| Background and Objectives: Hepatocellular carcinoma(HCC)is the fifth most common and third deadliest cancer worldwide,with increasing incidence in many countries.According to the statistics of the World Health Organization in 2020,liver cancer ranks second only to lung cancer in the death rate of cancer among men in Asia.In eukaryotic cells,variable splicing events occur all the time and are a key step that is tightly regulated in different tissues,stages of differentiation,and important cellular pathways.RNA splicing factors form mature m RNA through splicing and processing of pre-m RNA,so as to guide protein synthesis.Serine arginine-rich splicing factor family proteins(SRs)are concentrated in the spliceosome and play an important role in variable splicing.SRs family proteins have 12 members,all of which have similar domains,including 1-2 pre-m RNA recognition domain(RRM domain)at the N-terminal and arginine-rich serine domain(RS domain)at the C-terminal.Generally speaking,the RRM domain recognizes and splices pre-m RNA.The RS domain regulates different proteinprotein and protein RNA interactions through phosphorylation.Most SRs family proteins are located only in the nucleus,while some SRs family proteins,such as SRSF1,SRSF3 and SRSF7,are located between the nucleus and cytoplasm.The pathogenesis of tumor is very complex and involves many factors.Current studies have shown that there is a relationship between SRs and cancer,because SRs is the main regulator of pre-m RNA splicing,and the disorder of SRs will greatly destroy the stability of DNA and the normal expression pattern of proteins,thus leading to the abnormal biological function.According to literature reports,most SRs are located in the nuclear plaque,and the formation of the nuclear plaque is due to the liquid-liquid phase separation(LLPS)phenomenon in eukaryotic cells.In cells,LLPS is a physical phenomenon capable of forming extensive membraneless compartments.Phases can selectively recruit or reject certain types of molecules or proteins to perform their functions.At present,there are few studies on the correlation between SRSF4 and tumor.It has been reported that SRSF4 can regulate Caspase-8 in acute myeloid leukemia cells,thus affecting tumor apoptosis.I want to continue the study of SRSF4 in liver cancer,and explore whether SRSF4 affects the proliferation of liver cancer,its downstream regulation,whether the phase is involved in this process,and how the phase is involved.Methods and Results: Through structural light illumination ultra-high resolution microscopy(SIM)and immunofluorescence experiments,it was observed that the SRSF4-WT and C-terminus were expressed in the nucleus in the form of speckle and colocalization phenomenon with SC35,while the SRSF4-N terminus was speckled and had nucleation phenomenon,and the co-localization signal with SC35 was poor.FRAP experiment observed the fluidity of SRSF4-WT as well as the N-terminus and C-terminus.Western blotting assay,clonal formation assay and MTT assay showed that the proliferation rate of hepatocellular carcinoma cells slowed down after SRSF4 knockdown,overexpressed SRSF4-WT,and accelerated after N-terminal,and overexpressed Cterminal had no effect on the proliferation of hepatocellular carcinoma cells.The RNA sequencing results showed that AGR2 was one of the downstream regulated by SRSF4,which was then verified by RT-PCR,Western blot assay and immunofluorescence quantification.Conclusion:1.The splicing factor SRSF4 is located in the nuclear patch,the RS disordered region maintains the structure of SRSF4 and the location of the nuclear patch,and the N-terminal has the nucleation phenomenon when there is no C-terminal.2.The WT,N and C terminus of SRSF4 are all in phase.3.Knockdown and overexpression of SRSF4 can decrease or enhance the proliferation ability of hepatocellular carcinoma cells,and SRSF4-C terminal can indirectly affect the proliferation ability of hepatocellular carcinoma cells.4.AGR2 is one of the downstream regulated by SRSF4. |
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