Genomewide Small Regulatory RNA Library Screening Identifies A Tumorigenic Phenotype Of Mouse Liver Stem Cells And Its Underlying Mechanisms

Background:Liver as the body’s largest metabolism and digestive organ with a strong ability of regeneration.The intrahepatic stem cells are activated and invovled in the repair processes during liver damage happened,however the mismatch could be occurred when the regeneration cells participate in the processes of repairing,which could be one of the important mechanism of the origin of liver cancer.In order to explore the process of liver stem cells malignant transformation,we developed and constructed based on whole genome level of 19 random base RNA library n19 RNA,used for screening and identification non-coding RNA which regulating the malignant transformation of liver stem cells,and further study on the mechanism,in order to provide experimental and theoretical basis for treatment of liver cancer.Methods:(1)The stable expression n19 iHpn19 cells were generated that 19 random base RNA libraries retrovirus iffected mouse embryonic liver stem cells(iHpx).n19 i Hpn19 cells were amplificated and screened in vitro before subcutaneously nude mice.The masses were collected after9 weeks.The collected cells were amplificated and screening again in vitro.Then it’s subcutaneously in nude mice.Repeated amplification and screening 5 times in vivo and vitro,respectively.We named the each round cells as iHpn19A,iHpn19B,iHpn19C,i Hpn19D,iHpn19E,then used the i Hpx cells as control.(2)Cultured the 5 rounds cells and the control cells in vitro.The morphological changes were observed under the microscope.The ability of synthetic glycogen was tested by the use of Periodic Acid Schiff reaction(PAS).Indocyanine green uptake and release staining was used to detect the change of hepatic-related function.qPCR was used to detect the expression of stem cells stem and hepatic-relatedrelated genes.(3)Subcutaneous injection the 5 rounds cells and the control cells after amplification and screening in vitro/vivo.Then it’s carried out the pathological analysis after the tumor mass obtained at different time points.(4)Extraction the genomic DNA from 5 rounds cells,then do the high throughput RNA sequence to test the rich RNA fragment,named highly enriched fragments as the Hpx 1-4 after analysis the big data,Ensemble、Pubmed Blast and Wenn analyzed the common target LncRNAs,and do qPCR to verify the LncRNAs expression in each round cells.(5)Design and constructed siRNAs adenovirus which targeted BMP9CDS regions.The early and late osteogenesisin ability was detectd in vitro/vivo by infected iMads cells.it’s inorder to selcet the optimal siRNA.Results:(1)We constructed the innovation RNA library which target the whole-genome,the deep sequencing results verified the efficiency could up to 90%,and the diversity could up to 2.0~3.0 x 10~7,which could effectively transfect the target cells.The tumorigenesis hepatocyte-like were obtained by screening multiple rounds when the n19 RNA library was introduced in vitro and vivo.(2)In vitro experiments showed that compared with i Hpx cells,the morphology of screening cells have fibroblast-like changes gradually.In vivo experiments confirmed that iHpn19 cell proliferation rate increasesly with the screening rounds increased.(3)High throughput sequencing results and the data analysis showed that tumorigenesis hepatocyte-like cells have 4 enrichment RNA fragments,respectively named as Hpx 1,Hpx 2,Hpx3 and Hpx F4.Big data analysis found that there are 12 LncRNAs are common targeted by 4 RNA fragments,and qPCR investigate the 12 LncRNA expression level in 5rounds screening cells.(4)In the process of Ad BMP9-induced osteogenesis differentiation in i MADs.The experiments in vivo and in vitro confirmed that simB9-4 and simB9-7 could efficiently suppress the early and late stage of osteogenesis differentiation which induced by Ad BMP9.The high inhibit efficient were observed that when simB9-4 and simB9-7 combined used more than simB9-4 yor simB9-7 lonely.Conclusion:(1)Tumorigenesis hepatocyte-like cells successfully obtained by infecting RNA library in iHpx cells and screening in vitro and vivo,the imbalance of proliferation and differentiation of iHpx cells could lead to the liver cancer occurred.(2)Sequenceing RNA libraries,12 LncRNAs were predicted that play important role when imbalance proliferation and differentiation of iHpx cells.(3)In the process of Ad BMP9-induced osteogenesis differentiation of i MADs,the experiments in vivo and in vitro confirmed that simB9-4 and simB9-7 could efficiently suppress the early and late stage of osteogenesis differentiation which induced by AdBMP9,the high inhibit efficient was that simB9-4 and simB9-7 combined used.