Gross morphological observation
In the normal meniscus group, the meniscus was smooth and complete, with a white and shiny surface, without any signs of degeneration. In the degenerative meniscus group, the surface of meniscus was rough, the color was dim, the elasticity was poor, and there were some small defects and erosion (Fig. 1).
Figure 1. (a) In the normal meniscus group, the meniscus was crescent shaped, with complete and smooth surface, no tear, white color and good elasticity. And the medial part of the meniscus was thin and the lateral part was thick. (b) In the degenerative meniscus group, the color of the meniscus was light yellow, the elasticity was worse than that of the normal meniscus, the surrounding synovial membrane was congested and edema, the inner part was thinner with uneven wear, and the free edge was incomplete and cracked.
Histological examination
In the normal meniscus group, HE staining of the meniscus showed normal meniscus tissue. In the degenerative meniscus group, HE staining of the meniscus showed disorder, uneven staining and sparse arrangement of collagen fibers, disorder, reduction and swelling of chondrocytes, reduction or disappearance of cartilage lacunae, increased local fibers and hyaline changes, which were consistent with meniscal degeneration (Fig. 2).
Figure 2. (a-b) The HE staining of normal meniscus tissue. The chondrocyte nucleus was large and round, the cell distribution was regular, the collagen fibers were abundant, and the fiber bundles were thick and neat. (c-f) The HE staining of degenerative meniscus. The arrangement of collagen fibers was disordered, the staining was uneven, and the arrangement was sparse; the chondrocytes were arranged disorderly and reduced, and the visible cells were swollen, the cartilage lacuna was reduced or disappeared, and the local area was fibrotic and hyalinized. a: x40, b: x100, c: x40, d: x100, e: x200, f: x400.
Quality control
For RNA quantification and quality assurance performed by NanoDrop ND-1000, the ratio of A260/A280 for all samples was close to 2.0, and the ratio of O.D. A260/A230 was greater than 1.8. The result of Agilent 2100 Bioanalyzer showed that all RINs ≥ 6.3, indicating that the integrity of total RNA was good and there was no obvious degradation, so follow-up experiments could be carried out (Table 1).
Table 1. RNA quality control result
Sample ID
|
OD260/280 Ratio
|
OD260/230 Ratio
|
Conc. (ng/μl)
|
RIN
|
Pass or Fail
|
NM1
|
2.00
|
1.90
|
319.41
|
6.9
|
Pass
|
NM2
|
1.88
|
1.96
|
195.83
|
6.5
|
Pass
|
NM3
|
2.00
|
1.99
|
314.36
|
6.5
|
Pass
|
NM4
|
2.02
|
1.91
|
299.40
|
6.4
|
Pass
|
NM5
|
2.01
|
1.97
|
335.84
|
6.3
|
Pass
|
NM6
|
1.94
|
1.85
|
164.94
|
7.2
|
Pass
|
NM7
|
2.00
|
1.94
|
316.15
|
6.3
|
Pass
|
DM1
|
1.97
|
1.92
|
358.41
|
7.3
|
Pass
|
DM2
|
2.02
|
1.97
|
721.61
|
7.5
|
Pass
|
DM3
|
2.03
|
1.90
|
400.45
|
6.3
|
Pass
|
DM4
|
1.83
|
1.96
|
112.25
|
7.2
|
Pass
|
DM5
|
1.98
|
1.89
|
267.03
|
6.5
|
Pass
|
DM6
|
1.86
|
1.90
|
251.31
|
6.4
|
Pass
|
DM7
|
2.01
|
2.07
|
345.64
|
6.7
|
Pass
|
NM: normal meniscus; DM: degenerative meniscus
DEGs
There were 893 DEGs in the two groups, including 537 up-regulated genes and 356 down-regulated genes. The result was showed in the volcano plot and the dendrogram(Fig. 3). The top 10 most significant genes were cyp2c33, gcnt7, ncdn, exd3, MUC13, ppp1r3d, nphp3, upb1, CD81 and prph (Table 2).
Figure 3. (a) The volcano plot. Red and green represent DEGs; the downregulated DEGs are green, and the upregulated DEGs are red. (b) The dendrogram. The ordinate represents the grouping information of the sample. Red indicates high relative expression and green indicates low relative expression. NM: normal meniscus; DM: degenerative meniscus.
Table 2. The top 10 most significant genes.
ProbeName
|
P-value
|
FDR
|
Fold Change
|
Regulation
|
GeneSymbol
|
Rank
|
A_72_P526917
|
1.2593E-07
|
0.000537464
|
2.0593695
|
up
|
CYP2C33
|
1
|
A_72_P362758
|
1.38326E-07
|
0.000537464
|
3.2506906
|
up
|
GCNT7
|
2
|
A_72_P050406
|
6.10533E-07
|
0.001031403
|
2.5418055
|
up
|
NCDN
|
3
|
A_72_P258107
|
8.6355E-07
|
0.001198329
|
2.5599119
|
up
|
EXD3
|
4
|
A_72_P165116
|
9.46523E-07
|
0.001225904
|
2.046065
|
up
|
MUC13
|
5
|
A_72_P442485
|
1.08108E-06
|
0.001272895
|
2.2891991
|
up
|
PPP1R3D
|
6
|
A_72_P222892
|
1.13988E-06
|
0.001302652
|
2.0178437
|
up
|
NPHP3
|
7
|
A_72_P234107
|
1.23101E-06
|
0.001328635
|
2.5152315
|
up
|
UPB1
|
8
|
A_72_P496803
|
1.4119E-06
|
0.001443671
|
3.0181273
|
up
|
CD81
|
9
|
A_72_P352838
|
1.81842E-06
|
0.001613149
|
2.4487246
|
up
|
PRPH
|
10
|
GO analysis
A total of 55 biological processes enriched by the DEGs were obtained. The top 10 biological processes were cell response to hormone stimulus, response to hormone, sex determination, muscle fiber development, mehcheme morphogenesis, cell response to endogenous stimulus, C21 steroid hormone metallic process, neuropeptide signaling pathway, negative regulation, respectively of reactive oxygen species metallic process and regulation of nitric oxide biosynthetic process (Table 3).
Table 3. The enriched GO terms for the DEGs.
Term
|
Count
|
P-value
|
Regulation
|
Rank
|
cellular response to hormone stimulus
|
7
|
0.000311929
|
Up
|
1
|
response to hormone
|
7
|
0.001152029
|
Up
|
2
|
sex determination
|
2
|
0.001281445
|
Down
|
3
|
muscle fiber development
|
2
|
0.002753211
|
Down
|
4
|
mesenchyme morphogenesis
|
2
|
0.002753211
|
Down
|
5
|
cellular response to endogenous stimulus
|
8
|
0.003566943
|
Up
|
6
|
C21-steroid hormone metabolic process
|
2
|
0.003588081
|
Up
|
7
|
neuropeptide signaling pathway
|
3
|
0.003940886
|
Up
|
8
|
negative regulation of reactive oxygen species metabolic process
|
2
|
0.004359628
|
Up
|
9
|
regulation of nitric oxide biosynthetic process
|
2
|
0.007086781
|
Up
|
10
|
response to endogenous stimulus
|
8
|
0.007145167
|
Up
|
11
|
negative regulation of reproductive process
|
2
|
0.008950734
|
Down
|
12
|
nitric oxide biosynthetic process
|
2
|
0.009235862
|
Up
|
13
|
nitric oxide metabolic process
|
2
|
0.009235862
|
Up
|
14
|
reactive nitrogen species metabolic process
|
2
|
0.009235862
|
Up
|
15
|
gland development
|
4
|
0.010284414
|
Up
|
16
|
hormone metabolic process
|
3
|
0.011308564
|
Up
|
17
|
male gonad development
|
2
|
0.011498663
|
Down
|
18
|
development of primary male sexual characteristics
|
2
|
0.011498663
|
Down
|
19
|
regulation of reactive oxygen species biosynthetic process
|
2
|
0.011638114
|
Up
|
20
|
body fluid secretion
|
2
|
0.012931151
|
Up
|
21
|
striated muscle cell development
|
2
|
0.013594657
|
Down
|
22
|
hormone biosynthetic process
|
2
|
0.015695135
|
Up
|
23
|
male sex differentiation
|
2
|
0.016626965
|
Down
|
24
|
muscle cell development
|
2
|
0.016626965
|
Down
|
25
|
reproductive structure development
|
3
|
0.017568602
|
Down
|
26
|
reproductive system development
|
3
|
0.017988302
|
Down
|
27
|
cellular hormone metabolic process
|
2
|
0.018688631
|
Up
|
28
|
regulation of hormone levels
|
3
|
0.018844593
|
Down
|
29
|
reactive oxygen species biosynthetic process
|
2
|
0.020268615
|
Up
|
30
|
epithelial cell differentiation
|
3
|
0.020624915
|
Down
|
31
|
neurotransmitter biosynthetic process
|
2
|
0.021902613
|
Up
|
32
|
feeding behavior
|
2
|
0.023589533
|
Up
|
33
|
response to chemical
|
14
|
0.023617031
|
Up
|
34
|
cellular response to chemical stimulus
|
11
|
0.023892904
|
Up
|
35
|
hormone metabolic process
|
2
|
0.026281932
|
Down
|
36
|
bone remodeling
|
2
|
0.027117856
|
Up
|
37
|
striated muscle cell differentiation
|
2
|
0.033355701
|
Down
|
38
|
tissue development
|
5
|
0.035003106
|
Down
|
39
|
gonad development
|
2
|
0.035500212
|
Down
|
40
|
development of primary sexual characteristics
|
2
|
0.035500212
|
Down
|
41
|
mammary gland development
|
2
|
0.036791312
|
Up
|
42
|
mesenchyme development
|
2
|
0.038815337
|
Down
|
43
|
regulation of reproductive process
|
2
|
0.038815337
|
Down
|
44
|
muscle organ development
|
2
|
0.041089286
|
Down
|
45
|
response to oxygen-containing compound
|
6
|
0.041364725
|
Up
|
46
|
hormone-mediated signaling pathway
|
2
|
0.043140041
|
Up
|
47
|
neurotransmitter metabolic process
|
2
|
0.043140041
|
Up
|
48
|
regulation of reactive oxygen species metabolic process
|
2
|
0.043140041
|
Up
|
49
|
response to peptide
|
3
|
0.048362819
|
Up
|
50
|
body morphogenesis
|
1
|
0.048805944
|
Down
|
51
|
forebrain neuron differentiation
|
1
|
0.048805944
|
Down
|
52
|
regulation of interleukin-4 production
|
1
|
0.048805944
|
Down
|
53
|
positive regulation of mitotic nuclear division
|
1
|
0.048805944
|
Down
|
54
|
labyrinthine layer morphogenesis
|
1
|
0.048805944
|
Down
|
55
|
Pathway analysis
A total of 36 pathways were affected by the alteration of the gene expression. The top 10 pathways are Type II diabetes mellitus, Thyroid hormone synthesis, Taste transduction, Prolactin signaling pathway, Longevity regulating pathway, Ovarian steroidogenesis, Neuroactive ligand-receptor interaction, Inflammatory mediator regulation of TRP channels, Pantothenate and CoA biosynthesis and Cocaine addiction (Table 4).
Table 4. The enriched pathways for the DEGs.
Definition
|
Fisher P-value
|
regulation
|
Rank
|
Type II diabetes mellitus
|
0.000759595
|
Up
|
1
|
Thyroid hormone synthesis
|
0.002669959
|
Down
|
2
|
Taste transduction
|
0.002858715
|
Up
|
3
|
Prolactin signaling pathway
|
0.003352503
|
Up
|
4
|
Longevity regulating pathway
|
0.007875137
|
Up
|
5
|
Ovarian steroidogenesis
|
0.009215694
|
Up
|
6
|
Neuroactive ligand-receptor interaction
|
0.01021098
|
Up
|
7
|
Inflammatory mediator regulation of TRP channels
|
0.01098087
|
Up
|
8
|
Pantothenate and CoA biosynthesis
|
0.01205545
|
Up
|
9
|
Cocaine addiction
|
0.01468413
|
Down
|
10
|
Metabolism of xenobiotics by cytochrome P450
|
0.01528523
|
Down
|
11
|
Legionellosis
|
0.01844662
|
Down
|
12
|
AMPK signaling pathway
|
0.02220957
|
Up
|
13
|
Bile secretion
|
0.02225764
|
Up
|
14
|
Glycerolipid metabolism
|
0.02257206
|
Down
|
15
|
Chemical carcinogenesis
|
0.02329364
|
Down
|
16
|
Adipocytokine signaling pathway
|
0.0249136
|
Up
|
17
|
Amphetamine addiction
|
0.02627419
|
Down
|
18
|
Acute myeloid leukemia
|
0.02627419
|
Down
|
19
|
Adherens junction
|
0.03020581
|
Down
|
20
|
Drug metabolism - other enzymes
|
0.03062382
|
Up
|
21
|
Insulin signaling pathway
|
0.0323184
|
Up
|
22
|
African trypanosomiasis
|
0.03438215
|
Up
|
23
|
Dilated cardiomyopathy (DCM)
|
0.0360517
|
Up
|
24
|
Primary immunodeficiency
|
0.03632543
|
Up
|
25
|
Insulin secretion
|
0.03717241
|
Up
|
26
|
GABAergic synapse
|
0.03831094
|
Up
|
27
|
Phospholipase D signaling pathway
|
0.0386556
|
Up
|
28
|
Salmonella infection
|
0.03961531
|
Down
|
29
|
Protein digestion and absorption
|
0.04064128
|
Up
|
30
|
GnRH signaling pathway
|
0.04304219
|
Up
|
31
|
Progesterone-mediated oocyte maturation
|
0.04304219
|
Up
|
32
|
Protein digestion and absorption
|
0.04516019
|
Down
|
33
|
Morphine addiction
|
0.04551314
|
Up
|
34
|
Endocrine resistance
|
0.0467747
|
Up
|
35
|
Hematopoietic cell lineage
|
0.04706952
|
Down
|
36
|
Core gene network and Relevant miRNA analysis
Core network analysis yielded 40 core genes, of which the gene MDFI had the largest number of connections, indicating that it was in the most central position (Fig. 4, Table 5). Relevant miRNA analysis yielded 101 related miRNAs, among which miR-335-5p was the most connected related miRNA (Fig. 5, Table 6).
Table 5. The Correspondence and data source of the Core genes.
Gene 1
|
Protein 1
|
Gene 2
|
Protein 2
|
Relationship
|
Experiment
|
Source
|
MDFI
|
Q99750
|
GREM1
|
O60565
|
protein interaction
|
two hybrid array
|
PMID:unassigned1304
|
MDFI
|
Q99750
|
ZNF408
|
Q9H9D4
|
protein interaction
|
two hybrid array
|
PMID:19060904
|
MDFI
|
Q99750
|
ZNF408
|
Q9H9D4
|
protein interaction
|
two hybrid array
|
PMID:unassigned1304
|
MDFI
|
Q99750
|
ZNF408
|
Q9H9D4
|
protein interaction
|
two hybrid pooling approach
|
PMID:16189514
|
MDFI
|
Q99750
|
RIPPLY1
|
Q0D2K3
|
protein interaction
|
two hybrid array
|
PMID:unassigned1304
|
MDFI
|
Q99750
|
ADRA2C
|
P18825
|
protein interaction
|
two hybrid array
|
PMID:unassigned1304
|
SSX2IP
|
Q9Y2D8
|
ZNF408
|
Q9H9D4
|
protein interaction
|
two hybrid array
|
PMID:unassigned1304
|
IL2RG
|
|
PRL
|
|
activation
|
|
pathway_id:hsa04630
|
PRL
|
|
IL2RG
|
|
activation
|
|
pathway_id:hsa04630
|
LEF1
|
|
CTNND2
|
|
inhibition
|
|
pathway_id:hsa04310
|
CTNND2
|
|
LEF1
|
|
inhibition
|
|
pathway_id:hsa04310
|
CTNND2
|
Q9UQB3
|
TTR
|
P02766
|
protein interaction
|
two hybrid
|
PMID:21900206
|
ZNF408
|
Q9H9D4
|
MDFI
|
Q99750
|
protein interaction
|
two hybrid array
|
PMID:19060904
|
ZNF408
|
Q9H9D4
|
MDFI
|
Q99750
|
protein interaction
|
two hybrid array
|
PMID:unassigned1304
|
ZNF408
|
Q9H9D4
|
MDFI
|
Q99750
|
protein interaction
|
two hybrid pooling approach
|
PMID:16189514
|
ZNF408
|
Q9H9D4
|
SSX2IP
|
Q9Y2D8
|
protein interaction
|
two hybrid array
|
PMID:unassigned1304
|
RIPPLY1
|
Q0D2K3
|
MDFI
|
Q99750
|
protein interaction
|
two hybrid array
|
PMID:unassigned1304
|
ADRA2C
|
P18825
|
MDFI
|
Q99750
|
protein interaction
|
two hybrid array
|
PMID:unassigned1304
|
PDYN
|
|
ATF4
|
|
activation expression
|
|
pathway_id:hsa05030
|
PDYN
|
|
ATF4
|
|
activation expression
|
|
pathway_id:hsa05031
|
RAB33A
|
Q14088
|
RTN4
|
Q9NQC3
|
protein interaction
|
two hybrid pooling approach
|
PMID:16189514
|
RTN4
|
Q9NQC3
|
RAB33A
|
Q14088
|
protein interaction
|
two hybrid pooling approach
|
PMID:16189514
|
CFAP58
|
Q5T655
|
RIPPLY1
|
Q0D2K3
|
protein interaction
|
two hybrid array
|
PMID:unassigned1304
|
RSPH14
|
Q9UHP6
|
TRIM69
|
Q86WT6
|
protein interaction
|
two hybrid array
|
PMID:unassigned1304
|
TRIM69
|
Q86WT6
|
RSPH14
|
Q9UHP6
|
protein interaction
|
two hybrid array
|
PMID:unassigned1304
|
HTR2C
|
P28335
|
RTN4
|
Q9NQC3
|
protein interaction
|
ubiquitin reconstruction
|
PMID:28298427
|
SLC2A4
|
|
ADIPOQ
|
|
indirect relationship
|
|
pathway_id:hsa04930
|
TG
|
|
ATF4
|
|
activation expression
|
|
pathway_id:hsa04918
|
ADIPOQ
|
Q15848
|
FASN
|
P49327
|
protein interaction
|
two hybrid array
|
PMID:unassigned1304
|
AFDN
|
|
SSX2IP
|
|
binding/protein interaction
|
|
pathway_id:hsa04520
|
SSX2IP
|
|
AFDN
|
|
binding/protein interaction
|
|
pathway_id:hsa04520
|
SSX2IP
|
Q9Y2D8
|
ZNF408
|
Q9H9D4
|
protein interaction
|
two hybrid array
|
PMID:unassigned1304
|
ARID2
|
Q68CP9
|
CD14
|
P08571
|
protein interaction
|
anti tag coimmunoprecipitation
|
PMID:26496610
|
CD14
|
P08571
|
ARID2
|
Q68CP9
|
protein interaction
|
anti tag coimmunoprecipitation
|
PMID:26496610
|
CD14
|
P08571
|
CD81
|
P60033
|
protein interaction
|
fluorescent resonance energy transfer
|
PMID:11745332
|
ATF4
|
P18848
|
CEP83
|
Q9Y592
|
protein interaction
|
two hybrid array
|
PMID:unassigned1304
|
ATF4
|
P18848
|
TTR
|
P02766
|
protein interaction
|
two hybrid pooling approach
|
PMID:16189514
|
TTR
|
P02766
|
ATF4
|
P18848
|
protein interaction
|
two hybrid pooling approach
|
PMID:16189514
|
CEP83
|
Q9Y592
|
ATF4
|
P18848
|
protein interaction
|
two hybrid array
|
PMID:unassigned1304
|
ATXN2
|
Q99700
|
PABPC1
|
P11940
|
direct interaction
|
x-ray crystallography
|
PMID:20181956
|
ATXN2
|
Q99700
|
PABPC1
|
P11940
|
protein interaction
|
surface plasmon resonance
|
PMID:20181956
|
PABPC1
|
P11940
|
ATXN2
|
Q99700
|
direct interaction
|
x-ray crystallography
|
PMID:20181956
|
PABPC1
|
P11940
|
ATXN2
|
Q99700
|
protein interaction
|
surface plasmon resonance
|
PMID:20181956
|
CCR10
|
P46092
|
S100A10
|
P60903
|
protein interaction
|
pull down
|
PMID:26941067
|
S100A10
|
P60903
|
CCR10
|
P46092
|
protein interaction
|
proximity ligation assay
|
PMID:26941067
|
CD81
|
P60033
|
CD14
|
P08571
|
protein interaction
|
fluorescent resonance energy transfer
|
PMID:11745332
|
COBLL1
|
Q53SF7
|
DLG3
|
Q92796
|
protein interaction
|
anti tag coimmunoprecipitation
|
PMID:26496610
|
DLG3
|
Q92796
|
COBLL1
|
Q53SF7
|
protein interaction
|
anti tag coimmunoprecipitation
|
PMID:26496610
|
CYP17A1
|
|
ATF4
|
|
activation expression
|
|
pathway_id:hsa04927
|
CYP17A1
|
|
ATF4
|
|
activation expression
|
|
pathway_id:hsa04934
|
CYP17A1
|
|
CYP2E1
|
|
compound
|
|
pathway_id:hsa00140
|
CYP17A1
|
|
NR5A1
|
|
activation expression
|
|
pathway_id:hsa04927
|
CYP17A1
|
|
NR5A1
|
|
activation expression
|
|
pathway_id:hsa04934
|
CYP2E1
|
|
CYP17A1
|
|
compound
|
|
pathway_id:hsa00140
|
CYP2E1
|
|
EPHX1
|
|
compound
|
|
pathway_id:hsa00980
|
NR5A1
|
|
CYP17A1
|
|
Activation expression
|
|
pathway_id:hsa04927
|
NR5A1
|
|
CYP17A1
|
|
activation expression
|
|
pathway_id:hsa04934
|
CES1
|
|
CYP2E1
|
|
compound
|
|
pathway_id:hsa00983
|
EPHX1
|
|
CYP2E1
|
|
compound
|
|
pathway_id:hsa00980
|
GREM1
|
O60565
|
MDFI
|
Q99750
|
protein interaction
|
two hybrid array
|
PMID:unassigned1304
|
Table 6. The Correspondence and data source of miR-335-5p
MiRNA name
|
Target gene name
|
Experimental verification method
|
PMID
|
miR-335-5p
|
ABCC8
|
Microarray
|
18185580
|
miR-335-5p
|
ACE
|
Microarray
|
18185580
|
miR-335-5p
|
ADCY6
|
Microarray
|
18185580
|
miR-335-5p
|
ADGRE3
|
Microarray
|
18185580
|
miR-335-5p
|
AFDN
|
Microarray
|
18185580
|
miR-335-5p
|
CD14
|
Microarray
|
18185580
|
miR-335-5p
|
CFAP58
|
Microarray
|
18185580
|
miR-335-5p
|
CPEB4
|
Microarray
|
18185580
|
miR-335-5p
|
CYP2E1
|
Microarray
|
18185580
|
miR-335-5p
|
DLG3
|
Microarray
|
18185580
|
miR-335-5p
|
ESRP1
|
Microarray
|
18185580
|
miR-335-5p
|
EXD3
|
Microarray
|
18185580
|
miR-335-5p
|
GABBR1
|
Microarray
|
18185580
|
miR-335-5p
|
MACF1
|
Microarray
|
18185580
|
miR-335-5p
|
MDFI
|
Microarray
|
18185580
|
miR-335-5p
|
MLN
|
Microarray
|
18185580
|
miR-335-5p
|
MYOD1
|
Microarray
|
18185580
|
miR-335-5p
|
MYOT
|
Microarray
|
18185580
|
miR-335-5p
|
NCDN
|
HITS-CLIP
|
27418678
|
miR-335-5p
|
PNLIP
|
Microarray
|
18185580
|
miR-335-5p
|
PPP1R3D
|
Microarray
|
18185580
|
miR-335-5p
|
PTK2B
|
Microarray
|
18185580
|
miR-335-5p
|
SLC2A4
|
Microarray
|
18185580
|
miR-335-5p
|
SLC35A5
|
HITS-CLIP
|
23313552
|
miR-335-5p
|
SLC7A9
|
Microarray
|
18185580
|
miR-335-5p
|
SPTB
|
Microarray
|
18185580
|
miR-335-5p
|
TRIM40
|
Microarray
|
18185580
|
miR-335-5p
|
TRIM69
|
Microarray
|
18185580
|
miR-335-5p
|
TSPAN15
|
Microarray
|
18185580
|
miR-335-5p
|
ZNF609
|
Microarray
|
18185580
|
Real-time RT-PCR
In order to verify the reliability of the microarray results, the expression levels of the seven mRNAs (GCNT7, NCDN, EXD3, MUC13, PPP1R3D, NPHP3 and CD81) detected by RT-PCR were equivalent to the microarray data. And the difference of mRNA expression between the degenerative meniscus group and the normal meniscus group was statistically significant (P < 0.05) (Table 7).
Table 7. Primer and differential expression of mRNA
Gene
|
Primer( 5' - 3')
|
Sham
(x±s,n=7)
|
OA
(x±s,n=7)
|
P-value
|
GAPDH
|
F:5’ TCTCTGCTCCTCCCCGTT 3’
R:5’ CGGCCAAATCCGTTCACT 3’
|
|
|
|
GCNT7
|
F:5' GGCTTACACTGGCTTTAGGAG 3'
|
2.41±0.80
|
5.92±1.44
|
0.000
|
R:5' AGTTTGGCTTGTCTTGGATTTA 3'
|
NCDN
|
F:5' AGACCTGCTGTCACATCTTCCTC 3'
|
0.47±0.33
|
3.41±1.44
|
0.000
|
R:5' AGCGACGCCATCAGAGTGTT 3'
|
EXD3
|
F:5' GCATTTCAACGTGCGTGTCA 3'
|
0.26±0.17
|
1.33±0.41
|
0.000
|
R:5' CGAGCTGCTTCATCATGTCCTT 3'
|
MUC13
|
F:5' ATCTGCCAAGCGTGTCCATT 3'
|
0.02±0.02
|
0.14±0.11
|
0.014
|
R:5' TGCAATCACCAGGCTGAGAA 3'
|
PPP1R3D
|
F:5' GTCAAGGTGTTCAACGCGGG 3'
|
3.33±2.39
|
14.75±3.87
|
0.000
|
R:5' GCCAGTCCGAGAAGGTGTAG 3'
|
NPHP3
|
F:5' TGAACTGGGCGTGCTCTACTA 3'
|
1.97±0.94
|
5.04±1.72
|
0.001
|
R:5' TGCTCGTCTCCGAATGTCTAA 3'
|
CD81
|
F:5' ACTACCAGCCTCCTCTACCTG 3'
|
47.33±53.47
|
225.55±99.88
|
0.001
|
R:5' ACAGGCAAAGAGGATCACGA 3'
|
F: forword primer; R: reverse primer