLT2gene and the effect of miRNA-25/ANGPLT2on proliferation and invasion of HCT-116and SW620cells.Method:(1). Bioinformatics sofeware TargetScan was used to analyze the3’UTR of mRNA sequence of ANGPTL2and identification of the potential miRNA-25binding site.(2). Real-time PCR and Western blot were used to detect ANGPTL2mRNA and protein levels in HCT-116and SW620cells that transfected with miRNA-25mimics and miRNA-25inhibitors.(3). The wild-type and the mutant-type of the miRNA-25binding site in ANGPTL2mRNA3’untranslated region were cloned into the vector psi-CHECK2, respectively. Then the luciferase assay was used to detect the luciferase activity of the wild-type ANGPTL2-3’UTR-psi-CHECK2and the Mut-ANGPTL2-3’UTR-psi-CHECK2.(4). MTT and Colony formation assays were used to evaluate the cell proliferation ability of HCT-116and SW620cells that transfected with miRNA-25mimics or miRNA-25inhibitors.(5). Transwell experiments and Cell scratch experiments were used to detected the invasive ability of HCT-116and SW620cells that transfected with miRNA-25mimics or miRNA-25inhibitors.Results:(1). A potential miRNA-25binding site was identified by TargetScan in the3’untranslated region of human ANGPTL2gene.(2). Overexpression miRNA-25into HCT-116and SW620cells significantly reduced the ANGPTL2mRNA and protein levels; Whereas transfection of miRNA-25inhibitor into the HCT-116and SW620cells significantly increased the ANGPTL2mRNA and protein levels.(3). miRNA-25can significantly inhibit the luciferase activity of the wild-type ANGPTL2-3’UTR-psi-CHECK2, but have no effect on Mut-ANGPTL2-3’UTR-psi-CHECK2.(4). Compared to pre-con and blank control group, transfection of pre-miRNA-25into HCT-116and SW620cells during72h significantly slowed the proliferation of both cells. However, compared to negative and blank control group, transfection of anti-miRNA-25into HCT-116and SW620cells during72h significantly promoted the proliferation of the cells.(5). In HCT-116cells, the numbers of colonies of blank control, pre-con control and the pre-miRNA-25group were61.00±7.55,69.67±5.69and43.0±4.00; In SW620cells, the numbers of colonies of blank control, pre-con control and the pre-miRNA-25group were65.67±3.51,64.67±7.51and48.0±3.61.The difference was statistically significant (P<0.05). In HCT-116cells, the number of colonies of blank control, anti-Con and anti-miRNA-25were60.30±5.69,60.00±3.61and78.3±4.51; In SW620, the number of colonies of blank control, anti-Con and anti-miRNA-25were67.67±3.51,66.67±8.14and81.0±4.00. The difference was statistically significant (P<0.05).(6). Transwell experiments showed that the cell invasive ability of HCT-116and SW620cells that transfected with pre-miRNA was significantly reduced compared to the control miRNA-transfected cells or blank control cells. The difference was statistically significant (P<0.05). In contrast, the invasive ability of HCT-116and SW620that transfected with anti-miRNA-25was significantly increased compared to negative and blank control cells. The difference was statistically significant (P <0.05).(7).Cell scratch experiments showed that transfection of miRNA-25into HCT-116or SW620had much wider gaps compared to pre-con cells and blank control cells in24h and48h. In contrast, transfection of anti-miRNA-25into HCT-116or SW620had much narrower gaps compared to anti-con transfected cells and blank control cells in24h and48h.Conclusion:(1) miRNA-25can be directly bound to the miRNA-25binding sites in the3’untranslated region of ANGPTL2mRNA and negatively regulated ANGPTL2gene expression in HCT-116and SW620cells.(2). MiRNA-25is able to inhibit proliferation and invasion of colorectal cancer by targeting ANGPTL2gene, implied that miRNA-25/ANGPTL2cascade could be the diagnostic and therapeutic target for colorectal cancer.Main innovative points1. For the first time, we detected ANGPTL2expression in colon cancer tissues and cells, and disclosed the relationship between ANGPTL2expression and clinicopathological characteristics.2. For the first time, we investigated the critical roles of ANGPTL2in promoting proliferation and invasion of colon cancer.3. For the first time, we identified that miRNA-25could negatively regulate ANGPTL2gene by directly binding to miRNA-25binding site in the3’UTR of ANGPTL2mRNA.