| Objective:ARHGAP18 gene regulates cell morphology,migration and adhesion,participates in cell proliferation and apoptosis,and affects the occurrence and development of diseases.To investigate the role of ARHGAP18gene in the synthesis of fetal hemoglobin（HbF）,so as to provide a theoretical basis for the targeted treatment ofβ-thalassemia.Methods:1.The human chronic myeloid leukemia cell line K562 was cultured under suitable conditions in vitro.Real-time quantitative polymerase chain reaction（RT-PCR）was used to verify the expression abundance of ARHGAP18 gene in K562 cells.The A549 cell was used as the control.2.Specific siRNA design,construction of the lentiviral vector and screen a recombinant lentivirus with the best target knockdown efficiency:according to the ARHGAP18 mRNA sequence in Genebank,three siRNA target sequences targeting the ARHGAP18 coding region were designed and synthesized,and the unrelated control sequences were randomly designed as negative controls.Three recombinant green lentivirus-selective markers ARHGAP18 gene silencing recombinantlentivirus:shARHGAP18-KD1,shARHGAP18-KD2,sh ARHGAP18-KD3 and a negative control recombinant virus shCtrl were constructed.Three kinds of ARHGAP18-shRNA recombinant viruses were transfected into K562 cells,and the transfection efficiency was observed under a fluorescence microscope.RT-PCR was used to detect the transcription level of ARHGAP18,and a recombinant lentivirus with the best target knockdown efficiency was screened.Follow-up experiments were performed with the optimal interfering sequence and negative control sequence.3.Three recombinant virus group were set up,including experimental group（sh ARHGAP18-KD）,negative control group（shCtrl）and blank control group（CON）.Proliferation and apoptosis of K562 cells at different time points after knockdown of ARHGAP18 gene were detected by CCK-8 kit（Cell Counting Kit-8,CCK-8）and flow cytometry in vitro.The effect of specific silencing of ARHGAP18 on the biological characteristics of K562 cells was investigated in vitro.4.RT-PCR confirmed the expression of HBG1 and HBG2 mRNA after knockdown of ARHGAP18,and explored the role and molecular mechanism of ARHGAP18 in regulating HbF.HBG1 and HBG2 encode the gamma globin chain.Two gamma chains together with two alpha chains constitute HbF.Results:1.The expression level of ARHGAP18 gene in K562 cells was 7.41 times that of the control（P<0.01）.2.Under fluorescent microscope,the infection efficiency of recombinant lentivirus infected K562 cells was>80%.Quantitative RT-PCR revealed that the sh ARHGAP18-KD1 silenced 7.2%of ARHGAP18 in K562 cells（P>0.05）.Compared to the control group,sh ARHGAP18-KD2 silenced 69%of ARHGAP18 in K562 cells,demonstrating good targeting efciency（P<0.05）and sh ARHGAP18-KD3 silenced 65%of ARHGAP18 in K562 cells（P<0.05）.3.Un-infected K562 cells,shCtrl and sh ARHGAP18-KD2 K562 cells did not exhibit signifcant differences in proliferation at 5 days post-infection.The amount of apoptosis was signifcantly different among the three groups(shCtrl:4.80±0.25%,CON:2.91±0.18%,sh ARHGAP18-KD2:3.74±0.12%,CON vs shCtrl P=4.60×10-4,shCtrl vs sh ARHGAP18-KD2 P=2.80×10-3,CON vs sh ARHGAP18-KD2 P=2.80×10-3),but the values differed by less than 20%.4.The results of qRT-PCR showed that HBG1 expression increased by2.2-fold of(P=3.91×10-2),while HBG2 expression increased by 1.6-fold after differentiation(P=1.60×10-3)in cells transfected with sh ARHGAP18-KD2compared with shCtrl-transfected cells.Conclusions:1.The ARHGAP18 gene is highly expressed in K562 cells.2.shARHGAP18-KD2 has good targeting efficiency in K562 cells.Established a K562 cell line with relatively low expression of ARHGAP18.Knockdown of ARHGAP18 in K562 cells promoted apoptosis,the difference was statistically significant,but had no significant effect on cell proliferation.3.ARHGAP18 gene is related to HbF,and knockdown of ARHGAP18 gene can increase the expression of HBG1 and HBG2.ARHGAP18 gene is expected to be a therapeutic target forβ-thalassemia. |
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