| OBJECTIVE: To study the SMP30 related long non-coding RNA(lnc RNA)in hepatocellular carcinoma and the correlation between its expression and clinicopathological parameters.Preliminary study on the effects of target lnc RNA on proliferation,invasion,migration,apoptosis and cell cycle of hepatocellular carcinoma cells.And explore the relationship between target lnc RNA and its associated micro RNA,target gene and SMP30.It lays a foundation for further understanding of the role and mechanism of lnc RNA in the pathogenesis of hepatocellular carcinoma.METHODS: 1.Screening and validation of SMP30 related lnc RNA.The expression of lnc RNA in SK-Hep1-SMP30 and SK-Hep1-LV5 was detected by human lnc RNAs expression spectrum chip,and the lnc RNA of differential expression was analyzed,and the candidate lnc RNA was q RT-PCR verified in the above cells.The lnc RNA of different hepatocellular carcinoma cell lines were detected,and the target lnc RNA RP11-166B2.5 was selected.The expression of RP11-166B2.5 in 56 samples of paired hepatocellular carcinoma was detected by q RT-PCR,and the correlation between its expression level and the clinicopathologic parameters of patients was analyzed.The cell localization of RP11-166B2.5 was detected by nuclear separation experiment.2.Effects of RP11-166B2.5 on biological behavior of hepatocellular carcinoma cells.The effectiveness of over-expression RP11-166B2.5 lentivirus transfection of hepatocellular carcinoma cell lines SK-Hep1,Huh7,Hep G2 were verify by q RT-PCR.The effects of RP11-166B2.5 on proliferation,invasion,migration,apoptosis and cell cycle of hepatocellular carcinoma cell lines were detected by CCK8,clonal formation,Transwell and flow technique.3.Preliminary discussion on the relationship between RP11-166B2.5,micro RNA,target Gene and SMP30.Bioinformatics online predict micro RNA combined with RP11-166B2.5,and q RT-PCR to verify the expression level of the corresponding micro RNA in hepatocellular carcinoma cell lines and over-expression RP11-166B2.5 hepatocellular carcinoma cell lines.The target micro RNA was selected to detect their expression in paired hepatocellular carcinoma tissue samples,and the correlation between their expression level and RP11-166B2.5was analyzed.RP11-166B2.5’s target gene GSPT1 which was predicted by CIS mechanism,in the samples of hepatocellular carcinoma tissue were detected,and its correlation with RP11-166B2.5 and SMP30 was analyzed.RESULTS: 1.It was found that the expression of lnc RNA RP11-166B2.5 associated with SMP30 varied in different cell lines,and its low expression in hepatocellular carcinoma tissue samples was closely related to liver function.According to the differential expression multiples of lnc RNA in SK-Hep1-SMP30 and SK-Hep1-LV5 chips.This standard(Fold Change≥2 and P<0.05)assisted by the use of open database analysis,screening out 6 candidate lnc RNAs,and in SK-Hep1-SMP30 q RT-PCR verification,the final screening out of the lnc RNA RP11-166B2.5 as the target gene(the relative expression quantity is 1.68,P<0.005).The RP11-166B2.5 in 4 strains of different hepatocellular carcinoma cells(SK-Hep1,Huh7,97 L and Hep G2 cells)were tested q RT-PCR again,and the relative expression was found to be 1.68,2.58,1.63,0.82,statistical analysis showed that RP11-166B2.5 expressed high in Huh7 and 97 L,expressed low in Hep G2,and there was no significant difference in expression in SK-Hep1.Its target gene GSPT1 was low expression in SK-Hep1,97 L and Huh7 cells,while it was high in Hep G2 cells,and its relative expression was 0.20,0.62,0.89 and 1.69,respectively.RP11-166B2.5and SMP30 were low expression in 56 hepatocellular carcinoma tissue samples,the two were positively correlated,the relative expression was 0.41 and 0.36,and there were significant differences.Relative expression of RP11-166B2.5 in adjacent tissues of cancer and paired carcinoma with patient age,BCLC staging,glutamate transaminase(r=-0.341)and glutamate transaminase(r=-0.318)Close relationship(P<0.05),suggesting that RP11-166B2.5 expression decline can be used as a predictor of poor liver function.RP11-166B2.5’s protein coding ability is very weak,the score is-1.15,the mass nucleus separation shows that RP11-166B2.5 is mainly enriched in the nucleus,accounting for 99.77% of the total.2.RP11-166B2.5 inhibited the invasion and migration of hepatocellular carcinoma cells.Three kinds of RP11-166B2.5 expressed hepatocellular carcinoma cell lines SK-Hep1-RP11-166B2.5,Huh7-RP11-166B2.5 and Hep G2-RP11-166B2.5 were successfully constructed,and their effectiveness was verified by q RT-PCR.It’s an increase in the transcription level of SMP30 in SK-Hep1 and Huh7 cells expressing RP11-166B2.5,with a relative expression of 3.15 and 1.59,respectively,while the transcription level of Hep G2 in SMP30 decreased,with a relative expression of 0.84.The Western blot test SMP30 increased the level of translation in Huh7 cells that expressed RP11-166B2.5,and the level of translation decreased in Hep G2,which was consistent with the q RT-PC results,but could not be measured in SK-Hep1.The experimental results of CCK8 and clonal formation showed that RP11-166B2.5 had no obvious inhibitory effect on the proliferation of SK-Hep1,Huh7 and Hep G2 cells.The experimental results of transwell showed that RP11-166B2.5 had obvious inhibitory effect on the invasion and migration of SK-Hep1 and Huh7,but had no effect on the invasion ability of Hep G2,but inhibited the migration ability.And RP11-166B2.5 had no significant effect on the apoptosis and cell cycle distribution of hepatocellular carcinoma cell lines.3.The relationship between RP11-166B2.5,micro RNA,target Gene and SMP30.The 7 candidate mi RNA predicted by bioinformatics q RT-PCR verified,and it is determined that hsa-mi R-522-3p and hsa-mi R-224-3p are closely related to RP11-166B2.5.The relative expression of hsa-mi R-522-3p in SK-Hep1,Huh7 and Hep G2 cells who had expressed RP11-166B2.5 was 0.78,1.09 and 0.61,respectively,and was statistically significant.The relative expression of hsa-mi R-224-3p was 1.22,1.34 and 0.73,respectively,and was statistically significant only in Huh7-RP11-166B2.5.Hsa-mi R-522-3p showed high expression in 31 hepatocellular carcinoma tissues,with a relative expression of 2.105 and a moderate negative correlation with RP11-166B2.5(r=-0.402,P<0.05);has-mi R-224-3p was highly expressed in 31 hepatocellular carcinoma tissues,with a relative expression of 4.018(P<0.05),but no significant correlation with RP11-166B2.5.RP11-166B2.5’s target gene GSPT1 expressed low in SK-Hep1-RP11-166B2.5 and Huh7-RP11-166B2.5,with a relative expression of 0.57,0.67.The expression of GSPT1 protein detection in Huh7-RP11-166B2.5 by western blot was lower than that in Wild group and control group,which was consistent with q RT-PCR results.The relative expression of target gene GSPT1 in Hep G2-RP11-166B2.5 was 1.36,and there was no statistically significant difference between them.GSPT1 was low in 47 hepatocellular carcinoma tissues,the relative expression of cancer in the adjacent tissues of cancer was 0.39(P<0.001),GSPT1 and RP11-166B2.5showed a weak positive correlation(r=0.355,P<0.05),which was positively correlated with SMP30(r=0.536,P<0.001),prompting RP11-166B2.5 regulates the expression of SMP30 through GSPT1.CONCLUSION: The results of this study show that lnc RNA RP11-166B2.5 is highly expressed in hepatic cancer cells SK-Hep1 and Huh7,expressed in Hep G2,and is mainly distributed in the nucleus,the target gene is GSPT1;RP11-166B2.5 low expression in hepatocellular carcinoma tissue,and it was associated with the patient’s age,BCLC staging and glutamate transaminase glutamate transaminase.And over-expression RP11-166B2.5 inhibited the invasion and migration ability of hepatocellular carcinoma cell lines,and had no effect on cell proliferation,apoptosis and cell cycle.The expression of hsa-mi R-522-3p was low in SK-Hep1-RP11-166B2.5 and Hep G2-RP11-166B2.5,while it’s high in tumor tissue and moderate negative correlation with RP11-166B2.5.Has-mi R-224-3p was highly expressed in Huh7-RP11-166B2.5,GSPT1 was low in tumor tissue,was negatively correlated with RP11-166B2.5 and strongly correlated with SMP30. |
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