Analysis And Functional Study On LncRNA Expression Profile Microarray In Primary Biliary Cirrhosis Patients

Long non-coding RNA （lncRNA） refers to nucleotide greater than 200nt. Initially thought not having any biological functions, but more and more studies confirmed that lncRNA, which can form a specific spatial structure, could regulate the expression of specific genes in the epigenetic level, transcription and post-transcription level, widely involved in the physiology and pathological processes. Many studies have found abnormal expression or sequence mutations of lncRNA in liver cancer, stomach cancer, and prostate cancer, which contributed to the proliferation of tumor invasion and metastasis, and may be used as an independent predictor in diagnosis, staging, and prognosis. LncRNA also play an important role in immune cells differentiation, innate and adaptive immune response. Lnc-DC specifically expressed in human dendritic cells （Dendritic cells, DC）. It promotes the differentiation of monocytes to DC, and enhances the activation ability of T cells in combination with the transcription factor STAT3. Many immune-associated genes locate on the X chromosome, such as TNF-a, CD40L.FOXP3. Long non-coding RNA Xist involved in X chromosome inactivation. Changes in the structure or function of Xist can lead to abnormal expression of immune-related genes on the X chromosome, which may partly explain the female predominance in multiple autoimmune diseases.Primary biliary cirrhosis is a kind of organ-specific autoimmune disease, characterized by the bile duct injury and cholestasis. Disease-specific anti-mitochondrial antibody （AMA）, accompanied with increased Alkaline phosphatase （ALP）, glutamyl endopeptidase （GGT） and other indicators can be found in the serum of those patients. It usually occurs in middle-aged women. With the popularization and application of autoantibodies detected in recent decades, the incidence is rising year by year. Infection, environmental factors, genetic factors and epigenetic factors are likely involved in the pathogenesis of PBC, but the exact role remains unclear.Research of lncRNA in PBC is relatively few in either peripheral blood or liver tissue. Our study of expression profile in PBMC of patients with PBC can provide new ideas for the pathogenesis of PBC, and may also provide a new way for the clinical diagnosis and treatment of PBC.Part One:Analysis and RT-PCR validation of lncRNA expression microarray in PBC patientsIn this section, we collected peripheral blood of 30 PBC patients and 30 healthy groups, separated PBMC. Four cases from each group were selected for lncRNA expression profiling microarray detection. RT-PCR technology in a larger sample size was used to verify the microarray results. Compared to the healthy group,749 lncRNA were found to be abnormal expression, of which 541 was up-regulated, and 208 down-regulated. Besides, the expression level of 230 coding genes were also different, of which 184 was up-regulated and 46 down-regulated. We selected six differentially expressed lncRNA, uc003xrr, ucOlOnhz, p1102447, NR₀26777, ENST00000442026, ENST00000431157, and expanded the sample size to verify the reliability of microarray results. Luckily, results were consistent.Part Two:Bioinformatics analysis of microarray resultsThis section we mainly conducted Cis-/Trans-target genes GO and pathway analysis, and built lncRNA and mRNA co-expression networks and analyzed cell function and signaling pathways involved in these abnormal expressed lncRNA, in order to provide a theoretical basis for further exploration of lncRNA in the pathogenesis of PBC. The results suggested those lncRNA target genes were mainly associated with apoptosis and metabolic pathways. NR4A nuclear receptor subfamily widely participate in various signal transduction pathways, such as MAKP, NF-KB, PI3K/JNK, Wnt, and also regulate apoptosis, lipid metabolism, angiogenesis, cell proliferation, cell migration, DNA damage repair and other biological processes. We found that the expression level of NR4A3 gene was 0.22-fold down-regulated, P value was 0.0082. However, the expression level of lncRNA LOC441461, which was about 20000 bp downstream of NR4A3 gene, increased 2.56-fold, P value was 0.0015. Integrating results from three different databases, we confirmed LOC441461 as a non-coding RNA. Then, We predicted the interactive possibility of LOC441461 and PRC2 complex using CatRapid online software, and found that PRC2 complexes may interact with LOC441461 in the 439-554 of the nucleotide sequence. Interestingly, the Discriminative Power reached 94% in the location at 451-552, indicating very likely interactions between them. Therefore, we suspect that by recruiting PRC2 complex, LOC441461 increased the methylation level of NR4A3 gene promoter region, thus reduced the expression of NR4A3 in PBC patients and triggered a series of biological effects, and promoted the development of the disease.Part Three:Possible regulatory mechanism and function of linc-pbcThis part we examined linc-pbc and NR4A3 expression levels in PBC group, disease control group and the healthy control group, and found that both gene expression levels were consistent with the microarray results in the PBC group, P<0.001. Linc-pbc and NR4A3 showed a similar expression pattern to PBC in SLE patients, whereas no significant difference was found in HBV patients compared to the control group. We designed siRNA for linc-pbc to see if there is any change in the expression of NR4A3 and FOXP3 and cell apoptosis in transfected PBMC. After transfection, NR4A3 mRNA levels and protein levels were up-regulated, as well as FOXP3, indicating that linc-pbc may bind to PRC2 complex, and inhibit NR4A3 expression. Reduced NR4A3 futher downregulated FOXP3 expression and resulted in the reduction of immunosuppressive Treg cells in peripheral blood and liver tissue, and broke the equilibrium state of immune tolerance, and caused the disease. On the other hand, we noticed that after transfected with siRNA, apoptosis did not change significantly to the negative control group. One possible exploration was that these PBMC were isolated from healthy people, in whom damage repair mechanism may exist, and a single interference in linc-pbc may increase NR4A3 expression, but probably not enough to cause significant apoptosis. Or, NR4A3 only promoted apoptosis of auto-reactive lymphocytes.Conclusions:Compared to the healthy group,749 lncRNA were found to be abnormal expression in PBC patients, of which 541 was up-regulated, and 208 down-regulated. Besides, the expression level of 230 coding genes were also different, of which 184 was up-regulated and 46 down-regulated. The expression level of NR4A3 gene was 0.22-fold down-regulated, P value was 0.0082. However, the expression level of lncRNA LOC441461, which was about 20000 bp upstream of NR4A3 gene, increased 2.56-fold, P value was 0.0015. we hypothesized that by recruiting PRC2 complex, LOC441461 increased the methylation level of NR4A3 gene promoter region, thus decreased the expression of NR4A3 in PBC patients. Reduced NR4A3 futher downregulated FOXP3 expression and led to the reduction of immunosuppressive Treg cells in peripheral blood and liver tissue, breaking the equilibrium state of immune tolerance, thus promoted the occurrence and development of disease.