Noncoding RNAs In Colorectal Cancer Chemoresistance:Role And Its Regulatory Mechanism

Part 1.MicroRNA-218 enhances 5-fluorouracil-induced cytotoicity through suppressing BIRC5ObjectivesOne major reason for the failure of advanced colorectal cancer(CRC)treatment is the resistance to fluoropyrimidine(FU)-based chemotherapy.The enhanced ability of tumor cells to undergo anti-apoptosis process is the main contributor to drug resistance.Various reports showed that ectopic expression and function of miRNAs play key roles to mediate apoptosis by primarily down-regulating protein expression at the post-transcriptional level.To further explore the possible mechanisms and promote chemosensitivity of CRC treatment,we evaluated the prognostic effect of miR-218 in patients received 5-FU-based treatment and investigated the pro-apoptotic role of miR-218.MethodsPrimary tumor specimens and adjacent non-tumor sites were used to determine the miR-218 expression distribution and explore the potential prognostic value of miR-218 on the chemo-response to 5-FU-based treatment in CRC patients.Human CRC cells(HCT116 and HT29)were transfected with precursor miR-218 or negative control followed by assays to investigate the influence of miR-218 on cell apoptosis,cell proliferation and pathways involved in molecular mechanisms of chemoresistance to 5-FU.ResultsThe expression of miR-218 was significantly decreased in tumour tissues compared with paired normal tissues.Moreover,the CRC tissues in 68.3%(43 of 63)of cases had at least two-fold lower expression of miR-218.In addition,miR-218 expression level was much lower in patients who did not respond to 5FU treatment than those who experienced response to chemotherapy.ROC curve analysis was performed to establish the optimal cut-off value of miR-218(6×10-3)for distinguishing the responding and non-responding patients.Under these stratification criteria,patients were stratified into high(n = 34)and low(n = 29)miR-218 expression groups.The proportion of patients that responded to chemotherapy was significantly higher in the high miR-218 expression group than in the low miR-218 expression group.Kaplan-Meier survival analysis was performed to further investigate the effect of miR-218 on 5-FU treatment for CRC.The results indicated that high miR-218 expression was associated with long overall survival and progressive-free survival rate.Up-expression of miR-218 promoted apoptosis,inhibited cell proliferation in CRC cells.The anti-apoptotic gene-BIRC5,was identified as a direct target of miR-218 and the intrinsic apoptotic pathway triggered by miR-218 was through the silence of BIRC5.Gain and loss function assay indicated that miR-218 enhanced 5-FU-induced cytotoxicity and it has a strong synergistic effect with 5-FU on CRC cell growth.More importantly,western blotting showed that miR-218 silenced the 5-FU targeted enzyme,thymidylate synthase(TS).ConclusionIn this study,we demonstrated that high miR-218 predicted positive response to 5-FU-based treatments in CRC patients and discovered a novel mechanism mediated by miR-218 to promote apoptosis and to function synergistically with 5-FU to promote chemosensitivity by suppressing TS in CRC.These suggests a unique potential of miR-218 as a tumor suppressor and a novel candidate for developing miR-21 8-based therapeutic strategies in CRC.Part 2.MALAT1 is Associated with Poor Response to Oxaliplatin-Based Chemotherapy in Colorectal Cancer Patients and Promotes Chemoresistance through EZH2ObjectiveChemoresistance to oxaliplatin-based therapy has been a key barrier to the efficacy of colorectal cancer(CRC)treatment.One major reason for oxaliplatin chemoresistance in CRC is the acquisition of epithelial-mesenchymal transition(EMT)in cancer cells.The long non-coding RNA MALAT1,is a highly conserved nuclear ncRNA and a key regulator for metastasis development in several cancers.However,its role in oxaliplatin-induced metastasis and chemo-resistance is rarely known.In our study,we aim to investigate the prognostic role of MALAT1 in CRC patients receiving oxaliplatin-based therapy,and further explore the potential transcriptional regulation through interaction with EZH2 and miR-218 based on the established HT29 oxaliplatin-resistant cells.MethodsFor chemo-response study,221 serum samples and 48 primary tissues were collected from the patients who received standard Oxaliplatin-based chemotherapy,and 46 serum samples from patients who received standard FOLFOX chemotherapy were collected at Qilu Hospital of Shandong University between 2011 and 2015.QRT-PCR and QRT-PCR-D method previously established by us were used to determine the expression of mRNA expression in primary tissues and serum,respectively.Cell migration and invasion were assessed with Boyden chambers or modified Boyden chambers according to the protocol of the manufacturer.RNA immunoprecipitation(RIP)and Chromatin immunoprecipitation(ChIP)experiments were performed to investigate the potential interaction.Finally,the survival curves of CRC patients were estimated via the Kaplan-Meier method and the difference in survival curves was estimated using log-rank testing.ResultsMALAT1 expression level was much higher in patients who did not respond to treatment than those who experienced response to chemotherapy.We then stratified patients into a low(n=37)and a high(n=16)MALAT1 expressing group with an established cut-off value(0.432)by using a ROC curve analysis.In the validation group containing 168 serum samples of CRC patients,proportion of patients not responding to chemotherapy was significantly higher in the high MALAT1 expressing group than in the low group.The diagnostic sensitivity and specificity were 75.0%(72/96)and 61.1%(44/72).Furthermore,the Kaplan-Meier survival analysis indicated that high MALAT1 expression was associated with shorter OS and DFS in CRC patients.CRC cell line HT29 that had acquired resistance to oxaliplatin at the clinically relevant concentration of 2 μmol/L(HT29 OxR cells)were built.MALAT1 was significantly increased in HT29 OxR cells and MALAT1 silencing reversed oxaliplatin-induced EMT.Moreover,EZH2 directly interacts with 3’end of MALAT1,which subsequently suppressed the expression of E-cadherin.CRC cells attained increased migration and invasion ability after being treated with oxaliplatin,and this increased migration and invasion ability was partially suppressed by MALAT1 or EZH2 knockdown.Importantly,our results suggest that the interaction between MALAT1 and miRNA-218 has reciprocal effects.The Kaplan-Meier survival analysis showed that patients with low MALAT1 and high miR-218 expression had the longest OS,while the high MALAT1 and low miR-218 group showed the shortest for patients who received standard FOLFOX treatment.ConclusionsIn conclusion,the present work has identified that IncRNA MALAT1 was correlated with tumor metastasis and associated with poor response to oxaliplatin-based chemotherapy in CRC patients.MALAT1 mediates oxaliplatin-induced EMT and chemoresistance through EZH2 and interacting with miR-218.Thus,MALAT1 may be a novel functional biomarker and therapeutic target in CRC patients.Suppression of MALAT1 combining with miR-218 over-expression could be a future direction to develop a novel therapeutic strategy to enhance chemosensitivity to FOLFOX chemotherapy regimen.Part 3.LncRNA HOTAIR Contributes to 5-fluorouracil Resistance through Suppressing MiR-218 and Activating NF-κB/TS Signaling in Colorectal CancerObjectivesIn clinical situations,acquired drug resistance and enhanced metastasis frequently follow chemotherapeutic regimens,leading to treatment failure in tumor patients.Despite the extensive research on chemoresistance,the detailed mechanism underlying this phenomenon remains unclear.Long non-coding RNA HOTAIR has been considered as a pro-oncogene in multiple cancers.However,the precise functional mechanism of HOTAIR in chemoresistance is not well known.To further explore the possible mechanisms and promote chemosensitivity of CRC treatment,we evaluated the prognostic effect of HOTAIR in patients received 5-FU-based treatment and investigated the underlying regulatory mechanism of HOTAIR in 5FU resistanceMethodsThe small interfering RNAs(siRNAs)that specifically target human HOTAIR,EZH2,and VOPP1 mRNA were designated.The coding sequence of VOPP1 was amplified,cloned into PCDNA3.1 vector.The lentivirus vector containing HOTAIR short-hairpin RNA(Lv-ShHOTAIR)was amplified and cloned.Human CRC cells lines were transfected with small interfering RNAs or overexpressing precursor followed by assays to investigate the influence of HOTAIR and VOPP1 on cell proliferation,cell-cycle phase and pathways involved in molecular mechanisms of chemoresistance to 5-FU.RNA immunoprecipitation(RIP)and Chromatin immunoprecipitation(ChIP)experiments were performed to investigate the potential interaction.Western blot and immunofluorescence analysis were performed to detect the protein expression of NF-κB/TS signaling pathway.Primary tumor specimens and adjacent non-tumor sites were used to determine the HOTAIR expression distribution and explore the potential prognostic value of HOTAIR on the chemo-response to 5-FU-based treatment in CRC patients.ResultsHOTAIR negatively regulated miR-218 expression in CRC cells.RIP and ChIP assay showed that HOTAIR interacted with EZH2,and this interaction subsequently silenced miR-218-2.Both HOTAIR and miR-218 suppressed cell proliferation,and HOTAIR knockdown dramatically inhibited cell viability and induced G1-phase arrest by promoting miR-218 expression.Luciferase activity assay showed that VOPP1 was a functional target of miR-218.More importantly,the main downstream targets signaling of NF-κB,including the pathway involved in cell survival(p65-NF-KB,pAkt,pERK),cell cycle(E2F-1)and 5FU-targeted protein(thymidylate synthase,TS),were inactivated by HOTAIR through the suppression of miR-218 expression.Additionally,HOTAIR knockdown partially reversed 5FU resistance through promoting miR-218 and inactivating NF-κB signaling.Furthermore,HOTAIR restrained 5FU-induced cytotoxicity on CRC cells through promotion of TS expression.Clinical exploration of HOTAIR indicated that the HOTAIR expression level was much higher in CRC tissues from patients who did not respond to 5FU treatment than those from patients who experienced response to chemotherapy.ROC curve analysis was performed,and these patients were stratified into a low(n=56)and a high(n=96)HOTAIR expression group with an established cut-off value(4.01).The area under the curve(AUC)and diagnostic sensitivity and specificity reached 0.716,81.58%,and 55.26%with the established cut-off value,respectively.Furthermore,the proportion of patients not responding to chemotherapy was significantly higher in the high HOTAIR expression group than in the low expression group.Kaplan-Meier survival analysis indicated that high HOTAIR expression was associated with poor overall survival(OS)and recurrence-free survival(RFS)in CRC patients,and Cox regression univariate/multivariate analysis showed that HOTAIR expression level maintained its significance as independent prognostic factors for OS of CRC patients receiving 5FU treatment.ConclusionOur integrated approach demonstrated for the first time that HOTAIR contributes to CRC tumorigenesis and 5FU resistance through downregulation of miR-218 and activation of NF-κB signaling.This IncRNA directly recruits EZH2 and suppresses miR-218 by binding to its promoter,which provides a mechanistic foundation for the aberrant VOPP1 activation in CRC.This pro-resistant role of HOTAIR was further validated in an independent set of CRC patients who received standard 5FU treatment.Thus,HOTAIR may be a novel prognostic biomarker and therapeutic target in CRC patients.Suppression of HOTAIR could be a future direction for enhancing chemosensitivity to 5FU-based chemotherapy regimens.