Study On Imprinting Expression And The Roles Of A Novel Long Non-coding RNA IRAIN In Breast Cancer

IntroductionBreast cancer, a great menace to women‘s health, accounts for18%of allmalignant tumors of female patients. It has become the number one source of deathfor women between the age of35and65. Although the rapid development ofindividual target therapy and other treatments has significantly prolong the survival ofbreast cancer patients, we still have not any clue for solving the problem as resistanceto target therapy and no effective therapy for Triple Negative Breast Cancer. That ismainly because of the high heterogeneity of pathogenic factors, disease evolvement,therapeutic reaction and organ metastasis tendency in breast cancer. An in-depth studyat emergence and development of relative signal pathway and searching individualtherapy target is needed for the purpose of conquering breast cancer. In view of theimportant position of upstream members of PI3K/AKT/mTOR pathway such asestrogen receptor (ER)、Progesterone receptor（PR）and human epidermal growthfactor receptors (HER2), another upstream member, insulin-like growth factor1receptor (IGF-1R) arouses researchers‘great interest. It is a transmembrane tyrosinereceptor. After binding IFG ligands, it activates PI3K and MARP signal pathwaythrough autophosphorylation and then regulates cell‘s proliferation, differentiationand apoptosis. The pathway‘s frequent dysregulation in breast cancer evidences thatthe receptor-mediated signal transduction IGF-1R plays a very important part in thegrowth, evolvement, invasion of the cancer, the formation of tumor‘s blood vessel andmetastasis. As one of the most promising targets, the medicine of target therapyfocusing on IFG-1R came into being. Though the result of early experiment wasinspiring, most of the following ones came out as a failure, which shows that we haveinadequate knowledge on the signal pathway. We found it necessary to probe into theregulating mechanism further, and to search a biomarker which can predict the antiIGF-1R therapy effect with the objective of selecting the potentially beneficial target group. The most recent study suggests that the long non-coding RNA (LncRNA),previously regarded as a genomic transcription by-product‘or dark matter‘is able toregulate genomic expression through various application modes and consequentlyinvolves in tumorigenesis and metastasis. LncRNA is a general term for a class ofRNA molecules over a length of200nucleotides, which has become one of the hottestissues in modern molecular biology. Some LncRNA has been found in humangenome, but its regulating mechanism is still unknown. No publications ever revealhow LncRNA regulates IFG-1R worldwide. When the guest advisor of the projectProfessor Hu Jifan adopted the chromosome conformation approaches published on<Science> in a pilot study to search the regulating factor which involves in IGF-IR‘spromoter region, a novel LncRNA was found in IGF-1R locus, and thereafter calledIRAIN. In the light of the important status of IGF-1R pathway in breast cancer andthe fact of IRAIN‘s existence in IGF-1R locus, we presume that IRAIN play a certainrole in the breast cancer‘s tumorigenesis and growth, though the study of IRAIN inbreast caner is totally underdeveloped.ObjectivesWe aimed to illustrate the expression level and characteristics of a novelLncRNA-IRAIN in normal breast tissues and different breast cancer molecularsubtypes and the correlation between IRAIN and expression of IFG-1R. Furthermore,we are ready to confirm the key molecules in regulating IFG-1R pathway and themechanism, and to reveal the correlation between IRAIN and clinical pathologicalfactors and prognosis. Finally, we are subject to offer a novel thought andexperimental evidence to search for a biomarker with predictive and prognostic valueand individualized therapeutic target for breast cancer.Methods1. we were to confirm if IRAIN mRNA was expressed in breast cancer tissue bysemi quantitative reverse transcription polymerase chain reaction（RT-PCR）.2. IRAIN mRNA and IGF-1R mRNA were tested for the expression level andfeatures in normal breast tissue and different breast cancer molecular subtypes byapplying quantitative real time polymerase chain reaction(qRT-PCR).3. We mapped IRAIN transcription orientations using strand-specificRT-PCR(SSRT-PCR).4. We analyzed heterozygous state and allele expression features of IRAIN SNP site (rs8034564) in breast cancer cell lines and frozen human breast cancer tissueswith PCR-RELP and DNA sequencing approaches, for confirming if the gene wasmonoallelic imprinting expressed.5. We tracked the imprinting parental source with DNA sequencing approachand analyze the allele switch between normal tissue and breast cancer tissue, andprimary sites and metastasis.6. we analyzed initiation of DNA methylation status by IRAIN and explored themechanism of monoallelic imprinting expression and allele switch.7. We analyzed the correlation between the expression of IRAIN mRNA andIGF-1R mRNA and clinical pathological features of breast cancer in statisticalapproaches.Results1. Semi quantitative analysis showed that IRAIN expressed extensively in breastcancer with different levels. We used quantitative real time polymerase chain reaction(qRT-PCR) to examine the expression of IRAIN mRNA in different molecularsubtype breast cancer and normal breast tissues. The findings suggested that IRAINexpression lowers in all molecular subtypes. Among them, the Triple Negative BreastCancer became the most prominent in the reduction of IRAIN expression, and theHER2overexpression subtype also dropped considerably compared with the normalgroup. The difference of the both group was statistically significant. The expression ofIRAIN in Luminal subtype was also lower than the normal, but without statisticalsignificance. In pairwise comparison of the last three group suggested no significantdifference (P>0.05）. The expression of IGF-1R was the highest in Luminal subtypewhich had the best prognosis, and was close to that in the normal breast tissue, whilein poor prognostic types such as TNB and HER2+, the expression was significantlylower than that in the normal group and Luminal group (P<0.05). The differencebetween TNB and+subtype was not significant (P＞0.05）.2. We selected two different positions in IRAIN genes, and determined theorientation of IRAIN transcription in the tissue of several breast cancer patients usingSSRT-PCR, the findings were consistent. The transcription orientation of IRAIN wasthe opposite to that of ICF-1R mRNA. It was evident that IRAIN was transcribedantisense to IGF-1R in breast cancers.3. IRAIN was monoallelicly expressed in normal breast tissue, so was in cancer tissues. However, it was interesting to note the expression of the―A‖allele wasfavored over the―G‖allele. In18breast cancers that were heterozygous for thepolymorphic SNP,16tumour cDNAs (89%) expressed the―A‖allele alone. Only2tumors cDNAs (11%) expressed the―G‖allele. It is not clear yet that theadvantageous expression of―A‖allele is relevant to the LncRNA.4. In order to determine whether IRAIN was paternal allelic expressed ormaternal allelic expressed, we conducted family genealogy screening. The resultsuggested that IRAIN was paternally expressed and maternally imprinted.5. It was the first time we discovered the abnormal IRAIN allele-switch. Thatwas the IRAIN presenting allele expression from different parental source in thenormal tissue and cancer tissue in the same patient.6. The BSP analysis of DNA methylation status of IRAIN promoter rich in CpGinland demonstrated the abnormal DNA methylation in breast caner, which suggestedthe DNA methylation may involves in regulating IRAIN monoallelic imprintingexpression and allele-switch.7. IRAIN was expressed the highest in normal breast tissue, and significantlylower in triple negative breast cancer and HER2overexpression breast cancer,suggesting that IRAIN can be treated as antioncogene to regulate the growth of tumorcells. The expressiveness of IRAIN had no significant relation with menstruation,tumor size, lymphatic metastasis, histological grade, vascular tumor thrombus andproliferation index.Conclusions1. We discovered the extensive expression of a novel LncRNA IRAIN in IGFIRlocus. IRAIN was expressed the highest in a normal breast tissue, but much lower inaggressive triple negative breast cancer and HER2overexpression breast cancer,suggesting that IRAIN can be treated as antioncogene to regulate the growth of tumorcells.2. The transcription orientation of IRAIN was the opposite to that of ICF-1RmRNA. It was evident that IRAIN was transcribed antisense to IGF-1R in breastcancers.3. IRAIN was monoallelicly expressed in normal breast tissue, unequally. One ofthe allele was favorably expressed. Paternal allele expression and maternal imprintingexpression were closely related to abnormal DNA methylation in its promoter. It was one of the very few imprinting genes in human genome which discovered recently.4. we discovered for the first time that IRAIN allele-switch existed in breastcancer. That was not been seen in any international or domestic publications for breastcancer or other malignant tumors. If our postulation of IRAIN as an antioncogene wastrue, the allele-switch would become a new pattern of inactivation of antioncogenes.That was a hallmark incidence as important as the position of LOI in tumors.5IRAIN was expected to be a biomarker with predictive and prognostic valueand the target of individualized therapy.6. Research about IRAIN earned the recognition of authorities. IRAIN wascertified by the most authoritative organization, Gene Bank, National Center forBiotechnology Information in the USA, and was listed as Gene ID:104472848...