| Background:The insulin-like growth factor(IGF/IGFIR)signaling axis plays a critical role in development,growth,and metastasis of breast cancer,as well as the self renewal of cancer stem cells.In this axis,the IGFlR gene is upregulated in a variety of human malignancies and serves as a well-established target for cancer drug therapy In a previous study,we utilized a "RNA-guided chromatin conformation capture"(R3C)assay to identify IRAIN,an intragenic antisense long noncoding RNA(1ncRNA)in the IGFlR locus.As a putative tumor suppressor,IRAIN is downregulated in breast cancer cells,while IGFlR is overexpressed,leading to an abnormal IGFIR/IRAIN ratio that promotes tumor growth.To precisely target this aberrant pathway,we developed a novel"antisense IncRNA-meditated in situ cis competition"(ALIC)approach to therapeutically correct the elevated IGFlR/IRAIN bias in breast cancer cells.The IRAIn antisense IncRNA is overlapped with the IGFlR promoter and exon 1.We thgus proposed to use the CRISPR-Cas9 gene editing approach to insert a strong CMV promoter in front of IRAIN.The overexpressed antisense lncRNA competed in cis with the IGFlR sense mRNA.By activating the weak promoter of antisense IncRNA IRAIN and cis downregulating the sense IGFIR,we aimed to correct the aberrant IGFlR/IRAIN bias in breast cancers.This targeted normalization of IGFlR/IRAIN transcription would reverse the activated IGFlR signaling pathway in breast cancer cells and inhibited their tumor phenotypes.This ALIC approach may provide a useful strategy to develop novel precision medicine therapies for the treatment of breat cancersObjectives:We aimed to correct the elevated IGFlR/IRAIN bias in breast cancer cells,thus providing a useful strategy to target disease genes for the development of novel precision medicine therapiesMethods:1.The CRISPR-Cas9 gene editing system was used to target the weak promoter of IRAIN antisense lncRNA and replaced it with a strong cytomegalovirus(CMV)promoter;2.We constructed the targeting vector and the homologous template donor vector,and co-transfected them into MDA-MB-231 breast cancer cells.Puromycin was used to select the positive cells,and ganciclovir was then added to the cell medium to reduce cells that had random integration of the donor vector in the genome.3.The expression of IRAlN and IGFIR in selected cell clones was measued by real-time Quantitative PCR4.Western blot was used to detect the expression of IGFlR protein5.Tumor phenotypes,including cell proliferation,cell cycle,migration and clone formation,were measured by MTT,flow cytometry,transwell and plate assay.6.The status of DNA methylation of IGFlR promoter was analyzed by sodium sulfite seqeuncing.7.For comparison,lncNRA IRAlN was also overexpressed by lentivirus to study its trans effect on IGFIR8.IRAIN was overexpressed in the IRAIN knockdown cells as a rescue assay to determine its competitive inhibition of IGFlR9.To study the trans regulatroy mechanism of IRAIN,a"nuclear lncRNA reverse transcription-associated trap sequencing"(RAT-seg)assay was used to profile the genome-wdie target interactome for IRAIN lncRNAResuits:1.Targeted activation of IRAlN was accomplished by co-transfection of targeted vectors and donor vectors in MDA-MB-231 cell lines.The expression of gRNAs guided the Cas9 complex to the TRAIN promoter site and induced homologous recombinantion at the IRAIN promoter site.After puromycin and ganciclovir double select,stable cell clones were collected and identified by PCR sequencing to determine the success of the knock-in site.In selected clones,a strong CMV promoter(pCMV)was inserted in front of the weak IRAIN promoter in breast cancer cells.As expected,the expression of IRAIN weas significantly higher in selected clone cells than that in wild type cell lines without targeted modification.2.Targeted activation ofIRAINinhibited IGFlR expression.RT-PCR detected high expression of IRAIN while IGFlR was significantly down-regulated in clone cells.Western results showed that IGFlR protein expression was also reduced in targeted cells.These data suggest that IGFlR was significantly downregulated by cis competition of antisense IncRNA.3.Targeted inhibition of IGFIR by ALIC reduced the tumor potential of MDA-MB-231 cells,including cell proliferation,clone formation,migration and invasion.4.After targeted inhibition of IGFlR,cell cycle was analyzed by flow cytometry.There was a significant increase in S-phase cells(CTL 23.3%vs clonel 32.2%vs clone2 40.1%)and a decrease in G2-phase cells in targted clone cells.5.DNA methylation of the IGFlR promoter was detected by sodium sulfite sequencing.There was no significant difference between targeted cell clones and control cells,indicating that cis competition inhibition was independent of the epigenetic modification of the IGFlR promoter region.6.To explore the mechanisms underlying the role of IRAIN in breast cancer cells,we used RAT-seq to map the whole-genome target gene network for lncRNA-IRAIN.Interestingly,it was noted that IRAIN lncRNA interacted with multiple pathway genes,including those involving cell metastasis,signaling pathways and immortalization of cells.Among them,IRAIN downregulated its target gene NM23,a known metastatic suppressor gene.Conclusions:By activating the weak promoter of antisense IncRNA IRAIN using an ALIC approach,we showed that the intragenic activation of antisense IncRNA IRAIN potently competed in cis with the promoter of the IGFJR sense mRNA.Notably,the normalization of IGFlR/IRAIN transcrnption inhibited the IGFIR signaling pathway in breast cancer cells,decreasing cell proliferation,tumor sphere formation,migration,and invasion.Using RAT-seq,we uncovered a genome-wide IRAIN IncRNA-specific interactome,containing gene targets involved in cell metastasis,signaling pathways,and cell immortalization.These data suggest that aberrantly upregulated IGFIR in breast cancer cells can be precisely targeted by cis transcription competition,thus providing a useful strategy to target disease genes in the development of novel precision medicine therapies. |
