Feature And Function Of Pkm2 In Somatic Cell Reprogramming Process

ObjectiveCancer is a serious public health problem in the world. According to the GLOBOCAN, around 800 million people died of cancer. In our country, one person is diagnosed with cancer every six minutes, and there are 8550 people becoming cancer patients every day. Therefore, in-depth understanding of cancer and to find therapeutic targets for cancer diagnosis is very urgent.Organic survival is inseparable from the energy supply, including the tumor cells. Malignancies maintain aerobic glycolysis phenotype in a well-oxygenized environment(Warburg effect), which can meet the tumor cell viability and biosynthetic. It is widely accepted that specific expression of the M2 type pyruvate kinase(Pkm2) in tumor cells contributes to this aerobic glycolysis phenotype. Pkm2 expression is closely related to tumor stage, classification and grade of malignancy. But in the early stage of tumorigenesis, it’s not clear that how the Pkm2 plays its role and how the complex expression regulation, it is established, which may be due to unavailability of a good method to access the tumorigenesis process.Recent studies revealed the closely relationship of tumors and stem cells. Many signaling pathways are shared, such as Wnt, Notch, SHH. The ES markers as Oct4, Sox2, Nanog are highly expressed in many tumors. Therefore, we intend to bulid a platform to mimic tumorigenesis process in vitro by using reprogramming process, thus to explore the expression pattern and function of Pkm2, which may suggest the expression regulation of Pkm2 in tumorigenesis, and help to discover new potential treatment target.Methods:First, we prepare mouse embryonic fibroblast(MEF cells) from transgenic mice. Then we culture the MEF cells in ES medium containing doxycycline(Dox) to induce reprogramming, and obtain the induced pluripotent stem cells(iPS cells) successfully. Subsequently, shRNA are used for knocking down the expression level of Pkm and we observe the iPS clonogenic situation.In order to detect the change of pk expression pattern in reprogramming process, we collect samples in different time points during the induction process. The expression situation of Pkm1 and Pkm2 are both measured and analyzed by qPCR and western blot.Ptb, hnRANPA1, hnRNPA2B1 and hif-1αare Pkm2 related regulatory genes, which have been known in tumor cells. So we test the expression levels of these genes by qPCR. We knock down or overexpress these genes in reprogramming process to find the effect of these changes on reprogramming process and Pkm gene regulation.Among the PK isoforms, PKM2 is the only one to be allosterically regulated between a less active dimer(in tumor) and an active tetramer(in normol cell). So we use western blot to study the transformation of the two types in reprogramming process.Results:1. Pkm2 plays an important role in the process of reprogramming;2. In pluripotent cells, Pkm2 could enter the nucleus, thus may act as a transcription factor involved in gene regulation;3. The results shows that the expression, regulation and function of Pkm2 in reprogramming process are very similar with that in tumor cells, thus we conclude that study of Pkm2 in reprogramming process can reflect the process of tumorigenesis to some extent.