| Objective:Hepatocellular carcinoma(HCC)is one of the most common cancers in the world.The incidence of HCC is increasing year by year,and its treatment is lacking.In this study,we investigated the effects of baicalein on the proliferation,apoptosis and migration of hepatocellular carcinoma cell lines by regulating the expression of long noncoding RNA NKILA.The role and mechanism of NF-?B signaling pathway in the anti-hepatocarcinoma cells of LncRNA NKILA.It provides a new target for the anti-hepatocarcinoma treatment of baicalein,and provides a new idea for the anti-tumor effect of Chinese medicine.Methods:(1)Detection of expression level of NKILA in liver tissue: extract RNA from 35 pairs of liver cancer tissues and adjacent non-cancerous liver tissues,and detect the expression level of NKILA by qRT-PCR after reverse transcription;(2)The expression of NKILA detected by qRT-PCR in Vector plasmid,NKILA plasmid and shVector plasmid,shNKILA plasmid were 1.00±0.04,4.97±0.20,1.00±0.06,and 0.28±0.03 and the differences were statistically significant(P<0.05)(3)Detection of viability,apoptosis and migration of SMMC-7721 cells: establishment of up-regulatedcellexperimental groupand down-regulation of cell experimental group.The cell culture group was adjusted to set blankcontrol group 1,baicalein control group 1 and NKILA up-regulation group;down-regulation of cell experimental group and blank control group 2,baicalein control group 2,NKILA down-regulation group.The concentration of Baicalein was 50,100 ?mol/L,and the action time was 48 h.CellTiter-Glo? luminescence assay for cell viability,TUNEL for apoptosis,Transwell for cell migration;(4)NF-?B signaling pathway activation index I?B? phosphorylation,p65 nuclear translocation assay: The expressions of p-I?B?/ I?B? and P65 protein in all the group were analyzed by Western blot.Results:(1)Compared with adjacent tissues,the expression of NKILA was decreased(P<0.05).(2)The qRT-PCR detection of Vector plasmid,NKILA plasmid and shVector plasmid,shNKILA plasmid were 1.00±0.04,4.97±0.20,1.00±0.06,and 0.28±0.03 and the differences were statistically significant(P<0.05).(3)CellTiter-Glo? luminescence method for detecting cell viability results: the number of viable cells was expressed by the luminescence signal value,and the cell viability of the control group was set to 100%.The cell viability of the control group 1,the baicalein control group1 and the NKILAup-regulationgroupwere100.00±7.17,70.34±2.34,25.31±2.36 respe ctively.The results showed that the cell viability of the baicalein control group 1 was lower than that of the blank control group(P< 0.05),and the cell viability of the NKILA up-regulated group was significantly decreased,and the difference was statistically significant compared with the baicalein control group 1(P<0.05);The viability of the blank control group2,the baicalein controlgroup2,and the NKILA down-regulation group were100.00±4.05,67.67±2.60,96.67±2.91 respectively.The results showed that the cell viability of the NKILA down-regulated group was higher than that of the baicalein control group 2,the difference was statistically significant(P<0.05),while it was lower than baicalein control group 2,and there was no significant difference(P>0.05).(4)TUNEL detected apoptosis results: the apoptosis rate of the control group 1,baicalein control group1 and NKILA up-regulation group were2.75%±0.47%,12.31%±1.65%,42.00%±3.33%respectively.Theapopt osis rate of the control group 1 was higher than that of the blank control group(P<0.05),while the NKILA up-regulation group was significantly increased,and the difference was statistically significant compared with the baicalein control group 1(P<0.05);the apoptosis rate of the control group 2,baicalein control group 2 and NKILA down-regulation group were2.95%±0.45%,34.39%±2.90%,9.70%±1.30%respectively.Compared with the baicalein control group 2,the apoptosis rate of NKILA down-regulated group decreased,the difference was statistically significant(P<0.05),while it was higher than that of the blank control group2,the difference was statistically significant(P<0.05).(5)Transwell analysis and detection of migration results: The cell migration of blank control group 1,baicalein control group 1,NKILA up-regulation group were 94.00 ± 5.51,45.67 ± 4.06,11.00 ± 1.00 respectively,Compared with the blank control group 1,The cell migration of baicalein control group 1 was decreased(P<0.05),while the NKILA up-regulated group was significantly decreased,and the difference was statistically significant(P<0.05);The migration of blank control group 2,baicalein control group 2,the NKILA down-regulation group were 84.00±4.93,15.00±2.31,and 65.00±5.20 respectively.The results showed that the migration of the NKILA down-regulated group was higher than that of the baicalein control group,and the difference was statistically significant(P<0.05).The number of cell migration of the NKILA down-regulated group were lower than that in the blank control group 2,the difference was not statistically significant(P>0.05).(6)Western blot was used to detect the expression of p-I?B?/ I?B? and P65 protein: the amount of protein was expressed by gray value,and the gray value of the control group was set to 100%.The relative expression levels of p-I?B? in the control group 1,the baicalein control group1 and the NKILA up-regulationgroup were1.00±0.06,0.53±0.04,and 0.13±0.03 respectively,while the relative expression levels of I?B? were 1.00±0.05,1.54±0.05,2.07±0.05.Compared with the control group 1,the p-I?B? of the baicalein control group1 was decreased,while I?B? was increased(P<0.05).Compared with the baicalein control group1,the p-I?B? of NKILA up-regulated group was decreased,while I?B? was increased(P<0.05).That is,Baicalein has an inhibitory effect on I?B? phosphorylation,and this inhibition is more pronounced after up-regulation of NKILA.The relative expression levels of p-I?B? in the blank control group 2,baicalein control group2 and NKILA down-regulation group were 1.00±0.05,0.54±0.04,0.93±0.04,while I?B? were 1.00±0.06,1.47±0.03,1.12±0.04.the results showed that the expression of p-I?B? was significantly higher in the NKILA down-regulated group than in the baicalein control group 2,and the expression of I?B? was decreased(P<0.05).The expression of p-I?B? was lower in the NKILA-lowering group than in the blank control group 2,and the expression of I?B? was higher(P>0.05).That is,baicalein has an inhibitory effect on I?B? phosphorylation,and this inhibition is weakened after down-regulation of NKILA.At the same time,the P65 nuclear translocation was detected: the relative expression levels of p65 in the nuclei of the blank control group 1,the baicalein control group1 and the NKILA up-regulation group were 1.00±0.05,0.52±0.03,0.25±0.01 respectively,while the cytoplasmic p65 were 1.00±0.05,1.84±0.09,2.78±0.12.Compared with the control group 1,the nuclei p65 of the baicalein control group1 was decreased,while cytoplasmic p65 was increased(P>0.05).Compared with the baicalein control group1,the nuclei p65 of the NKILA up-regulation group was decreased,while cytoplasmic p65 was increased(P>0.05).In other words,Baicalein inhibited p65 nuclear translocation,and this inhibition was more pronounced after up-regulation of NKILA.The relative expression levels of p65 in the blank control group 2,baicalein control group 2 and NKILA down-regulation group were 1.00±0.07,0.45±0.03,0.78±0.06 respectively,while the cytoplasmic p65 were 1.00±0.05,1.77±0.09,1.34±0.04.the results showed that the expression of nuclei p65 was increased in the NKILA down-regulated group compared with the control baicalein group 2,and the expression of cytoplasmic p65 was decreased(P<0.05);Compared with the blank control group 2,The nucleus p65 of the NKILA down-regulated group was low and the cytoplasmic p65 expression was high(P>0.05).In other words,baicalein inhibited p65 nuclear translocation,and this inhibition was attenuated after down-regulation of NKILA.Conclusion:1.baicalein has anti-hepatocarcinoma effect;2.NKILA enhanced the inhibitory effect of baicalin on the proliferation,migration and the promotion of apoptosis of hepatocellular carcinoma cell;3.NKILA increases baicalein on hepatocellular carcinoma I?B? phosphoric acid,p65 nuclear translocation,and NKILA enhances the anti-cancer effects of baicalein in hepatocellular carcinoma via the regulation ofNF-?B signaling. |
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