- Email: [email protected]
Differential gene and protein expression in gastrocnemius and tibialis anterior muscle following tibial and peroneal nerve injury in rats
Journal Pre-proof Differential gene and protein expression in gastrocnemius and tibialis anterior muscle following tibial and peroneal nerve injury in rats Yaofa Lin, Zheng Xie, Jun Zhou, Gang Yin, Haodong Lin PII:
S1567-133X(19)30096-1
DOI:
https://doi.org/10.1016/j.gep.2019.119079
Reference:
MODGEP 119079
To appear in:
Gene Expression Patterns
Received Date: 9 June 2019 Revised Date:
18 November 2019
Accepted Date: 19 November 2019
Please cite this article as: Lin, Y., Xie, Z., Zhou, J., Yin, G., Lin, H., Differential gene and protein expression in gastrocnemius and tibialis anterior muscle following tibial and peroneal nerve injury in rats, Gene Expression Patterns (2020), doi: https://doi.org/10.1016/j.gep.2019.119079. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
1
Differential gene and protein expression in gastrocnemius and tibialis anterior
2
muscle following tibial and peroneal nerve injury in rats
3
Yaofa Lin1, Zheng Xie1, Jun Zhou1, Gang Yin1, Haodong Lin1
4
1 Department of Orthopedic Surgery, Shanghai General Hospital, Shanghai Jiaotong
5
University School of Medicine, Shanghai 200080, P. R. China
6
Corresponding author: Haodong Lin, M.D.PhD
7
Department of orthopedic Surgery
8
Shanghai General Hospital
9
Shanghai Jiaotong University School of Medicine
10
Haining Road 100
11
Shanghai 200080
12
People’s Republic of China
13
Telephone: 86-21-81885627
14
Fax: 86-21-63520020
15
Email: [email protected]
16
Running title: Differential expression following nerve injury in rats
17 18 19 20
Abstract
1
21
Peripheral nerve injury is encountered quite commonly in the clinic, and
22
treatment results are often not satisfactory. Therefore, promoting nerve regeneration
23
and functional recovery is a primary goal of neuroscience research. Recovery of
24
corresponding target muscle can differ following peripheral nerve injury, but the
25
reasons are unknown. Herein, we investigated differential gene and protein expression
26
in gastrocnemius and tibialis anterior muscle following tibial and common peroneal
27
nerve injury using RNA sequencing and proteomics approaches, and analysed the
28
results by bioinformatics. In total, 1794, 1765, 1656 and 2006 differential genes and
29
398, 400, 959 and 472 differential proteins were identified in gastrocnemius and
30
tibialis anterior muscles at 1, 7, 14 and 21 days after surgery, related to activation of
31
51 signalling pathways. Differential expression of these genes and proteins may
32
contribute to the degree of recovery of target organs following peripheral nerve injury.
33
The findings provide a foundation for investigating regeneration mechanisms
34
following peripheral nerve injury.
35 36
Keywords: Wallerian degeneration; Gastrocnemius muscle; Tibialis anterior muscle;
2
37
RNA sequencing; Proteomics.
38
3
39
Introduction
40
Peripheral nerve injury is encountered quite commonly in the clinic, treatment is
41
often difficult, and damaged function may not be fully recovered, resulting in
42
impaired sensory and motor functions
43
satisfactory, promoting nerve regeneration and functional recovery is a major goal of
44
neuroscience research. In our previous preliminary study, we found functional
45
recovery is highly variable following repair of different peripheral nerves [2]. This
46
phenomenon has also been reported by other scholars [3-5]. In one study [6], the degree
47
of functional recovery after ulnar nerve injury was much lower than that of median
48
and radial nerves. Another study compared the repair of 393 different peripheral nerve
49
injuries, and identified a large difference in regeneration potential [7].
[1]
. Because treatment results are often not
50
Peripheral nerve regeneration is a complex and highly coordinated process. And
51
there are two main theories on the mechanism; nerve chemotaxis regeneration and
52
contact-oriented regeneration. The chemotaxis theory is generally more widely
53
accepted [8]. The basic premise of the nerve chemotaxis regeneration theory is that
54
during nerve regeneration, axons of newly-formed nerves are directed to grow by the
4
55 56
release of chemical substances at distal nerves or target tissues [9].
Several genes are up- or down-regulated during the process of Wallerian
57
degeneration at distal nerves following sciatic nerve injury in rats
58
differences in physiological changes occurring in different target muscles after
59
corresponding
60
high-throughput RNA sequencing (RNA-seq) and proteomics approaches were
61
employed to explore differences in gene and protein expression in gastrocnemius and
62
tibialis anterior muscles following tibial and common peroneal nerve injury.
peripheral
nerve
injuries
have
not
been
[10]
. However,
studied.
Herein,
63 64
Materials and Methods
65
Animal preparation and surgical procedures
66
Sixteen adult male Sprague-Dawley (SD) rats weighing 200 ± 20 g were provided
67
by the Shanghai JieSiJie Experimental Animal Co., Ltd., Shanghai, China (license No.
68
SCXK(Hu) 2013-0006). Surgical procedures were approved by the Committee on
69
Ethics of Biomedicine, Changzheng Hospital, China.
70
The rats were equally and randomly divided into four groups according to survival
5
71
time (1, 7, 14 and 21 days). Then, they were anesthetised by intraperitoneal injection
72
with 2.5% sodium pentobarbital (30 mg/kg), and fixed in the prone position A 2 cm
73
incision was made in a posterior medial position to expose the tibial nerve and the
74
common peroneal nerve on the right side, and the two nerves were cut 1 cm below the
75
piriformis with microsurgical scissors simultaneously. Accurate end-to-end suture
76
was performed immediately with 11-0 nylon threads. At 1, 7, 14 and 21 days after
77
surgery, rats were anesthetised again, and gastrocnemius and tibialis anterior muscle
78
were excised, immediately placed in liquid nitrogen, and stored at -80°C until further
79
use.
80
RNA-seq
81
Frozen gastrocnemius and tibialis anterior muscle tissue was rapidly ground into
82
granules on ice, total RNA was isolated using TRIzol reagent (Invitrogen, Grand
83
Island, NY, USA), and cDNA was synthesised using a SuperScript II RT kit according
84
to the manufacturer’s instructions. A double-end sequencing library was constructed
85
according to the Illumina (Illumina, San Diego, CA, USA) operating manual. An
86
Agilent 2100 Bioanalyzer and ABI Step One Plus Real-time PCR System (Applied
6
87
Biosystems, Waltham, MA, USA) were used to analyse the constructed library.
88
Sequencing was performed using an Illumina HiSeq 2000 Sequencer (USA) and raw
89
readings were obtained. The SOAP denovo program was then employed to obtain
90
unigenes.
91 92
Proteomics
93
Muscle tissue was ground into a powder in liquid nitrogen, and protein cleavage
94
and extraction were performed using RIPA Lysis Buffer (Thermo Fisher Scientific).
95
protein quantification using the Pierce BCA Protein Assay kit (Thermo Fisher
96
Scientific), and protein digestion was carried out with trypsin (V5280; Promega,).
97
samples were dissolved in 0.1% formic acid (FA; Sigma) and 2% acetonitrile (Fisher),
98
thoroughly shaken, centrifuged at 13,200 rpm for 10 min at 4°C, and the supernatant
99
was taken for mass spectrometry identification.
100 101 102
Bioinformatics analysis
Known reference gene sequences and annotation files were used as database
7
103
libraries, and sequence alignment was used to identify genes in each sample. The
104
number of reads for each gene was determined using htseq [11], and cufflinks [12] was
105
employed to calculate gene expression based on fragments per kilobase of transcript
106
per million mapped reads (FPKM) values. Differential gene expression was calculated
107
using the negative binomial (NB) distribution test in DESeq software. The NB test
108
results revealed differences in the number of reads, and differential gene expression
109
was estimated using basemean values with default settings (p <0.05 and differences
110
>2). Similarly, we used the UniProt Rattus norvegicus database to search for and
111
identify proteins. Finally, differentially expressed genes (DEGs) and differentially
112
expressed proteins (DEPs) were subjected to gene ontology (GO) analysis and Kyoto
113
Encyclopedia of Genes and Genomes (KEGG) pathway analysis.
114 115
Results
116
Differential gene expression
117
At 1, 7, 14 and 21 days after surgery, 728, 368, 846 and 780 DEGs were
118
up-regulated in gastrocnemius and tibialis anterior muscle, while 1066, 1397, 810 and
8
119
1226 DEGs were down-regulated (Table 1).
120 121
Functional analysis of DEGs
122
GO enrichment analysis of DEGs
123
GO enrichment analysis of DEGs was performed to investigate their functions.
124
At 1 day after surgery, the Biological Process category was enriched with terms
125
related to collagen fibril organisation and extracellular matrix organisation (Figure
126
1-a). The Molecular Function category was enriched with terms mainly involved in
127
collagen binding, heparin binding, and calcium ion binding. At 7 days after surgery,
128
Biological Process terms were mainly associated with extracellular matrix
129
organisation, ossification, and collagen fibril organisation, while Molecular Function
130
terms were mainly related to heparin binding, calcium ion binding and collagen
131
binding (Figure 1-b). At 14 days after surgery, the top Biological Process terms were
132
cardiac muscle contraction and response to wounding, while principal Molecular
133
Function terms were calcium ion binding and heparin binding (Figure 1-c). At 21 days
134
after surgery, the most enriched Biological Process terms were related to the transition
9
135
between fast and slow fibers, sarcomere organisation, and cardiac muscle contraction,
136
while the top Molecular Function terms were heparin binding and calcium ion binding
137
(Figure 1-d).
138 139
KEGG enrichment analysis of DEGs
140
KEGG analysis identified 51, 49, 50 and 49 differentially regulated signalling
141
pathways in gastrocnemius and tibialis anterior muscles at 1, 7, 14 and 21 days after
142
surgery. Throughout the entire postoperative period from 1 to 21 days, 51 signalling
143
pathways were altered. At 1 day after surgery, PI3K-AKt signalling, cGMP-PKG
144
signalling and calcium signalling pathways were most affected, and the most enriched
145
DEGs were related to axon guidance, focal adhesion and phagosomes (Figure 2-a). At
146
7 days, PPAR signalling and chemokine signalling pathways were altered, and DEGs
147
related to cytokine-cytokine receptor interactions, cell adhesion molecules (CAMs),
148
herpes simplex infection, and phagosomes were the most enriched (Figure 2-b). On
149
day 14, PPAR signalling, insulin signalling, and AMPK signalling pathways were
150
most affected, and DEGs related to herpes simplex infection, influenza A, and
10
151
regulation of lipolysis in adipocytes were the most enriched (Figure 2-c). On day 21,
152
cGMP-PKG signalling, calcium signalling, and PPAR signalling were the main
153
pathways altered, and DEGs associated with CAMs, vascular smooth muscle
154
contraction, and cytokine-cytokine receptor interactions were the most enriched
155
(Figure 2-d).
156 157
Proteomics analysis
158
Analysis of DEPs
159
At 1, 7, 14 and 21 days after surgery, 254, 272, 581 and 300 DEPs were
160
up-regulated in gastrocnemius and tibialis anterior muscle, and 144, 128, 378 and 172
161
DEPs were down-regulated (Table 2).
162 163
GO enrichment analysis of DEPs
164
GO enrichment analysis of DEPs was performed to investigate their functions.
165
We obtained the top 10 significantly enriched subcategories in Biological Process,
166
Cell Component, and Molecular Function categories (p <0.05). At 1, 7, 14 and 21
11
167
days after surgery, the top Biologic Process terms were organonitrogen compound
168
metabolic process and small molecule metabolic process. The top Cell Component
169
terms were cytoplasm and cytoplasmic parts, and the top Molecular Function terms
170
were related to protein binding (Figure 3).
171 172
KEGG enrichment analysis of DEPs
173
KEGG pathway annotation was performed using Kobas 3.0[13]. We analysed
174
genomics, chemical molecules, and biochemical systems categories, including
175
pathway, drug, disease, genes and genome subcategories. At 1 day after surgery,
176
VEGF signalling and Rap1 signalling were the main signalling pathways affected
177
(Figure 4-a). At 7 days after surgery, MAPK signalling, Ras signalling, and Fox0
178
signalling were the main signalling pathways affected (Figure 4-b). At 14 days, HIF-1
179
signalling and ECM-receptor interactions were the main signalling pathways affected
180
(Figure 4-c). At 21 days, pathways related to complement and coagulation cascades,
181
adrenergic signalling in cardiomyocytes, and insulin signalling were the most altered
182
(Figure 4-d).
12
183 184
Discussion
185
Regeneration of peripheral nerves is a complex process, and the mechanism of
186
regeneration is still unclear. Many scholars have found the rate of regeneration of
187
different peripheral nerves to be inconsistent, indicating that the potential for nerve
188
regeneration may differ
189
injury depends on whether the defect at the severed end is well bridged, as well as the
190
speed of nerve regeneration and whether regenerated axons can accurately grow into
191
target organs. Chemotaxis plays a crucial role in the function of many biological
192
systems, especially the nervous system [14-16]. During nerve regeneration, axons of
193
newly generated nerves are directed to grow by chemical substances released by distal
194
nerves and target organs [9]. Thus, whether regenerated axons can accurately grow into
195
distal target organs is largely dependent on the chemicals released by distal nerves or
196
target organs.
[3-7]
. The outcome of functional recovery following nerve
197
Nerve regeneration after injury often involves the regulation of many cells and
198
factors, and is not determined by a single or even several genes and proteins [17].
13
199
However, it is not clear how many chemicals are released from distal nerves or target
200
organs after peripheral nerve injury, and exactly which substances are related to nerve
201
regeneration remains poorly understood. In recent years, powerful gene and protein
202
chip technologies have been applied to investigate the expression and functions of
203
genes in diverse organisms [18,19]. In one study 6076 DEGs were identified during
204
Wallerian degeneration and regeneration after sciatic nerve injury in rats [20], and in
205
another [21], the ability of nerve regeneration declined with age. This is mainly because
206
secretion of endogenous neurotrophic factors decreases after nerve injury. Whether
207
the chemicals released by target organs during Wallerian degeneration and
208
regeneration are different in different peripheral nerve injuries remains to be
209
determined. Similarly, exactly which chemokines and neurotrophins affect the precise
210
growth of regenerating axons into target organs after nerve injury, which ultimately
211
results in differences in the recovery of nerve function, also requires further research.
212
Differential factors related to nerve regeneration could be identified using
213
bioinformatics approaches, which could prove helpful for developing new methods to
214
promote peripheral nerve regeneration, and identifying associated targets.
14
215
In this study, we assessed differential gene and protein expression in
216
gastrocnemius and tibialis anterior muscle after tibial nerve and common peroneal
217
nerve injury at different time points by RNA-seq and proteomics. We identified 1794,
218
1765, 1656 and 2006 differential genes and 398, 400, 959 and 472 differential
219
proteins at 1, 7, 14 and 21 days after surgery, respectively. GO enrichment analysis of
220
these DEGs and DEPs found that the Biological Process category mainly included
221
terms related to collagen fibril organisation, extracellular matrix organisation, and the
222
transition between fast and slow fibers. The most enriched Molecular Function terms
223
were calcium ion binding and heparin binding. KEGG enrichment analysis identified
224
51 altered signalling pathways, including cGMP-PKG, calcium, and PPAR signalling
225
pathways. GO enrichment analysis of DEPs revealed that the Biological Process
226
category was dominated by organonitrogen compound metabolic process and small
227
molecule metabolic process terms. The top cell component category terms were
228
cytoplasm and cytoplasmic parts, and the most enriched Molecular Function
229
subcategory was protein binding. KEGG enrichment analysis showed that VEGF,
230
Rap1, MAPK, Ras, Fox0, HIF-1, and insulin signalling pathways were the most
15
231
enriched. We initially explored differences in gene and protein expression in the
232
corresponding target muscles after different peripheral nerve injuries. The results
233
revealed numerous differential genes and proteins in gastrocnemius and tibialis
234
anterior muscle after surgery, involving a variety of signalling pathways. Differential
235
expression of these genes and proteins may help to explain differences in the degree
236
of recovery of target muscles after different peripheral nerve injuries. These findings
237
provide a new basis for the study of regeneration mechanisms after peripheral nerve
238
injury.
239 240 241 242
Funding: This work was supported by the National Natural Scientific Foundation of
243
China [grant number 81572146], the Program of Outstanding Medical Talent of
244
Shanghai Municipal Health Bureau [grant number 2017BR034], the Shuguang
245
Program of Shanghai Education Development Foundation and Shanghai Municipal
246
Education Commission [grant number 15SG34].
16
247 248
Conflict of interest
249
None
250
17
251
References
252
1. Panagopoulos GN, Megaloikonomos PD, Mavrogenis AF. The present and future
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for peripheral nerve regeneration. Orthopedics 2017, 40(1): e141-e156.
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10. Gordon T. Neurotrophic factor expression in denervated motor and sensory
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Schwann cells: relevance to specificity of peripheral nerve regeneration. Exp
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14.Bicknell BA, Pujic Z, Feldner J, Vetter I, Goodhill GJ. Chemotactic responses of
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309 310 311 312 313
314
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Tables
316
Table 1. Differential gene expression in gastrocnemius and tibialis anterior
317
muscle following peripheral nerve injury Time post-surgery (days)
Up_diff
Down_diff
Total_diff
1
728
1066
1794
7
368
1397
1765
14
846
810
1656
21
780
1266
2006
318
Up_diff, number of significantly up-regulated genes; Down_diff, number of
319
significantly down-regulated genes; Total_diff, total differential genes.
320 321
Table 2. Differentially expressed proteins in gastrocnemius and tibialis anterior
322
muscle following peripheral nerve injury (fold change >2 or <0.5) Time post-surgery (days)
Up_diff
Down_diff
Total_diff
1
254
144
398
7
272
128
400
14
581
378
959
21
300
172
472
323
Up_diff, number of significantly up-regulated proteins; Down_diff, number of
324
significantly down-regulated proteins; Total_diff, total differential proteins.
325
22
326
Figure legends
327
Figure 1. GO enrichment analysis of the top 10 differential genes at 1
328
21 days after surgery. Figure1-a: 1 day; Figure1-b: 7 days; Figure1-c: 14 days;
329
Figure1-d: 21 days;
7
14
330 331
Figure 2. KEGG enrichment analysis of the top 20 terms in gastrocnemius and
332
tibialis anterior muscle at 1
333
PI3K-AKt, cGMP-PKG, and calcium signalling pathways are the most significantly
334
altered pathways. Figure1-b: 7 days, PPAR and chemokine signalling pathways are
335
the most significantly altered pathways. Figure1-c: 14 days, PPAR, insulin, and
336
AMPK signalling pathways are the most significantly altered pathways. Figure1-d: 21
337
days, cGMP-PKG, calcium, and PPAR signalling pathways are the most significantly
338
altered pathways.
339
Note: The x-axis indicates the enrichment score. The larger the bubble, the greater the
340
number of differential genes. Bubbles are coloured red-blue-green-yellow with
341
increasing enrichment (based on p-values).
7
14
21 days after surgery. Figure1-a: 1 day,
23
342 343
Figure 3. GO enrichment analysis of the top 10 differential proteins at 1, 7, 14
344
and 21days after surgery. Organonitrogen compound metabolic process and small
345
molecule metabolic process are the top terms in the Biological Process category. The
346
main terms in the Cell Component category are cytoplasm and cytoplasmic parts, and
347
protein binding is the dominant subcategory in the Molecular Function is category. a,
348
1 day after surgery; b, 7 days after surgery; c, 14 days after surgery. d, 21 days after
349
surgery.
350 351
Figure 4. KEGG pathway enrichment analysis of gastrocnemius and tibialis
352
anterior muscle at 1
353
Rap1 signalling pathways are the main signalling pathways involved. Figure4-b: 7
354
days, MAPK, Ras, and Fox0 signalling pathways are the main signalling pathways
355
involved. Figure4-c: 14 days, The HIF-1 signalling pathway and ECM-receptor
356
interactions are the main pathways involved. Figure4-d: 21 days, Complement and
357
coagulation cascades, adrenergic signalling in cardiomyocytes, and the insulin
7
14
21 days after surgery. Figure4-a: 1 day, VEGF and
24
358
signalling pathway are the main pathways involved.
25
Research highlights •
This article is the first to explore the expression of genes and proteins in the corresponding target muscles after different peripheral nerve injuries.
•
This article is the first to explore the differential expression of genes and proteins in the corresponding muscles of the tibial and the common peroneal nerves after injury in the same horizontal plane.
•
This article explores the expression of differential genes and proteins after different nerve injuries by RNA sequencing and proteomics techniques.
•
This article suggests that different nerves have different gene and protein expressions in their respective muscle tissues after injury.
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S1567-133X(19)30096-1
DOI:
https://doi.org/10.1016/j.gep.2019.119079
Reference:
MODGEP 119079
To appear in:
Gene Expression Patterns
Received Date: 9 June 2019 Revised Date:
18 November 2019
Accepted Date: 19 November 2019
Please cite this article as: Lin, Y., Xie, Z., Zhou, J., Yin, G., Lin, H., Differential gene and protein expression in gastrocnemius and tibialis anterior muscle following tibial and peroneal nerve injury in rats, Gene Expression Patterns (2020), doi: https://doi.org/10.1016/j.gep.2019.119079. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
1
Differential gene and protein expression in gastrocnemius and tibialis anterior
2
muscle following tibial and peroneal nerve injury in rats
3
Yaofa Lin1, Zheng Xie1, Jun Zhou1, Gang Yin1, Haodong Lin1
4
1 Department of Orthopedic Surgery, Shanghai General Hospital, Shanghai Jiaotong
5
University School of Medicine, Shanghai 200080, P. R. China
6
Corresponding author: Haodong Lin, M.D.PhD
7
Department of orthopedic Surgery
8
Shanghai General Hospital
9
Shanghai Jiaotong University School of Medicine
10
Haining Road 100
11
Shanghai 200080
12
People’s Republic of China
13
Telephone: 86-21-81885627
14
Fax: 86-21-63520020
15
Email: [email protected]
16
Running title: Differential expression following nerve injury in rats
17 18 19 20
Abstract
1
21
Peripheral nerve injury is encountered quite commonly in the clinic, and
22
treatment results are often not satisfactory. Therefore, promoting nerve regeneration
23
and functional recovery is a primary goal of neuroscience research. Recovery of
24
corresponding target muscle can differ following peripheral nerve injury, but the
25
reasons are unknown. Herein, we investigated differential gene and protein expression
26
in gastrocnemius and tibialis anterior muscle following tibial and common peroneal
27
nerve injury using RNA sequencing and proteomics approaches, and analysed the
28
results by bioinformatics. In total, 1794, 1765, 1656 and 2006 differential genes and
29
398, 400, 959 and 472 differential proteins were identified in gastrocnemius and
30
tibialis anterior muscles at 1, 7, 14 and 21 days after surgery, related to activation of
31
51 signalling pathways. Differential expression of these genes and proteins may
32
contribute to the degree of recovery of target organs following peripheral nerve injury.
33
The findings provide a foundation for investigating regeneration mechanisms
34
following peripheral nerve injury.
35 36
Keywords: Wallerian degeneration; Gastrocnemius muscle; Tibialis anterior muscle;
2
37
RNA sequencing; Proteomics.
38
3
39
Introduction
40
Peripheral nerve injury is encountered quite commonly in the clinic, treatment is
41
often difficult, and damaged function may not be fully recovered, resulting in
42
impaired sensory and motor functions
43
satisfactory, promoting nerve regeneration and functional recovery is a major goal of
44
neuroscience research. In our previous preliminary study, we found functional
45
recovery is highly variable following repair of different peripheral nerves [2]. This
46
phenomenon has also been reported by other scholars [3-5]. In one study [6], the degree
47
of functional recovery after ulnar nerve injury was much lower than that of median
48
and radial nerves. Another study compared the repair of 393 different peripheral nerve
49
injuries, and identified a large difference in regeneration potential [7].
[1]
. Because treatment results are often not
50
Peripheral nerve regeneration is a complex and highly coordinated process. And
51
there are two main theories on the mechanism; nerve chemotaxis regeneration and
52
contact-oriented regeneration. The chemotaxis theory is generally more widely
53
accepted [8]. The basic premise of the nerve chemotaxis regeneration theory is that
54
during nerve regeneration, axons of newly-formed nerves are directed to grow by the
4
55 56
release of chemical substances at distal nerves or target tissues [9].
Several genes are up- or down-regulated during the process of Wallerian
57
degeneration at distal nerves following sciatic nerve injury in rats
58
differences in physiological changes occurring in different target muscles after
59
corresponding
60
high-throughput RNA sequencing (RNA-seq) and proteomics approaches were
61
employed to explore differences in gene and protein expression in gastrocnemius and
62
tibialis anterior muscles following tibial and common peroneal nerve injury.
peripheral
nerve
injuries
have
not
been
[10]
. However,
studied.
Herein,
63 64
Materials and Methods
65
Animal preparation and surgical procedures
66
Sixteen adult male Sprague-Dawley (SD) rats weighing 200 ± 20 g were provided
67
by the Shanghai JieSiJie Experimental Animal Co., Ltd., Shanghai, China (license No.
68
SCXK(Hu) 2013-0006). Surgical procedures were approved by the Committee on
69
Ethics of Biomedicine, Changzheng Hospital, China.
70
The rats were equally and randomly divided into four groups according to survival
5
71
time (1, 7, 14 and 21 days). Then, they were anesthetised by intraperitoneal injection
72
with 2.5% sodium pentobarbital (30 mg/kg), and fixed in the prone position A 2 cm
73
incision was made in a posterior medial position to expose the tibial nerve and the
74
common peroneal nerve on the right side, and the two nerves were cut 1 cm below the
75
piriformis with microsurgical scissors simultaneously. Accurate end-to-end suture
76
was performed immediately with 11-0 nylon threads. At 1, 7, 14 and 21 days after
77
surgery, rats were anesthetised again, and gastrocnemius and tibialis anterior muscle
78
were excised, immediately placed in liquid nitrogen, and stored at -80°C until further
79
use.
80
RNA-seq
81
Frozen gastrocnemius and tibialis anterior muscle tissue was rapidly ground into
82
granules on ice, total RNA was isolated using TRIzol reagent (Invitrogen, Grand
83
Island, NY, USA), and cDNA was synthesised using a SuperScript II RT kit according
84
to the manufacturer’s instructions. A double-end sequencing library was constructed
85
according to the Illumina (Illumina, San Diego, CA, USA) operating manual. An
86
Agilent 2100 Bioanalyzer and ABI Step One Plus Real-time PCR System (Applied
6
87
Biosystems, Waltham, MA, USA) were used to analyse the constructed library.
88
Sequencing was performed using an Illumina HiSeq 2000 Sequencer (USA) and raw
89
readings were obtained. The SOAP denovo program was then employed to obtain
90
unigenes.
91 92
Proteomics
93
Muscle tissue was ground into a powder in liquid nitrogen, and protein cleavage
94
and extraction were performed using RIPA Lysis Buffer (Thermo Fisher Scientific).
95
protein quantification using the Pierce BCA Protein Assay kit (Thermo Fisher
96
Scientific), and protein digestion was carried out with trypsin (V5280; Promega,).
97
samples were dissolved in 0.1% formic acid (FA; Sigma) and 2% acetonitrile (Fisher),
98
thoroughly shaken, centrifuged at 13,200 rpm for 10 min at 4°C, and the supernatant
99
was taken for mass spectrometry identification.
100 101 102
Bioinformatics analysis
Known reference gene sequences and annotation files were used as database
7
103
libraries, and sequence alignment was used to identify genes in each sample. The
104
number of reads for each gene was determined using htseq [11], and cufflinks [12] was
105
employed to calculate gene expression based on fragments per kilobase of transcript
106
per million mapped reads (FPKM) values. Differential gene expression was calculated
107
using the negative binomial (NB) distribution test in DESeq software. The NB test
108
results revealed differences in the number of reads, and differential gene expression
109
was estimated using basemean values with default settings (p <0.05 and differences
110
>2). Similarly, we used the UniProt Rattus norvegicus database to search for and
111
identify proteins. Finally, differentially expressed genes (DEGs) and differentially
112
expressed proteins (DEPs) were subjected to gene ontology (GO) analysis and Kyoto
113
Encyclopedia of Genes and Genomes (KEGG) pathway analysis.
114 115
Results
116
Differential gene expression
117
At 1, 7, 14 and 21 days after surgery, 728, 368, 846 and 780 DEGs were
118
up-regulated in gastrocnemius and tibialis anterior muscle, while 1066, 1397, 810 and
8
119
1226 DEGs were down-regulated (Table 1).
120 121
Functional analysis of DEGs
122
GO enrichment analysis of DEGs
123
GO enrichment analysis of DEGs was performed to investigate their functions.
124
At 1 day after surgery, the Biological Process category was enriched with terms
125
related to collagen fibril organisation and extracellular matrix organisation (Figure
126
1-a). The Molecular Function category was enriched with terms mainly involved in
127
collagen binding, heparin binding, and calcium ion binding. At 7 days after surgery,
128
Biological Process terms were mainly associated with extracellular matrix
129
organisation, ossification, and collagen fibril organisation, while Molecular Function
130
terms were mainly related to heparin binding, calcium ion binding and collagen
131
binding (Figure 1-b). At 14 days after surgery, the top Biological Process terms were
132
cardiac muscle contraction and response to wounding, while principal Molecular
133
Function terms were calcium ion binding and heparin binding (Figure 1-c). At 21 days
134
after surgery, the most enriched Biological Process terms were related to the transition
9
135
between fast and slow fibers, sarcomere organisation, and cardiac muscle contraction,
136
while the top Molecular Function terms were heparin binding and calcium ion binding
137
(Figure 1-d).
138 139
KEGG enrichment analysis of DEGs
140
KEGG analysis identified 51, 49, 50 and 49 differentially regulated signalling
141
pathways in gastrocnemius and tibialis anterior muscles at 1, 7, 14 and 21 days after
142
surgery. Throughout the entire postoperative period from 1 to 21 days, 51 signalling
143
pathways were altered. At 1 day after surgery, PI3K-AKt signalling, cGMP-PKG
144
signalling and calcium signalling pathways were most affected, and the most enriched
145
DEGs were related to axon guidance, focal adhesion and phagosomes (Figure 2-a). At
146
7 days, PPAR signalling and chemokine signalling pathways were altered, and DEGs
147
related to cytokine-cytokine receptor interactions, cell adhesion molecules (CAMs),
148
herpes simplex infection, and phagosomes were the most enriched (Figure 2-b). On
149
day 14, PPAR signalling, insulin signalling, and AMPK signalling pathways were
150
most affected, and DEGs related to herpes simplex infection, influenza A, and
10
151
regulation of lipolysis in adipocytes were the most enriched (Figure 2-c). On day 21,
152
cGMP-PKG signalling, calcium signalling, and PPAR signalling were the main
153
pathways altered, and DEGs associated with CAMs, vascular smooth muscle
154
contraction, and cytokine-cytokine receptor interactions were the most enriched
155
(Figure 2-d).
156 157
Proteomics analysis
158
Analysis of DEPs
159
At 1, 7, 14 and 21 days after surgery, 254, 272, 581 and 300 DEPs were
160
up-regulated in gastrocnemius and tibialis anterior muscle, and 144, 128, 378 and 172
161
DEPs were down-regulated (Table 2).
162 163
GO enrichment analysis of DEPs
164
GO enrichment analysis of DEPs was performed to investigate their functions.
165
We obtained the top 10 significantly enriched subcategories in Biological Process,
166
Cell Component, and Molecular Function categories (p <0.05). At 1, 7, 14 and 21
11
167
days after surgery, the top Biologic Process terms were organonitrogen compound
168
metabolic process and small molecule metabolic process. The top Cell Component
169
terms were cytoplasm and cytoplasmic parts, and the top Molecular Function terms
170
were related to protein binding (Figure 3).
171 172
KEGG enrichment analysis of DEPs
173
KEGG pathway annotation was performed using Kobas 3.0[13]. We analysed
174
genomics, chemical molecules, and biochemical systems categories, including
175
pathway, drug, disease, genes and genome subcategories. At 1 day after surgery,
176
VEGF signalling and Rap1 signalling were the main signalling pathways affected
177
(Figure 4-a). At 7 days after surgery, MAPK signalling, Ras signalling, and Fox0
178
signalling were the main signalling pathways affected (Figure 4-b). At 14 days, HIF-1
179
signalling and ECM-receptor interactions were the main signalling pathways affected
180
(Figure 4-c). At 21 days, pathways related to complement and coagulation cascades,
181
adrenergic signalling in cardiomyocytes, and insulin signalling were the most altered
182
(Figure 4-d).
12
183 184
Discussion
185
Regeneration of peripheral nerves is a complex process, and the mechanism of
186
regeneration is still unclear. Many scholars have found the rate of regeneration of
187
different peripheral nerves to be inconsistent, indicating that the potential for nerve
188
regeneration may differ
189
injury depends on whether the defect at the severed end is well bridged, as well as the
190
speed of nerve regeneration and whether regenerated axons can accurately grow into
191
target organs. Chemotaxis plays a crucial role in the function of many biological
192
systems, especially the nervous system [14-16]. During nerve regeneration, axons of
193
newly generated nerves are directed to grow by chemical substances released by distal
194
nerves and target organs [9]. Thus, whether regenerated axons can accurately grow into
195
distal target organs is largely dependent on the chemicals released by distal nerves or
196
target organs.
[3-7]
. The outcome of functional recovery following nerve
197
Nerve regeneration after injury often involves the regulation of many cells and
198
factors, and is not determined by a single or even several genes and proteins [17].
13
199
However, it is not clear how many chemicals are released from distal nerves or target
200
organs after peripheral nerve injury, and exactly which substances are related to nerve
201
regeneration remains poorly understood. In recent years, powerful gene and protein
202
chip technologies have been applied to investigate the expression and functions of
203
genes in diverse organisms [18,19]. In one study 6076 DEGs were identified during
204
Wallerian degeneration and regeneration after sciatic nerve injury in rats [20], and in
205
another [21], the ability of nerve regeneration declined with age. This is mainly because
206
secretion of endogenous neurotrophic factors decreases after nerve injury. Whether
207
the chemicals released by target organs during Wallerian degeneration and
208
regeneration are different in different peripheral nerve injuries remains to be
209
determined. Similarly, exactly which chemokines and neurotrophins affect the precise
210
growth of regenerating axons into target organs after nerve injury, which ultimately
211
results in differences in the recovery of nerve function, also requires further research.
212
Differential factors related to nerve regeneration could be identified using
213
bioinformatics approaches, which could prove helpful for developing new methods to
214
promote peripheral nerve regeneration, and identifying associated targets.
14
215
In this study, we assessed differential gene and protein expression in
216
gastrocnemius and tibialis anterior muscle after tibial nerve and common peroneal
217
nerve injury at different time points by RNA-seq and proteomics. We identified 1794,
218
1765, 1656 and 2006 differential genes and 398, 400, 959 and 472 differential
219
proteins at 1, 7, 14 and 21 days after surgery, respectively. GO enrichment analysis of
220
these DEGs and DEPs found that the Biological Process category mainly included
221
terms related to collagen fibril organisation, extracellular matrix organisation, and the
222
transition between fast and slow fibers. The most enriched Molecular Function terms
223
were calcium ion binding and heparin binding. KEGG enrichment analysis identified
224
51 altered signalling pathways, including cGMP-PKG, calcium, and PPAR signalling
225
pathways. GO enrichment analysis of DEPs revealed that the Biological Process
226
category was dominated by organonitrogen compound metabolic process and small
227
molecule metabolic process terms. The top cell component category terms were
228
cytoplasm and cytoplasmic parts, and the most enriched Molecular Function
229
subcategory was protein binding. KEGG enrichment analysis showed that VEGF,
230
Rap1, MAPK, Ras, Fox0, HIF-1, and insulin signalling pathways were the most
15
231
enriched. We initially explored differences in gene and protein expression in the
232
corresponding target muscles after different peripheral nerve injuries. The results
233
revealed numerous differential genes and proteins in gastrocnemius and tibialis
234
anterior muscle after surgery, involving a variety of signalling pathways. Differential
235
expression of these genes and proteins may help to explain differences in the degree
236
of recovery of target muscles after different peripheral nerve injuries. These findings
237
provide a new basis for the study of regeneration mechanisms after peripheral nerve
238
injury.
239 240 241 242
Funding: This work was supported by the National Natural Scientific Foundation of
243
China [grant number 81572146], the Program of Outstanding Medical Talent of
244
Shanghai Municipal Health Bureau [grant number 2017BR034], the Shuguang
245
Program of Shanghai Education Development Foundation and Shanghai Municipal
246
Education Commission [grant number 15SG34].
16
247 248
Conflict of interest
249
None
250
17
251
References
252
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309 310 311 312 313
314
21
315
Tables
316
Table 1. Differential gene expression in gastrocnemius and tibialis anterior
317
muscle following peripheral nerve injury Time post-surgery (days)
Up_diff
Down_diff
Total_diff
1
728
1066
1794
7
368
1397
1765
14
846
810
1656
21
780
1266
2006
318
Up_diff, number of significantly up-regulated genes; Down_diff, number of
319
significantly down-regulated genes; Total_diff, total differential genes.
320 321
Table 2. Differentially expressed proteins in gastrocnemius and tibialis anterior
322
muscle following peripheral nerve injury (fold change >2 or <0.5) Time post-surgery (days)
Up_diff
Down_diff
Total_diff
1
254
144
398
7
272
128
400
14
581
378
959
21
300
172
472
323
Up_diff, number of significantly up-regulated proteins; Down_diff, number of
324
significantly down-regulated proteins; Total_diff, total differential proteins.
325
22
326
Figure legends
327
Figure 1. GO enrichment analysis of the top 10 differential genes at 1
328
21 days after surgery. Figure1-a: 1 day; Figure1-b: 7 days; Figure1-c: 14 days;
329
Figure1-d: 21 days;
7
14
330 331
Figure 2. KEGG enrichment analysis of the top 20 terms in gastrocnemius and
332
tibialis anterior muscle at 1
333
PI3K-AKt, cGMP-PKG, and calcium signalling pathways are the most significantly
334
altered pathways. Figure1-b: 7 days, PPAR and chemokine signalling pathways are
335
the most significantly altered pathways. Figure1-c: 14 days, PPAR, insulin, and
336
AMPK signalling pathways are the most significantly altered pathways. Figure1-d: 21
337
days, cGMP-PKG, calcium, and PPAR signalling pathways are the most significantly
338
altered pathways.
339
Note: The x-axis indicates the enrichment score. The larger the bubble, the greater the
340
number of differential genes. Bubbles are coloured red-blue-green-yellow with
341
increasing enrichment (based on p-values).
7
14
21 days after surgery. Figure1-a: 1 day,
23
342 343
Figure 3. GO enrichment analysis of the top 10 differential proteins at 1, 7, 14
344
and 21days after surgery. Organonitrogen compound metabolic process and small
345
molecule metabolic process are the top terms in the Biological Process category. The
346
main terms in the Cell Component category are cytoplasm and cytoplasmic parts, and
347
protein binding is the dominant subcategory in the Molecular Function is category. a,
348
1 day after surgery; b, 7 days after surgery; c, 14 days after surgery. d, 21 days after
349
surgery.
350 351
Figure 4. KEGG pathway enrichment analysis of gastrocnemius and tibialis
352
anterior muscle at 1
353
Rap1 signalling pathways are the main signalling pathways involved. Figure4-b: 7
354
days, MAPK, Ras, and Fox0 signalling pathways are the main signalling pathways
355
involved. Figure4-c: 14 days, The HIF-1 signalling pathway and ECM-receptor
356
interactions are the main pathways involved. Figure4-d: 21 days, Complement and
357
coagulation cascades, adrenergic signalling in cardiomyocytes, and the insulin
7
14
21 days after surgery. Figure4-a: 1 day, VEGF and
24
358
signalling pathway are the main pathways involved.
25
Research highlights •
This article is the first to explore the expression of genes and proteins in the corresponding target muscles after different peripheral nerve injuries.
•
This article is the first to explore the differential expression of genes and proteins in the corresponding muscles of the tibial and the common peroneal nerves after injury in the same horizontal plane.
•
This article explores the expression of differential genes and proteins after different nerve injuries by RNA sequencing and proteomics techniques.
•
This article suggests that different nerves have different gene and protein expressions in their respective muscle tissues after injury.
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