Expression profile of lncRNA induced by DPP4 in human aortic smooth muscle cells and calcification gene prediction

doi:10.21203/rs.3.rs-1656423/v1

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Expression profile of lncRNA induced by DPP4 in human aortic smooth muscle cells and calcification gene prediction

https://doi.org/10.21203/rs.3.rs-1656423/v1

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Arterial media calcification caused by diabetes is an important cause of cardiovascular calcification. Dipeptidyl peptidase-4 (DPP4) is associated with diabetic arterial media calcification. While, long non-coding RNA(lncRNA) is closely related to the cardiovascular diseases and calcification caused by diabetes. Experiment have shown that LncRNA can regulate DPP4 activation to modulate senescence and lncRNA ENST00000540293 may associated with human periodontal ligament cells development. our previous studies showed that DPP4 can promote the calcification of human aortic smooth muscle cells (HASMCs) by activating the ERK/NF-kB pathway, but it is unknown whether it is related to the regulation of lncRNA. Calcification of HASMCs was induced by DPP4. There was a significant difference in the expression of lncRNAs and mRNAs between normal and calcified cells detected by gene chip technology. Based on the results of microarray detection, we found that lncRNA ENST00000540293 may be involved in vascular calcification induced by DPP4 through regulating target genes.

Dipeptidyl peptidase 4

Vascular Calcification

RNA Chip

Long non-coding RNA

Vascular calcification is a common pathological phenomenon in coronary atherosclerotic heart disease, chronic kidney disease, diabetes and other diseases¹, Its pathological basis is an expression of osteoblast-specific genes in smooth muscle cells². Previous studies have shown that vascular calcification is a very complex pathological process, which is regulated by many factors. Recent studies have found that vascular calcification is an adjustable and active biological process, which is similar to bone formation and involves the expression of some bone formation related proteins, such as alkaline phosphatase (ALP), osteocalcin (OCN), Runx2 and BMP-2³. Vascular calcification is common in patients with diabetes, and the important pathological change of vascular complications is intima-media calcification. Medial calcification and cardiovascular disease caused by diabetes are one of the main causes of death. Vascular calcification is closely related to the high incidence and mortality of cardiovascular disease. Therefore, it is of great significance to study the treatment and intervention of diabetic vascular calcification.

Dipeptidyl peptidase 4 (DPP4) is a highly glycosylated type II transmembrane protein composed of 766 amino acids, which belongs to the family of serine proteases. One of its catalytic substrates is incretin. The treatment of type 2 diabetes with DPP4 inhibitors (Letins) establishes the role of incretin in blood glucose control. Low molecular weight DPP4 inhibitors exert their hypoglycemic effect by promoting glucose-dependent insulinotropic polypeptide (GIP) and inhibiting the degradation of glucagon-like peptide 1 (GLP-1). DPP4 is widely expressed in a variety of cells, such as endothelial cells and immune cells. DPP4 also plays an important role in the regulation of cardiovascular function⁴. Meanwhile, DPP4 not only plays an important role in reducing blood sugar, but also has unique functions in other aspects⁵. Nina Wronkowitz et al demonstrated that soluble DPP4 induces inflammation and proliferation of human smooth muscle cells via protease-activated receptor 2⁶. Yu et al demonstrated that soluble DPP4 can induce oxidative stress in human umbilical vein endothelial cells through mannose 6-phosphate/insulin-like growth factor receptor II⁷. A.M.Lambeir et al found that the expression of DPP4 in visceral tissues of obese and diabetic patients was higher than normal people⁸. Studies have shown that DPP4 and its inhibitors are closely related to vascular calcification⁹. DPP4 may lead to vascular calcification¹⁰. While,DPP4 inhibitors may lowered the levels of fasting serum glucose, triglyceride (TG),TNF-α and decreased the calcium deposits¹¹, and also attenuated vascular calcification and osteogenic trans-differentiation in vascular smooth muscle cells via multiple steps, such as: downregulation of PiT-1 expression and suppression of reactive oxygen species generation, phospho-PI3K/AKT, and the Wnt signaling pathway¹².The above data suggest that diabetes and its concomitant diseases may be closely related to the regulation of DPP4.

Long non-coding RNA (lncRNA) is a functional RNA molecule with a length of more than 200bp in non-coding RNA¹³. LncRNA is located in the cytoplasm or nucleus. They regulate gene expression through epigenetic, transcriptional and post-transcriptional regulation in the form of RNA, but lack the ability of protein coding. It has been found that lncRNA plays an important role in regulating cell growth, development, distribution and targeted differentiation¹⁴. Intergenic lncRNA(lincRNA) and antisense lncRNA are one of the hotspots in the current research. LincRNA ENST00000540293 is an intergenic noncoding RNA located on chromosome 11 and contains 1000 bases, and may associated with human periodontal ligament cells development¹⁵. Our RNA microarray results show that the lncRNA ENST00000540293 nearby gene is NM-002421, and the protein encoded by NM-002421 is matrix metalloproteinase 1 (MMP-1) (Table 1)¹⁶. Previous studies have shown that matrix metalloproteinases are closely related to the occurrence and development of vascular calcification^17,18. Our preliminary experiments in vitro experiments showed that DPP4 can promote the calcification of HASMCs by activating the ERK/NF-kB pathway, and the DPP4 inhibitors (sitagliptin) can inhibit ERK1/2 and NF-kB signaling pathway so that inhibits aortic vascular calcification in type 2 diabetic mice also confirmed in vivo. Although DPP4 is closely related to cardiovascular disease, the exact mechanism of DPP4 promoting HASMCs calcification is not completely clear¹¹. Therefore, in this study, HASMCs were cultured with DPP4 to establish calcification model, so as to study the mechanism of DPP4 promoting osteogenic differentiation of vascular smooth muscle cells.

2.1 The culture of HASMCs

The primary human aortic vascular smooth muscle cell (HASMCs,no.6110) and culture medium (no.1101)were obtained from Science Cell Research-Laboratory (USA). SMCM medium was composed of 500ml basic medium, 10ml fetal bovine serum (FBS, no.0010), 5ml smooth muscle cell growth factor (SMCGS, no.1152) and 5ml penicillin / streptomycin (Pmax S, no.0503).The 3rd to 8th generation cells were selected for the experiment and cultured in an incubator at 37℃with 5% humidified CO2.

2.2 Determination of intracellular calcium concentration

The cell intracellular calcium concentration was determined using a spectrophotometer at 570 nm. Transferring 800ul of the buffer to another 1 ml cuvette and adding 100 ul of the reaction solution by pipette. They were evenly mixed and incubated at room temperature for 5 minutes. Taking 100 μl mixture liquid in 96-well plates with 3-6 multiple holes in each group. The standard curve was plotted with the optical density (OD) on the y axis and the standard calcium concentration (mmol/l) on the x axis and get the calcium concentration.

2.3 Western blotting

The levels of the all target proteins was measured by the fluorescence imaging system Image Studio, and the expression level of protein was quantified by measuring the greyscale of bands and normalized to β-actin. Briefly,the concentration of protein was detected using a BCA kit (Beyotime Biotechnology, Shanghai,China). Then, Approximately 20μg of protein was loaded onto a 10% SDS-PAGE gel and blotted onto PVDF membrane.After blocking with 5% BSA, the membranes were incubated with primary antibodies at 4°C overnight. The membranes were washed thrice by TBST and incubated with secondary antibodies for 1 h at room temperature.Such as Osteoprotegerin (OPG), Bone Morphogenetic Protein 2 (BMP-2), Osteopontin (OPN), Runt Related Transcription Factor 2 (RUNX2), α Smooth Muscle actin (α-SMA), Matrix Metalloproteinase 1 (MMP-1) were detected by western blotting with their respective antibodies(anti-OPG,OPN,BMP-2 ,MMP-1 all from Abcam; anti-α-SMA from R&D Systems;anti-RUNX2 from Cell Signaling Technology).

2.4 Alizarin red staining for Calcification Detection

Cell calcification nodules were detected by Alizarin red kit（Sigma USA)）.Control group and experimental group cells were inoculated on 12-well plates. Cleaning solution was added to each well to clean the growing cell surface, fixed solution was added and incubated at room temperature for 15 minutes, then staining solution was added and incubated at room temperature for 5 minutes or until orange-red was visible. The staining solution was removed and dried in air at room temperature for about 10 minutes. Then transparent solution was added and immediately observed under light microscope: the positive cells of calcium deposition were orange-red.

2.5 F-actin staining

The HASMCs (2×10⁵ cells/well) were inoculated on a 6-well plate overnight and then treated with the tested compound for 72 hours. Cells were washed with PBS and fixed with 4% Formaldehyde, Permeabilized in 0.2% Triton X-100 at room temperature for 5 minutes, then stained with DAPI (Beyotime,USA) and Rhodamine Phalloidin (1:200,Sigma,MO,USA) to mark the cyteblast and cytoskeleton respectively. Pictures were acquired by Photometrics CoolSNAPHQ2 CCD camera on the Olympus IX71-Applied Precision Delta Vision restoration microscope (Applied Precision, USA) and deconvolved using Delta Vision algorithms (Applied Precision, USA).

2.6 Detection of intracellular ALP activity

For ALP activity measurement,the calcified HASMCs were solubilized with RIPA lysis buffer (Beyotime Biotechnology, Shanghai,China). After centrifugation, the supernatants were examined with the ALP activity kit (Beyotime Biotechnology,Shanghai,China) .The optical density (OD) value of each group was determined by 450nm wavelength. The standard curve was made and the corresponding ALP activity of each group was calculated.

2.7 RNA extraction and real-time quantitative polymerase chain reaction.

Total RNA was extracted using TRI zol reagent (Invitrogen;Thermo Fisher Scientific, Inc). RNA quantity and quality were measured by Nano Drop ND-1000. RNA integrity was assessed by standard denaturing agarose gel electrophoresis. Based on the predicted consequences, we selected the target linRNA with the most expression change between the control group and DPP4 intervened group and designed PCR primers (Table 2). RT‑qPCR was performed using SYBR Green Real‑Time PCR Master mix (Invitrogen;Thermo Fisher Scientific, Inc),and was run at 95˚C for 5 min, followed by 40 cycles at 95˚Cfor 10 sec, 65˚C for 20 sec and 72˚C for 30 sec. The characteristics of the linRNAs and mRNAs are presented in Table 1 and Table 3. Data were normalized to β-actin and the relative level of gene expression was calculated using the 2^-ΔΔCtmethod.

2.8 Microarray

Arraystar Human LncRNA Microarray V4.0 is designed for the global profiling of human lncRNAs and protein-coding transcripts, which is updated from the previous Microarray V3.0. About 40,173 lncRNAs and 20,730 coding transcripts can be detected by our third-generation lncRNA microarray.

2.9 RNA labeling and array hybridization

Sample labeling and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology) with minor modifications. Briefly, mRNA was purified from total RNA after removal of rRNA (mRNA-ONLY Eukaryotic mRNA Isolation Kit, Epicentre). Then, each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3' bias utilizing a random priming method (Arraystar Flash RNA Labeling Kit, Arraystar). The labeled cRNAs were purified by RNeasy Mini Kit (Qiagen). The concentration and specific activity of the labeled cRNAs (Pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000. 1 μg of each labeled cRNA was fragmented by adding 5 μl 10 × Blocking Agent and 1 μl of 25 × Fragmentation Buffer, then heated the mixture at 60°C for 30 min, finally 25 μl 2 × GE Hybridization buffer was added to dilute the labeled cRNA. 50 μl of hybridization solution was dispensed into the gasket slide and assembled to the LncRNA expression microarray slide. The slides were incubated for 17 hours at 65°C in an Agilent Hybridization Oven. The hybridized arrays were washed, fixed and scanned with using the Agilent DNA Microarray Scanner (part number G2505C).

2.10 LncRNA transient transfection.

VSMCs were interference with 50 nm lncRNA ENST00000540293 Smart Silencer (siRNA ) and negative control (NC-siRNA) using Smart Silencer NC at a density of 4 x10⁵ cells for 48 hours, according to the manufacturer's protocol¹⁹. The inhibitory efficiency was tested by real-time qPCR, and the effective siRNA was picked for the further experiments. The lncRNA ENST00000540293 siRNA and NC-siRNA were synthesized by RiboBioCo (Guangzhou, China).

Statistical analysis.

Data are presented as the mean ± standard deviation (n=3) and were analyzed using a Student's t-test. All statistical analysis was performed with SPSS 20.0 (IBM Corp, Armonk, NY, USA). P value < 0.05 was accepted as significant.

1.Effect of DPP4 on HASMCs calcification protein

The 5rd to 7th generation HASMCs were cultured with 200ng/ml DPP4 for 7 days. The total cell protein was extracted and the expression of OPG, OPN, BMP-2 and RUNX2 was detected by western blotting. The expression of OPG, OPN, BMP-2 and RUNX2 protein in DPP4 group was significantly higher than control group (Fig.1A). The results confirm that DPP4 can induce HASMCs calcification.

2.Calcification of HASMCs induced by DPP4

After cultured with 200ng/ml DPP4 for 14 days, the cells in the control group showed typical multi-layer smooth muscle cells, and the alizarin red staining was negative, while the orange red calcified nodules in the DPP4 group were significantly more than those in the control group.The alizarin red staining was strongly positive (Fig.1Ban Fig.1D),and calcium content was measured(Fig.1C), which further confirmed that DPP4 could induce HASMCs calcification.

3.Expression profile of lncRNA and mRNA in HASMCs after DPP4 intervention

After adding 200ng/ml DPP4 to HASMCs, the total RNA was extracted to detect its quality, concentration and integrity.Differentially expressed lncRNAs and mRNAs with statistical significance between the two groups were identified through P-value/FDR filtering. Differentially expressed lncRNAs and mRNAs between the two samples were identified through Fold Change filtering( Fig.2A and Fig.2B).

4.DPP4 inhibits the expression of lncRNA ENST00000540293 and MMP-1 mRNA in HASMCs

The differentially expressed lncRNAs or mRNAs between the two groups were screened by P-value/FDR, and further screened by Fold Change. The results suggest that DPP4 can inhibit the expression of lncRNA ENST00000540293 and MMP-1 mRNA (Table 1 and Table3 ).To confirm the reliability and validity of the microarray data. LncRNAs and mRNAs chip screening results were verified by RT-qPCR.

5.Effect of inhibiting the expression of HASMCs lncRNA ENST00000540293 on calcification proteins.

In order to clarify the relationship between inhibition of lncRNA ENST00000540293 expression and calcification-related protein expression, HASMCs was divided into control group, transfection control group(include Transfection Reagent only), siRNA control group and siRNA group and cultured for 48 hours. The expression of calcification-related proteins BMP-2, OPG, OPN and RUNX2 was detected by Western blot. The results showed that the expression of calcitonin in siRNA group was significantly higher than the other groups (Fig.3). It was proved that inhibiting the expression of lncRNA ENST00000540293 could promote the expression of HASMCs calcitonin.

6. Morphological analysis and calcium content determination of calcification of HASMCs after inhibition of lncRNA ENST00000540293 expression.

In order to further analyze the cell morphology, intracellular calcium content and the activity of alkaline phosphatase (ALP) after inhibiting the expression of lncRNA ENST00000540293 in HASMCs. HASMCs were cultured for 72 hours and stained with alizarin red. The intracellular calcium content, activity of ALP of each group was determined. The results showed : the area of calcified nodules and orange calcium nodules increased significantly in SiRNA group(Fig.4A and Fig.4B),the activity of ALP and intracellular calcium content in SiRNA group increased significantly (***P<0.001)(Fig. 4C and Fig.4D). Previous studies have proved that there is a positive correlation between intracellular ALP activity, intracellular calcium content and HASMCs calcification. The results showed that inhibition of epidermis of lncRNA ENST00000540293 could promote the calcification of HASMCs.

7.Inhibition of lncRNA ENST 00000540293 expression promotes phenotypic transformation of HASMCs.

The mechanism of differentiation of HASMCs into osteoblast-like cells is similar to that of bone formation²⁰.In order to further verify that inhibiting the expression of lncRNA ENST00000540293 can promote the differentiation of VSMCs into osteoblasts, we detected the expression of α-SMA by Western blot(Fig.5A). The expression of α-SMA protein in siRNA group was significantly lower than other groups (**P <0.01). At the same time, the results of F-actin immunofluorescence staining were observed by laser confocal microscope(Fig.5B). F-actin (red) fluorescence intensity, myofilament expression and aggregation in siRNA group were significantly decreased, further indicate that HASMCs had a tendency to transform into osteoblasts. In short, it can be speculated that after lncRNA ENST00000540293 silencing, the phenotype of HASMCs changes from contractile type to synthetic secretory type, and finally into osteoblast-like cells.

8. Silencing lncRNA ENST00000540293 promoted the expression of MMP-1 and ERK_1/2 in HASMCs.

Studies have shown that DPP4 regulate the expression of MMP-1 protein through ERK1/2 signal pathway²¹.And lncRNA may play a role by regulating coding genes near transcription or other signal way²². Our previous gene chip microarray subarray analysis showed that there were many differentially expressed lncRNAs near mRNAs related to tissue development, in which (Nearby Gene), a gene attached to lncRNA ENST00000540293, was a protein encoded by NM-002421, which was matrix metalloproteinase-1 (Table 1). It was found that after inhibiting the expression of lncRNA ENST00000540293, MMP-1 protein and ERK_1/2phosphorylate in siRNA group was significantly higher than the other groups detected by Western blot(Fig.6A and Fig.6B),and the difference was statistically significant (**p<0.01,***p<0.001).

Vascular calcification is a pathological process in which HASMCs change from contractile type to synthetic secretory type and finally to osteoblast-like phenotype under various pathological actions, which mediates the abnormal deposition of calcium in the vascular wall. Vascular calcification exists widely in diabetes, atherosclerosis, hypertension, chronic renal failure and other diseases, and the degree of calcification is positively correlated with the severity of the disease²³^,2⁴. Previous studies have shown that the occurrence and development of vascular calcification is closely related to cell degeneration and necrosis, and it is a passive and degenerative vascular terminal lesion process, while　recent studies have found that vascular calcification is a complex biological process that is controllable, active, preventable and reversible similar to bone formation²⁵. Changes in extracellular conditions and phenotypic transformation of vascular smooth muscle cells play a key role²⁶. At the same time, vascular calcification is similar to intramembranous osteogenesis, which usually leads to an increase in vascular hardness, a decrease in vascular compliance, and an increase in the incidence and mortality of cardiovascular events, more common in diabetes, chronic kidney disease²⁷^,2⁸．DPP4 also known as T cell surface antigen CD26, containing 110kDa glycoprotein, is a serine protease widely expressed on the surface of all kinds of cells. As a new type of fat factor, it can decompose and metabolize glucagon-like peptide-1 (GLP-1) to regulate blood sugar, and also plays an important role in the pathogenesis of diabetes and cardiovascular disease²⁹^,3⁰. At the same time, it has been found that DPP4 as an agonist of protease activated receptor 2 (PAR2), can directly activate the cascade of MAPK and NF-kB signals involved in PAR2, and induce the proliferation and inflammation of HASMCs⁶.

Long non-coding RNA is a functional RNA transcribed by RNA polymerase II. It is located in the nucleus or cytoplasm. Due to the lack of the ability to encode proteins, it was once considered to have no biological function. At present, it is believed that it plays a similar role in the regulation of gene expression. Such as chromosome silencing, chromatin modification, transcriptional activation, transcriptional interference, nuclear transport and other important regulatory processes³¹^-3⁵. Among them, lncRNA ENST00000540293 is differentially expressed in human dental follicle cells and periodontal ligament cells, and is closely related to the osteogenic differentiation of human periodontal ligament cells^15,3⁶. In this experiment, the calcification of HASMCs induced by DPP4 was used to screen out lncRNAs and mRNAs with obvious differences. The results showed that there were 25 differentially expressed inter-gene lncRNA (8 up-regulated and 17 down-regulated), and the most down-regulated inter-gene lncRNA was ENST00000540293, while lincRNA ENST00000540293 nearby gene was NM-002421, which encoded matrix metalloproteinase-1 (MMP-1). MMP-1 as the prototype of all interstitial collagenases, is also known as collagenase-1, fibroblast collagenase and interstitial collagenase³⁷. It is closely related to the regulation of cell microenvironment, and can change cell function to regulate cell differentiation, such as osteogenic differentiation of human periodontal ligament cells and calcification of HASMCs³⁸^-4¹.

In order to further determine whether the calcification of HASMCs promoted by DPP4 is related to the regulation of lncRNA ENST00000540293. We constructed smart silencer transducers silencing lncRNA ENST00000540293 expression and infected HASMCs, to detect the changes of Runx2, BMP-2, OPG and OPN proteins. The expression of calcification proteins was significantly increased and alizarin red staining also showed a significant increase in calcified nodules after lncRNA ENST0000054029 silenced. The determination of intracellular calcium content and alkaline phosphatase activity showed the same results.Also,α-SMA which as the surface marker protein of vascular smooth muscle cells also decreased significantly after lncRNA ENST0000054029 gene knockdown.And the immunofluorescence results also showed that the fluorescence intensity of F-actin, myofilament expression and aggregation decreased significantly. It is suggested that DPP4 can induce the decrease of contractile phenotype of HASMCs, which further confirms the tendency of HASMCs to osteoblast.

DPP4 is an enzyme that targets the incretin hormone GLP-1 for degradation,is also an important drug target of diabetes and new adipokines, which needs to be cleaved, exfoliated and released from the cell membrane to regulate the circulatory system⁴². MMP-1, MMP-2 and MMP-14 and MMP-9 are involved in the cleavage and shedding of DPP4,and the increased expression of MMP-1 can promote the shedding of DPP4 on the membrane of HASMCs under hypoxia⁴³. DPP4 inhibitor Alogliptin reverses vascular remodelling by downregulating matrix metalloproteinase 1 expression through inhibition of the ERK1/2/NF-κB signal pathway³¹.The results suggest that DPP4 inhibits the expression of lncRNA ENST00000540293 and MMP-1mRNA. However, inhibiting the expression of lncRNA ENST00000540293 can promote the expression of MMP-1 protein and phosphorylation of ERK1/2 protein.

Therefore, we speculate that DPP4 promotes HASMCs calcification by inhibiting lncRNA ENST00000540293, which may be related to the regulation of ERK1/2 signal pathway or MMP-1 protein. In this study, we are unable to investigate all mechanisms of DPP4 silencing lncRNA ENST00000540293 expression and activating ERK1/2 or MMP-1 and their mutual regulation. And Whether DPP4, MMP-1 and lncRNA ENST00000540293 can be used as key targets for the treatment of diabetic vascular calcification remains to be further studied.

Acknowledgements

Not applicable.

Authors' contributions

All authors contributed to the study conception and design. The first draft of the manuscript was written by Tongjien X; material preparation, data collection and analysis were performed by Hao C, Xiongfei X, Xiaolei S, Huqiang H, Hong Z, Sen S and Yong L was responsible for project administration, review, and editing. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

The study was supported by research grants from the National Natural Science Foundation of China (Fund No.81270358); the Science & Technology Department of Sichuan Province, Grant 2019JY0691.

Availability of data and materials

The data were obtained from the NCBI GEO database (Number :GSE189923).https://www.ncbi.nlm.nih.gov/geo/.

Consent for publication.

Not applicable.

Competing interests

The authors declare that there is no competing interests regarding the publication of this article.

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Table 1. The characteristics of the lincRNAs induced by DPP4.

top 10 downregulated LincRNAs

Seqname	Chrom	GeneSymbol	Fold Change	P-value	RNA length	Nearby Gene Symbol
ENST00000540293	chr11	AP000619.5	5.4691462	0.018646883	1000	MMP1
uc001phf.3	chr11	AB231724	5.3684649	0.000377659	157	MMP1
uc002ywn.1	chr21	AL109792	4.0931103	0.002062516	2900	KCNJ6
T240875	Chr3	G055454	3.9343595	0.003993263	8371	LYZL4
T240875	Chr3	G055454	3.9343595	0.003993263	8371	VIPR1
T046285	Chr10	G010780	3.4726155	0.012499379	1603	CEP55
ENST00000412427	Chr1	RP11-380J14.1	3.3043558	0.002605688	4676	ATP13A2
ENST00000452326	Chr22	AC000067.1	3.2458684	0.012123994	287	C22orf29
ENST00000452326	Chr22	AC000067.1	3.2458684	0.012123994	287	CDC45
uc002xuq.1	Chr20	AK055386	2.9207398	0.008848319	3404	KCNB1

top 8 upregulated LincRNAs

Seqname	Chrom	Gene Symbol	Fold Change	P-value	RNA length	Nearby Gene Symbol
ENST00000450216	Chr22	CTA-150C2.13	4.1538977	0.033626171	678	APOBEC3B
ENST00000450216	Chr22	CTA-150C2.13	4.1538977	0.033626171	678	APOBEC3A
ENST00000607314	Chr8	RP11-1002K11.1	3.3245744	0.000171600	1835	NRG1
NR_003133	Chr1	GBP1P1	3.035663	0.017197101	1234	LRRC8B
ENST00000520544	Chr8	RP11-760H22.2	2.2794756	0.006961939	490	COL14A1
ENST00000562082	Chr10	RP11-119F7.5	2.0858913	0.009993759	2295	STOX1
T365864	Chr9	G086505	2.0357136	0.033876979	1819	TRAF1
ENST00000608605	Chr3	RP11-804H8.6	2.0171033	0.006191725	2060	CISH

Table 2.The specific primers of LncRNA ENST00000540293,MMP-1 andβ-actin were designed.

Gene name	Primers sequence
LncRNA ENST00000540293	Forward	5'-CTGGACCAGGTATCGGAGGAGATG-3'
LncRNA ENST00000540293	Reverse	5'-GGTAGCCACTCAGCAGTTGATCC-3'
MMP-1	Forward	5'-AGATTCTACATGCGCACAAATC-3'
MMP-1	Reverse	5'-CCTTTGAAAACCGGACTTCAT-3'
β-actin	Forward	5'-CCTGGCACCCAGCACAAT-3'
β-actin	Reverse	5'-GCCGATCCACACGGAGTA-3'

Table 3. The characteristics of the mRNAs induced by DPP4

top 10 downregulated mRNAs

Seqname	Chrom	RNA length	Fold Change	P-value	Gene Symbol	type
NM_144634	chr3	916	7.7044098	0.001159771	LYZL4	protein_coding
NM_198404	chr13	2133	5.2321373	0.003622246	KCTD4	protein_coding
NM_003280	chr3	705	5.1611148	0.002562575	TNNC1	protein_coding
NM_002240	chr21	2790	4.9792082	0.008725652	KCNJ6	protein_coding
NM_005582	chr5	2725	4.8654311	0.003843377	CD180	protein_coding
NM_003783	chr1	3548	4.7786871	0.001342022	B3GALT2	protein_coding
NM_145060	chr18	2893	4.6475968	0.011197155	SKA1	protein_coding
NM_021158	chr20	2554	4.5764374	0.011266314	TRIB3	protein_coding
NM_002421	chr11	2081	4.2991079	0.028861407	MMP1	protein_coding
NM_002849	chr12	3516	4.2792469	0.004702521	PTPRR	protein_coding

top 10 upregulated mRNAs

Seqname	Chrom	RNA length	Fold Change	P-value	Gene Symbol	type
NM_000064	chr19	5148	27.6095442	0.002442785	C3	protein_coding
NM_001080400	chr19	6353	11.528119	0.001785257	PLIN4	protein_coding
NM_000668	chr4	2707	11.3082146	0.015208381	ADH1B	protein_coding
NM_000014	chr12	4678	11.2869199	0.000195162	A2M	protein_coding
NM_000669	chr4	1769	11.1388959	0.00879313	ADH1C	protein_coding
NM_013244	chr12	1934	9.4263368	0.011624943	MGAT4C	protein_coding
NM_000587	chr5	4034	7.0028976	0.023068264	C7	protein_coding
NM_004000	chr1	1486	6.8544351	0.020855768	CHI3L2	protein_coding
NM_000041	chr19	1234	6.70272	0.000195164	APOE	protein_coding
NM_005525	chr1	1477	6.6150899	0.017043214	HSD11B1	protein_coding

No competing interests reported.

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Expression profile of lncRNA induced by DPP4 in human aortic smooth muscle cells and calcification gene prediction

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