Long Non-coding RNA GS1-293C5.1 Inhibiting The Metastasis Of Liver Cancer Cells And Its Mechanisms

Objective: To test the expression of long-chain non-coding RNA(lnc RNA)in liver cancer tissue samples and to analyze the correlation between its expression level and clinicopathological parameters.Preliminary purposes of this study are to investigate the effects of target lnc RNA on proliferation,adhesion,invasion,migration,apoptosis and cell cycle and to explore the relationship between target lnc RNA and its target gene.This is expected to lay a foundation for further understanding of the role of lnc RNA in the development of hepatocellular carcinoma.Methods:(1)Screening and verification of lnc RNA related to hepatocellular carcinoma.The screening of differentially expressed lnc RNA in human SK-Hep-1-LV5 and SK-Hep-1-SMP30 are carried out using expression profile chips and q RT-PCR verification of the candidate lnc RNA was in SK-Hep-1-SMP30 cells,The expression of candidate lnc RNA was detected in different liver cancer cell lines and the target lnc RNA GS1-293C5.1 was screened.The q RT-PCR was employed to detect the expression level of 50 cases of hepatocellular carcinoma and paired adjacent tissues and the correlations among the clinical pathological indicators were analyzed.The CPAT database was used to predict the coding capacity of lnc RNA GS1-293C5.1 and the subcellular localization of lnc RNA GS1-293C5.1 was determined by cytoplasmic nucleus separation experiments.(2)The effect of lnc RNA GS1-293C5.1 on the biological behavior of liver cancer cells.The lnc RNA GS1-293C5.1 stable overexpression cell line was constructed by lentivirus transfection in SK-Hep-1,Huh7,SMMC-7721,Hep3 B cell lines,and the expression efficiency of lnc RNA GS1-293C5.1 was verified by q RT-PCR.CCK8,cell clone formation experiment,homogeneity and heterogeneity adhesion experiment,transwell invasion,transwell migration experiment and flow cytometry were used to detect cell proliferation,adhesion,invasion,migration,apoptosis and cell cycle changes.(3)The prediction of lnc RNA GS1-293C5.1target genes and preliminary discussion on the mechanism of action.Bioinformatics conducted lnc RNA GS1-293C5.1 GO enrichment and KEGG pathway analysis and used RNAfold and RNAcofold prediction free energy sizes in the Vienna RNA server to determine the relationship between lnc RNA GS1-293C5.1 and the target gene PON2 m RNA.The q RT-PCR was used to verify the expression of target gene PON2 in wild-type liver cancer cell lines and overexpressed liver cancer cells.The q RT-PCR and Western Bolt were used to detect the transcription and translation of PON2 in wild-type liver cancer cell lines and overexpressed liver cancer cell lines.Finally,the expression of PON2 in liver cancer tissue was detected by q RT-PCR,and the correlation between its expression and lnc RNA GS1-293C5.1 was analyzed.Results:(1)It has been found that the expression of lnc RNA GS1-293C5.1is different in different liver cancer cells,and that its low expression in liver cancer tissue samples is closely related to postoperative recurrence.Six candidate lnc RNAs have been selected from the SK-Hep-1-LV5 and SK-Hep-1-SMP30 differential expression profiling chips constructed by the research group in the early stages and the six candidate lnc RNAs have been selected in SK-Hep-1-SMP30 cells.We have performed q RT-PCR verification to eliminate lnc RNA that do not match the prediction of the chip,and finally determined that lnc RNA RNA GS1-293C5.1 is the target gene.The lnc RNA GS1-293C5.1 has been detected in SK-Hep-1,Huh7,SMMC-7721,Hep3 B,Hep G2,the relative expression levels of the five liver cancer cell lines relative to normal liver cells HL-7702 are: 0.42,1.10,0.39,1.06,1.18 times,and low expression of lnc RNA RNA GS1-293C5.1is detected in liver cancer tissues.The relative m RNA expression level is 0.52.The relative low expression of lnc RNA GS1-293C5.1 in cancer and paired adjacent tissues is related to the recurrence of liver cancer after eradication.The protein coding ability of lnc RNA RNA GS1-293C5.1 is very weak,with a score of 0.00025.The cytoplasmic nucleus separation shows that GS1-293C5.1 is mainly concentrated in the nucleus,accounting for 76.21% of the total.(2)The lnc RNA GS1-293C5.1 affects the adhesion of liver cancer cells and has an inhibitory effect on invasion and migration.In this study,four GS1-293C5.1overexpressing liver cancer cell lines SK-Hep-1-GS1-293C5.1,Huh7-GS1-293C5.1,SMMC-7721-GS1-293C5.1,Hep3B-GS1-293C5.1 have been successfully constructed;CCK8,clone formation experiment results show that lnc RNA GS1-293C5.1 has no significant inhibitory effect on the proliferation of SK-Hep-1,Huh7,SMMC-7721 and Hep3 B cells;adhesion experiments have found that overexpression of lnc RNA GS1-293C5.1 can increase the selfadhesion of SK-Hep-1,Huh7 and SMMC-7721 liver cancer cells,and weaken the heterogeneous adhesion of Huh7 cells;Transwell invasion and migration results show that lnc RNA GS1-293C5.1 is inhibitory effected on SK-Hep-1and Huh7 cells,while the migration ability of SMMC-7721 is inhibited,but the invasion effect is not obvious.(3)The relationship between lnc RNA GS1-293C5.1 and the target gene PON2.Bioinformatics analysis suggests that lnc RNA is mainly enriched in biological processes such as protein modification,negative regulation of gene expression and cell adhesion,and involves pathways such as RNA translocation,RNA degradation and m RNA monitoring;Vienna RNA server predicts the minimum free energy of PON2 m RNA and lnc RNA GS1-293C5.1.It has been observed that the minimum free energy of the combination of the both is smaller than the free energy of their forming the secondary structure alone.The q RT-PCR detected relative expression levels of PON2 target genes in SK-Hep-1,Huh7,SMMC-7721,Hep3 B,and their m RNA expression relative to normal liver cancer cell line HL-7702 are: 0.62,3.19,0.71,1.42;and the relative expression in overexpression liver cancer cell lines SK-Hep-1-GS1-293C5.1,Huh7-GS1-293C5.1,SMMC-7721-GS1-293C5.1,Hep3B-GS1-293C5.1 are 0.37,1.21,0.23,0.73,compared with normal liver cells HL-7702,the difference is statistically significant(P<0.05).Western bolt has detected that the expression of PON2 protein was low in SK-Hep-1 and SMMC-7721,while the high expression of Huh7 and Hep3 B,they are consistent with the q RT-PCR results.The PON2 was is not significantly different from wild-type cells SK-Hep-1,SMMC-7721 and overexpression cells SK-Hep-1-GS1-293C5.1 and SMMC-7721-GS1-293C5.1.Huh7-GS1-293C5.1 and Hep3B-GS1-293C5.1 have lower PON2 protein expression than wild-type Huh7 and Hep3 B,suggesting that there may be a negative regulatory relationship between GS1-293C5.1 and PON2 in Huh7 and Hep3 B.The q RT-PCR have showed that PON2 is highly expressed in hepatocellular carcinoma tissues,the relative expression is 1.75,and lnc RNA GS1-293C5.1 was negatively correlated with the m RNA expression of PON2(r=-0.31,P=0.03).Conclusion: The preliminary results of this research indicate that lnc RNA GS1-293C5.1 is weakly expressed in hepatoma cells SK-Hep-1 and SMMC-7721 and moderately expressed in Huh7 and Hep3 B.The subcellular localization is mainly located in the nucleus and the predicted target was PON2.The expression level of lnc RNA GS1-293C5.1 in liver cancer tissues of patients with recurrence after liver cancer eradication is lower than that of patients without recurrence,Which suggest that the low expression of lnc RNA GS1-293C5.1 may be an indicator of recurrence.The overexpression of lnc RNA GS1-293C5.1 can improve the homogenous adhesion ability of liver cancer cell lines and reduce the heterogeneity of Huh7 cells Adhesion;overexpression of lnc RNA GS1-293C5.1inhibits the invasion and migration of liver cancer cell lines,and has no effect on cell proliferation,apoptosis,and cell cycle.The overexpression of lnc RNA GS1-293C5.1 reduced the PON2 m RNA transcription level and PON2 protein amount of Huh7 and Hep3 B,combined with bioinformatics prediction may be due to the related role of lnc RNA GS1-293C5.1 and PON2 in the secondary structure.The expression of lnc RNA GS1-293C5.1 in liver cancer tissue is low,while the target gene PON2 is highly expressed in liver cancer tissue.Correlation analysis has suggested that the expression of the two is inversely correlated.