Role Of LncRNA Activated By Transforming Growth Factor Beta In The Progression Of Hepatitis C Virus-related Liver Fibrosis

Hepatitis C virus(HCV) infection remains a global threat to public health. According to World Health Organization(WHO), about 185 million people globally have HCV infection. Approximately 500,000 people die each year from hepatitis C-related liver diseases. In recent years, the rate of HCV infection increases in China, and 220,000 new cases were reported in 2013. Liver cirrhosis and hepatocellular carcinoma(HCC) represent the major causes of HCV-associated death. Liver fibrosis is a key sequelae of chronic HCV infection and an important pathological process leading to HCC. Recently, Butt et al found that liver fibrosis may develop even in the early stage of HCV infection. In addition, FIB-4 index(fibrosis index based on the 4 factor), the serum biomarker of liver fibrosis, was increased persistently over the period of 11 years. Thus, early diagnosis and effective treatment are important for preventing progression of HCV-related diseases and improving the quality of life of the patients. At present, accurate diagnosis biomarker and effective therapeutic target are still lacking. Therefore, further revealing novel genes for regulating liver fibrosis are the essential steps for early diagnosis and molecular-targeted therapy of HCV-raltated hepatic fibrosis.Long non-coding RNA(lnc RNA) is a novel class of m RNA-like transcripts with sizes longer than 200 bp, with little or no function of protein-coding but plays pivotal roles in regulating gene expression at epigenetics, transcriptional and post-transcriptional levels. Increasing evidence revealed that differential expression and dysfunction of lnc RNAs were closely related to diverse hepatic pathological processes, such as hepatocyte regeneration, inflammation, immune response and the development of HCC. In addition, lnc RNAs could regulate the function of HSC, and contribute to the development and progression of liver fibrosis. Lnc RNA-activated by transforming growth factor-beta(lnc RNA-ATB) is known to be participated in the initiation, progression and metastasis of malignant neoplasm, and is positively correlated with the development of liver cirrhosis and HCC. However, the role of lnc RNA-ATB in HCV-related liver fibrosis remains largely unknown.In the present study, we used cell model of HCV-related liver fibrosis to explore the role of lnc RNA-ATB in HCV-related hepatic fibrosis and the regulated genes or signaling pathways, and to clarify the underlying mechanism of lnc RNA-ATB in HCV-related liver fibrosis. The data will will provide scientific evidence for the development of diagnostic markers and therapy for HCV-related hepatic fibrosis. Part 1 Expression of lnc RNA-ATB in plasma and liver tissue of patients with HCV-related liver fibrosisObjective: To examine the changes of lnc RNA-ATB in plasma and liver tissue of patients with HCV-related liver fibrosis, and further analyze the diagnostic value of plasma lnc RNA-ATB in HCV-related hepatic fibrosis.Method: Thirty-six patients with chronic HCV infection and 15 age- and gender-matched healthy subjects, all from the Third Hospital of Hebei Medical University(Shijiazhuang, China) from September 2014 through June 2015, were included in this study. Chronic HCV infection was confirmed using serology and liver biopsy. Plasma and serum were isolated from peripheral blood and stored at-80oC for further analysis. Liver tissues were obtained from HCV patients through liver biopsy. Hepatic fibrosis was graded into mild(F<2; n=14) and moderate to severe(2?F<4; n=22) according to METAVIR scoring system. Normal liver tissues obtained from five cases of liver transplant donors were used as healthy controls. Written informed consents were obtained prior to sample collection. The study was approved by the Human Ethics Committee of the Third Hospital of Hebei Medical University. Serum alanine aminotransferase(ALT) and aspartate aminotransferase(AST) levels were measured by the Olympus biochemical autoanalyzers. A small portion of the liver tissues was fixed in 10% formalin, and subsequently processed for hematoxylin and eosin(H&E) staining(for routine histology), Masson trichromatism(for steatosis staging), inflammation analysis, and fibrosis. Alpha-smooth muscle actin(?-SMA) and alpha-1 type I collagen(Col1A1) was detected by immunohistochemical staining. Hepatic expression of lnc RNA-ATB was detected by in situ hybridization assay. Total RNA in plasma was extracted by Trizol LS method, and quantitative real-time PCR(q PCR) was used to detect the expression of lnc RNA-ATB.Results:1. General characteristic of the patientsA total of 15 healthy subjects(8 males and 7 females, average age 51.2 ± 11.4 years), 14 HCV patients with mild liver fibrosis(F<2)(6 males and 8 females, average age 48.8 ± 11.6 years), 22 HCV patients with moderate to severe liver fibrosis(2?F<4)(12 males and 10 females, average age 52.5± 7.2 years) were included in this study. There was no significant difference in age between the three groups(F=0.596,p=0.555).2. Serum ALT and AST levels in healthy controls and HCV patientsThe median serum ALT levels from healthy control group, HCV patients(F<2) group, and HCV patients(2?F<4) group were 17.0 U/L, 33.0 U/L, and 50.0 U/L, respectively. The median serum AST levels from healthy control group, HCV patients(F<2) group and HCV patients(2?F<4) group were 15.0 U/L, 32.0 U/L, and 43.5 U/L, respectively. There were significant differences in ALT and AST levels between the three groups(?2=23.79, p=0.000; ?2=23.33, p=0.000, respectively). Furthermore, post-hoc comparison showed that serum ALT and AST in HCV patients(F<2) group was higher than that in healthy control group(p=0.000, p=0.000, respectively). Serum ALT and AST in HCV patients(2?F<4) group was significantly higher than that in healthy control group(p=0.000, p=0.000, respectively). However, there was no significant difference of serum ALT between HCV patients(2?F<4) group and HCV patients(F<2) group(p=0.107). However, serum AST in HCV patients(2?F<4) group was significantly higher than that in HCV patients(F<2) group(p=0.000).3. Histolopathological features of liver tissues from HCV patientsThe healthy controls showed normal liver histology. The liver sections of HCV patients(F<2) group showed mild hepatocyte ballooning, hepatocyte steatosis and inflammatory infiltration, with mild or no proliferation of fibrous tissue in portal area. In contrast, HCV patients(2?F<4) exhibited moderate hepatocyte steatosis, inflammatory infiltration, and bridging fibrosis formation.4. Hepatic expressions of ?-SMA, Col1A1 and lnc RNA-ATBLittle expression of ?-SMA, Col1A1 and lnc RNA-ATB was found in the liver tissues from healthy controls. In the liver tissues from HCV patients(F<2), increased expression of ?-SMA in the activated HSCs and fibrotic areas, increased expression of Col1A1 in the fibrotic areas, and increased expression of lnc RNA-ATB in hepatocytes cytoplasm were observed. In the liver tissues of HCV patients(2?F<4), rich expression of ?-SMA in the activated HSCs and fibrotic areas, increased expression of Col1A1 in fibrotic areas, and increased expression of lnc RNA-ATB in hepatocytes cytoplasm were observed, and these changes were positively associated with the progression of the liver injury.5. The level of plasma lnc RNA-ATB and the correlation with stages of liver fibrosisUsing q RT-PCR analysis, we revealed that compared to healthy control subjects, there was a significant increase in the plasma level of lnc RNA-ATB in HCV patients(Z=-5.562, P=0.000), and the increased plasma level of lnc RNA-ATB was closely correlated with the stage of liver fibrosis(r=0.785, P=0.000, Figure 2B). In addition, the plasma level of lnc RNA-ATB was markedly higher in HCV patients with moderate to severe fibrosis than in patients with mild fibrosis(Z=-3.960, P=0.011).6. Analysis of diagnosis value of plasma lnc RNA-ATB level in liver fibrosis.Receiver Operating Characteristics Analysis showed that the area under the curve was 0.877 for the lnc RNA-ATB level in the prediction of the fibrosis stage in HCV patients(P=0.000, Figure 2D), suggesting that lnc RNA-ATB may be a potential biomarker with high sensitivity and specificity for forecasting the progression of liver fibrosis in HCV patients.Conclusion:1. The progression of the liver injury and development of fibrosis were accompanied by a parallel increase in the expression of hepatic lnc RNA-ATB.2. Significantly increased plasma level of lnc RNA-ATB was found in HCV patients, and the level of plasma lnc RNA-ATB was positively correlated with the stage of liver fibrosis.3. Lnc RNA-ATB may be a potential biomarker with high sensitivity and specificity for forecasting the progression of liver fibrosis in HCV patients. Part 2 lnc RNA-ATB and HSC activation in vivoObjective: To observe the changes of lnc RNA-ATB in activated HSCs in vivo and clarify the role of lnc RNA-ATB on the activation of HSCs.Method: Hep G2 cells were transfected by pc DNA3.1-HCV core plasmid. Hep G2-CORE cells stably expressing HCV core protein were screened and validated. Furthermore, knockdown of TGF?1 in Hep G2-CORE cells were performed using si RNA transfection. The m RNA and protein expressions of HCV core were detected by real-time PCR and Western blot assay. The TGF?1 level in cell culture supernatants was measured by enzyme linked immunosorbent assay(ELISA). Human hepatic stellate cell line LX-2 cells were treated with cell culture supernatants from Hep G2-CORE cells and recombinant human TGF?1(rh TGF?1) of various doses for various durations. The expression of ?-SMA and Col1A1 was detected by q PCR, Western blot assay and Dual-fluorescent immunocytochemistry strain, respectively. Cell counting Kit-8(CCK-8) was used to detect cell proliferation. Lnc RNA-ATB was detected by q PCR.Results:1 Establishment of Hep G2-CORE cell lineHep G2-CORE cells were confirmed to express the m RNA and protein of HCV core; and the TGF?1 level in cell culture supernatants from Hep G2-CORE cell was significantly increased compared to control cells(Hep G2-NC). Knockdown of TGF-?1 in Hep G2-CORE cells with si-TGF?1 could significantly decrease TGF?1 m RNA expression, and reduce TGF?1 level in cell culture supernatants;2 Cell culture supernatants from Hep G2-CORE cells promoted the activation of HSCs;Cell culture supernatants as conditioned media from Hep G2-CORE cells significantly increased the expression of ?-SMA and Col1A1 at m RNA and protein levels in LX-2 cells. In contrst, cell culture supernatants derived from Hep G2-CORE cells which lack TGF?1 expression mildly increased the expression of ?-SMA and Col1A1 in LX-2 cells.3. rh TGF?1 promoted the activation and proliferation of HSCsTreatment of LX-2 cells with rh TGF?1 led to a does- and time-dependent increase of the expression of ?-SMA and Col1A1 at the m RNA and protein levels. LX-2 cell treated with 10 ng/ml of rh TGF?1 for 48 h showed dramatic activation and proliferation.4. Lnc RNA-ATB was upregulated in activated HSCsLnc RNA-ATB was significantly upregulated in LX-2 cells treated withthe cell culture supernatants of Hep G2-CORE cells. Rh TGF?1 could increasethe expression of lnc RNA-ATB in the activated LX-2 cells at dose- andtime-dependent manners.Conclusion:1. Conditioned media from Hep G2-CORE cells stably expressing HCV core protein could promote the activation of HSCs.2. Knockdown of TGF-?1 in Hep G2-CORE cells with si-TGF?1 could markedly decrease the expression of TGF?1 at the m RNA level and reduce the level of TGF?1 in cell culture supernatants. The conditioned medium from the si-TGF?1-treated Hep G2-CORE cells was less potent in activating LX-2 cells.3. rh TGF?1 promoted the activation and proliferation of HSCs.4. Lnc RNA-ATB was upregulated in activated HSCs. Part 3 Mechanisms by which lnc RNA-ATB function as competingendogenous RNA(ce RNA) in the regulation of gene expressionObjectives:(1) To predict the potential binding sites of lnc RNA-ATB on mi RNAs and other target genes,(2) to explore the mechan isms of lnc RNA-ATB as a ce RNA in the regulation of gene expression.Method: The online softwares in Segal Laboratory of Computational Biology(genierget gene of mi RNAUsing the bioinformatic analysis, lnc RNA-ATB was found to share the common MRE of mi R-425-5p with TGF-? type II receptor(TGF-?RII) and SMAD2. The binding site of mi R-425-5p(CACAGUA) is present in lnc RNA-ATB, TGF-?RII and SMAD2.2. Verification of the binding sites by dual luciferase reporter assaymi R-425-5p mimic could significantly reduce the lnc RNA-ATB-, TGF-?RII and SMAD2 3'-UTR-dependent luciferase activity but did not affect the luciferase activity of the mutant reporter. In contrast, mimic controls had no effect on wild type or mutant reporter luciferase activity.3. mi R-425-5p regulates gene expression at post-transcriptional levelEctopic expression of mi R-425-5p in LX-2 cells could significantly up-regulate the endogenous level of mi R-425-5p but not the level of lnc RNA-ATB and the m RNA of TGF-?RII and SMAD2. However, overexpression of mi R-425-5p in LX-2 cells led to significant reduction of TGF-?RII and SMAD2 at the protein level. On the contrary, knockdown of mi R-425-5p could down-regulate the endogenous level of mi R-425-5p but not the level of Lnc RNA-ATB and the m RNA of TGF-?RII and SMAD2, and could increase the protein expression of TGF-?RII and SMAD2.4. mi R-425-5p could suppress the HSC activationOverexpression of mi R-425-5p could significantly decrease the expression of ?-SMA and Col1A1 at the m RNA and protein levels. However, mi R-425-5p inhibitor promoted ?-SMA and Col1A1 expression both at m RNA and protein levels.Conclusions1. Lnc RNA-ATB shares the common binding sites of mi R-425-5p with TGF-?RII and SMAD2;2. Dual luciferase reporter assay verified that mi R-425-5p could bind to lnc RNA-ATB, TGF-?RII and SMAD2.3. Modulation of mi R-425-5p could induce the change of endogenous mi R-425-5p, regulate TGF-?RII and SMAD2 expression at protein level, and alter the expression of ?-SMA and Col1A1 at the m RNA and protein levels. Part 4 Role and mechanism of targeted modulation of lnc RNA-ATB inthe activation of HSCObjective: To clarify the effect and molecular mechanism of lnc RNA-ATB on HSC activation through overexpression or knockdown of lnc RNA-ATB.Methods: LX-2 cells were transfected with pc DNA3.1-ATB, pc DNA3.1-ATB-mut and their respective controls. The expression of ?-SMA, Col1A1, TGF-?RII and SMAD2 was detected by q RT-PCR and Western blot assay. Cell counting Kit-8(CCK-8) was used to detect cell proliferation. TGF-?RII and SMAD2 protein expression was determined by dual-fluorescent immunocytochemistry straining. The expression of lnc RNA-ATB and mi R-425-5p was detected by q RT-PCR. LX-2 cells were transfected by si-ATB and the respective controls. q RT-PCR and Western blot assay were used to detect the expression of ?-SMA, Col1A1, TGF-?RII and SMAD2. Proliferation of LX-2 cells was detected by cell counting Kit-8(CCK-8). TGF-?RII and SMAD2 protein expression was determined by dual-fluorescent immunocytochemistry straining. Lnc RNA-ATB and mi R-425-5p were detected by q RT-PCR.Results:1. Lnc RNA-ATB promotes the activation and proliferation of LX-2 cells.Ectopic expression of lnc RNA-ATB remarkably increased the expression of ?-SMA and Col1A1 in LX-2 cells at the m RNA and protein levels. These changes were associated with increased proliferation of LX-2 cells.2. Lnc RNA-ATB regulates TGF-?RII and SMAD2 expression by binding to mi R-425-5p.Overexpression of lnc RNA-ATB in LX-2 cells increased the protein expression of TGF-?RII and SMAD2 with a concommittant down-regulation of mi R-425-5p. Treatment of LX-2 cells with exogenous mi R-425-5p could partially attenuate the upregulation of TGF-?RII and SMAD2 at the protein level. These data were confirmed by immunofluorescent staining.3. Knockdown of lnc RNA-ATB could suppress HSC activation and proliferation.Among the three si RNAs of lnc RNA-ATB we designed, si-ATB-3 exhibited the strongest inhibitory effect on lnc RNA-ATB expression, and hence was used to knockdown lnc RNA-ATB in the subsequent studies. Inhibition of lnc RNA-ATB led to reduced activation of LX-2 cells as indicated by decreased expression of ?-SMA and Col1A1 at the m RNA and protein levels. Further, inhibition of lnc RNA-ATB resulted in significant suppression of LX-2 proliferation.4. Knockdown of lnc RNA-ATB inhibits the expression of TGF-?RII and SMAD2 by up-regulating mi R-425-5p expressionKnockdown of lnc RNA-ATB decreased the protein expression of TGF-?RII and SMAD2 but increased mi R-425-5p expression. Inhibition of mi R-425-5p could blunt the suppression of TGF-?RII and SMAD2, which were further confirmed by immunofluorescent staining.Conclusions:1. Overexpression of lnc RNA-ATB could up-regulate the expression of TGF-?RII and SMAD2 by decreasing endogenous mi R-425-5p, leading to HSC activation and proliferation.2. Knockdown of lnc RNA-ATB could down-regulate TGF-?RII and SMAD2 expression through increasing mi R-425-5p, resulting in the activation and proliferation of HSC.