Function And Mechanism Of Igf1r Related Rioncoding Rna-irain In Malignant Tumor

Insulin-like growth factor1receptor (IGF1R) is a member of insulin-like growth factorsystem, after binding its ligands (IGF1and IGF2), autophophorylation activates PI3K andMAPK signal pathway, resulting in cell proliferation, differentiation and apoptosis. IGFIR isfrequently overexpressed in both solid tumors and hematopoietic malignancies, participatingin the regulation of cancer cell proliferation, survival, metabolism and metastasis. It isidentified that IGF1R and its ligands dysregulated in acute leukemia, multiple myeloma,breast cancer, prostate cancer, ovarian cancer, endometrial carcinoma, cervical carcinoma, nonsmall cell lung cancer, Ewings sarcoma. IGF1R dysregulation was reported to related withtumor progression, metastasis, resistance to chemotherapy and radiotherapy. IGF1R signalingpathway plays a critical role in tumorigenesis and progression.Long noncoding RNA is a general term for a class of RNA molecules over a length of200nucleotides. Recent reports states that lncRNA regulated gene expression and function byepigenetic mechanism, resulted in tumorigenesis and metastasis.However, little is known regarding the molecular mechanisms underlying IGF1R genedysregulation in cancer. To address the mechanisms underlying the dysregulation of IGF1R inleukemia cells and solid tumors. We discovered a novel intragenic long noncoding RNA(lncRNA) within the IGF1R locus, named IRAIN.Aim:We aimed to reveal structure, function and regulation mechanism of this novellncRNA-IRAIN.Methods:1. SSRT-PCR (SSRT) assay was used to map the transcription of the IRAIN lncRNA.2. Characterization of the IRAIN lncRNA by5’ and3’ racing.3. Northern blot was used to examine the RNA and other splicing transcripts.4. Gene expression by real-time qRT-PCR.5. Examination of genomic imprinting by enzyme cutting and sequencing.6. Promoter DNA methylation. 7. Knockdown of IRAIN lncRNA by shRNA.8. Lentiviral expression of IRAIN lncRNA.9. Cell growth, invasion and migration assays10. Chromosome conformation capture11. Reverse transcription-associated trap assay12. RNA-Chromosome conformation capture13. Statistical analysisResults:1. IRAIN structure: we characterized IRAIN as a5366bp noncoding RNA, initiatedfrom a promoter located in the IGF1R intron1. Using this SSRT assay, we found that thisnoncoding RNA was detected only when cDNA was synthesized using5_-oligonucleotides，indicated that this noncoding RNA is transcribed in the antisense direction as compared withthe IGF1R codingRNA.2. IRAIN expression: Using a normal hematopoietic stem cell line HSC2as a standard,we found that IRAIN was downregulated in leukemia cell lines and breast cancer cell lines，as compared with the IGF1R sense coding RNA，especially in HL60，U266，MDA-MB-231，T47D.3. IRAIN genomic imprinting: We first examined if IRAIN was monoallelicallyexpressed in tissues. Kg-1and Kg-1a cells expressed the IRAIN lncRNA in a monoallelicmanner, as only the ‘A’ allele was detected in both the Alu I and Sac II sites. Refering toimprinting allele，We searched three families. In one healthy family and two leukemiapatients families, the father carried both the A and G alleles and the mother had the G allele.The child was informative at the polymorphic site, carrying both the A and G alleles ingenomic DNA. In the cDNA sample, however, we detected the expression of IRAIN lncRNAonly from the A allele that was inherited from the father, demonstrating that this lncRNA ispaternally expressed and maternally suppressed.4. Function of IRAIN: We virally expressed the full-length5.3kb IRAIN lncRNA inT47D tumor cells and silenced the IRAIN expression in breast cancer cell lines. As comparedwith thevector control, expression of the full-length5.4kb IRAIN lncRNA significantlyinhibited tumor cell migration).No differences was seen in IGF1R expression in cell growthand invasion after overexpression or silencing by lentivirus plasmid.5．Mechanism of IRAIN imprinted expression: Promoter DNA methylation was found to be related to genomic imprinting phenomenone. After treated with dy-methylation drugs5-azacytidine，mild increase of IRAIN expression was seen by qRT-PCR.6．Mechanism of IRAIN involved formation of chromatin spatial structure: Werevealed a long distance intragenic chromasome confirmation capture that are150kb apart.Using primers from these remote regions, we found that the IGF1R promoter DNA interactedwith the putative intronic enhancer DNA. We knocked down IRAIN lncRNA with twoshRNAs. Both shRNAs significantly decreased the IRAIN lncRNA. These data suggest theinvolvement of the lncRNA in the formation and/or maintenance of the long-rangeintrachromosomal loop.7. Clinical significance of IRAIN: By examine the expression of IRAIN in low risk andhigh risk acute myeloid leukemia patients，We found IRAIN expression was higher in lowrisk patients than in high risk patients，indicating an tumor suppressor role in gene regulation.Conclusions：1. A novel lncRNA-IRAIN is found. IRAIN is a5366bp antisense long noncoding RNA,which initiated from intron1of IGF1R.2. IRAIN is genomic imprinted gene, which expresses paternal allele. Promoter DNAmethylation is related to genomic imprinting.3. IRAIN regulates IGF1R gene promoter and enhancer interaction, without which longintragenic chromotin confirmation disappears.4. IRAIN expresses lower in tumor cell lines compared to that of IGF1R expression, andit also expresses lower in acute myeloid leukemia with high risk, suggesting that IRAIN maybe a tumor suppressor in malignancies.5. Future studies will needed to make sure the function and mechanism of this novellncRNA-IRAIN， and it will promote targeted therapy and prognosis surveillance formalignang tumors.