| Objective To investigate the migration and invasion of hepatocellular carcinoma(HCC)from lnc RNA levels,the differentially expressed gene between Huh7 cell line knocked out GPC-3 and its control group were screened by high-throughput sequencing technology,which provides new strategies for the mechanism of HCC development and its clinical diagnosis and treatment.Methods1.The differentially expressed gene between Huh7 cell line knocked out GPC-3(Huh7-KO)and its control group were subjected to RNA digital expression spectrum sequening(RNA-Seq)and bioinformatic analysis,and lnc RNA with significant differential expression were screened by q PCR.2.A total of 60 pairs of HCC tissue and mathched peritumor samples were collected.The 60 patients were followed up to investigate and collect the clinical and pathological data of the patients.3.The expression of lnc145801 in 60 pairs of tumor and mathched peritumor tissue was validated by q PCR.4.Combined with clinical data of patients,SPSS24.0 software was used to statistically analyze the correlation between the expression of lnc145801 and clinicopathological characteristics such as tumor size,number,envelope boundary,tumor differentiation,HBV DNA,AFP,hepatocirrhosis,microvascular invasion,recurrence,and the relationship with postoperative recurrence and prognosis.5.The expression of lnc145801 in different HCC cell lines was validated by q PCR.6.Si RNA was transiently transfected into HCC cell lines with low-expression of lnc145801 through lipofectamine 3000,and identified by q PCR.7.Pc DNA3.1-lnc145801 was transiently transfected into HCC cell lines with high-expression of lnc145801 HCC cell lines through lipofectamine 3000,and verified by q PCR.8.The effects of lnc145801 on cell migration,invasion and proliferation were performed by cell functional experiments such as Transwell,scratch test and CCK-8.Results1.High-throughput sequencing and bioinformatic analysis predicted that 2033 genes were different,including 372 lnc RNAs(192 up-reguatted and 80down-regulated)and 1661 m RNAs(853 up-reguatted and 798 down-regulated).Five lnc RNAs(lnc145801,lnc324563,lnc211673,ENST00000641912,ENST00000602938)with significant differential expressions were screened out.2.It was verified by q PCR that among the 5 differentially expressed lnc RNAs,lnc145801 was significantly overexpressed in untreated Huh7 cell lines,and the expression in liver cancer tissues was significantly higher than that in adjacent liver tissues(P = 0.0001).This result is consistent with the results of high-throughput sequencing and bioinformatics prediction(fold change ≥ 2.0 or ≤ 0.5,P < 0.05).3.Kaplan-Meier analysis showed that patients with higher expression of lnc145801 always had a shorter DFS and OS than those with lower expression of lnc145801 postoperatively.Cox’s multivariate proportional hazards model confirmed that higher expression of lnc145801 was an independent poor prognostic factor for DFS and OS.Correlation analysis of clinicopathological characteristics showed that the tumor recurrence of patients was related to the expression level of lnc145801 in HCC tissues.4.The HCC cell lines MHCC97 H and LM3 highly express lnc145801.The migration,invasion and proliferation decreased in MHCC97 H and LM3,which were down expression of lnc145801(P < 0.05).5.After successfully constructing the overexpression vector pc DNA3.1-lnc145801 and transfecting the HCC cell lines Hep G2 and SMCC7721 with low expression of lnc145801,the migration,invasion and proliferation ability of Hep G2 and SMCC7721 were significantly enhanced(P < 0.05).Conclusions1.Through high-throughput sequencing,bioinformatics analysis and clinical liver cancer specimen verification,obtain a long-chain non-coding RNA highly expressed in HCC tissues---lnc145801.2.Liver cancer patients with high expression of lnc145801 are more prone to relapse and have a shorter survival period after operation.Analysis of the multi-factor Cox’s proportional risk model indicates that high expression of lnc145801 is an independent risk factor for tumor-free survival and overall survival.3.lnc145801 can promote the migration,invasion and proliferation of HCC cells.Knocking down lnc145801 can be used as a potential indicator to prolong HCC survival rate and reduce HCC metastasis. |
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