Analysis Of Chimeric RNA In Infant Brain Tissues And Function Of DUS4L-BCAP29

Chimeric or fusion RNAs are transcripts composed of exon fragments from different genes.Bioinformatics analyses and second generation sequencing technologies have rapidly developed in recent years.These new technologies are of high accuracy and high throughput,and thus are widely used in chimeric RNAs detections.Declining cost also greatly facilitates discovery of chimeric RNAs in transcriptomes.The widespread detection of chimeric RNAs has far exceeded previous expectations,providing a new hot spot of discovery in transcriptomics.Chimeric RNAs has drawn much attention in the diagnosis and treatment of cancer.In recent years,more chimeric RNAs have been found in non-cancerous tissues and cells,some of which may play important roles in normal physiology.Mining chimeric RNAs from normal tissues will help us better understand the composition and functions of genome.Human umbilical cord mesenchymal stem cells(hUC-MSCs)are pluripotent cells derived from h UC tissue.Because they are easy to isolate,culture,and are self-renewable,with multiple differentiation potentials,h UC-MSCs have become a favorite seed cell for cell transplantation and tissue engineering,and are widely used in regenerative medicine research.In vivo studies have shown that only a small number of transplanted MSCs can differentiate into neuronal cells,therefore,the improvements of both MSCs neuronal differentiation capability and functional neuron differentiation efficiency are unmet needs.In the pluripotent state,cell transcription and epigenetic conditions are distinctly different from that of adult cells.Pluripotent stem cell gene expression is co-regulated by transcription factors and other regulatory factors.Consequently,discovery of key factors that regulate the differentiation of h UC-MSCs to neuronal cells,and exploration of the neural differentiation mechanisms induced by microenvironments in vivo and in vitro are critical in order to improve the clinical effects of h UC-MSCs.Current studies of regulatory factors focus on small molecule compounds,epigenetics,mi RNAs,and Inc RNAs.However,the effects of chimeric RNA in h UC-MSCs proliferation and differentiation into neuron cells have not yet been reported.Objective:Here,we analyze the characteristics of chimeric RNAs in brain tissue and explored the key chimeric RNAs related to neural differentiation.In addition,we identify key chimeric RNAs that proliferate h UC-MSCs and promote h UC-MSCs to differentiate into neuron-like cells and explore the mechanism.These results will provide a new direction and theoretical basis for adult stem cell differentiation at the fusion genome level.Methods:1 Identification of chimeric RNAs in DLPFCs of infant brain tissuesBecause there is a lack of published hUC-MSCs and neural differentiation RNA sequencing(RNA-seq)data,we used RNA-seq data of dorsolateral prefrontal cortex(DLPFC)from infant brain tissue from SRA(Sequence Read Archive),that most closely matches the developmental stage of the human umbilical cord we work with,in order to identify key chimeric RNAs of h UC-MSCs involved in neural differentiation.We used SOAPfuse to discover,identify,and characterize chimeric RNAs from these RNA-seq data,including type,quantity,and distribution on chromosomes,as well as functional clustering of parent genes.According to the analysis of bioinformatics,we then verified 22 chimeric RNAs,including DUS4L-BCAP29,in h UC-MSCs and their neural differentiated cells using RT-PCR and Sanger sequencing,allowing us to analyze their fusion junctions,the relative expression of the chimeric RNAs to parent genes,and the distance between the neighboring genes.Furthermore,we predicted the splicing site at the second last exon of the upstream parent gene and the second exon of the downstream parent gene of the chimeric RNA.We next predicted transcriptional repressor CTCF binding sites in the region between parent genes.We examined the formation mechanism of DUS4L-BCAP29: To confirm its transcriptional read-through nature,we used a reverse primer that annealed to the second exon of BCAP29 to detect a primary transcript fragment between the last intron and the last exon of DUS4 L.To avoid DNA contamination,DNase I treatment and “no reverse transcriptase” control were used.2 Expression and function of DUS4L-BCAP29 in normal tissues and cellsDUS4L-BCAP29 has been reported in both gastric and prostate cancers.However,by analyzing RNA-Seq datasets,we detected the recurrent DUS4LBCAP29 in infant brain tissue.To confirm the finding that the chimeric RNA exists in non-cancerous tissues,we designed primers flanking the fusion junction site,and used RT-PCR to detect the fusion in our collection of normal tissues and normal cell lines.To test whether the fusion RNA is expressed at a much higher level in cancer vs.non-cancer cell lines,we used q RT-PCR to quantify the difference of expression in several gastric and prostate non-cancer(GES-1 and RWPE-1),and cancer lines(SGC-7901,HGC-27,LNCa P,and PC3).Previously,a si RNA(si DUS4L-BCAP29)targeting the fusion was used to demonstrate its role in gastric cancer.However,the si RNA targeting sequence lies in the common region of the fusion and wild type DUS4 L,thus difficult to justify its specificity on the fusion transcript as reported.Unfortunately,we failed to develop such a specific si RNA for DUS4L-BCAP29.To determine whether the previously reported reduced cell proliferation was truly due to the chimeric RNA silencing,we designed another si RNA targeting only the wild type DUS4 L.Since the fusion is expressed in non-neoplastic tissues and cells,we tested the loss-of-function system and gain-of-function system in GES-1 and RWPE-1 cells to detect whether DUS4L-BCAP29 affected the proliferation and migration.We then performed microarray analyses on the GES-1 samples transfected with the control si RNA,or si DUS4L-BCAP29,or si DUS4 L.3 Effect of lentivirus-mediated DUS4L-BCAP29 overexpression on h UC-MSCsThe hUC-MSCs were transfected with DUS4L-BCAP29 overexpressing lentivirus.The proliferation of h UC-MSCs was detected with CCK-8 and Ed U staining.The cell cycle was determined by flow cytometry with PI staining.Western blot was used to detect apoptosis proteins.In order to detect DUS4L-BCAP29 product,western blot analysis using Flag tag was performed.The experiment was divided into an untreated control group(CON)and DUS4L-BCAP29 overexpression group(LV-DUS4L-BCAP29).After transfection and differentiation of the two groups of cells,the expression of Doublecortin(DCX)and Neuron Specific Enolase(NSE)were detected by immunofluorescence.The expression of Neurogenin1(Ngn1),Neurogenin2(Ngn2)and mammalian achaete-scute homologue-1(Mash1)were detected with q RT-PCR.Expression of phosphorylated p38 MAPK and phosphorylated STAT3 were detected using western blot for cells in both groups.Results:1 Abundant chimeric RNAs in DLPFCs of infant brain tissues1.1 A total of 599 chimeric RNAs were identified in DLPFC samples from 9 infant brain tissues.Chimeric RNAs were identified on each chromosome.Chromosome 1 contained the most chimeric RNAs(72 chimeric RNAs),while chromosome 18 had the fewest,with only 3 chimeric RNAs.The number of chimeric RNAs was related to the number of genes in the same chromosome(R = 0.8357,p <0.0001).1.2 We searched gene ontology terms for the parental genes involved in chimeric RNAs.The predominant functions of upstream parent genes forming chimeric RNA are protein polymerization and post translational protein folding.Downstream parent gene functions are mainly related to small molecule or protein metabolism,and glutamate secretion.1.3 Chimeric RNAs are classified according to the chromosomal location of their parent genes: INTERCHR,INTRACHR-SS-0GAP,INTRACHR-OTHER.The proportions of the three types of chimeric RNA in this study were 46%,51% and 3%,respectively.Chimeric RNAs were also classified according to the junction position relative to the exon of parental genes: E / E,E / M or M / E,M / M.The proportions of these three types in this study were 36%,21% and 43%,respectively.Regarding reading frames,chimeric RNAs can be classified as: in-frame,frame-shift,both,NA.The proportions of these four types in this study were 15%,21%,1%,and 63%,respectively.1.4 We verified 22 in-frame chimeric RNAs from hUC-MSCs and their neutrally differentiated cells with RT-PCR,and 10 chimeric RNAs were correctly amplified and sequenced.Among the 10 chimeric RNAs,9 of them used the second-to-last exon of their 5' parental genes fused to the second exon of the 3' gene.The lengths of these chimeric RNA genes were less than the 54 Kb average genomic distance between adjacent parent genes.Bioinformatics methods predicted that there was a variable splicing site near the second to last exon of the upstream parent gene and the second exon of the downstream parent gene of these chimeric RNAs.Additionally,there was at least one possible CTCF binding site in the intergenic regions of each of the chimeric RNAs.1.5 q RT-PCR results revealed that the chimeric RNA DUS4L-BCAP29 was highly expressed in neurogenic differentiated h UC-MSCs(p<0.05),with no significant change in the expression of DUS4 L.RT-PCR could detect transcripts from the intergenic region.These results indicated that DUS4L-BCAP29 may be a product of cis-splicing of adjacent genes..2 DUS4L-BCAP29 is widely expressed in normal tissues and cells and is involved in cell proliferation and other functions2.1 DUS4L-BCAP29 was indeed detected in multiple normal tissues,ranging from heart to testis.Furthermore,it is expressed in diverse non-cancerous cell lines,including mammary gland(MCF10A),lung epithelial(Beas2B and 16HBE),and foreskin fibroblast(HFF).It is the same fusion form,involving the first seven exons of the DUS4 L gene,and the last seven exons of BCAP29 as previously reported.The junction sequence is also identical to that reported in the gastric cancer study.In the gastric cells,contradictory to the previous report,DUS4L-BCAP29 is expressed at a comparable level in GES-1 as in SGC-7901,and even lower in HGC-27.In the prostate cells,the fusion is indeed expressed at lower levels in RWPE-1,than in LNCa P and PC3 cells.We then compared the chimeric RNA expression in clinical samples.No statistical difference was observed between the 21 gastric prostate cancer and normal matched pairs.Similarly,no statistical difference was seen in 18 prostate cancer and 18 non-cancer prostate tissue samples.2.2 Since DUS4L-BCAP29 is expressed in non-neoplastic tissues and cells,we tested the loss-of-function system in GES-1 and RWPE-1 cells.As expected,si DUS4L-BCAP29,but not si DUS4 L silenced DUS4L-BCAP29 in both cell lines,whereas both si RNAs silenced the wild type transcript to a similar extent.Both cell lines showed significantly reduced migration ability when transfected with either si RNA,and to a similar extent.We thus suspect that the effect of this loss-of-function system is mainly due to the silencing of wild type DUS4 L.We then performed microarray analyses on the GES-1 samples transfected with the control si RNA,or si DUS4L-BCAP29,or si DUS4 L.Consistent with the above observation,we noticed that the most significantly enriched Gene Ontology(GO)term is negative regulators of apoptosis,and this term is shared by both si DUS4L-BCAP29,and si DUS4 L.Interestingly,a few GO terms unique to the si DUS4L-BCAP29 were enriched,suggesting that the chimeric RNA may have some unique functions.In Gain-of-function system,we observed enhanced proliferation rates in both cell lines,and significantly increased cell motility,at least in GES-1 cells.These results argue that the increased proliferation and motility effects of DUS4L-BCAP29 are not specific to cancer cells.3 DUS4L-BCAP29 can effectively promote h UC-MSCs proliferation rates and differentiate into neuron-like cells3.1 Compared with the control group,the number of h UC-MSCs actively overexpressing DUS4L-BCAP29 significantly increased(p<0.05)and cell proliferation was faster(p <0.05);PI staining showed that compared with the control group,the overexpression of DUS4L-BCAP29 led to an increase in the percentage of cells in S phase(p <0.05).Western blot analysis of apoptosis proteins showed that the expression of caspase3 decreased(p <0.05)and the expression of Bcl-2 increased(p <0.05).3.2 Western blot did not detect a fusion protein corresponding to the DUS4LBCAP29 chimeric RNA,indicating that it may be non-coding.3.3 Under the same neuronal induction medium conditions,compared with the control group,the neuronal differentiation efficiency of the DUS4L-BCAP29 overexpression group was higher.Immunofluorescence results demonstrated that the expression of DCX and NSE were significantly increased(p <0.05).q RT-PCR results showed that the expression of neural differentiation-related genes(Ngn2,Mash1,etc.)was significantly increased(p <0.05).3.4 Western blot results showed that there was no change in phosphorylated p38 MAPK expression and phosphorylated STAT3 expression decreased(p <0.05)in the DUS4L-BCAP29 overexpression group.Conclusion:1.We have discovered 599 chimeric RNAs from brain samples of infants.The INTRACHR-SS-0GAP type chimeric RNA is the most predominant.The 10 correctly identified chimeric RNAs are almost fused by the fragment of first to the second last exon of upstream parent gene and second to the last exon of downstream parent.The chimeric RNA DUS4L-BCAP29 are highly expressed after neural differentiation and may be the product of cis-splicing of adjacent genes.2.DUS4L-BCAP29 is widely expressed in normal tissues and cells.DUS4LBCAP29 cannot be used as a gastric and prostate cancers biomarker.3.DUS4L-BCAP29 can promote the proliferation of hUC-MSCs,increase the proportion of cells in S phase and enhance the anti-apoptotic property of cells.DUS4L-BCAP29 may function as a non-coding RNA.DUS4L-BCAP29 can effectively promote h UC-MSCs to differentiate into neuron-like cells and could serve as a much needed improvement of MSC neuronal differentiation capability.This process may be related to the fact that DUS4L-BCAP29 inhibits phosphorylated STAT3 protein expression.