Screening Sensitivity Markers Of Preoperative Concurrent Chemoradiotherapy For Locally Advanced Rectal Cancer By MicroRNA Microarray

Part 1:Long follow-up results from a phase II clinical trial:Preoperative chemoradiotherapy after 5-fluorouracil and oxaliplatin for local advanced rectal cancerObjective:To observe the long follow-up results for patients with locally advanced rectal cancer with preoperative chemoradiotherapy after 5-fluorouracil and oxaliplatin in the treatment of.Methods:From Feb.2002 to Nov.2006,58 patients with locally advanced rectal cancer were participated in this clinical study.The primary end points included tumor regression,anus preserving rate,toxicity and side effects.The secondary end points included cumulative incidence of local recurrence and distant metastasis,disease-free survival rate and overall survival rate.Results:58 patients participated in the study and completed preoperative chemoradiotherapy.53(53/58)patients received TME operation,1(1/58)received R1 resection and two(2/58)received R2 resections.The evaluation of the patient’s postoperative specimens was based on the tumor regression grading standard.The total effective rate was 32/55(58%),of which the complete response percent(pCR)was 11/55(20%),and only detected in vivo The ratio of circulating tumor cells was 21/55(38%).In terms of toxicity,grade 4 toxicity and postoperative death did not occur,and 9 patients(16%)had grade 3 toxicity.Diarrhea and proctitis are the most common side effects.2 patients(3.4%)occurred rectitis(Grade-2).4 patients(6.8%)occurred in rectitis(grade 3),and 10 patients(17%)occurred in Grade 2 diarrhea and grade 3 diarrhea in 3 patients(5%).No Severe neurotoxicity was occurred,and mild neurotoxicity was observed in 9 patients(16%)with grade 1 sensory neuropathy.Myelosuppression was mild,with grade 3 neutropenia and thrombosis in one patient.Two patients(3%)developed grade 3 fatigue.All patients completed concurrent chemoradiotherapy without modifying the main scheme.The median follow-up period reached 138 months(range 109-151 months).32(55.2%)patients died during the time;the other 28(87.5%)died of tumor-progress cause.Among the 51(87.9%)patients received R0/R1 complete resection,only one had local recurrence,21(38.2%)patients end up with distant metastasis,and 4 had both distant and local recurrence.2(3.6%)patients received R2 resection died of distant tumor metastasis.6 patients with distant metastasis received multiple organ metastasis among the 27 patients.The most common site of metastasis(n=14)was the lung,followed by the liver(n=11),bones(n=5),retroperitoneal lymph nodes(n=3),and ovaries(n=1).The total incidence of in situ recurrence in 5 years was 12.1%,while the 10 years was 12.1%,respectively.The median in situ recurrence periods was 22 months(range from 7-43 months).Morover,the total incidence of distant metastasis in 5 years was 51.3%,while the 10 years was 53.4%,respectively.The median distant metastasis time was 16 months(range from 6-74 months).The 5 years-disease-free-survival(5y-DFS)rate was 47.2%.The 10 years DFS was 45.1%.The 5 y-overal-survival(OS)rate was 54.7%.The 10 y-overal-survival(OS)was 43.2%..The 5y-overall survival rate of those intention to treat(ITT)population was 50.0%.The l0y-overall survival rate of those intention to treat(ITT)population was 39.5%.Among various probable prognostic factors,like anal distance,gender,age,T-classification,tomor N-classification and complete remission of Pathology,to predict local recurrence,distant metastasis,disease-free survival and overall survival.Results univariate analysis showed that complete remission was related to local recurrence,distant metastasis,disease-free survival and total survival(P<0.05).Conclusions:After preoperative chemoradiotherapy and total mesorectal resection,distant metastasis is still the main failure mode of local advanced rectal cancer.CRC may has one independent prognostic factor,which is pathological complete remission,for after preoperative radiotherapy and chemotherapy.Part 2:Biomarker Selection for Predicting the Sensitivity of Preoperative Concurrent Chemoradiotherapy in Locally Advanced Rectal CancerObjective:Our study was to discover the deep association between miRNA-519b-3p and response to preoperative chemoradiotherapy in LARC.Methods:RT q-PCR was conducted to anylysis the miRNA-519b-3p expression in bloods of patients with LARC.HCT116 or SW480 cells were all transient transfected with miRNA-519b-3p inhibitor or mimics.Results:Where miRNA expression levels were assessed based on microarray data(GSE98959),miRNA-519b-3p was the most prominent miRNA upregulated(P<0.05).Real-time PCR results showed that miRNA-519b-3p still showed higher expression levels in responders(n=21)than non-responders(n=34).Chemoradiotherapy responders with LARC had higher miRNA-519b-3p.The level of miRNA-519b-3p was tested by in situ hybridization(ISH)in rectal cancer in responders and non-responders.The results showed that the expression level of miRNA-519b-3p was significantly higher in LARC responders than in the non-responder group.The Receiver Operate Charecteristic Curve(ROC)was analyzed based on miRNA-519b-3p expression,and the AUC of miRNA-519b-3p was 0.87.The sensitivity and specificity of miRNA-519b-3p were 100%and 69%,respectively.Furthermore,the AUC,sensitivity and specificity of miRNA-519b-3p based on 55 LARC patients were 0.91,100%and 81%,respectively.HCT1 16 or SW480 cells were respectively transfected in vitro with miRNA-519b-3p mimic or inhibitor,and miRNA-519b-3p was up-regulated or down-regulated.The CCK8 clone formation assay showed that the miRNA-519b-3p mimic made HCT116 and SW480 cells more sensitive to chemoradiotherapy,while the miRNA-519b-3p inhibitor gave the opposite result.The CCK-8 method results told that miRNA-519b-3p overexpression inhibited HCT116 and SW480 cell proliferation after CRT treatment.After CRT treatment,the effect of miRNA-519b-3p on apoptosis of HCT116 and SW480 cells was evaluated by FACS.The miRNA-519b-3p mimetic increased the apoptosis of HCT116 and SW480 cells after chemoradiotherapy,while the miRNA-519b-3p inhibitor reduced cell death.Conclusions:miRNA-519b-3p is a biomarker for predicting the sensitivity of preoperative chemoradiotherapy for locally advanced rectal cancer.Part 3:miRNA-519b-3p is the Enhancer to the Response of Preoperative Chemoradiotherapy in Patients with Locally Advanced Rectal Cancer by Targeting ARID4BObjective:Explore the mechanism of miRNA-519b-3p of improving preoperative chemoradiotherapy response in locally advanced rectal cancer patients by targeting ARID4B.Methods:The correlation between the chemoradiotherapy response and the expression level of miRNA-519b-3p was verified in vitro.Dual luciferase reporter assay was conducted to vetify the binding site of miRNA-519b-3p and ARID4B.The miRNA-519b-3p were up-regulated or down-regulated by recombinant plasmid.We constructed heterotopic xenograft tumor nude mice to study the efficacy of miRNA-519b-3p on ARID4B..We used the online tools of TargetScan and miRanda to perform target gene bioinformatics analysis of miRNA-519b-3p.Results:Chromatin remodeling protein AR1D4B,TargetScan total score-0.61,miRanda mirSVR score-0.9906,became our target molecule,and predicted binding sites in the 3’UTR of ARID4B.Overexpression of miRNA-519b-3p significantly inhibited luciferase activity in HCT116 and SW480 cells transfected with 3’UTR-WT plamid(p<0.05),and vice versa.However,this mutation in the binding site eliminates this trend.There was a significant negative correlation between miRNA-51 9b-3p and ARID4B(r=-0.55 and P<0.001).qRT-PCR analysis showed that miRNA-519b-3p overexpression significantly reduced the level of ARID4B in HCT116 and SW480 cells.The expression of ARID4B in LARC patients found that the expression level of ARID4B was much higher in non-responders than in responders(P<0.05).After transfected with the miRNA-519b-3p mimics,,the ARID4B expression more in both HCT116 and SW480 cells,the mRNA and protein levels of ARID4B returned to the control level.The miRNA-519b-3p mimic makes HCT116 and SW480 cells more sensitive to chemoradiotherapy.miRNA-519b-3p also reduced the sensitivity of ARID4B.Overexpression of ARID4B,can stimulated the proliferation of both HCT1 16 and SW480 cells after chemo radiotherapy,Apoptosis of both HCT116 and SW480 cells after chemoradiation decreased.In nude mouse xenograft body,miRNA-519b-3p reduction inhibited tumor progress in vivo,and win back the recovery of ARID4B expression reversed this effect.Conclusions:ARID4B was confirmed to be targeted by miRNA-519b-3p.ARID4B was also involved in the response to pCRT in LARC patients.miRNA-519b-3p has the rising ability,which can rise the response of preoperative chemo radiotherapy in patients with LARC by targeting ARID4B.