Functional Study Of Demethylase Kdm1b In Human Dermal Fibroblasts During Reprogramming

Kdm1b is an epigenetic modifier with demethylase activity to H3K4 and H3K9 histones.Recent studies have reported that Kdm1 b is highly expressed in types of tumor cells,promoting proliferation and inhibiting apoptosis,which is closely related to the occurrence and development of tumors.Kdm1 b has also been reported to induce the expression of multipotent factors SOX2 and NANOG in tumors.However,the function of Kdm1 b in somatic reprogramming is rarely reported.Induced pluripotent stem cells（iPSCs）have attracted much attention in the field of regenerative medicine due to their unique properties,but their low generative efficiency has always been a great obstacle to their clinical application and further development.In this study,we aim to use Kdm1 b to improve the efficiency of somatic reprogramming into iPSCs and elucidate the role of Kdm1 b in the reprogramming process.Firstly,the overexpressed plasmid of Kdm1 b was constructed by molecular cloning and the sequence was verified by DNA gel electrophoresis and sequencing.Then,the lentivirus vector of Kdm1 b was packaged by the constructed plasmid.The optimal conditions for packaging,concentration and purification of lentivirus were explored.The intracellular transcription and protein expression of Kdm1 b were verified by q PCR and western blot analysis.And then infected human dermal fibroblasts（HDF）with lentivirus of Kdm1 b in combination with traditional Yamanaka factors（OCT4,SOX2,KLF4 and c-MYC,OSKM）The combination of five transcription factors（OSKMK）with Kdm1 b produced more iPSCs-like clones than the combination of four transcription factors in HDF,which were determined by alkaline phosphatase（AP）staining to be AP-positive clones.These cells were further injected into mice to generate teratoma which was determined containing three dermal layers,which verified the cloned cells as iPSCs.Further,we studied the mechanism of Kdm1 b promoting the formation of iPSCs.Through Western blot,we not only verified the previously reported demethylase activity of Kdm1 b against H3K4 Me,H3K4Me2 and H3K9Me2,but also found that Kdm1 b have catalytic activity against H3K36me2 and H3K27 Ac,which are thought to promote the formation of iPSCs.At the same time,through the overexpression of Kdm1 b and RNA-Sequence（RNA-Seq）analysis,we found that a large number of differential genes were enriched in proliferation,metabolism and pluripotency genes and other related pathways.In order to further confirm the results of RNA sequencing,we analyzed the cell proliferation capacity after the overexpression of Kdm1 b through CCK-8,and found that Kdm1 b could indeed promote cell proliferation.Meanwhile,western blot analysis showed that the protein levels of cyclepromoting such as p-AKT1,Cycin D1 and ERK1/2 in HDF were significantly increased after overexpression of Kdm1 b,while the expression levels of cycle-inhibiting proteins such as P16 and P21 were significantly inhibited.XF-metabolic analysis showed that Kdm1 b promoted both glycolysis and oxidative phosphorylation,leading to HDF enter a mixed metabolic state.In cell apoptosis experiments,we found that overexpression of Kdm1 b could improve the anti-apoptotic ability of HDF.Meanwhile,western blot results also showed that the expression levels of p53,Caspase-3,Caspase-9 and other apoptotic genes were decreased,and the expression levels of Bcl-2,β-Catein and other anti-apoptotic genes were increased.The results in cell experiments were further supported.Furthermore,q PCR was used to verify the expression of pluripotency related factors detected in RNA-Seq,confirming that the expression levels of core pluripotency factors SOX2 and NANOG,as well as epigenetic factors Bmi1,Ctcf,Ezh2,Kdm2 b and Wdr5 increased after the overexpression of Kdm1 b.These results suggest that Kdm1 b can directly promote the formation of iPSCs by promoting the expression of multiple pluripotency related genes.As a demethylase,Kdm1 b can regulate the epigenetic modification of various histones,and also affect the acetylation status of H3K27 Ac.Kdm1b inhibits apoptosis by inhibiting p53-related pathways,promotes cell proliferation,regulates cell metabolism,and promotes the expression of core pluripotent factors SOX2 and NANOG,as well as epigenetic factors Bmi1,Ctcf,Ezh2,Kdm2 b and Wdr5,thus improving the generation efficiency of i PS cells.Therefore,we believe that Kdm1 b is an important epigenetic factor associated with pluripotency and plays an important role in cell reprogramming.