| Objective:hepatocellular carcinoma(HCC)is a kind of primary liver cancer and is one of the most common malignancies worldwide,especially in China and southeast Asia,and it is a common disease causing death due to cancer.Tumor metastasis is the leading cause of tumor-related death.However,the mechanism that drives the tumor metastasis is not fully understood.Long non-coding RNA snaR(Lnc RNA snaR)is a tumor activator.Studies have found that Lnc RNA snaR can play a carcinogenic role in a variety of cancers,but no studies have reported on HCC.According to some reports,Epithelial mesenchymal transition(EMT),matrix metalloproteinase(MMPs),transforming growth factor-beta 1(TGF-β1),extracellular regulated protein kinases(ERK),JAK kinase/signal transducer and activator of the transcription 3(JAK/STAT3)play an important role in the migration and invasion of tumors.Here,the purpose of this study was to explore the effects of LncRNA snaR on HCC migration and invasion and its mechanisms:1.Correlation and prognosis evaluation of LncRN A snaR for HCC disease progression;2.Effects of LncRNA snaR and TGF-β1,ERK and JAK/STAT3 signaling pathways on EMT,tumor migration and invasion of HCC cells.Methods:1.Clinical relative studies:(1)A total of 233 HCC patients admitted to the department of hepatology and biliary surgery of the second affiliated hospital of Kunming Medical University from January 201 1 to December 2013 were collected.32 male and 24 female HCC patients with an average age of 49.2±6.4 years were selected after screening for inclusion and exclusion criteria.The procedures of specimen collection and informed consent were discussed and approved by the ethics committee of the second affiliated hospital of Kunming Medical University.HE determined the tissue types:HCC tissue,para-cancinoma tissue,and normal liver tissue.(2)The expression levels of LncRNA snaR in the HCC tissues,plasma of HCC patients and healthy controls were detected by Q-RT-PCR.The expression level of TGF-β1 in plasma of HCC patients and control group was detected by ELISA.The expression levels of LncRNA snaR and TGF-β1 in plasma were analyzed by Pearson correlation analysis.(3)The correlation between the LncRNA snaR expression and clinicopathological data of HCC disease were retrospectively analyzed.(4)Kaplan-meier analysis was used to determine the correlation between LncRNA snaR expression and the clinical end points of HCC patients,and the curves of over survival(OS)and disease-free survival(DFS)were plotted.2.Studies on the effects of LncRNA snaR on the migration and invasion of HCC cells:(1)Cultured Human-hepatocellular carcinoma cell line HepG2 and SMMC7721,normaL liver cell line HL7702.LncRNA snaR gene sequences were obtained by molecular cloning,and LncRNA snaR expression plasmids were constructed using pcDNA3.1 and synthesize LncRNA snaR siRNA,which were transfected into HepG2,SMMC7721and HL7702 cells,respectively.LncRNA snaR expression levels were detected by Q-RT-PCR.(2)HCC cells were treated with gene overexpression or si-RNA techniques,and the effect of LncRNA snaR on HCC cell migration and invasion ability was detected by Transwell assay.3.Effects of LncRNA snaR on EMT of HCC cells:(1)The effects of LncRNA snaR on EMT signature protein and MMPs protein expression were detected by Western Blot,;(2)The effects of LncRNA snaR on E-cadherin of HCC cells were detected by immunofluorescence assay.4.Studies on the effects of LncRNA snaR and TGF-β1 on HCC cell migration and invasion:the project from the regulating mechanism of signal transduction pathways,using gene expression or si-RNA technology,(1)Transwell experiment were used to detect LncRNA snaR and TGF-β1 on the HCC cell migration and invasion ability;(2)The effect of LncRNA snaR on TGF-β1 protein expression was detected by Western Blot assay;(3)The interaction between LncRNA snaR and TGF-β1 was detected by RIP assay.5.The effects of LncRNA snaR,ERK and JAK/STAT3 signaling pathways on HCC cell migration and invasion:This project from the regulating mechanism of signal transduction pathways,the use of a gene expression or si-RNA technology,(1)Western Blot test TGF-β 1 on ERK,STAT3 protein expression levels;(2)Detect LncRNA snaR,ERK,JAK/STAT3 influence on E-cadherin of HCC cells by immunofluorescence experiments;(3)Time course experiment were used to detect the effects of LncRNA snaR on,ERK,JAK/STAT3 signal pathways of HCC cells;(4)Western Blot assay detected the interaction between ERK and JAK/STAT3;(5)Transwell assay detected the effect of ERK and JAK/STAT3 on the migration and invasion of HCC cells induced by LncRNA snaR.Results:1.Clinical relative studies showed that:(1)The expression of LncRNA snaR in HCC tissues and plasma was increased compared with the control group,(p<0.05).In addition,compared with the control group,tthe expression of TGF-β1 in HCC plasma was increased(p<0.05).Moreover,the high plasma LncRNA snaR in HCC patients was positively correlated with the level of TGF-β1.(2)The expression levels of LncRNA snaR in HCC tissues and plasma were significantly correlated with distant tumor metastasis.(3)High expression of LncRNA snaR in HCC patients was significantly correlated with shortened overall survival(OS)(p=0.01,HR=0.36)and disease-free survival(DFS)(p=0.01,HR=0.41).2.Studies on the effects of LncRNA snaR on the migration and invasion of HCC cells showed that overexpression of LncRNA snaR could promote the migration and invasion of HCC cells;si-RNA interference with LncRNA snaR weakened the migration and invasion of HCC cells(p<0.05).3.Studies on the effect of LncRNA snaR on EMT of HCC cells showed that:(1)Overexpressed LncRNA snR significantly increased the expression levels of N-cadherin and Vimentin,while overexpressed LncRNA snaR significantly inhibited the expression levels of E-cadherin in HepG2 cells(p<0.05).On the contrary,interfering LncRNA snaR with siRNA significantly reduced the expression levels of N-cadherin and Vimentin,while interfering LncRNA snaR with siRNA greatly promoted the expression of E-cadherin in HepG2 cells(p<0.05).Immunofluorescence detection showed that the overexpression of LncRNA snaR reduced the expression level of E-cadherin compared with the control group(p<0.05).(2)Overexpression of LncRNA snaR in HCC cells HepG2 can significantly increase the expression levels of MMP9 and MMP2(p<0.05),while interference of siRNA with LncRNA snaR can significantly reduce the expression levels of MMP9(p<0.05),but interference of siRNA with LncRNA sna can’t significantly reduce the expression levels of MMP2(p>0.05).4.Studies on the effects of LncRNA snaR and TGF-β1 on the migration and invasion of HCC cells:(1)Transwell assay showed that TGF-β1 could promote the migration and invasion of HepG2 cells;si-snaR can inhibit the migration and invasion of liver cancer cells induced by TGF-β1(p<0.05).(2)Transwell test showed that,compared with the control group,the migration and invasion ability of HepG2 cells in the LncRNA snaR group were significantly enhanced(p<0.05).However,the effect of LncRNA snaR on the migration and invasion of HCC cells HepG2 was significantly reduced after treatment with TGF-β1 inhibitor SD 208 at a dose of 10 ng/ml(p<0.05).(3)Compared with the control group,TGF-β1 expression was significantly up-regulated in LncRNA snaR overexpressed HepG2 cells(p<0.05).In contrast,HepG2 cells were treated with exogenous TGF-β1 at concentrations of 10 and 30ng/ml,and the expression ofLncRNA snaR was not significantly affected by exogenous TGF-β1(p>0.05).Moreover,after si-RNA interference with LncRNA snaR,TGF-β1 protein expression level significantly decreased(p<0.05,).(4)The interaction between LncRNA snaR and TGF-β1 was verified by RNA immunoprecipitation(RIP)and antibodies against TGF-β1 in HepG2 cells.5.Studies on the effects of LncRNA snaR,ERK and JAK/STAT3 signaling pathways on HCC cell migration and invasion found that:(1)Western Blot showed that,compared with the control group,the protein levels of ERK and STAT3 were significantly increased after the addition of exogenous TGF-β1.(2)Immunofluorescence experiments showed that,compared with the control group,when exogenous LncRNA snaR was added,the overexpression of LncRNA snaR reduced the expression level of E-cadherin(p<0.05).ERK inhibitor(U0126)inhibited ERK activity and JAK inhibitor(AG490)inhibited JAK/STAT3 signaling pathway activity,which saved E-cadherin degradation(p<0.05).(3)Cell time course assay showed that phosphorylation of ERK(phospho-ERK,P-ERK)increased in a time-dependent manner after transfection with LncRNA snaR overexpression vector,and reached its peak at 1 hour,nearly 1.4 times higher than the control group(P<0.05).Similarly,phosphorylation of STAT3 Tyr705(phospho-STAT3 Tyr705,P-STAT3 Y705)peaked nearly 15 minutes after transfection with LncRNA snaR overexpressed vectors,increasing by nearly 1.5 times compared with the control group(P<0.05).In the same way,phosphorylation of STAT3 Ser727(phospho-STAT3 Ser727,P-STAT3 S727)is at a peak laterly,most at 1 hour after stimulation,similar to the ERK peak(P<0.05).No significant changes in total ERK and STAT3 protein levels were observed in this study(p>0.05).(4)Western Blot analysis showed that,compared with LncRNA snaR overexpression group,the treatment of HCC cells with JAK inhibitor AG490 resulted in significantly lower phosphorylated ERK expression(p<0.05).Besides,in the pretreatment of AG490 and the transfection group of snaR-vector(A+snaR),overexpression of LncRNA snaR could save the reduced expression level of phosphorylated ERK in HCC cells induced by JAK inhibitor AG490(p<0.05).In addition,compared with the LncRNA snaR overexpression group,phosphorylation of STAT3 Ser727(P-STAT3 S727)with ERK inhibitor U0126 was significantly reduced(P<0.05),and phosphorylation of STAT3 Tyr705(P-STAT3 Y705)was also observed to be reduced(P<0.05).Besides,in the U0126 pretreatment group and the snaR-vector transfection group(U+snaR),overexpression of LncRNA snaR also reduced the levels of phosphorylation of STAT3 Ser727(P-STAT3 S727)and phosphorylation of STAT3 Tyr705(P-STAT3 Y705)in HCC cells induced by ERK inhibitor U0126(P<0.05).(5)Transwell assay showed that U0126 blocking ERK significantly reduced the role of LncRNA snaR in promoting liver cancer cell migration compared with the control group(figure p<0.05).Similarly,blocking JAK with XL019 can significantly reduce the role of LncRNA snaR in promoting HCC cell migration(p<0.05).Furthermore,blocking STAT3 with C188-9 also significantly reduced the role of LncRNA snaR in promoting HCC cell migration(p<0.05).In addition,we conducted Matrigel invasion assay,and the results showed that U0126 blocking ERK significantly reduced the effect of LncRNA snaR on the invasion of HCC cells compared with the control group(p<0.05).Similarly,blocking JAK with XL019 can significantly reduce the role of LncRNA snaR in promoting the invasion of HCC cells(p<0.05).Moreover,blocking STAT3 with C1 88-9 also significantly reduced the role of LncRNA snaR in promoting the invasion of HCC cells(p<0.05).Conclusions:1.The expression level of LncRNA snaR in HCC tissues and plasma was increased compared with the control group,and the expression of TGF-β1 in HCC plasma was increased compared with the control group.LncRNA snaR in the plasma of HCC patients was positively correlated with the level of TGF-β1.The expression levels of LncRNA snaR in HCC tissues and plasma were significantly correlated with distant tumor metastasis.High level of LncRNA snaR is related to shorted OS and DFS and overexpression of LncRNA snaR may be a factor for poor prognosis of HCC patients.2.Overexpression of LncRNA snaR can promote the migration and invasion of HCC cells;The interference of siRNA with LncRNA snaR weakens the migration and invasion of HCC cells.3.LncRNA snaR is involved in the EMT of HCC cells,and the overexpression of LncRNA snaR promotes the expression of MMP2 and MMP9 in HCC cells.4.The regulation of LncR snaR on the migration and invasion ability of HCC cells may be achieved through TGF-β1.5.LncRNA snaR promotes the migration and invasion of HCC cells by activating ERK and JAK/STAT3 pathways in HCC cells. |
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