| Part ?:Study on the Mechanism of Long Non-coding RNA SNHG3 in Hepatocellular CarcinomaBackgroundLiver cancer is one of the main causes of tumor death due to about 840,000 newly diagnosed cases and 740,000 deaths each year,of which hepatocellular carcinoma(HCC)accounts for about 85%-90%.There are many causes of liver cancer,including alcoholism,aflatoxin-contaminated diet,and hepatitis virus infection.According to the report of the Global Cancer Research Agency,the etiology distribution of liver cancer has regional differences.In China,chronic hepatitis B is the main reason,liver cells are chronically stimulated by inflammation and can develop Liver cirrhosis(LC)and HCC,known as "trilogy of hepatitis B virus infection".HCC has an insidious onset,a high degree of malignancy,rapid progress,and a poor prognosis.In recent years,treatment methods for HCC have developed rapidly,including systemic chemotherapy,radiofrequency ablation,and microwave ablation,but the prognosis of patients with advanced HCC is still unideal.According to previous research reports,the occurrence and development of HCC is an extremely complex process,which may involve a variety of genes and signal pathway abnormalities.Therefore,it is particularly important to study the molecular mechanism of HCC development and find new targets for treatment.Long non-coding RNA(LncRNA)which is longer than 200 nt and has no protein-coding function.A large number of studies have shown that LncRNAs play an important role in tumor development and can affect many biological functions in tumor progression,including epithelial-mesenchymal transition(EMT),invasion,metastasis,proliferation,apoptosis,and Drug resistance.It has been reported that lncRNA-Small nucleolar RNA host gene 3(SNHG3)can participate in the development of malignant tumors.SNHG3 is abnormally expressed in a variety of malignant tumors and is associated with disease progression,such as colorectal cancer,lung adenocarcinoma,and glioma.Abnormal SNHG3 expression is also related to the prognosis of malignant tumors.For example,increased expression of SNHG3 in ovarian cancer often indicates a poor prognosis of ovarian cancer.SNHG3 expression is elevated in HCC and plays a role in the progress of HCC,but the exact molecular mechanism of SNHG3 in HCC is still unclear.Therefore,it is important to further study the mechanism of SNHG3 to determine whether SNHG3 can be used as a new therapeutic target.MicroRNA(miRNA)is a highly conserved small non-coding RNA,and its biological role in malignant tumors is widely recognized,including cell proliferation,invasion,apoptosis,metabolism and signal transduction.According to reports,miRNA-326(miR-326)is involved in apoptosis,invasion,embryonic development,tumor growth,immune regulation,resistance to chemotherapy drugs,and tumorigenesis.MiR-326 acts as a tumor suppressor in glioma,endometrial cancer and cervical cancer by targeting different mRNAs.MiR-326 is lowly expressed in HCC,and can promote apoptosis,inhibit cell invasion and tumor growth.Research reports that many lncRNAs as competitive endogenous RNAs(ceRNAs)can competitively bind miRNAs through microRNA response elements(MREs),thereby reducing the miRNA's inhibitory effect on target mRNAs.That is,miRNA can lead to gene silencing by binding to mRNA,and lncRNA can compete with miRNA as ceRNA to bind miRNA,thereby inhibiting gene silencing caused by miRNA.However,the relationship between SNHG3 and miR-326 in HCC has not been reported.Objective1.To investigate the expression of SNHG3 in peripheral blood mononuclear cells of patients with chronic hepatitis B,liver cirrhosis and hepatocellular carcinoma.2.To explore the expression of SNHG3 in HCC tissues and HCC cells.To analyzed the correlation between the expression levels of SNHG3 in HCC tissues and clinical pathological parameters3.To find the miRNAs that SNHG3 can bind.4.To investigate the relationship between SNHG3 and miR-326 and the effects of their interaction on the biological functions of HCC.5.To identify the regulatory mechanism of SNHG3 on SMAD3 as a miR-326 targeted mRNA.6.To study the biological functions of SNHG3 through SMAD3/ZEB1 signal pathway.Methods1.To detect expression levels of SNHG3 in peripheral blood mononuclear cellsA total of 124 study subjects were recruited in this experiment,including 40 patients with hepatocellular carcinoma,30 patients with liver cirrhosis,30 patients with chronic hepatitis B,and 24 healthy controls.They are patients admitted to the department of hepatology,Qilu Hospital of Shandong University from May 2016 to December 2017.QRT-PCR was used to detect the expressions of SNHG3 in peripheral blood mononuclear cells.2.To detection of SNHG3 expression in tissue specimens and HCC cell lines47 pairs of HCC tissues and adjacent normal tissues were collected.These specimens were from inpatients in Qilu Hospital of Shandong University from August 2017 and January 2018.QRT-PCR was used to detect the expressions levels of SNHG3 in these tissues.Chi-square was used to test the relationship between the expression levels of SNHG3 in the tumor tissues and the clinical pathological parameters of patients.QRT-PCR was used to detect the expression levels of SNHG3 in HepG2 cells and HCCLM3 cells compared with normal hepatocyte L02 cells.3.To analyze the relationship between SNHG3 and miR-326MiRNA combined with SNHG3 was predicted by biological software.HCC cells were co-transfected with SNHG3-MUT or SNHG3-WT and miR-326 mimic or negative control(NC-mimic).The luciferase activities were tested by Daul-Luciferase reporter assay.QRT-PCR was used to measure expression levels of miR-326 in 47 pairs of HCC tissue specimens and and adjacent normal tissues.Pearson correlation was used to analyze the relationship between expression levels of SNHG3 and miR-326.QRT-PCR was used to measure the expression levels of miR-326 in HCCLM3 and HepG2 cells compared with normal hepatocyte L02 cells.4.To analyze the effect of SNHG3 and miR-326 interaction on the biological function of liver cancer cells.The HCCLM3 cells were transfected with si-SNHG3 or co-transfected with si-SNHG3 and miR-326 inhibitor(anti-326).MTT assay,transwell assay,flow cytometry were used to tested the proliferation,migration,apoptosis abilities of HCCLM3 cells.The EMT related mRNA and protein E-cadherin,Vimentin,and Snail in HCCLM3 cells were tested by qRT-PCR and western blot.The HepG2 cells were transfected with pLVX-SNHG3 or co-transfected with pLVX-SNHG3 and miR-326 mimic.MTT assay,transwell assay,flow cytometry were used to tested the proliferation,migration,apoptosis abilities of HCCLM3 cells.The EMT related mRNA and protein E-cadherin,Vimentin,and Snail in HCCLM3 cells were tested by qRT-PCR and western blot.5.To study the regulation of miR-326 targeted mRNA by SNHG35.1 To find and detect the target mRNA for miR-326The mRNA contains the sequence complementary to miR-326 was predicted by biological software.HCC cells were co-transfected with SMAD3-MUT or SMAD3-WT and miR-326 mimic or negative control(NC-mimic).The luciferase activities were tested by Daul-Luciferase reporter assay.QRT-PCR was used to detect the expression levels of SMAD3 in 47 pairs of HCC tissues and adjacent normal tissues.QRT-PCR and western blot were used to detect the mRNA and protein levels of SMAD3 in HCC cell lines and normal hepatocyte L02 cells.The HepG2 cells were transfected with miR-326 mimic,the HCCLM3 cells were transfected with miR-326 inhibitor,qRT-PCR and western blot were used to measure the expression of SMAD3 at mRNA and protein levels.5.2 To analyze the regulation of SMAD3 by SNHG3The HepG2 cells were transfected with pLVX-SNHG3 or co-transfected with miR-326 mimic,qRT-PCR and western blot were used to detect the expression of SMAD3 at mRNA and protein levels.The HCCLM3 cells were transfected with si-SNHG3 or co-transfected with miR-326 inhibitor,qRT-PCR and western blot were used to detect the expression of SMAD3 at mRNA and protein levels.6.To study the biological functions of SNHG3 via SMAD/ZEB1 signaling pathway6.1 To measure the expression levels of ZEB1QRT-PCR was used to detect the expression levels of ZEB1 in 47 pairs of HCC tissues and adjacent normal tissues.QRT-PCR and western blot were used to detect the mRNA and protein levels of SMAD3 in HCC cell lines and normal hepatocyte L02 cells.6.2 To study the regulation of ZEB1 by SNHG3The HepG2 cells were transfected with pLVX-SNHG3,qRT-PCR and western blot were used to detect the expression of ZEB1 at mRNA and protein levels.The HCCLM3 cells were transfected with si-SNHG3,qRT-PCR and western blot were used to detect the expression of ZEB1 at mRNA and protein levels.6.3 To study SNHG3 exerts biological functions through ZEB1The HepG2 cells were transfected with pLVX-SNHG3 or co-transfected with si-ZEB1,MTT assay was used to test the proliferation ability of cells.Western blot was used to measure the related proteins of EMT.The HCCLM3 cells were transfected with si-SNHG3 or co-transfected with pLVX-ZEB1,MTT assay was used to test the proliferation ability of cells.Western blot was used to measure the related proteins of EMT.6.4 To study that SNHG3 might work through SMAD3/ZEB1 signaling pathway in vivoThe HCC cells interfered with SNHG3 were constructed for subcutaneous implant tumor experiments in nude mice.The volumes of tumors were measured by calipers.The mice were dissected 33 days later to observe the growth status of the subcutaneous implant tumors in nude mice.Ki-67 and Tunel staining were used to test the proliferation and apoptosis abilities of SNHG3 in vivo.Western blot was used to detect the protein levels of E-cadherin,Vimentin,Snail,SMAD3,and ZEB1.Results1.Expression of SNHG3 in peripheral blood mononuclear cellsThe expression levels of SNHG3 in peripheral blood mononuclear cells of patients with hepatocellular carcinoma were significantly higher than that of liver cirrhosis,chronic hepatitis B and healthy controls.There was no significant difference in the expression levels of SNHG3 in peripheral blood mononuclear cells from liver cirrhosis,chronic hepatitis B and healthy controls.2.The expression level of SNHG3 in HCC tissues and HCC cell linesThe expression level of SNHG3 in HCC tissues was significantly higher than that in adjacent noncancerous tissues.Chi-square test showed that the expression level of SNHG3 was related to TNM stage,lymph node metastasis and distant metastasis,not related with age and sex.The expression levels of SNHG3 in HepG2 cells and HCCLM3 cells were significantly higher than that in normal liver cells L02.3.SNHG3 could bind to miR-326Bioinformatics analysis showed that miR-326 contained the potential binding sequence for SNHG3.Luciferase activities were significantly lower in HepG2 cells cotransfected with SNHG3-WT and miR-326 mimic than those in cells cotrnsfected with SNHG3-WT and NC mimic,but the luciferase activities were not alterd with the mutant SNHG3 vector.The expression levels of miR-326 were lower in HCC tissues compared with NCs.Pearson correlation analysis indicated the expression trend of SNHG3 in 47 pairs of tissues was opposite to that of miR-326.The relative expression levels of miR-326 levels were lower in HCCLM3 cells and HepG2 cells compared with normal hepatocyte(L02).4.SNHG3 as a ceRNA of miR-326 promotes the abilities of proliferation,migration and EMT and inhibits apoptosis of HCC cellsSNHG3 knockdowned with si-SNHG3 significantly reduced proliferation,migration and EMT abilities of HCCLM3 cells and conversely increased apoptosis,while these effects were abolished by anti-326.Overexpression of SNHG3 significantly increased proliferation,migration and EMT abilities of HepG2 cells,but these enhancing effects of SNHG3 were not detected in cells co-transfected with miR-326 mimic.By contrast,overexpression of SNHG3 significantly decreased apoptosis,while miR-326 mimic restored the degree of apoptosis.5.SNHG3 regulated the targeted mRNA of miR-3265.1 The targeted mRNA of miR-326 was SMAD3By searching the Targetscan website,the 3'-UTR of SMAD3 mRNA contains the sequence complementary to miR-326.The luciferase activities were significantly lower in HepG2 cells co-transfected with SMAD3-WT and NC mimic.The expression levels of SMAD3 mRNAs were significantly higher in in human HCC tissues compared with the matched adjacent noncancerous tissues.The expression levels of SMAD3 in HCC cell lines were higher than in normal hepatocyte cells(L02)in mRNA and protein levels.5.2 MiR-326 inhibited the expression of SMAD3The effect of miR-326 was analysed on the expression levels of SMAD3 mRNA and protein;miR-326 mimic decreased SMAD3 expression in HCCLM3 cells,whereas anti-326 increased SMAD3 expression in HepG2 cells.5.3 SNHG3 promotes the expression of SMAD3 through sponging miR-326The SMAD3 mRNA level was elevated by SNHG3 overexpression,which was reduced by miR-326 mimic.SNHG3 knockdown decreased the SMAD3 mRNA level,which was reversed by anti-326.6.SNHG3 may function via SMAD/ZEB1 signaling pathway6.1 The expression levels of ZEB1The expression levels of ZEB1 mRNAs were significantly higher in in human HCC tissues compared with the matched adjacent noncancerous tissues.The expression levels of ZEB1 in HCC cell lines were higher than in normal hepatocyte cells(L02)in mRNA and protein levels.6.2 The regulation of ZEB1 by SNHG3The expression levels of ZEB1 mRNA and protein were increased by SNHG3 overexpression in HepG2 cells.The expression levels of ZEB1 mRNA and protein were decreased by SNHG3 knockdown in HCCLM3 cells.6.3 SNHG3 functions via ZEB1Si-ZEB1 resumed the effects of SNHG3 overexpression on proliferation and EMT in HepG2 cells.The abilities of proliferation and EMT were restored when we overexpressed ZEB 1 after silencing SNHG3 in HCCLM3 cells.6.4 SNHG3 works through SMAD3/ZEB1 signaling pathway in vivoThe results showed that SNHG3 knockdown significantly inhibited tumor growth in mice orthotopic tumor model.SNHG3 knockdown alleviated the staining intensity of proliferating cell nuclear antigen Ki-67.In addition,Tunel staining demonstrated that silencing SNHG3 led to increased apoptosis.Western blot showed that SNHG3 depletion reduced SMAD3,ZEB1,Vimentin,and Snail protein levels and increased E-cadherin levels.ConclusionIn this experiment,we found that the expression levels of SNHG3 in peripheral blood mononuclear cells of patients with hepatocellular carcinoma was significantly higher than that of liver cirrhosis,chronic hepatitis B and healthy controls.SNHG3 expression was elevated in both 47 tissue specimens of human HCC and hepatocellular carcinoma cell lines,and the expression levels of SNHG3 in human HCC tissues were related to TNM stage,lymph node metastasis,and distant metastasis.SNHG3 and miR-326 had the same binding sequence and could bind to each other.The expression levels of miR-326 were reduced and had a negative correlation with the expression levels of SNHG3 in human HCC tissues.At the same time,the expression levels of miR-326 in HCC cell lines were also reduced.SNHG3 could promote the proliferation,migration,EMT and inhibit apoptosis of HCC cells,and miR-326 inhibits the biological functions of SNHG3.SMAD3 is the targeted mRNA of miR-326 which is involved in the regulation of SMAD3 by SNHG3.SNHG3 regulates ZEB1,a downstream gene of SMAD3,to affect the biological functions of HCC cells.SNHG3 functioned as a competing endogenous RNA(ceRNA)for miR-326 to promote HCC progression via the SMAD3/ZEB1 signaling pathway.The findings may provide novel targets for the diagnosis and treatment of HCC.Part ?:Long non-coding RNA SNHG3 Affects the Prognosis of Acute-On-Chronic Hepatitis B Liver Failure through NLRP3BackgroudAcute-on-chronic hepatitis B liver failure(ACHBLF)refers to the rapid deterioration of liver function in a short period of time due to certain acute injuries during the progression of chronic hepatitis B.Jaundice and coagulopathy are the main clinical manifestations,and ascites and/or hepatic encephalopathy appear within four weeks.ACHBLF can occur at any stage of chronic HBV infection and progresses rapidly.Due to the lack of effective clinical treatments,the mortality rate of ACHBLF is as high as 50%-90%.At present,there are more than 20 international methods for assessing the prognosis of liver failure,of which the end-stage liver disease score(Model for end-stage liver disease,MELD)is the most commonly used method for evaluating the prognosis of liver failure.Due to differences in factors,MELD is not ideal for evaluating the prognosis of ACHBLF.Therefore,it is of great significance to find a more effective ACHBLF prognostic evaluation method.ACHBLF undergoes triple blows at different times,including immune injury,ischemic hypoxic injury and endotoxemia.In the course of liver failure,inflammatory factor-mediated immune injury plays an important role.Cytokine imbalance is gaining more and more attention as a known pathological mechanism of ACHBLF.Due to HBV infection and cytotoxicity-mediated immune disorders,it further promotes the progress of ACHBLF.In addition,adrenal insufficiency will occur during the development of liver failure,so the early use of glucocorticoids has become one of the methods for the treatment of early chronic liver failure with acute hepatitis B.Glucocorticoids can prevent liver cell necrosis and progressive acute deterioration by protecting cell membranes,lysosomal enzymes and mitochondria.Glucocorticoids are a double-edged sword,and correct application can greatly improve the survival rate of patients.However,whether glucocorticoid use affects cytokine imbalances in patients with ACHBLF has not been studied.In previous studies,we confirmed that lncRNA SNHG3 and miR-326 can promote hepatocellular carcinoma progression through SMAD3 signaling pathway in hepatocellular carcinoma.However,miR-326 can also act as a key proinflammatory factor through nuclear factor kappa(nuclear factor kappa)-B,NF-?B)signaling pathway is involved in various inflammatory processes.NF-?B can regulate the expression of NLRP3 as an important transcription factor.NLRP3(Nucleotide-binding oligomerisation domain-like receptors(NLRs)Family Pyrin Domain Containing 3)belongs to the NLRs protein family.After activation,it activates caspase-1 to make the cytokine interleukin 1? precursor(pro-interleukin(IL)-1?,pro-IL-1?)and interleukin 18 precursors(pro-interleukin(IL)-18,pro-IL-18)become active IL-1? and IL-18.IL-1? and IL-18 are important pro-inflammatory molecules,and their dysregulation can cause cytokine imbalance.At present,the expression status of lncRNA SNHG3 and miR-326 in ACHBLF and the relationship between NLRP3 and ACHBLF are unclear.Therefore,this study first detected the expression levels of SNHG3 and miR-326 in patients with ACHBLF,and then detected the expression status of pro-inflammatory cytokines IL-1?,IL-18 and NLRP3,and then analyzed glucocorticoids for IL-1?,IL-18 and NLRP3 expression and the relationship with prognosis.Objective1.To determine the expression of SNHG3 and miR-326 in peripheral blood mononuclear cells from patients with ACHBLF,CHB and HCs.2.To define the expression of pro-inflammatory cytokines in plasma and peripheral blood mononuclear cells from patients with ACHBLF,CHB and HCs3.To clarify the change of NLRP3 expression and its correlation with clinical data.4.To study the effects of glucocorticoids on NLRP3 and its related cytokines.Method1.Use qRT-PCR to detect the expression of SNHG3 and miR-326 in peripheral blood mononuclear cells from patients with ACHBLF,CHB and HCs.2.ELISA and qRT-PCR were used to detect the expression of proinflammatory cytokines in plasma and peripheral blood mononuclear cells of patients with ACHBLF,CHB and HCs3.qRT-PCR was used to detect the expression of NLRP3 in peripheral blood mononuclear cells from patients with ACHBLF,CHB and HCs.Spearman linear correlation was used to analyze the relationship between NLRP3 mRNA levels and related clinical indicators.4.The expression levels of NLRP3 and its related genes in the survival and death groups during glucocorticoid treatment was detected by qRT-PCR.Results1.The expression of SNHG3 in peripheral blood mononuclear cells of patients with ACHBLF is significantly lower than that of patients with CHB and HCs;the expression of miR-326 in peripheral blood mononuclear cells of patients with ACHBLF is significantly higher than that of patients with CHB and HCs.2.The expression of IL-1? and IL-18 in plasma is the largest among various pro-inflammatory cytokines.The expression of IL-1? and IL-18 in peripheral blood mononuclear cells of patients with ACHBLF is significantly higher than that of CHB and HCs patients.3.The expression of NLRP3 mRNA in peripheral blood mononuclear cells of patients with ACHBLF was significantly higher than that of patients with CHB and HCs,and was positively correlated with total bilirubin(TBIL)and MELD score(model for end-stage liver disease),and Prothrombin activity(PTA),albumin(Albumin,ALB)are negatively correlated with Alanine aminotransferase(ALT),Aspartate aminotransferase(AST),Creatinine(Cr)no clearly relevant.4.On the 7th and 28th days after receiving glucocorticoid treatment,the expression levels of NLRP3,caspase-1,IL-1? and IL-18 in peripheral blood mononuclear cells in the survival group were significantly lower than those in the death group.On the 7th and 28th days of glucocorticoid treatment,TBIL and PTA in the survival group were also significantly lower than those in the death group.ConlusionMiR-326 regulates NLRP3 in the ACHBLF through the NF-?B signaling pathway.NLRP3 expression was increased in patients with ACHBLF and was positively correlated with the clinical severity of the patients.Glucocorticoids can affect patient prognosis by regulating NLRP3. |
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