The Expression Level Of SLFN11 Gene Affects The Sensitivity Of Human Colorectal Cancer Cell Line To SN38

Objective: Colorectal cancer (CRC) is one of the most common malignancies with a highrate of cancer mortality worldwide. Chemotherapeutic regimens have been found to bebackbone of therapy for advanced-stage cancer. Although irinotecan (CPT-11), atopoisomerase inhibitor, is one of the most important drugs in the treatment of advancedand/or metastatic colorectal cancer (CRC), resistance is a critical drawback to its clinicaleffectiveness. There is no recognized predictor of the efficacy of CPT-11until now. Therefore,it is of great importance to find efficacy predictors of capable of selecting those patients whowould benefit most from CPT-11.Schlafen-11(SLFN11)，a member of Schlafen family, has been identified as highlycorrelate to irinotecan(CPT-11) sensitivity in some malignant cells. Schlafen family wasoriginally identifed to regulate T cell development/differentiation in the thymus of mouse bySchwarz in1998. The limited studies of Slfn genes suggest that they have important functionin cell growth and differentiation. As an important negative regulator of cell growth gene,SLFN gene family is likely to be a potential tumor suppressor gene. SLFN11expressed inmany kinds of tumor cell lines with wide expression range. SLFN11expression varieswidely in clinical colon and ovarian adenocarcinoma specimens. Furthermore, mean SLFN11expression in cancer specimens was lower than that that in normal tissues. These resultsprovide a basis for the studies of SLFN11functions in vivo and in vitro. Some researchersrecently screened a large panel of cancer cell lines transcriptome to find SLFN11sensitizesome malignant cells to topoisomerase inhibitors. The mechanism by which this occurs hasn’tbeen elucidated fully. It is necessary to further validate SLFN11function and investigate itsrelated mechanism.This study aimed to identify the role of SLFN11in predicting the response of coloncancer cells to CPT-11in order to develop predictive biomarkers at the transcript expressionlevel and to investigate the related mechanism further.Methods:(1) The mRNA and protein expression of SLFN11in eight human colon cancer cell lines weredetected by real time quantitative PCR and Western blot analysis respectively. The highexpression level cell lines and the low ones were selected to further study. (2) The small RNA interference (siRNA) targeting at SLFN11gene was constructed and wastransiently transfected into colon cancer cell line. The effect of SLFN11silence was detectedby real time quantitative PCR and Western blotting.(3)SLFN11overexpression was done through the way of gene cloning and prokaryoticexpression, plasmid containing SLFN11construction and cells transfection. The transfectvector include EXN91mAb labeled gold nanoparticles(AuNP), conventionalLipofectamine2000and control vector. The effect of SLFN11overexpression was examinedby conversion rate and expression level.(4) The cell lines were treated with different concentrations of SN-38for48h. Then theinfluence of SLFN11expression level on growth inhibiton induced by SN-38were examinedby MTT assay.(5) After80nM SN-38treatment，SLFN11effects on apoptosis rate and cell cycle arrestwere determined by flow cytometry.Results:(1) We detected SLFN11mRNA and protein levels in eight human colon cancer cell lines.SLFN11protein expression was similar to SLFN11mRNA levels. SLFN11expression washighest in the LS174T and SW480cell lines; in contrast, it was lowest in the HCT-8andHCT116cell lines. LS174T, SW480, HCT-8and HCT116cell lines were selected to furtherstudy.(2) The expression of SLFN11gene of the above four cells were efficiently blocked bysiRNA, which the best inhibition rates were all more than50%reduction at mRNA andprotein levels respectively. SLFN11expression was upregulated most effectively by EXN91mAb-AuNP-SLFN11transfection in HCT8cells with more than90%cell conversion ratesand11.9fold SLFN11expression level increase. Only10%cell conversion rates wereobserved in Lipofectamine2000-SLFN11transfecting HCT8cells.(3) After treatment with SN-38at different concentrations for48h， the cell growthinhibition effect of SLFN11was observrd. Silencing SLFN11gene significantly decreasedSN-38induced anti-proliferation compared with negative control silence (mock) in LS174Tand SW480cells (p<0.01, p <0.01). However, silencing SLFN11didn’t have effect ongrowth inhibition induced by SN-38in two low SLFN11level cells(p>0.05, p>0.05).SLFN11upregulated by EXN91mAb-AuNP-SLFN11transfection markedly increased SN-38induced growth inhibition by5.1fold in HCT8cells with low SLFN11level.(4) After24-h treatment with80nM SN-38, the effect of SLFN11interference on cell cycledistribution was examined by flow cytometry. Following SN-38treatment, the percentage of cells in the G0/G1phase decreased significantly in LS174T SLFN11-silenced cells andSW480SLFN11-silenced cells compared with their corresponding control-silenced cellsrespectively (p<0.05, p<0.05). Moreover, SLFN11overexpression in HCT8cells caused earlyS cycle arrest in response to SN-38: with significant increase in G0/G1phase and decrease inS phase (p<0.01, p>0.05).（5） After24-h treatment with80nM SN-38, The proportion of SN-38treatedSLFN11-siRNA-transfected LS174T cells that underwent apoptosis was decreasedsignificantly compared with SN-38–treated control siRNA-transfected cells (p<0.05). Thesimilar significant change in the SLFN11-siRNA-transfected SW480cells compared tocontrol transfections was detected (p <0.05). SLFN11overexpression significantly enhancedthe effect of SN-38–induced apoptosis in HCT-8cells compared to their controlcounterparts(p <0.01).（6）SLFN11silencing/overexpression had no effect on cell cycle distribution and apoptosiscompared with their corresponding controllers in the absence of SN-38(p>0.05).Conclusions:(1) SLFN11gene expression in human colorectal cancer cell lines has a wide distribution, ishighly expressed in LS174T, SW480cell lines, and lowly expressed in HCT8HCT116celllines.(2) SLFN11gene expression levels has significant effect on colon cancer cells in response toSN-38. SLFN11expression downregulation can decrease SN-38induced growth inhibition,early S cell cycle arrest and apoptosis in colon cancer cell lines. We first reported thatSLFN11upregulation might improve SN-38effect.(3) EXN91mAb mediated AuNP vector might be acted as a highly efficient SLFN11gene-targeted delivery systems and help upregulate SLFN11gene expression.(4) SLFN11might be a novel predictive biomarker for CPT-11treatment in CRC. SLFN11also might be a potential contributor to targeted therapy to improve chemosensitivity.