| Research background:Colorectal cancer(CRC)is one of the most common malignant tumors of the digestive tract in the world.It is the fourth most common cause of death in cancer patients,and its incidence is increasing significantly.CRC is prone to liver metastasis,which is the leading cause of poor prognosis and death.Therefore,it is necessary to investigate the molecular mechanism of invasion and metastasis of CRC.micro RNAs(mi RNAs)is a class of endogenous gene encoding length of 19-25 nucleotides single stranded non-encoding small RNA molecules by degrading the target messenger RNA(m RNA)or inhibiting translation,which can play a regulatory role in a variety of biological processes such as cell proliferation,differentiation,metabolism and death.It was found that mi RNAs could also act as a tumor suppressor gene or oncogene in the processes of tumorigenesis and metastasis.microRNA-140-5p(mi R-140)is a cartilage specific expression mi RNA,and its down-regulated expression has been shown to be involved in the osteoarthritis(OA)pathogenesis.Recent studies have shown that mi R-140 is involved in tumorigenesis and development.Studies have reported that the expression of mi R-140 is decreased in colorectal cancer,hepatocellular carcinoma(HCC),non-small cell lung cancer(NSCLC),esophageal cancer,pancreatic cancer,bile duct carcinoma,osteosarcoma,hypopharyngeal squamous cell carcinoma and glioma,which inhibitsthe migration and invasion of tumor cells and plays the role of tumor suppressor.In addition,mi R-140 can inhibit the formation of cancer stem cells in breast cancer and colon cancer.However,some studies have found that mi R-140 increased from grade II to grade IV in the process of glioma.It is also reported that mi R-140 is highly expressed in invasive ductal carcinoma and metastasis of breast cancer,which can enhance the tumor progression and metastasis.Therefore,some contradictories still exist on the role of mi R-140 in the tumor progression and metastasis and the underlying mechanism is uncertain.Studies have confirmed that ADAMTS5(a disintegrin metalloproteinase with thrombospondin motifs 5)and IGFBP5(insulin like growth binding protein 5)are the direct targets of mi R-140.The overexpression of ADAMTS5 was positively correlated with the progression of malignant glioma,laryngeal squamous cell carcinoma,breast cancer,head and neck squamous cell carcinoma(HNSCC)and non small-cell lung cancer.IGFBP5 can promote the occurrence of prostate cancer,breast cancer,colon cancer,and its high level can promote the progression of breast cancer.However,the role of ADAMTS5 and IGFBP5 in the invasion and metastasis of human colon cancer has not been reported.Objectives:(1)To investigate the role and mechanism of mi R-140 on the invasion and metastasis of CRC.(2)To investigate the expressions of ADAMTS5 and IGFBP5 in human CRC samples and their clinicopathologic significance.Methods:(1)The level of mi R-140 in human CRC and adjacent normal colorectal tissues was detected by real-time quantitative PCR(q RT-PCR).The relationship between mi R-140 expression and CRC stage and distant metastasis was analyzed with clinical pathological characteristics.(2)Human CRC cell lines HCT116 and RKO were transfected with mi R-140 mimic(mi R-140)and non-specific mi RNA(NC),inhibitor(Anti-mi R-140)and non-specific mi RNA inhibitor(Anti-NC),or small interfering RNA(si RNA)against ADAMTS5 or IGFBP5(si ADAMTS5 and si IGFBP5 respectively)using lipofectamine 2000.(3)q RT-PCR was used to measure the levels of mi R-140,ADAMTS5 and IGFBP5 m RNA in the CRC cells.Western blot was used to detect the expression of ADAMTS5 and IGFBP5 protein and m RNA.(4)Scratch-wound assay and Transwell migration and invasion assays were used to evaluate the effect of mi R-140 on the capabilities of migration and invasion from gain-of andloss-of-function analysis.(5)Immunohistochemistry was performed to determine the expressions of ADAMTS5 and IGFBP5 protein in 60 paired of human CRC specimens and the corresponding normal colorectal tissues,and the relationship between ADAMTS5 and IGFBP5 expression and colon cancer progression and distant metastasis was analyzed.Results:(1)mi R-140 was significantly reduced in human CRC tissues,compared to the corresponding normal colorectal mucosa(P = 0.037).Its level was negatively correlated with CRC stage(P = 0.045)and distant metastasis(P = 0.031).No significance was found between mi R-140 expression and the clinicopathologic factors,such as age,gender,tumor size and differentiation(P> 0.05).(2)Western blot analysis showed that the ectopic expression of mi R-140 downregulated ADAMTS5 and IGFBP5 protein levels compared to the Control and NC groups(P< 0.05),whereas no difference was found when compared to the positive controls,si ADAMTS5 and si IGFBP5 groups(P> 0.05).The q RT-PCR analysis showed that the m RNA levels of ADAMTS5 and IGFBP5 were also reduced significantly(P< 0.05).(3)The wound healing assay showedthat the migrating ability was dramatically attenuated by mi R-140 compared with that in the control and NC groups,whereas no significance was found when compared with that of the ADAMTS5 and IGFBP5 si RNA transfected cells.The number of HCT116 cells migrating through Transwell chamber without matrigel in the mi R-140 group was 63.7±4.5,remarkably lowered than that in the control(103.2±3.5)and NC(107.5±6.1)groups(P< 0.05 for both),but no significant difference between the mi R-140 and si ADAMTS5(58.9±3.8)and si IGFBP5(57.8±2.6)groups(P> 0.05).Similar results were also obtained in RKO cells.Transwell chamber with matrigel assay showed that number of cells penetrating through themembrane was 54.6±2.9 in the mi R-140 group,significantly lower than that in the control(91.2±6.1)and NC(89.6±3.9)groups(P< 0.05 for both),while ADAMTS5 and IGFBP5 si RNA transfection had a similareffect(45.2±2.3 and 47.9±3.6,P > 0.05).The results from RKO cells were consistent with the data from HCT116 cells.Knockdown of mi R-140 increased the level of ADAMTS5 and IGFBP5 protein expressions,and partially reversed the inhibition of the cell migration and invasion mediated by mi R-140.Co-transfection of mi R-140 inhibitor and ADAMTS5 and IGFBP5 si RNA had no significant effect on the ADAMTS5 and IGFBP5 protein expressions and the abilities of cell migration and invasion.(4)The expressions of ADAMTS5 and IGFBP5 were increased in human CRC samples,and their overexpressions were positively associated with CRC progression(P = 0.041 and P = 0.037 respectively)and distant metastasis(P = 0.027 and P = 0.021 respectively).Conclusions:(1)ADAMTS5 and IGFBP5 are negatively regulated by mi R-140 at posttranscriptional level in the CRC cells.(2)mi R-140 inhibits the migration and invasion of CRC cells,possibly through downregulating ADAMTS5 and IGFBP5.(2)mi R-140 downregulation and ADAMTS5 and IGFBP5 overexpressions contribute to the progression and distant metastasis of CRC.mi R-140 might be a potential therapeutic candidate for the treatment of CRC. |
