Exploration On The Regulatory Mechanism Of LncRNA CUST-49525 In Cytotoxicity Induced By 1,4-Benzoquinone

Objective:To investigate the regulatory role of Lnc RNA CUST-49525 in K562 cytotoxicity induced by 1,4-benzoquinone.Methods:The growth curve of K562 cells was drawn by cell counting method,and the half inhibitory rate(IC₅₀)of 1,4-benzoquinone on K562 cells was measured by CCK-8 method.Determine that infected dose accord to the IC50;The effect of 1,4-benzoquinone on the apoptosis and cell cycle of K562 cells was detected by flow cytometry.The effects of 1,4-benzoquinone on the m RNA expression levels of Lnc RNA CUST-49525,mi R-2861,SOCS3 and CDKN1A m RNA K562 cells were detected by real-time fluorescent quantitative PCR.The expressions of protein SOCS3 and CDKN1A were detected by western blot.According to the expression situation of Lnc RNA CUST-49525,lentivirus was used as the vector to construct a stable overexpressed K562 cell line of Lnc RNA CUST-49525,and the effects of 1,4-benzoquinone mediated by Lnc RNA CUST-49525 on the proliferation,apoptosis and cycle of K562 cells were detected by CCK-8method and flow cytometry.The expression levels of mi R-2861,SOCS3,and CDKN1A m RNA in K562 cells overexpressed by 1,4-benzoquinone to Lnc RNA CUST-49525 were determined by real-time fluorescent quantitative PCR.Results:（1）IC₅₀ of K562 cells treated with 1,4-benzoquinone for 24h has 20.24μmol/L.After K562 cells have treated with 1,4-benzoquinone for 24h,the number and morphology of cells have significantly changed under the microscope.Compared with the control group,the relative proliferation rates of K562 cells in the 20μmol/L and 40μmol/L dosage groups have significantly reduced（F=196.8,P<0.01）.（2）Flow cytometry have shown that 1,4-benzoquinone in each dose group could induce apoptosis in a concentration-dependent manner（F=70.23,P<0.01）.Compared with the control group,the G1 phase proportion of K562 cells in the 20μmol/L and 40μmol/L dose groups have increased（F=10.33,P<0.01）,and the S phase proportion has decreased（F=34.81,P<0.01）.（3）Real-time fluorescent quantitative PCR results have shown that the m RNA expression levels of Lnc RNA CUST-49525,mi R-2861,SOCS3 and CDKN1A m RNA K562 cells in the 20μmol/L and 40μmol/L dose groups were increased compared with the control group.Western blot results have shown that the expression levels of SOCS3 and CDKN1A were increased in each dose group in a dose-effect relationship.（4）K562 cells stably overexpressing Lnc RNA CUST-49525 have successfully constructed.Compared with the control group K562 cells,the overexpression efficiency of CUST-49525detected by PCR was significantly increased（t=45.32,P<0.01）.After overexpression,the m RNA expressions of mi R-2861 and SOCS3 m RNA K562 cells have decreased,while the m RNA expression of CDKN1A has increased.K562 cell mi R-2861 and SOCS3 and CDKN1A m RNA expression increased in the CUST-49525-OE+1,4-BQ-exposed group relative to the CUST-49525-OE group.（5）Compared with the CUST-49525-NC+1,4-BQ group,the relative proliferation rate of K562 cells in the CUST-49525-OE+1,4-BQ-infected group has further reduced（F=391.281,P<0.01）,the total apoptosis rate has significantly increased（F=113.58,P<0.01）,the cells blocked in G1 phase have increased（F=73.10,P<0.01）,and the proportion of cells in G1/S phase has significantly decreased（F=65.43,P<0.01）.（6）Real-time fluorescent quantitative PCR results have shown that compared with the CUST-49525-NC+1,4-BQ group,the m RNA expression levels of mi R-2861 and SOCS3 m RNA K562 cells of the CUST-49525-OE+1,4-BQ group have decreased,while the m RNA expression level of CDKN1A has increased.Conclusion:Lnc RNA CUST-49525 has promoted CDKN1A m RNA expression by inhibiting mi R-2861 and SOCS3m RNA and mediated the toxic effect of 1,4-benzoquinone on K562 cells.