| BackgroundAlzheimer’s disease (AD), the most common cause of dementia in the elderly, is a progressive and fatal neurodegenerative disorder. The disease is a global dilemma, with over 30 million patients worldwide and an economic burden exceeding half a trillion USD. Epidemiological studies suggest that 11% of those aged 65 years and older, and almost one third of those 85 and older, have some form of the disease. Currently, it affects approximately 10.6 million people in the USA and Europe, with projected estimates reaching epidemic proportions (15.4 million) by the year 2030.Alzheimer’s disease, which is characterized by progressive loss of synapses and neurons in the hippocampus and cortex, is associated with gradual decline inshort-term memory and cognitive functions. In molecular levels, AD is characterized by deposition of insoluble amyloid beta peptide to form plaques out of neuron and aggregation of highly phosphorylated Tau protein to form neurofibrillary tangles. AD is a serious public health challenge. However, current therapeutic strategies are very limited for AD patients and do not modify disease course. Therefore, investigation of new therapeutic targets that address multiple different facets of the AD phenotype and related pathophysiology must be actively sought to help to address this problem.Advances in genome-wide analysis of the eukaryotic transcriptome have revealed that up to 90% of the human genome are transcribed, however only 1-2% of those transcripts encode for proteins (～20,000 protein encoding genes). The vast majority of the genome, previously regarded as "junk DNA" or"transcriptional noise", was transcribed largely as non-protein-coding RNA (non coding RNA, ncRNA). Noncoding RNA were divided into two classes based on nucleotide length-small RNA and long non-coding RNA. LncRNA was defined as noncoding RNA molecules greater than 200 nt, and lacked any protein-coding potential. LncRNA was mostly transcribed by RNA polymerase II, and are often capped, poly-adenylated, and spliced. Recent studies revealed that lncRNA-related dysfunction had been found to play critical roles in various diseases, and neurodegenerative diseases were becoming increasingly evident. For example, M.A. Faghihi et al found that BACE1-antisense transcript (BACE1-AS) had extensive complementarity with BACE1 mRNA and enhanced BACE1 mRNA stability by protecting it from degradation by miR-485-5p. BACE1 protein levels and activity increased with brain aging; in AD, high BACE1 levels correlated with elevated BACE1-AS abundance, suggesting that BACE1-AS could play a role in AD development or progression. Also highly abundant in individuals with AD, lncRNA-17A was associated with a defect in the alternative splicing of GABABR2 (y-aminobutyric acid B receptor 2). S. Massone et al revealed that overexpression of lncRNA-17A repressed GABABR2 variant A, promoted expression of variant B, and favored the accumulation of peptides Aβ42 and Aβ40, which derived from cleavage of the amyloid precursor protein (APP) and closely implicated in AD pathogenesis. These findings indicated that lncRNA-17A may play a role in GABA signaling and Aβ production. Also in AD, E. Mus et al found that BCYRN1 decreased by more than 60% in cortical areas between the ages of 49 and 86, but upregulated in AD brains compared to age-matched normal brains. However, lncRNAs studies are still at a relatively early stage. Their characteristics, conservation, functions, and action mechanisms remain to be further studied.With the development of gene chip technology, rapid and high sensitivity detection of expression changes of low abundance RNA came true. Our research group tested both lncRNA and mRNA in normal cells and AD model cells with Arraystar LncRNA chip technology respectively, the expression profiles analysis showed:compared to normal cells, there are 2650 differentially expressed lncRNAs, of which 997 up-regulated,1653 down-regulated; and 1209 differentially expressed mRNAs, of which 606 up-regulated,603 down-regulated in AD model cells. Then we used quantitative PCR to selectively validate 9 lncRNAs named RP11-642D21.2, ZBTB20-AS1, RP11-354P11.2, RP11-543N12.1, RP1-77H15.1, RP11-121G22.3, RP11-10O22.2, RP11-414H23.3, RP11-79E3.2 and 5 mRNAs named MAPT, PPFIA2, CDH13, ZFPM2, ZBTB20, consistent with the results of gene chip. Finally, we determined to further study the expression correlativity of lncRNA RP11-543N12.1 and CDH13 according to NCBI, lncRNA database and relevant literature.CDH13 codes for T-cadherin (also known as truncated, H-or heart cadherin), belongs to the cadherin superfamily, a group of genes encoding calcium-dependent cell adhesion molecules, with important roles in a wide range of tissues. The CDH13 gene was mapped to 16q24 in human chromosome, comprises 14 exons (two of them non-coding) and one promoter that is rich in CpG islands and can display a high degree of methylation, especially in various cancers. CDH13 is an atypical member of the cadherin family of cell adhesion molecules. Unlike classical cadherins, it lacks a transmembrane domain, is attached to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor and has low adhesive properties. T-cadherin was found first in the chick embryo brain tissue and subsequently was cloned through Polymerase Chain Reaction. A variety of studies have demonstrated the functional role of CDH13 in cell proliferation, the cell cycle, and epigenetic silencing in cancer, and CDH13 is now recognized as a tumor suppressor gene. The expression of CDH13 is downregulated through methylation in breast, ovarian, lung cancer and other cancers, so it is supposed as a potent predictor of poor prognosis in them. CDH13 is believed to suppress the development of neural cells and induce G2 arrest in astrocytomas via p21. Several studies showed that CDH13 has also been associated with some metabolic syndrome. There are little studies on neurodegenerative disorder according to current reports, this study may be the first to show that CDH13 was upregulated in AD cell model and explore the possible regularory effect of long noncoding RNA on CDH13 in Alzheimer’s disease.Objective:To explore the regulatory function of lncRNA RP11-543N12.1 to the expression of CDH13.Methods:1. Firstly, cell model of Alzheimer’s disease was constructed. Then, differentially expressed lncRNAs and mRNAs were screened through Microarray.2. Some significant differences lncRNA and mRNA were confirmed by Real-time PCR.3. Construction the expression vector of pcDNA3.1 (+)-RP 11-543N 12.1, then RP11-5 43N12.1-siRNA and pcDNA3.1(+)-RP11-543N12.1 were transfected into SH-SY5Y cells respectively. In order to detect the effect of knocking down RP11-543N12.1 in cells, SH-SY5Y cells were divided into six groups:MOCK, MOCK+Aβ25-35, negative control RNA, negative control RNA+Aβ25-35, RP11-543N12.1-siRNA, RP11-543 N12.1-siRNA+Aβ25-35. In order to detect the effect of overexpressing RP11-543N12.1 in cells, SH-SY5Y cells were divided into six groups: MOCK, MOCK+Aβ25-35, pcDNA3.1(+), pcDNA3.1(+)+Aβ25-35, pcDNA3.1(+)-RP11-543N-12.1, pcDNA3.1(+)-RP11-543N12.1+Aβ25-35. SH-SY5Y cells were cultured for 24 hours and then treated with Aβ25-35(20μmol/L) for 48 hours.4. The RNA expression of RP11-543N12.1 and CDH13 were detected by Real-time PCR, the proliferation of SH-SY5Y cells were detected by MTT method, the protein expression of CDH13 were valued by Western blotting, the nucleus morphological changes were detected by fluorescence microscope after staining with Hoechst33258 and the percentage of apoptosis was determined by flow cytometer.Results:1. In normal cultivation cells treated with Aβ25-35(20μmol/L) for 48 hours,the expression of total Tau and phosphorylated Tau(P-tau) were increased(P<0.01), indicating that cell model of Alzheimer’s disease was successfully constructed.2650 differentially expressed IncRNAs were screened through Microarray, in which 997 upregulated and 1653 downregulated; 1209 differentially expressed mRNAs were screened through Microarray, in which 606 upregulated and 603 downregulated.2.9 IncRNAs and 5 mRNAs were confirmed selectively, and the results had statistical significance(P<0.05), in accordance with Microarray. The expressional correlation of lncRNA RP11-543N12.1 and CDH13 were regarded as the objects for further study.3. Compared with control SH-SY5Ycells, in cells treated with Aβ25-35, the proliferation was inhibited(P<0.05), RNA level of CDH13 was upregulated(P<0.01), active form expression of CDH13 protein was upregulated(P<0.01) too, apoptotic nuclei were observed and cellular apoptosis rate was increased(P<0.05). In cells transfected with RP11-543N12.1-siRNA and treated with Aβ25-35 for 48 hours, cellular survival rate, RNA expression and protein active form expression of CDH13, and cellular apoptosis rate were all close to the normal levels. Nuclei apoptosis disappeared. In cells transfected with pcDNA3.1(+)-RP11-543N12.1 and treated with A025-35 for 48 hours, cellular survival rate was significantly reduced(P<0.01), RNA expression, protein active form expression of CDH13, and cellular apoptosis rate were all increased. The nucleus morphological changes were observed.Conclusion:LncRNA RP11-543N12.1 can regulate the expression of CDH13. Knock-down RP11-543N12.1 can downregulate the expression of CDH13 and overexpress RP11-543N12.1 can upregulate the expression of CDH13.That means the expressions of RP11-543N12.1 and CDH13 have positive correlation. |
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