Expression status and clinical significance of lncRNA APPAT in the progression of atherosclerosis

Background Long non-coding RNAs (lncRNAs) have been reported to modulate cardiovascular diseases, and expression dynamics of lncRNAs in the bloodstream were proposed to be potential biomarkers for clinical diagnosis. However, few cardiovascular diseases-related circulating lncRNAs were identified and their prediction power has not been investigated in depth. Here we report a new circulating lncRNA, atherosclerotic plaque pathogenesis associated transcript (APPAT), and evaluated its role and predicting ability in atherosclerotic development. Methods APPAT was analyzed and screened by high-throughput sequencing, and then detected in vitro and in vivo. Immunofluorescence-fluorescence in situ hybridization (IF-FISH) was utilized to explore distribution and subcellular location of APPAT. The expressing alteration of APPAT in samples of healthy and pathological coronary artery was explored further. We also assessed the level of circulating APPAT in blood samples from healthy individuals, and patients with angina pectoris (AP) or myocardial infarction (MI). Additionally, we predicted and validated microRNA targets of APPAT, then showed the expression level of a candidate target which was primarily measured in human VSMCs cell line, coronary artery, and blood samples. Lastly, we examined the potential indicating ability of APPAT for the risk of AP or MI. Results APPAT showed significant reduction in ox-LDL treated human VSMCs in vitro. It enriched in contractile VSMCs of artery tunica media and mainly existed in cytoplasm. Significant down-regulation of APPAT was found in coronary artery samples with severe stenosis. More importantly, we observed decreased expression of APPAT in blood samples accompanying disease progression. ROC and correlation analyses further verified the relatively high predicting ability of APPAT. We also observed the predicted miRNA exhibited opposite expression direction to that of APPAT. Conclusions This study revealed that circulating lncRNA-APPAT may perform an important function and have some indicating ability on the development of atherosclerosis.

109 LncRNA identification and expression 110 All successfully assembled reads were combined by Cuffcompare software. Then qualified 111 transcripts (≥200 bp, or ≥2 exons, ≥3 reads coverage) were screened out. Comparison 112 between transcripts and reference rabbit lncRNAs was performed by Cuffcompare software. 113 RNA transcripts like tRNA, rRNA, snoRNA, snRNA, pre-miRNA, and pseudogenes were also 114 detected and discarded. Software CNCI, CPC, PFAM, and phyloCSF were used to assessing the  The expression levels of lncRNAs in each sample were evaluated using Cuffdiff (v2.1.1). 118 Fragments per kilo-base of exon per million fragments (FPKM) for expressing evaluation 119 followed the previous method (Trapnell et al. 2010). The threshold of "P <0.05, FC (fold 120 change) >2 or <0.5, and False discovery rate (FDR) <0.05" was used to judge the significance of 121 gene expression differences between control and ox-LDL-treated group. FDR was generated by 122 Cuffdiff.  160 Prediction and primary validation of miRNA targets for APPAT 161 The pictar, miRDB, lncBase and mirna22 online databases were used for predicting 162 miRNAs targets of APPAT. The intersection of these predicting results assemblages was 163 calculated. Then candidate miRNAs were primarily selected as which were at least included in 164 three databases. And further screening work was based on an integrated consideration of hybrid 165 ability, number of biding site, sequence identity, and research background of them. The 166 expressing change of miRNAs in human VSMCs was detected after ox-LDL treatment.  The high-throughput RNA-seq technique was used to analyze the expression of lncRNAs in 179 ox-LDL treated VSMCs of rabbit. A total of 42,533,007 clean reads were generated after 180 discarded those reads with poly-N >10%, adapters, or any other type of contaminants. All clean 181 reads were mapped to the reference genome of rabbit, and the final mapping rate was ranged 182 from 71.39 to 74.23% in all rabbit VSMCs samples. The Cufflinks results indicated that 147,153 183 transcripts were assembled at the first step.

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Then sequences were qualified and the rest transcripts were blasted with latest 185 transcriptome of rabbit lncRNAs as well as known types of RNAs. 3630 presumed lncRNAs 186 transcripts were detected and all of these lncRNAs were recognized as novel sequence without 187 any report before. After protein-coding predicting analyses, 2037 novel lncRNAs were identified 188 with no protein-coding ability for following analysis (Fig. 1A).

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The expression level of lncRNA transcripts were estimated, and a total of 28 lncRNA 190 transcripts differentially expressed in ox-LDL treated rabbit VSMCs group comparing to control 191 group, including 17 up-regulated transcripts and 11 down-regulated (FDR <0.05, P <0.05).
195 Selection and validation of lncRNAs from rabbit and human 196 2 up-regulated (TCONS_02288701, TCONS_02225105) and 2 down-regulated 197 (TCONS_00489746, TCONS_02443383) lncRNAs were selected for validation of RNA 198 sequencing data using qPCR. All of these selected lncRNA transcripts were successfully 199 amplified using designed primers (Tabel S2) and exhibited statistically differential expression 200 between ox-LDL-treated and control groups (n=3, P <0.01) (Fig. 1B). The expression pattern of 201 these 4 lncRNA were consistent with their RNA sequencing data.

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These 4 selected rabbit lncRNAs sequences were chosen and compared with human 203 sequence data on the NCBI website. Within the blast result, human lncRNAs with relatively high 204 alignment identity were selected and amplified from total RNA production of human VSMCs 205 using designed primer respectively (Table S3). Finally, ENST00000620272, corresponding to 206 rabbit lncRNA transcript TCONS_00489746, was successfully amplified from human VSMCs 207 and prepared for the subsequent study. ENST00000620272, renamed as APPAT, was a long 208 intergenic non-protein coding RNA, which firstly generated from sequencing data of human 209 blood sample. The protein-coding potential of APPAT was assessed using PhyloCSF and gained 210 a score <20 which met the threshold as lncRNA (Lin et al. 2011;Pauli et al. 2012).

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The expression level of APPAT was checked by qPCR. A similar expressing trend was 212 found between APPAT and TCONS_00489746. The former showed significantly decrease (n=4, 213 P <0.01) in ox-LDL treated group compared with control group in human VSMCs (Fig 1C).
214 Distribution and location of APPAT in tissue and subcellular level 215 Since VSMCs was the major constituent of tunica media of artery, and APPAT could be 216 successfully amplified from cultured VSMCs, samples of coronary artery was collected to 217 determine the distribution and subcellular location of APPAT. We utilized designed 5'Cy3-probe 218 for RNA IF-FISH to investigate visuospatial information of APPAT within human coronary 219 artery (Table S4). On the IF-FISH images of coronary artery slides, APPAT intriguingly located 220 in VSMCs of tunica media of coronary artery but no obviously stain in tunica intima and 221 adventitia (Fig 2A, B). By contrast, the atherosclerotic plaque area on slide exhibited negative 222 fluorescent signals of APPAT probe as the α-SMA probe did (Fig. 2B). APPAT and α-SMA 223 exhibited similarly distributing and co-expression pattern. Furthermore, it could be distinguished 224 under high magnification that APPAT was mainly localized in the cytoplasm but not the nuclear 225 area (Fig. 2C).  (Fig. 3A). Samples of coronary artery with Ⅲ-Ⅳ stenosis (Fig. 3B) 231 were selected as pathology group after forensic pathological diagnose (J.Chen & Zhou 2015).

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A statistically significant down-regulation was detected in the arteriostenosis group (P 233 <0.01) (Fig. 3C). The sharp decline of APPAT in arteriostenosis group seemed identical with the 234 severity of pathological artery stenosis.
235 Detection of APPAT in circulating blood of patients with coronary artery disease 236 For patients characteristics, see Table 1. The level of APPAT in extracellular circulating 237 blood was investigated for whether it was detectable in blood sample, as well as existence of any 238 dysregulation in atherosclerosis pathophysiologic process. Total RNA production was extracted 239 and successfully amplified in all three groups. An obviously decreasing tendency of circulating 240 APPAT was found from normal, to AP group, then to MI group (Fig. 3D). The level of APPAT 241 was slightly decreased in AP group without statistical significance, but sharply declined in the 242 MI group. This result exhibited the down-regulation of APPAT was identical with the 243 pathological progress of MI, since AP was usually treated as the early stage of coronary heart 244 disease (P <0.01).
245 The effect of diabetes and hypertension on APPAT level in circulating blood 246 The clinical diagnosis of hypertension and diabetes of those patients with MI were 247 investigated. Patient with MI was further classified as MI (none of hypertension or diabetes) 248 group with MI+hypertension group and MI+diabetes group respectively. No significant 249 differences of APPAT level was found between MI (n=23) and MI+hypertension group (n=24) 250 (P >0.05) (Fig. S1A). And similar situation found in MI (n=13) and Mi+diabetes group (n=34) 251 (P >0.05) (Fig. S1B).  Manuscript to be reviewed 271 and calculated the intersection of these predicting results assemblages. Then candidate miRNAs 272 were primarily selected as which were at least included in three databases (Fig. S2). And further 273 screening work was based on an integrated consideration of hybrid ability, number of biding site, 274 sequence identity, even the research background of them. Three miRNAs, miR-135b, miR-647 275 and miR-1229 were selected as candidate targets of APPAT (Table S5)  The expression level of miR-647 in samples with stenosis (n=28) was almost three folds 283 than that of normal group (n=21) (Fig. 5B). Furthermore, a statistically significant up-regulation 284 of circulating miR-647 was found in the MI group (n=47) compared with the normal subjects 285 (n=43) (Fig. 5C). The altering tendency was coincident with the founding of coronary artery 286 sample.

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The correlation analyses between level of circulating miR-647 and circulating APPAT in 288 blood samples of myocardial infraction group was calculated. The level of circulating APPAT 289 was negatively correlated with that of circulating miR-647(r=-0.7908, P <0.01) (Fig. 5D).