Preliminary Study On The Effect Of X-linked Rsk4 Gene On Cell Proliferation And Its Mechanism

Objective:The effect of Rsk4 gene on cell proliferation and expression of some cell cyclerelated genes was studied by knockout method.Methods:(1)Purchase of female,male and female heterozygous C57BL/6 mice with knockout Rsk4(Ribosomal S6 protein kinase 4)gene using CRISPR-Cas9 and normal wild mice without knockout gene.The male and female were interbred to obtain homozygous or heterozygous generation mice,and more Rsk4 gene knockout progeny mice were raised and bred to observe and statistic the phenotype of Rsk4 gene deletion in live animals.(2)Primary cells were extracted from skin tissues of Rsk4 gene knockout mice,and cell lines of different Rsk4 gene knockout mice were established.(3)The established Rsk4 gene knockout cells were used for plate cloning,CCK-8 to detect the proliferation activity of each genotype,and flow cytometry to detect the effect of each Rsk4 gene knockout on cell cycle distribution.(4)RNA was extracted from each group of cells,and the expression profile of Rsk4 gene in each genotype was detected by reverse transcription-polymerase chain reaction(RT-PCR).(5)The expression of Cyclin A1,Cyclin E1 and Cdk2(Cyclin dependent kinase 2)were detected by RT-PCR.Results:(1)Deletion of Rsk4 gene affected the birth and survival of mice,and Rsk4 gene knockout mice had low birth rate and high death rate.(2)Some Rsk4 gene knockout mice had no ability to induce or conceive.(3)Phenotype of Rsk4 gene knockout mice: the Rsk4 gene knockout newborn mice were larger than their litter counterparts;renal cyst;overgrowth of teeth and jaw;cervical lymph node enlargement,identified by histology as plasma cell tumor,spleen also has plasma cell tumor,involving liver and kidney;large tumor of testis and epididymis,suspected to be benign;several female mice had ovarian tumors.Skin tumor;osteosarcoma and abdominal giant tumor.All the above phenotypes suggest that Rsk4 gene acts as a tumor suppressor gene in mice,and its deletion may lead to the occurrence of proliferative lesions or tumors.The incidence of all these abnormal phenotypes did not appear to be significantly different among the three knockout genotypes,especially between homozygous and heterozygous genotypes,suggesting that Rsk4 deletion has a strong dominant feature.(4)In the female heterozygous cells with Rsk4 knockout,only the Rsk4 knockout m RNA was expressed,but the wild allele was not expressed.(5)Rsk4 gene has three new and different variable spliceosome.DNAstar analysis of the open reading box of the new variable spliceosome showed that it could encode different proteins from the wild type m RNA of Rsk4 gene,suggesting the complex regulation and other unknown functions of Rsk4 gene.(6)In vitro experiments,homozygous deletion of Rsk4 gene resulted in accelerated cell proliferation compared with the unknockout group,and there was no significant difference between heterozygous cells and wild type cells.The cell cycle distribution of different knockout types was different by flow cytometry.(7)Ccna1 expression was different in different Rsk4 knockout types,and low expression was found in homozygous Rsk4 knockout cells.Conclusions:(1)In animals,the homozygous or heterozygous deletion of Rsk4 gene increases the probability of tumor occurrence,suggesting that Rsk4 gene is a dominant tumor suppressor gene.(2)Rsk4 gene has three new variable splicing sites,suggesting that Rsk4 gene has complex expression regulation and function.(3)Rsk4 gene has an effect on cell proliferation and cell cycle distribution,and Rsk4 gene knockout homozygous can promote cell proliferation by increasing the proportion of cells entering S phase in vitro.Ccna1 expression of cell cycle related gene was decreased in Rsk4 knockout cells.