| Objective: The research about the roles of long non-coding RNAs(lnc RNAs)has drawn more attention over the past dacade.Few researchs demonstrate that lnc RNAs involved participate in a variety of biological processes by regulating the expression of corresponding genes.However,the relation between lnc RNAs and liver fibrosis remains unclear.In the current study,we have screened out the differentially expressing lnc RNAs in liver model mice and control mice by microarray analysis and elucidated,preliminaryly,the role in liver fibrosis.Methods: 1.Establish the mice model of liver fibrosis by carbon tetrachloride(CCl4): Twenty SPF male balb/c mice aged at 8 weeks were divided into two groups randomly.The mice of experimental group,CCl4 group,were intraperitoneally injected with CCl4 twice every week for six weeks.And the mice of the other group,control group,were intraperitoneally injection of olive oil at the same time.All the mice were sacrificed two days after the last injection.2.Liver tissues from mice of CCl4 group and control group were examined by means of macroscopic morphology,Sirius red staining and Haematoxylin & eosin staining,immunohistochemistry(?-SMA and Col1?1).3.Hydroxyproline kit was used to determine the content of hydroxyproline in CCl4 group and control group.4.The expression of ?-SMA and col1?1 in liver tissues of CCl4 group and control group was detected by immunofluorescence technique and q RT-PCR to verify the success of liver fibrosis mice model.5.The liver tissues of 5 mice from control group and 5 mice from CCl4 group were selected and tested by microarray analysis.6.Compare the data of CCl4 group with control group and sort out differentially expressed lnc RNAs and m RNAs.7.Screen lnc RNAs and m RNAs according to the fold change,GO analysis,Pathway analysis and lnc RNA-m RNA co-expression network.8.CCl4 model of balb/c and C57 mice were established respectively to validate the data of microarray analysis by q RT-PCR.9.q RT-PCR was used to detect the expression of lnc RNAs in different cells(HSC,HC and kuffer)and different organs(liver,heart,lung,kidney,brain,intestine,testis,spleen).10.The transcription potential of lnc RNAs was measured in Coding Potential Calculator.11.We cloned the predicted ORF of lnc RNA into pc DNA3.1(+)vector with a C-terminal EGFP tag.And the transcription potential of lnc RNA was measured by luciferase activity assays.12.5'-RACE and 3'-RACE analyses were used to determine the transcriptional initiation and termination sites of lnc RNAs.13.Cytoplasmic and nuclear RNA isolation were performed to verify the location of lnc RNA.14.The expression of lnc RNAs was detected in primary HSCs and HCs of fibrotic mice and during the activation of primary hepatic stellate cells in vitro.15.The expression of lnc RNAs was detected in TGF-?-stimulated primary hepatic stellate cells,hepatocytes and AML12 cells.16.The expression of lnc RNA was detected in liver tissues and blood of patients from liver fibrosis.17.After the treatment of recombinant TGF?,the expression of pro-fibrosis genes in primary HSCs with overexpress or knockdown lnc RNAs were detected by q RT-PCR,Western Blot and Confocal.Results: 1.Compared with control group,livers of CCl4 group were harder and bigger.And there were more nodules on liver surfaces in CCl4 group.2.HE staining and the Sirius red staining show that,compared with the control group,the liver cells of CCl4 group arrange disorder,and the liver lobular structure is destroyed.3.The concentration of hydroxyproline in mouse livers increased from 104 ?g/g to 397 ?g/g after CCl4 injection.4.Immunohistochemical staining,Immunofluorescence and q RT-PCR revealed that CCl4-treated livers exhibited increased expression of ?-SMA and col1?1.5.Five CCl4-treated mouse livers and five normal mouse livers inspection up to standard of microarray analysis.6.We found 266 lnc RNAs and 1007 m RNAs were up-regulated,and 447 lnc RNAs and 519 m RNAs were down-regulated in fibrotic livers by Microarray analysis.7.GO analysis,Pathway analysis and lnc RNA-m RNA co-expression network were performed to sort out 10 lnc RNAs and 10 m RNAs to verify the microarray results.Moreover,NONMMUT045304(lnc-LFAR1)and ENSMUST00000158992 were selected to the following study.8.The q RT-PCR results of the CCl4 model(balb/c and C57 mouse)consistent with Microarray data.9.q RT-PCR results showed that lnc-LFAR1 was enriched in mice liver.Both lnc-LFAR1 and ENSMUST00000158992 were detected in HSCs,HCs and Kuffers? 10.According to the Coding Potential Calculator(CPC),lnc-LFAR1 harbors short open reading frame(ORF)of 55 aa and ENSMUST00000158992 harbors short open reading frame(ORF)of 68 aa.Whereas,the full length transcripts of lnc-LFAR1 and ENSMUST00000158992 have no protein coding potential.11.Immunofluorescence showed that the full length transcripts of lnc-LFAR1 and ENSMUST00000158992 have no protein-coding potential.12.With the use of 5'-and 3'-rapid amplification of c DNA ends(RACE),we found that lnc-LFAR1 was a 734-nucleotide transcript with poly A tail,and ENSMUST00000158992 was a 325-nucleotide transcript without poly A tail.13.Cell fractionation followed by q RT-PCR showed that NONMMUT045304 located both in the cytoplasm and the nucleus,while ENSMUST00000158992 mainly located in the nucleus.14.The expression of lnc-LFAR1 and ENSMUST00000158992 was increased in primary HSCs and HCs of fibrotic mouse and during the activation of primary hepatic stellate cells in vitro.15.The expression of lnc-LFAR1 and ENSMUST00000158992 was increased in TGF?-stimulated primary HSCs and HCs of normal mice and AML12 cells.16.The expression of ENSMUST00000158992 was increased in the blood of patients of liver fibrosis.17.Consistent with result of overexpression,cells infected with lnc RNA-sh RNAs express a lower expression level of fibrosis-related genes in primary HSCs.Moreover,knockdown of lnc RNA dramatically decreased TGF?-induced up-regulation of these fibrosis-related genes in primary HSCs.Conclusions: 1.CCl4-induced mouse liver fibrosis model was successfully constructed.2.The data of microarray analysis showed 266 lnc RNAs and 1007 m RNAs were upregulated,and 447 lnc RNAs and 519 m RNAs were downregulated in fibrotic livers of the mice.3.Lnc-LFAR1 is enriched in mice livers and down-regulated in mice fibrotic livers.Hoever,ENSMUST00000158992 is up-regulated in mice fibrotic liver tissue.4.Lnc-LFAR1 is a734-nucleotide transcript with poly A tail which is located in both the cytoplasm and the nucleus,and ENSMUST00000158992 is a 325-nucleotide transcript without poly A tail and located in nucleus mainly.And both of them have no protein coding potential.5.Lnc-LFAR1 and ENSMUST00000158992 promote the development of liver fibrosis by promoting the expression of fibrosis associated genes and the activation of HSCs. |
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