Mechanisms Of Long Non-coding RNA LINC01093 In Inhibiting The Proliferation And Metastasis Of Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is one of the most common malignancies which seriously endangers human health.Most patients with HCC are diagnosed at the advanced stage,which have no chance of surgery and suffer a very poor prognosis.Therefore,it is important to clarify the mechanism of its occurrence,development and evolution,and search for early diagnostic markers in HCCObjectives:1.To explore the expression of long noncoding RNA(IncRNA)LINC01093 and its prognosis value in HCC;2.To investigate the effect of IncRNA LINC01093 on the biological behavior in HCC;3.To investigate the molecular mechanism of IncRNA LINC01093 in inhibiting proliferation and metastasis of HCC cellsMethods1.Relative expression levels of LINC01093 in 70 pairs of HCC and adjacent noncancerous liver tissues were determined by qPCR;Kaplan-Meier analysis and Cox regression analysis were also performed to explore the prognostic value of LINC01093 in HCC;2.Subcellular fractionation and fluorescence in situ hybridization assay were used to determine the subcellular distribution of LINC01093 in HCC cells;3.The 5' and 3' end sequence of LINC01093 was amplified by RACE assay.Stable cell lines with LINC01093 overexpression or knockdown were constructed to explore the effects of LINC01093 on HCC biological behavior;4.Transwell assays were conducted to evaluate migratory and invasive abilities of HCC cells in vitro,tail-vein metastasis models were established to determine the effect of LINC01093 on HCC metastasis in vivo'5.CCK8 and colony formation assays were performed to evaluate proliferative abilities of HCC cells in vitro:xenograft experiment with subcutaneous implanted tumor was utilized to analyze the effects of LINC01093 on tumor proliferation in vivo:6.RNA pulldown assay combined with mass spectrum and RIP experiment were applied to identify the proteins directly bind with LINC01093;7.Truncated RNA pulldown and RIP assays were used to identify the specific domain of LINC01093 and IGF2BP1 binding with each other;8.Mutant LINC01093 and IGF2BP1 were constructed respectively to further determine the combination locus of each other;9.qPCR and western blot experiments were applied to explore the effect of LINC01093 on GLI1 and its downstream target genes;10.Actinomycin D and luciferase reporter assay were conducted to detect the effect of LINC01093 on GLI1 mRNA stability;11.qPCR and western blot assay were applied to explore the relationship between LINC01093 and GLI1 as well as its downstream target genes in HCC tissuesResults:1.Liver-specific lncRNA LINC01093 was downregulated in HCC tissues,and its expression level was negatively correlated with cancer embolus and TNM stage in patients with HCC;2.LINC01093 could be used as an independent prognostic indicator for overall survival in HCC patients;3.LINC01093 inhibited the proliferation and invasion of HCC cells;4.LINC01093 bound with IGF2BP1 to prevent the combination of IGF2BP1 with GLI1 mRNA and promotes GLI1 mRNA degradation;5.GLI1 was required for LINC01093 regulation of HCC cell progression;6.LINC01093 expression level was negative correlated with GLI1 expression in HCC tissues.Conclusion:LncRNA LINC01093 can inhibit the proliferation and invasion in HCC LINC01093 directly binds IGF2BP1,interfering with interaction between IGF2BP1 and GLI1 mRNA,which leads to degradation of GLI1 mRNA,further affects expression of GLI1 downstream molecules involved in HCC progression.Moreover,LINC01093 is a promising prognostic indicator for HCC patients.