CN108653738A - Applications of the miR-145 and MKK4 in treating or preventing cartilage damage, regression and osteoarthritis - Google Patents
Applications of the miR-145 and MKK4 in treating or preventing cartilage damage, regression and osteoarthritis Download PDFInfo
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Abstract
The present invention relates to applications of the miR 145 and MKK4 in treating or preventing cartilage damage, regression and osteoarthritis.Specifically, the present invention relates to the expression of up-regulation miR 145 or active reagent and/or lowering expression or active the reagent cartilage degeneration caused by preparing the degradation for inhibiting cartilage cell epimatrix, relief from osteoarthritis of MKK4 or treating or alleviate the application in cartilage damage or the drug of osteoarthritis.
Description
Technical field
The present invention relates to applications of the miR-145 and MKK4 in treating or preventing cartilage damage, regression and osteoarthritis.
Background technology
Osteoarthritis (OA) is one kind using synovitis, articular cartilage retrogression pathological changes and periarticular osteoproliferation as pathology
The chronic progressive osteoarthropathy of feature, although osteoarthritis influences entire joint structure, including soft from articular cartilage onset
Muscle outside bone sending down the fishbone, ligament, synovial membrane, capsular ligament and joint finally leads to joint deformity and work(because of articular cartilage whole depigmentation
It can lose.
Articular cartilage is a kind of rich in extracellular matrix, the tissue without blood vessel, impassivity, and cartilage cell only accounts for its volume
2%-3%, the cartilage matrix of remaining horn of plenty include mainly Type Ⅱ collagen, high molecular aggrecan (aggrecan)
And extremely hydrophilic hyaluronic acid.In osteoarthritis early stage, articular cartilage shows of short duration self-regeneration behavior, including life
The long factor, such as bone morphogenesis protein-2 (BMP-2), insulin-like growth factor-i (IGF-1), bone morphogenesis protein-7
(BMP-7), the synthesis of inhibin β A/ activins (TGF superfamily members) and cartilage epimatrix increases, and mainly has II type glue
Original, type Ⅳ collagen, Ⅸ Collagen Type VI, Ⅺ Collagen Type VI, aggrecan.But with the aggravation of osteoarthritis pathology process, divide
The cell factor of solution property sharply increases, while inducing a large amount of extracellular matrix degrading enzyme and generating, and cartilaginous tissue is eventually striking to degrade.Normally
The performance of cartilaginous tissue function needs cartilaginous tissue to be in a metabolic homeostasis, i.e., with Col2a1 (Type Ⅱ collagen), Aggrecan
(proteoglycans), hyaluronic acid synthesis based on anabolism with MMP3 (matrix metalloproteinase 3), MMP13 (matrix metals
Protease 13), the catabolism based on Adamts-5 (depolymerizing protein sample metalloprotein 5) secretions be in the state of dynamic equilibrium.
The ECM segments that the degradation of early stage cartilage matrix generates, which occur, in osteoarthritis (OA) can stimulate synovial cell to generate synovial membrane
Inflammation, to discharge a large amount of inflammatory factors to articular cavity, research shows that play a significant role in cartilage homeostasis destructive process
Inflammatory factor has TNF-α, IL-1 β, IL-6, IL-8, IL-17 etc., these inflammatory factors by activate cartilage cell in NF- κ B,
The signal paths such as MAPK, cause the transcription factors such as p-P65, p-c-Jun and p-ATF2 enters core, to inhibit Sox-9,
The isogenic expression of Col2a1, Aggrecan, while opening the isogenic expression of MMP3, MMP13, Adamts5, and in the time and
Dose-dependence is further exacerbated by the destruction of cartilage matrix.The activation of the Inflammatory Pathways such as NF- κ B, MAPK is made in Cascaded amplification
With, therefore the target molecule for intervening inflammatory reaction should be the molecules upstream in Inflammatory Pathway, such as Traf3, Traf6, TAB2,
IKK α and MAP kinase kinases (MKKs).Inflammatory factor signal paths such as NF- κ B, MAPK in activating cartilage cell
While, it can also cause a series of change of miRNA expressions.
Invention content
The application first aspect provide up-regulation miR-145 expression or active reagent and/or lower MKK4 expression or
Active reagent prepare cartilage degeneration caused by inhibiting the degradation of cartilage cell epimatrix, relief from osteoarthritis or treatment or
Alleviate the application in the drug in osteocartilaginous and subchondralo bone injury or arthritis.
In one or more embodiments, the reagent is nucleic acid molecules, carbohydrate, lipid or small molecule chemical combination
Object.
In one or more embodiments, the expression that the reagent of the expression of the up-regulation miR-145 is miR-145 carries
Body.
In one or more embodiments, the reagent for lowering MKK4 expression is the carrier for knocking out MKK4.
In one or more embodiments, the osteoarthritis be spontaneous osteoarthritis, joint caused by aging not
Stablize the osteoarthritis of induction, the osteoarthritis of tears of anterior cruciate ligament induction or sections inner side Meniscectomy and inside pair is tough
Osteoarthritis with cross-section induction.
The application second aspect provides the reagent for detecting miR-145 expressions and/or detects the expression water of MKK4
Application of the flat reagent in the kit for preparing monitoring cartilage degeneration, cartilage damage or osteoarthritis.
In one or more embodiments, the reagent for detecting MKK4 expressions includes detection MKK4mRNA
The reagent used during the reagent and detection MKK4 protein contents that are used in horizontal process.
The application third aspect, which provides, a kind of inhibiting cartilage caused by the degradation of cartilage cell epimatrix, relief from osteoarthritis
Regression or treatment or the method for alleviating cartilage damage or osteoarthritis, the method includes either one or two following steps
Combination:
(a) expression of miR-145 in object or the step of activity are raised;With
(b) expression of MKK4 in object or the step of activity are lowered.
In one or more embodiments, the method includes giving the object:Raise the expression or work of miR-145
Property reagent and/or lower MKK4 expression or active reagent.
In one or more embodiments, the reagent is nucleic acid molecules, carbohydrate, lipid or small molecule chemical combination
Object.
In one or more embodiments, the expression that the reagent of the expression of the up-regulation miR-145 is miR-145 carries
Body.
In one or more embodiments, the reagent for lowering MKK4 expression is the carrier for knocking out MKK4.
The application fourth aspect provides a kind of method for diagnosing osteoarthritis or monitoring osteoarthritic condition development, the side
Method includes detecting the expression step of miR-145 and/or MKK4 in object body fluid.
In one or more embodiments, the detection MKK4 expressions include detection MKK4mRNA levels and detection
Camk2d albumen.
In one or more embodiments, the mRNA is detected using Northern hybrid methods, RT-PCR or RNA-seq
Level.
In one or more embodiments, the content of albumen is detected using Western blot or ELISA.
In one or more embodiments, the body fluid is peripheral blood.
Description of the drawings
Fig. 1:TNF-α inhibits cartilage cell's anabolism, while promoting catabolism.(A) rat OA models are grouped:It does evil through another person
Art group (sham groups, 6), OA groups (taking knee joint respectively at hand 15 days after operation, 30 days, 45 days, 60 days, 90 days), qRT-PCR inspections
It surveys several inflammatory factors (IL-1 β, TNF-α, IL-6, IL-10, IL-17A, IL-22) and vegf expression is horizontal.(B)TNF-α
(10ng/ml) handles rat primary cartilage cell 24 hours, qRT-PCR detect sox-9, col2a1, aggrecan, MMP-3,
The expression of MMP-13 and Adamts-5, GAPDH is as internal reference.(C) TNF-α (10ng/ml) stimulation cartilage cell 15 minutes,
The phosphorus of Western blot analysis NF- κ B and MAPK signal path downstream molecules (JNK, p38, Erk, p65, c-Jun and ATF2)
Acidification is horizontal.(D) TNF-α (10ng/ml) processing cartilage cell 48 hours, Westernblot analyzes sox-9, col2a1, MMP-
3, the expression of MMP-13, Adamts-5 and COX-2, GAPDH is as internal reference.(E) TNF-α (10ng/ml) processing cartilage is thin
Born of the same parents' different time (0 day, 12 hours, 1 day, 2 days, 3 days, 5 days), Western blot analysis sox-9, col2a1, MMP-3,
The expression of MMP-13, Adamts-5 and COX-2 change.(F) various concentration TNF-α (0,1,5,10,20,40ng/ml)
Handle cartilage cell 48 hours, Western blot analyze sox-9, col2a1, MMP-3, MMP-13, Adamts-5 and COX-2
Expression variation.(G) following grouping is done to cartilage cell respectively and does respective handling:DMSO groups, TNF-α processing group
(DMSO groups, BAY11-7082 groups, SP600125 groups, SB203580 groups and PD98059 groups), qRT-PCR detections sox-9,
The expression of col2a1, aggrecan, MMP-3, MMP-13 and Adamts-5.(H) mapk pathway inhibitor pre-processes cartilage
Cell 2 hours, TNF-α handle 48 hours, Westernblot analyze sox-9, col2a1, MMP-3, MMP-13, Adamts-5 and
The expression of COX-2.(I) TNF-α processing cartilage cell 48 hours, cellular immunofluorescence detects col2a1, MMP-3, MMP-
13, Adamts-5 protein levels.Scale, 20 μm.(J) TNF-α processing cartilage cell 20 minutes, cellular immunofluorescence detects p-
P65, p-c-Jun and p-ATF2's enters core situation.Scale, 10 μm.*P<0.05,**P<0.01,***P<0.001.NS, without significantly
Sex differernce.All P values are based on Student ' s test.Data are indicated with mean+SD, represent 3 independent experiments.
Fig. 2:TNF-α inhibits the expression of miR-145 in cartilage cell.(A) rat OA models are grouped:Sham-operation group (sham
Group, 9), OA groups (taking knee joint respectively at hand 15 days after operation, 30 days, 45 days, 60 days, 90 days, 6-9 is only), OA+CC-5013 groups
(7-9 is only), ELISA detects TNF-α and the content of C2C in serum.(B) caused by microarray detection TNF-α stimulation cartilage cell
MiRNA express spectras change, and thermal map shows the miRNA of maximum 15 miRNA and 18 downwards of modulation.(C) qRT-PCR is tested
Demonstrate,prove the expression variation of TNF-α stimulation lower miR-92a, miR-23b and miR-145.(D) TNF-α of various concentration acts on cartilage
Influence of the cell to the expression of miR-92a, miR-23b and miR-145.(E) qRT-PCR detects patient's OA cartilaginous tissue
The differential expression of miR-145 in (10) and normal articular cartilage (10), elisa assay patient OA cartilaginous tissue and normal soft
TNF-α content in bone tissue, the morphological differences of H&E staining analysis patient OA cartilaginous tissue and normal articular cartilage, scale, 20 μ
m.(F) TNF-α and miR-145 correlation analysis in patient's OA cartilaginous tissue.(G) the OA rat knee joints cartilage groups of operation induction
Knit middle TNF-α and miR-145 correlation analysis.(H) qRT-PCR detections are from the soft of sham groups, OA groups and OA+CC-5013 groups
MiR-145 expressions in bone tissue.(I) NF- κ B and MAPK signal pathway inhibitor (BAY11-7082, SP600125,
SB203580 and PD98059) after 2 hours, TNF-α stimulates 24 hours processing cartilage cell, and qRT-PCR detects miR-145 expression
It is horizontal.(J) p65siRNA and its negative control are transfected respectively into cartilage cell, qRT-PCR and Western blot after 48 hours
Detect p65 expressions.(K) p65siRNA, traf2siRNA, tradd siRNA and negative control transfect thin into cartilage respectively
Born of the same parents, Western blot detect p65 and p-p65, and GAPDH is as internal reference.(L) p65siRNA, traf2siRNA, tradd
SiRNA and negative control are transfected respectively into cartilage cell, TNF-α stimulation are given after 48 hours, qRT-PCR is detected after 24 hours
MiR-145 expressions.*P<0.05,**P<0.01,***P<0.001.NS, there was no significant difference.All P values are based on
Student’s test.Data are indicated with mean+SD, represent 3 independent experiments.
Fig. 3:MiR-145 inhibits the generation of cartilage matrix degrading proteinase caused by TNF-α.(A) utilize RNAiMax with
By miR-145 analogs, inhibitor is transfected into cartilage cell the final concentration of 20nM, extracts total serum IgE, qRT- within 24 hours after transfection
PCR detects miR-145 expressions, and U6 is as internal reference.(B) after transfecting 24 hours, annexinV-FITC/ propidium iodides are carried out
Double dyes, flow cytometer showed.(C) flow cytometer showed quantitative result.(D) miR-145 analogs are transfected, 24 hours after inhibitor, TUNEL dyes
Color is observed and is taken pictures under laser confocal microscope.Scale, 10 μm.(E) respectively by miR-145 analogs, inhibitor transfect into
Cartilage cell gives TNF-α stimulation for 24 hours after transfection, and total serum IgE and total protein, Western blot detections are extracted after 24 hours
Sox-9, col2a1, MMP-3, MMP-13, Adamts-5 and COX-2.(F) qRT-PCR detects MMP-3, MMP-13, Adamts-4
And the expression of Adamts-5.(G) protein expression level of cellular immunofluorescence detection MMP-3, MMP-13 and Adamts-5.
Scale, 50 μm.(H) respectively by miR-145 analogs, inhibitor is transfected into cartilage cell, is given respectively within 24 hours after transfection
TNF-α stimulates different time (0 hour, 3 hours, 6 hours, 12 hours), qRT-PCR to detect MMP-3, MMP-13 and Adamts-5
Expression.*P<0.05,**P<0.01,***P<0.001.NS, there was no significant difference.All P values are based on Student ' s
test.Data are indicated with mean+SD, represent 3 independent experiments.
Fig. 4:3 ' UTR regions of MKK4 contain the binding site of miR-145.(A) utilize RNAiMax that miR-145 is similar
Object, inhibitor are transfected into cartilage cell, extract total serum IgE and total protein within 48 hours after transfection, qRT-PCR detects map2k4 and expresses water
It is flat.(B) Western blot detect MKK4 expressions.(C) the miR-145 analogs of various concentration gradient are inhibited respectively
Agent transfects cartilage cell's (miR-145 analogs:5nM、10nM、20nM;MiR-145 inhibitor:10nM, 20nM, 40nM), transfection
TNF-α stimulation is given within 24 hours afterwards, total protein is collected after 24 hours, and Western blot detection MKK4 and p-MKK4 express water
It is flat.(D) miR-145 is overexpressed plasmid and struck respectively using Lipofectamine2000 and subtract plasmid transfection into cartilage cell, turned
Total serum IgE and total protein are collected within 48 hours after dye, qRT-PCR detects map2k4 expressions.(E) Western blot detect MKK4
Expression.(F) TNF-α processing cartilage cell's different time points (0,1,3,6,12,24 hour), qRT-PCR detect map2k4
Expression.(G) the miR-145 analogs (5nM, 10nM, 20nM) and its negative control of various concentration are transfected into cartilage respectively
Cell, gives TNF-α stimulation different time (as shown in the figure) for 24 hours after transfection, and Western blot detections MKK4 expresses water
It is flat.(H) WT MKK4 3'UTR and MT MKK4 3'UTR luciferase reporter gene carrier schematic diagrames.(I) by WT MKK4
3'UTR, MT MKK4 3'UTR luciferase reporter genes carriers and empty carrier respectively with miR-145 analogs, inhibitor and
Its negative control cotransfection in human osteosarcoma cell line SW1353, fluorescence intensity after 24 hours.(J) different from SD rats
(heart, liver, spleen, lung, kidney, small intestine, brain, muscle, tendon, cartilage, bone and synovial tissue) is organized to extract total serum IgE, qRT-PCR detections
Map2k4 expressions.(K) total protein, Western are extracted from patient's OA cartilaginous tissue and self normal articular cartilage respectively
Blot detects MKK4, p-MKK4, p-c-Jun, p-ATF2 and COX-2 expression, and GAPDH is as internal reference.(L) by cross-section interior
Collateral ligaments and unstable (DMM) OA rat models of meniscus of robert's ligament structure operation induction, sham groups are to do evil through another person
Art group is drawn materials for 2 months after operation, paraffin embedding, slice, and carries out histologic analysis, and immunohistochemistry detects MKK4 and p-MKK4
Level, scale, 50 μm.(M) patient's OA cartilaginous tissue and self normal articular cartilage, paraffin embedding, slice, immunohistochemistry inspection are taken
It is horizontal to survey MKK4, scale, 50 μm.*P<0.05,**P<0.01,***P<0.001.NS, there was no significant difference.All equal bases of P values
In Student ' s test.Data are indicated with mean+SD, represent 3 independent experiments.
Fig. 5:The phosphorylation of JNK, p38 caused by miR-145 negative regulation TNF-α.(A) utilize RNAiMax by miR-145 classes
Transfected respectively into cartilage cell like object and its negative control, give within 48 hours after transfection TNF-α stimulation different time points (0,15,
30,60,90,120 minutes), collect total protein, Western blot detection NF- κ B and MAPK access key signal molecules (p-
JNK, Total-JNK, p-Erk, Erk, p-p38, p38, p-p65, p65, I κ B α, p-c-Jun, p-ATF2 and p-MKK4),
GAPDH is as internal reference.(B) it utilizes RNAiMax to transfect miR-145 inhibitor and its negative control into cartilage cell respectively, turns
TNF-α stimulation different time points (0,15,30,60,90,120 minute) are given within 48 hours after dye, total protein, Western are collected
Blot detects NF- κ B and MAPK access key signal molecules (p-JNK, Total-JNK, p-Erk, Erk, p-p38, p38, p-
P65, p65, I κ B α, p-c-Jun, p-ATF2 and p-MKK4), GAPDH is as internal reference.(C) by miR-145 analogs, inhibitor
And its negative control is transfected respectively into cartilage cell, giving within 48 hours after transfection TNF-α stimulates 20 minutes, cellular immunofluorescence inspection
That surveys p-c-Jun and p-ATF2 enters core situation, observes and takes pictures under laser confocal microscope, scale, 10 μm.(D) it uses
SP600125+SB203580 or DMSO processing cartilage cell 2 hours, then distinguishes miR-145 inhibitor and its negative control
Transfect into cartilage cell, TNF-α stimulation is given in transfection after 24 hours, collect total serum IgE after 24 hours, qRT-PCR detect sox-9,
The expression of col2a1 and aggrecan.(E) qRT-PCR detects the expression of MMP-3, MMP-13 and Adamts-5.*P<
0.05,**P<0.01,***P<0.001.NS, there was no significant difference.All P values are based on Student ' s test.Data are with flat
Means standard deviation indicates, represents 3 independent experiments.
Fig. 6:It strikes and subtracts the generation that MKK4 inhibits cartilage matrix degrading proteinase caused by TNF-α.(A) it utilizes
Lipofectamine2000 will be transfected for the 3 of MKK4 siRNA and its negative control into cartilage cell, 48 after transfection respectively
Hour collects total serum IgE and total protein, and qRT-PCR detects map2k4 rna levels, and Western blot detect MKK4 protein levels.
(B) siRNA#1 is transfected into cartilage cell, total serum IgE and total protein is collected after 48 hours, qRT-PCR detects map2k4 rna levels.
(C) Western blot detect MKK4 protein levels.(D) respectively that si-MKK4, si-HGK and its negative control transfection cartilage is thin
Born of the same parents give TNF-α stimulation for 48 hours after transfection, collect total protein after 24 hours, Western blot detections sox-9, col2a1,
MMP-3, MMP-13, Adamts-5 and COX-2 expression.(E) it uses respectively and contains empty carrier (pGLVH1) or sh-MKK4 plasmids
Slow virus infect cartilage cell with different MOI (50,100), collect within 72 hours after infection total serum IgE and total protein, qRT-PCR inspections
Map2k4 rna levels are surveyed, Western blot detect MKK4, p-MKK4 and p-c-Jun protein level.(F) respectively with containing free
The slow-virus infection cartilage cell of carrier (pGLVH1) or sh-MKK4 plasmids, when giving TNF-α for 48 hours after infection stimulates different
Between (0,6,12,24 hour), qRT-PCR detect sox-9, col2a1, aggrecan, MMP-3, MMP-13 and Adamts-5 expression
It is horizontal.(G) respectively with the slow-virus infection cartilage cell containing empty carrier (pGLVH1) or sh-MKK4 plasmids, 48 is small after infection
When give TNF-α stimulation, collect total protein after 24 hours, Western blot detections sox-9, col2a1, MMP-3, MMP-13,
Adamts-5, COX-2, MKK4, p-MKK4, p-c-Jun and p-ATF2 expression, GAPDH is as internal reference.(H) by miR-145
Inhibitor and its negative control are transfected respectively into cartilage cell, after transfection 24 hours respectively with containing empty carrier (pGLVH1) or
The slow-virus infection cartilage cell of sh-MKK4 plasmids gives TNF-α stimulation for 48 hours after infection, total serum IgE is collected after 24 hours,
QRT-PCR detects the expression of MMP-3, MMP-13 and Adamts-5.*P<0.05,**P<0.01,***P<0.001.NS, nothing
Significant difference.All P values are based on Student ' s test.Data are indicated with mean+SD, represent 3 times independently in fact
It tests.
Fig. 7:It strikes and subtracts the generation that HGK inhibits cartilage matrix degrading proteinase caused by TNF-α.(A) use SP600125 or
DMSO handles cartilage cell 2 hours, and TNF-α stimulates different time points (0,15,30,60,90,120 minute), collects total protein,
Western blot detect p-JNK, Total-JNK and p-c-Jun.(B) use SB203580 or DMSO processing cartilage cells 2 small
When, TNF-α stimulates different time points (0,15,30,60,90,120 minute), collects total protein, and Western blot detect p-
P38, p38 and p-ATF2.(C) si-MKK4, si-HGK and its negative control are transfected into cartilage cell respectively, 48 hours after transfection
TNF-α stimulation different time points (0,15,30,60,90,120 minute) are given, total protein is collected, Western blot detect p-
JNK, Total-JNK, p-p38, p38, p-c-Jun, p-ATF2 and p-MKK4.(D) SP600125+ of various concentration is used
SB203580 Combined Treatments cartilage cell (0,2.5,5,10,20,40 μM), TNF-α is given after 2 hours to stimulate 15 minutes, collects
Total protein, Western blot detect p-JNK, Total-JNK, p-p38, p38, p-c-Jun, p-ATF2 and p-MKK4.(E) sharp
It will be transfected into cartilage cell, 48 after transfection for the 3 of HGK siRNA and its negative control respectively with Lipofectamine2000
Hour collects total serum IgE and total protein, and it is horizontal that qRT-PCR detects map4k4RNA.(F) Western blot detect HGK albumen water
It is flat.(G) si-HGK and its negative control are transfected into cartilage cell, give within 48 hours after transfection TNF-α stimulation different time (0,6,
12,24 hours), qRT-PCR detects sox-9, col2a1, MMP-3, MMP-13 and Adamts-5 expression.*P<0.05,**P
<0.01,***P<0.001.NS, there was no significant difference.All P values are based on Student ' s test.Data are with average value ± mark
Quasi- difference indicates, represents 3 independent experiments.
Fig. 8:It is overexpressed the generation that MKK4 promotes cartilage matrix degrading proteinase caused by TNF-α.(A) respectively with containing free
The slow virus of carrier (pEF-1a) or Len-MKK4 plasmids infects cartilage cell with different MOI (50,100), 72 hours after infection
Total serum IgE and total protein are collected, qRT-PCR detects map2k4 rna levels.(B) Western blot detect MKK4, p-MKK4 and
P-c-Jun protein levels.(C) thin with the slow-virus infection cartilage containing empty carrier (pEF-1a) or Len-MKK4 plasmids respectively
Born of the same parents, give within 48 hours after infection TNF-α stimulation different time (0,6,12,24 hour), qRT-PCR detect sox-9, col2a1,
Aggrecan, MMP-3, MMP-13 and Adamts-5 expression.(D) it uses respectively and contains empty carrier (pEF-1a) or Len-MKK4
The slow-virus infection cartilage cell of plasmid gives TNF-α stimulation for 48 hours after infection, total protein, Western is collected after 24 hours
Blot detects sox-9, col2a1, MMP-3, MMP-13, Adamts-5, COX-2, MKK4, p-MKK4, p-c-Jun and p-ATF2
Expression, GAPDH is as internal reference.(E) miR-145 analogs and its negative control are transfected respectively into cartilage cell, transfection
Respectively with the slow-virus infection cartilage cell containing empty carrier (pEF-1a) or Len-MKK4 plasmids after 24 hours, 48 is small after infection
When give TNF-α stimulation, collect total serum IgE after 24 hours, qRT-PCR detects sox-9, col2a1, aggrecan, MMP-3, MMP-
13 and Adamts-5 expressions.*P<0.05,**P<0.01,***P<0.001.NS, there was no significant difference.All equal bases of P values
In Student ' s test.Data are indicated with mean+SD, represent 3 independent experiments.
Fig. 9:It is overexpressed the generation that MKK4 promotes cartilage matrix degrading proteinase caused by TNF-α.(A) zoopery is illustrated
Figure:The male SD rat of 8 week old is taken, medial collateral ligament and robert's ligament are cut off, joint cavity injection is administered after a week, often
Week administration 2 times, continuous injection 8 weeks.(B) sarranine green staining analysis DMSO groups, SP60012 groups, SB203580 group articular cartilages soon
The degradation situation of matrix, scale, 50 μm.(C) injection PBS, miR-145 agonist (miR-145Agomir), miR-145 inhibit
Agent (miR-145Antagomir) experimental group knee joint sarranine green coloration result soon, scale, 50 μm.(D) Len-sh-MKK4 is injected
Slow virus and Len-MKK4 slow virus experimental group knee joints sarranine green coloration result soon, scale, 50 μm.
Specific implementation mode
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment)
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Present document relates to the method for the degradation of regulation and control object cartilage cell epimatrix, this method include regulation and control miR-145 and/or
The expression of MKK4 or the step of activity.
Herein, " object " can be mammal, preferably people.MiR-145 and MKK4 be usually mammal especially
It is the corresponding molecule of people.
The protein sequence and its coded sequence of these molecules can be found from corresponding database such as Genbank.For example,
The sequence of miR-145 can be obtained from miRbase databases, and the number of logging in is MIMAT0000851;The sequence of MKK4 can be from NCBI
Database obtains, and the number of logging in is NC_005109.4.It should be understood that miR-145 and MKK4 from different plant species source are in ammonia
There may be certain differences on base acid sequence or nucleotide sequence, even from these molecules of same species Different Individual
There may be certain sequence differences, but as long as the biological function of these molecules is identical as biological function described herein
Or it is similar, this kind of molecule is included within the scope of the present invention.
The degradation of " regulation and control " cartilage cell epimatrix includes inhibiting degradation and the promotion cartilage cell of cartilage cell epimatrix
The degradation of epimatrix.For needing to inhibit the phenomenon that the degradation of cartilage cell epimatrix, can by raise miR-145 expression or
Activity and/or lower MKK4 expression or activity and realize.It the case where for needing to promote the degradation of cartilage cell epimatrix, can
It is realized by the expression of downward miR-145 or activity and/or by raising expression or the activity of MKK4.
In general, miR-145 and MKK4 expression or active up-regulation or downward can be realized by giving suitable reagent.Example
Such as, it lowers or the reagent of miR-145 and MKK4 gene expression doses is inhibited to may include but be not limited to:Promote miR-145 degradations
Reagent inhibits the reagent of MKK4mRNA translations for the siRNA of MKK4 genes, knocks out the reagent of miR-145 and MKK4 genes,
With the guiding nucleic acid of specific recognition MKK4 genes and reagent etc. to reduce its expression is sheared to it.In certain realities
It applies in scheme, can be knocked out miR-145 and/or MKK4 full genomes by giving targeting vector, to realize expression
It reduces.In certain embodiments, the reagent for lowering the expression of miR-145 is miR-145 inhibitor.In certain embodiments
In, miR-145 inhibitor is the viral vectors (such as slow virus carrier) for the reverse complements for expressing miR-145.
It is the miR-145 that those can specifically bind intracellular mature to be suitable for the invention reverse complements, to
Check the reverse complements of the effect of miR-145.Its antisense complementarity sequence can be designed according to the miR-145 expressed by object
Row.It is suitable for the invention the mammal that is suitable for that viral vectors (such as slow virus carrier) can be well known in the art, especially
It is the viral vectors of human administration.
It can be the respective specific antibody of these albumen to lower or inhibit the reagent of MKK4 protein actives.
Herein, the activity for reducing MKK4 albumen includes making host cell compared with control cell (wild-type cell),
MKK4 albumen activity reduce at least 30%, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%,
At least 90%, or even do not express these albumen completely or can't detect the activity of these albumen completely.
Also its activity can be made to reduce by introducing mutation in MKK4 albumen.Herein, MKK4 albumen activity especially
Refer to the promotion or inhibit cartilage cell that it is played in this central axes MKK4-JNK/p38-c-Jun/ATF-2 described herein
The activity of the degradation of epimatrix.Therefore, in certain embodiments, introduced in the functional domain of MKK4 albumen and cause its corresponding activity
The mutation for weakening or losing.Mutation can be 1 or several even more (such as 10 or more, 20 or more, 30 or more)
The insertion, deletion or substitution of a amino acid.It can make its encoded MKK4 albumen by donation in the reagent of MKK4 genes
Functional domain in exist cause its related biological activities weaken or lose mutation.The sequence of MKK4 genes can be changed in this kind of reagent
Row cause encoded MKK4 albumen to there is corresponding mutation, to the activity or loss of activity weakened.For example, can lead to
The MKK4 genes in the MKK4 genes replacement wild-type cell of homologous recombination technique mutation are crossed, weak activity is expressed so as to cause it
Or inactive MKK4 albumen.
The up-regulation that miR-145 and MKK4 are respectively expressed can be by being overexpressed these molecules by reality in host or host cell
The up-regulation of its existing expression.For example, being suitable for expressing these molecules in host cell using this field conventional technique structure
Expression vector, and be transferred in host cell by conventional method so that expression vector expresses the molecule in host cell, from
And realize the up-regulation of its expression.In certain embodiments, the expression of the upstream gene of these controllable molecules, to improve this
The expression of class molecule.For example, in certain embodiments, the viral vectors of object representation miR-145 mature sequences can be given (such as
Slow virus carrier), to improve expressions of the miR-145 in subject cell.
In general, the raising of protein active refers to compared with wild type, protein active has at least 20%, at least 30%, at least
40%, at least 50%, at least 60% raising;Or compared with wild-type cell, the protein active in host cell has at least
20%, at least 30%, at least 40%, at least 50%, at least 60% raising.
Therefore, it includes giving the object of needs that the present invention, which inhibits the method for the degradation of cartilage cell epimatrix,:Raise miR-
145 expression or active reagent;And/or lower the expression of MKK4 or active reagent.
It includes giving the object of needs that the present invention, which promotes the method for the degradation of cartilage cell epimatrix,:Lower miR-145's
Expression or active reagent;And/or expression or the active reagent of up-regulation MKK4
The reagent of this paper can be nucleic acid molecules, carbohydrate, lipid, micromolecular compound and albumen.Herein, core
Acid molecule can be such as siRNA, expression vector or targeting vector.Micromolecular compound is often referred to using being chemically synthesized
The lower chemical synthesis compound of molecular weight.Albumen includes specific antibody.For example, reagent can be and MKK4 protein-specifics
In conjunction with antibody.Antibody can be polyclonal antibody, monoclonal antibody or scFv.Method well known in the art can be used to prepare
The antibody of MKK4 albumen, especially monoclonal antibody.Commercially available antibody can also be used.In certain embodiments, above-mentioned side of the invention
Method includes being overexpressed the viral vectors (such as adenovirus vector) of miR-145 or being overexpressed the reverse complements of miR-145
Viral vectors (such as adenovirus vector) is injected into articular cavity, to inhibit or promote the degradation of cartilage cell epimatrix.
Mentioned reagent give mode and dosage can be according to different objects, different illnesss and different medicine types
It gives.For example, in certain embodiments, administration route includes but not limited to:Direct naked RNA injection methods;Liposome RNA
Direct injection;Gold coating rna gene rifle blast technique;Breeding unsoundness bacterium or replication defective adenoviral carry Plasmid DNA method;Electricity
Perforation method, etc..The dosage given should be that object can be made to generate the required amount for treating or preventing effect.It can be according to actual treatment
Or prevent situation, divide the reagent or pharmaceutical composition for giving the present invention several times.
When being given in the form of pharmaceutical composition, pharmaceutically acceptable excipient can be also contained in pharmaceutical composition.
For example, when preparing pharmaceutical composition, usually the reagent is mixed with excipient, or with figuration dilution agent or Bao Ke with
In carrier existing for capsule or anther sac, form of nanoparticles.When excipient plays diluent, it can be solid, partly consolidate
The medium of body or fluent material as excipient, carrier or active constituent.Therefore, pharmaceutical composition can be tablet, pill, powder
Agent, solution, syrup, sterilizing injecting solution etc..The example of suitable excipient includes lactose, glucose, sucrose, sorb
Alcohol, mannitol, starch, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, nano particle etc..Pharmaceutical composition may be used also
Contain other ingredients, such as wetting agent, emulsifier, preservative (such as methyl hydroxybenzoate and propyl ester and sweetener.It is above-mentioned each
Amount of the reagent in pharmaceutical composition should treat or prevent effective amount.
In general, the reagent should be that treatment or prevention are a effective amount of in the content of pharmaceutical composition;Specific content is given
Pharmaceutical quantities are contemplated that the factors such as administration route, patient health situation consider.
The method of the degradation of inhibition cartilage cell epimatrix as described herein, which can be used for treating, benefits from the outer base of cartilage cell
The various diseases of the degradation of matter or the various diseases for alleviating the degradation for benefiting from cartilage cell epimatrix, such as relief from osteoarthritis
Caused cartilage degeneration.Therefore, the above method of the present invention can be used for treating cartilage damage, osteoarthritis or relief from osteoarthritis
Symptom.Term " treatment " and " alleviation " have the meaning that this field usually understands.For example, the object state of an illness has to a certain extent
It releives, it is believed that having achieved the purpose that " to alleviate ".Herein, osteoarthritis includes but not limited to spontaneous bone caused by aging
Arthritis, the osteoarthritis of joint instability induction, the osteoarthritis or sections inner side meniscus of tears of anterior cruciate ligament induction
The osteoarthritis of excision and the cross-section induction of medial collateral ligament.
Therefore, in certain embodiments, the present invention provide up-regulation miR-145 expression or active reagent and/or under
The expression or active reagent for adjusting MKK4 are drawn in the drug for the degradation for preparing inhibition cartilage cell epimatrix, relief from osteoarthritis
Application in the drug of the cartilage degeneration risen or treatment or alleviation cartilage damage or the drug of osteoarthritis.
By detecting the expression of miR-145 and/or MKK4 genes in object body fluid, outside also diagnosable and cartilage cell
The relevant disease of degradation of matrix, especially cartilage damage or osteoarthritis, or monitor the progression of the disease of the disease.Therefore, originally
Invention also provides a kind of method for diagnosing osteoarthritis or monitoring osteoarthritic condition development, and the method includes detecting subject
In liquid the step of the expression of miR-145 and/or MKK4.
Expression described in detection object body fluid includes the level and the corresponding protein content of detection for detecting mRNA.It can be used
The method of this field routine carries out the detection.For example, the detection for mRNA, can be used Northern hybrid methods, RT-
PCR or RNA-seq;Western blot or ELISA can be used in detection for protein content.For example, existing examination can be used
Agent box detects the content or level of GAP-associated protein GAP.When detecting protein level, corresponding antibody can be used to carry out ELISA inspections
It surveys.These are all well-known to those skilled in the art.Body fluid can be such as peripheral blood.
Therefore, the reagent for detecting miR-145 expressions is also provided herein and/or for detecting MKK4 expressions
Reagent preparing answering for the kit of the diagnosis and monitoring degradation of cartilage cell epimatrix, cartilage damage or osteoarthritis
With.
Reagent for above-mentioned detection includes detecting the reagent used during its mRNA level in-site and detecting its albumen to contain
The reagent used during amount.This kind of reagent for detection can be nucleic acid molecules, carbohydrate, lipid, small molecule
Compound and albumen (such as antibody).For example, when being detected or being monitored using ELISA, the reagent includes but not limited to phase
Answer the specific antibody of albumen.When detecting or monitoring mRNA level in-site, the reagent includes but not limited to corresponding primer sequence
And/or probe sequence, also include the reagent carried out needed for PCR or other biological experiment, such as corresponding enzyme, solvent and reaction
Object etc..In certain embodiments, this kind of reagent further includes for example for developing the color or other being convenient for quantitatively or qualitatively object
Matter.
In certain embodiments, the disclosure also provides a kind of detection kit, and the detection kit contains:For examining
Survey the reagent of miR-145 expressions and/or the reagent for detecting MKK4 expressions.
Preferably, the reagent for these detections includes detecting the reagent used during its mRNA level in-site and detecting it
The reagent used during protein content, more preferably Northern hybrid methods, RT-PCR or RNA-seq detect mRNA level in-site
And Western blot or ELISA detect reagent used when protein content, including but not limited to primer and probe, and
Such as the reagent needed for progress PCR.
The present invention will be hereafter illustrated in a manner of specific embodiment.The experiment side of actual conditions is not specified in the following example
Method, such as Sambrook usually according to normal condition,《Molecular cloning:Lab guide》(New York, United States:Cold spring harbor laboratory
Publishing house, 1989) condition described in, or carry out according to the normal condition proposed by manufacturer.For the usage and dosage of reagent, remove
It is non-to be otherwise noted, otherwise used according to conventional usage and dosage.
Furthermore, it is to be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and below (such as embodiment)
It can be combined with each other between each technical characteristic of middle specific descriptions, to constitute preferred technical solution.Term used herein,
Unless otherwise stated, its meaning for usually understanding with this field.
One, materials and methods
1, the part meniscus of acquisition and the operation induction of osteoarthritic condition human articular cartilage extracts (PMM) rat OA
The foundation of model
The acquisition of human articular cartilage mostlys come from the kneed total joint replacement operation of patients with osteoarthritis, same patient
Undamaged cartilaginous areas as normal control, this experiment takes 10 patients with osteoarthritis cartilaginous tissues altogether.Ethics is ratified by upper
The extra large attached 9th the People's Hospital Medical Ethics Committee of medical college of university of communications is completed, and all participants obtain informed consent.
The used SD experimental rats of this experiment are purchased from Shanghai Slac Experimental Animal Co., Ltd., the raising of animal and
Experiment flow passes through health science research institute animal welfare to be ratified with administration committee.4% chloral hydrate anesthesia SD rats,
Remove knee joint position hair, 75% alcohol disinfecting;Cut knee joint skin in front, it can be seen that white tendon tissue;It cuts
Tendon is pushed knee joint in the outer part and fixed by muscle on the inside of tendon, and other side integumentary musculature is also fixed, and cut-out inside is secondary tough
Band opens capsular ligament, is subject to and sees medial meniscus;Medial meniscus is provoked with blade, this month plate both ends will be connected
Ligament is cut off, and causes partial meniscectomy;Sham-operation group only opens skin and muscle at knee joint, does not cut off any ligament,
Suture knee joint muscle and skin.One week after surgery joint cavity injection SP600125 (JNK pathway inhibitors, 10 μM)
(S1460;Selleck, USA), SB203508 (P38 pathway inhibitors, 10 μM) (S1076;Selleck,USA)、CC-5013
(TNF-α secretion inhibitor, 10 μM) (S1029;Selleck, USA), miR-145 agonists (50 μM), miR-145 antagonists
The slow virus (1 of (200 μM), unloaded slow virus (Len-C) and expression sh-MKK4 (Len-sh-MKK4) or MKK4 (Len-MKK4)
×109PFU, 20 μ l) or corresponding isometric solvent (DMSO or PBS).2 times a week, continuous injection 7 weeks.
2, SD rat primaries cartilage cells is separately cultured
Sprague Dawley (SD) rat (7 week old or so) that cervical dislocation execution weight is 180g or so, 75%
Alcohol impregnates 5-10 minutes.In superclean bench, the cartilaginous tissue of knee joint and hip joint is detached under aseptic condition, avoids getting
Subchondral bone, PBS is cleaned twice, and is cut into 2mm3-3mm3The fragment of size.0.25%Trypsin-EDTA is used in incubator
(GibcoTM, Invitrogen) 37 DEG C of digestion 10mins, the complete culture solution containing 10% fetal calf serum (FBS) terminate digestion simultaneously
It is cleaned twice with PBS, to remove serum deprivation.In incubator with 0.02% 5 hours of Type Ⅱ collagen enzymic digestion cartilage fragment, during which
It was rocked every 30 minutes once, to accelerate to digest, in the cell inoculation under digesting to 10cm culture dishes.With containing 10%FBS, 50U/
Ml penicillin, the DMEM/F12=1 of 50mg/ml streptomysins (HyClone, Logan, Utah):1(Invitrogen,
Carlsbad, CA) complete culture solution culture, in 37 DEG C, containing 5%CO2Constant incubator in.When cell growth to 90%~
When 95%, culture medium is abandoned in suction, is cleaned twice with PBS, then with 0.25%Trypsin-EDTA (GibcoTM, Invitrogen) 37
DEG C digestion 1 minute, gently pats culture dish and all floats to cell, the DMEM/F12=1 containing 10%FBS in right amount is added later:1
Complete culture solution terminates reaction, is aspirated repeatedly with pipettor several times, and room temperature 300g is centrifuged 5 minutes and collected cell, after abandoning supernatant
The DMEM/F12=1 containing 10%FBS is added:1 complete culture solution be resuspended cell, if then in one pass three ratio plant respectively in
In dry culture dish.The program of cell cryopreservation is:Vitellophag → frozen stock solution (DMSO:DMEM/F12:FBS=1:2:7) it is resuspended thin
40mins → -20 DEG C, 15mins → -80 DEG C, liquid nitrogen container is transferred to after staying overnight by born of the same parents → adjustment cell concentration → 4 DEG C.Or utilize journey
Sequence cooling box (uses the preceding suitable isopropanol of injection):Cell → adjustment cell concentration → -80 are resuspended in vitellophag → frozen stock solution
DEG C, stay overnight → move to liquid nitrogen container.It when cell recovery, is taken out from liquid nitrogen container, screws cryopreservation tube lid, be put into 37 DEG C of water rapidly
Bath.Until dissolving, cell suspension is sucked out, the DMEM/F12=1 containing 10%FBS is slowly added to:1 complete culture solution, and constantly
It rocks.It aspirates repeatedly several times, room temperature 300g centrifuges 5mins, supernatant is abandoned, with the DMEM/F12=1 containing 10%FBS:1 training completely
Sedimentation cell is resuspended in nutrient solution, plants in culture dish.
3, oligonucleotides and plasmid transfection
It is respectively that miR-145 analogs is (identical with miR-145 sequences artificial synthesized using Lipofectamine 2000
Double stranded oligonucleotide acid fragment), miR-145 inhibitor (the single strand oligonucleotide acid fragment with miR-145 sequence complementations), siRNAs
(p65 siRNA, TRADD siRNA, Traf2 siRNA, MKK4 siRNA and HGK siRNA) is transfected into Primary chondrocyte,
The final concentration of 20nM of transfection of wherein miR-145 analogs and miR-145 inhibitor, and the final concentration of 40nM of the transfection of siRNA.
SiRNA uses preceding brief centrifugation, with RNase-free H2O or sterilizing ddH2O is configured to 20 μM of storing liquids (such as
Following table), packing preserves, and avoids multigelation (being no more than 5 times as possible).In experimentation, product is placed on ice.
The configuration reference of 20 μM of storing liquids
siRNA(nmol) | 0.25 | 0.5 | 1 | 2 | 5 | 20 |
Volume of dissolution (μ l) | 12.5 | 25 | 50 | 100 | 250 | 1000 |
SiRNA sequence (SEQ ID NO:45-62):
4, histology and immunohistochemistry
Reagent and instrument used by 4.1 experiments
PBS buffer solution:9g NaCl, 1.44g Na2HPO4·7H2O, 0.25g KH2PO4·2H2O, with 1000mL ultra-pure waters
Configuration, pH value are transferred to 7.2, spare after high-temperature sterilization.4% paraformaldehyde:PBS is prepared, and adjusts pH value to 7.0.12.5%EDTA is de-
Calcium liquid:PBS is prepared, and adjusts pH value to 7.0.Antigen retrieval buffers:A liquid:0.1M citric acids:21.01g citric acids are dissolved in 1L distilled water.B
Liquid:0.1M sodium citrates:29.41g sodium citrates are dissolved in 1L distilled water.9ml A liquid and 41ml B liquid are added in 450ml distilled water
It is configured to working solution.3% hydrogenperoxide steam generator:30% hydrogenperoxide steam generator dilutes 10 times with distilled water.5%BSA:PBS is prepared.
All antibody MKK4 (ab131351;Abcam, Cambridge, UK), p-MKK4 (sc-101795;Santa Cruz), p-c-
Jun(ab13671;Abcam), (9221 p-ATF2;Cell Signaling Technology), p-JNK (9912;Cell
Signaling Technology), p-P38 (ab47363;Abcam), MMP-3 (sc-6839;Santa Cruz), MMP-13
(sc-30073;Santa Cruz) and ADAMTS-5 (sc-83186;Santa Cruz) (being prepared with the PBS containing 5%BSA).DAB
Colour reagent box;Inverted microscope (Nikon, Japan);Paraffin slicing machine (Leica SM2400, Germany);Paraffin wax embedding
(Thermo Shandon, Britain);Infrared drying oven (766-2, Shanghai);Paraffin (Leica, Germany).
4.2HE is dyed and sarranine green dyeing soon
Take out knee joint within postoperative 8 weeks, 4% paraformaldehyde fixes 12 hours, subsequent 12.5%EDTA decalcifications two months, tissue
Dehydration embeds, slice.The HE dyeing of human articular cartilage tissue's row, the green dyeing soon of rat articular tissue row sarranine, neutral gum envelope
Piece, room temperature dries 1-2 days after mounting, in microscopically observation, and carries out OARSI scorings.
4.3 immunohistochemistry
1) slice is placed in 15min in dimethylbenzene I;2) slice is placed in 15min in dimethylbenzene II;3) slice is placed in 100% second
5min in alcohol I;4) slice is placed in 5min in 100% ethyl alcohol II;5) slice is placed in 5min in 95% ethyl alcohol I;6) slice is placed in
5min in 95% ethyl alcohol II;7) slice is placed in 5min in 85% ethyl alcohol;8) slice is placed in 5min in 75% ethyl alcohol;9) PBS rinses
Three times, each 3min;10) slice is immersed in the antigen retrieval buffers for being heated to boiling, keeps 10min;11) natural cooling
Afterwards, PBS rinses three times, each 3min;12) 3% hydrogenperoxide steam generator is added dropwise, incubation 10 minutes is protected from light at room temperature, in blocking
The activity of endogenous peroxidase enzyme;13) PBS rinses three times, each 3min;14) 5%BSA is added dropwise, room temperature closes half an hour;15)
Primary antibody, 4 DEG C overnight.Negative control replaces primary antibody with PBS, remaining step is identical;16) PBS rinses three times, each 3min;17)
The secondary antibody of HRP labels, room temperature 1 hour;18) PBS rinses three times, each 3min;19) PBS, every slice plus 2 drops or 100 are removed
The DAB developing solutions of μ l Fresh, microscopically observation 3-5min;20) color development stopping in tap water is immersed;21) hematoxylin contaminates
Liquid dyes 5min, and tap water rinses;22) 1% hydrochloride alcohol solution breaks up 5s;23) tap water rinses, and returns blue 30min-1h;24)
It is sliced PBS gently rinses;25) slice is placed in 1min in 75% ethyl alcohol;26) slice is placed in 1min in 85% ethyl alcohol;27) it is sliced
It is placed in 2min in 95% ethyl alcohol I;28) slice is placed in 2min in 95% ethyl alcohol II;29) slice is placed in 2min in 100% ethyl alcohol I;
30) slice is placed in 2min in 100% ethyl alcohol II;31) slice is placed in 2min in dimethylbenzene;32) neutral gum mounting;33) mounting
Room temperature dries 1-2 days afterwards, in microscopically observation.
5,3 ' UTR vector constructions of MKK4
Reagent and experimental facilities needed for 5.1 experiments
LB culture mediums:Peptone (tryptone), yeast extract (yeast extract), OXOID companies (English);Limit
Property restriction endonuclease processed, Phusion archaeal dna polymerases connect reaction solution, common archaeal dna polymerase, Fermentas companies;Plasmid extraction
Kit, DNA gel purification kit, DNA product purification kit, DH5 α competent cells, Tiangeng biochemical technology (Beijing)
Co., Ltd;Agarose, PCR instrument, BioWest companies;Nucleic acid-protein analyzer, Eppendorf companies;Multifunctional enzyme mark
Instrument, PE companies;Primer synthesizes, Guangzhou Ribo Bio Co., Ltd.;Sequencing, Suzhou gold only intelligence biotechnology are limited
Company.
5.2 experimental procedure
Using PCR method, according to 3 ' UTR sequence information design its amplimer of MKK4 (people), with 293T cellular genomes
DNA is 3 ' UTR sequences of template PCR amplifications MKK4 genes, using two restriction enzymes of XbaI/XbaI by target gene piece
Section is cloned into GV249 carriers (Shanghai Ji Kai Genetic Biotechnologies Co., Ltd) Dual-Luciferase report carrier, and used draws
Object sequence (SEQ ID NO:It is 63-64) as follows:
5’-GATCGCCGTGTAATTCTAGAACATATTCATGAAATGTGG-3’;
5’-CCGGCCGCCCCGACTCTAGAGCTGTGCAAGCTCTTCTC-3’。
Mutant primer designs:The primer for designing two sequences complete complementary, makes the site that need to be mutated in the interposition of primer
It sets, respectively there are 10-15 base, G/C content >=40%, Tm value >=78 DEG C of primer in both sides.
PCR;Agarose gel electrophoresis;It is tapped and recovered;Digestion;Connection;Conversion;Identification.
6, luciferase reporter gene is tested
Prepare passive 1 × PLB of lysis buffer:5 × Passive lysis buffers (PLB) of 1 times of volume are added to 4 times
In the distilled water of volume.It is uniformly mixed.It is stored in 4 DEG C (≤1 months).LARⅡ:With Luciferase Assay Buffer II
Dissolved freeze-dried powder Luciferase Assay Substrate.It is stored in -20 DEG C (≤1 months).Stop&Reagent:It takes
2.1ml 50×Stop&Substrate is added to 105ml'sBuffer.In the brown of offerIn Reagent bottles, shake 10 seconds.Cell cracking;It is shone with manual or fluorescent equipped with single autosampler
Instrument is detected.
7, cellular immunofluorescence
Training liquid is abandoned in suction, and (day before cell is with 5 × 104A/hole kind is in 24 orifice plates), PBS is washed 2 times;4% paraformaldehyde room temperature
Fix 10 minutes;PBS rinses 2 times (quick), PBS are washed 2 times, each 5 minutes;Confining liquid (5%BSA, 0.3%triton-X) room temperature
Closing 1 hour;4 DEG C of primary antibodies (1:200) dyeing (1%BSA, 0.3%triton-X) PBS is washed 3 times, each 5 minutes overnight;Secondary antibody
It is incubated 2 hours (1:1000) it, is slowly rocked on shaking table;PBS is washed 3 times, each 5 minutes;Hochest33342 (1ug/ml) dyeing 5
Minute, PBS is washed 2-3 times, each 5 minutes;It takes pictures.
8, Apoptosis detects
8.1TUNEL dyeing
1) PBS or HBSS washed once.
If 2) cell pastes loosely, cell can be made to paste more firm with drying sample.
3) cell is fixed 30-60 minutes with 4% paraformaldehyde.
4) it washed once with PBS or HBSS.
5) PBS of the X-100 containing 0.1%Triton is added, ice bath is incubated 2 minutes.
6) it prepares TUNEL and detects liquid
7) it is washed 2 times with PBS or HBSS.
8) 50 μ l TUNEL are added to detect liquid on sample, 37 DEG C are protected from light incubation 60 minutes.Pay attention to:It should be noted in week when incubation
It encloses and is moistened with holdings such as the paper of the sufficient water of leaching or absorbent cotton, to reduce the evaporation that TUNEL detects liquid to the greatest extent.
9) PBS or HBSS is washed 3 times.
10) under the microscope with fluorescence microscopy after anti-fluorescent quenching mounting fluid-tight piece.The excitation wavelength range that can be used is
450-500nm, launch wavelength ranging from 515-565nm (green fluorescence).
8.2 flow cytometer detection Apoptosis
1) cell is collected, ice-cold PBS is washed 2-3 times;
2) 1 × annexin-binding buffer are prepared;
3) the PI working solutions of 100ug/ml are prepared;
4) cell is resuspended with 1 × annexin-binding buffer (concentration is up to 1 × 106);
5) add 5ul 488Annexin V and 1ul 100ug/ml PI;
6) it is incubated at room temperature 15 minutes;
7) after being incubated, 400ul 1 × annexin-binding buffer are added, gently mixing, is put on ice;
8) flow cytomery (530nm and 575nm).
9, slow virus carrier structure and packaging
Len-C, Len-sh-MKK4 and Len-MKK4 are purchased from Shanghai Ji agate biology Co., Ltd, titre 1x109TU/
ml.Liquid is changed after infecting Primary chondrocyte with MOI=50-100 within 12 hours, and corresponding analysis is carried out after continuing culture 48-72 hours
Detection.
Len-sh-MKK4 slow virus builds:
Carrier information:LV3(pGLVH1/GFP+Puro);
The primer sequence is:
5’-GATCCGGCAGATAATGGCAGTTAATTCAAGAGATTAACTGCCATTATCTGCCTTTTTTG-3’(SEQ
ID NO:65);
5’-AATTCAAAAAAGGCAGATAATGGCAGTTAATCTCTTGAATTAACTGCCATTATCTGCCG-3’(SEQ
ID NO:66)。
Len-MKK4 slow virus builds:
Carrier information:LV-5(pEF-1a/GFP+Puro);
The primer sequence is:
5’-TTAAGCTTATGGCGGCTCCGAGCC-3’(SEQ ID NO:67);
5’-CGGGATCCTCAGTCGACATACATGGGA-3’(SEQ ID NO:68)。
10, reverse transcriptase chain reaction and real-time quantitative PCR
1) reverse transcription reaction system is prepared according to below table:
Reagent | Volume |
RNA(500ng) | x |
5 × M-MLV buffer solutions | 2 |
dNTP | 0.5 |
MiR-145RT primers | 1 |
M-MLV RTase | 0.5 |
DEPC water | Total volume complements to 10 μ l |
Reverse transcription PCR program:16 DEG C, 30min --- 42 DEG C, 1h --- 85 DEG C, 5min.
2) cDNA reverse transcription reaction systems are prepared according to below table:
Reagent | Volume |
RNA(1μg) | x |
Random primer | 1 |
DEPC water | Total volume complements to 10 μ l |
After mixing, 70 DEG C, 5min.It is immediately placed on ice, 1min or more.It is continuously added in reaction system:
Reagent | Volume |
5 × M-MLV buffer solutions | 5 |
dNTP | 2 |
M-MLV RTase | 0.5 |
DEPC water | Total volume complements to 25 μ l |
37 DEG C, 1h --- 95 DEG C, 5min.
3) after 10 times of RT product dilutions quantitative fluorescent PCR reaction is done by following reaction condition:
Reagent | Volume |
SYBR Premix Ex Taq Mix(2×) | 5 |
Forward primer | 0.2 |
Reverse primer | 0.2 |
RT products | 1 |
ROX | 0.2 |
dd H2O | 3.4 |
Real-time PCR programs:95 DEG C, 30s --- 95 DEG C, 5s;60 DEG C, 20s;72 DEG C, 30s (40 wheel) --- dissociation
Curve --- 4 DEG C.
The primer sequence (SEQ ID NO:It is 1-44) as follows:
11, western blot
Instrument and reagent used in 11.1 experiments
RIPA lysates (Shen energy lottery industry, Shanghai);PMSF:According to 1:100 ratio is added to RIPA lysates (Shen Nengbo
Coloured silk, Shanghai);BCA protein quantifications kit (PIERCE, Rockford, Ill);BSA protein standard substances (PIERCE,
Rockford,Ill);Microplate reader (Safire 2TM,TECAN);Running buffer:Tris 3.03g,Glycine
14.4g, SDS 1g are dissolved in 1L distilled waters;Transfer buffer:Tris 3.03g, Glycine 14.4g, methanol 200ml,
It is dissolved in 800ml distilled waters, 4 DEG C of pre- cold standbies;TBST buffer:Tris 2.42g, NaCl 8g, are dissolved in 1L distilled waters, are added
1ml Tween-20;Pvdf membrane (Millipore), 0.22 μm;5%BSA (TBST preparations);Primary antibody, secondary antibody:Sox-9(1:
1000,ab26414;Abcam, Cambridge, MA, USA), Col2a1 (1:1000,BS1071;Bioworld
Technology, St.Louis Park, MN, USA), MMP-3 (1:500;sc-6839;Santa Cruz, CA, USA), MMP-
13(1:500;sc-30073;Santa Cruz, CA, USA), Adamts-5 (1:500;sc-83186;Santa Cruz,CA,
USA), (1 COX-2:1000,ab15191;Abcam, Cambridge, MA, USA), JNK, phospho-JNK, Erk,
phosphor-Erk、p38、phospho-p38、p65、phospho-p65、IκBα、phospho-c-Jun、phosphor-
ATF2、MKK4、phospho-MKK4、HGK(1:1000, it all is from Cell Signaling Technology, Danvers,
MA, USA), GAPDH (1:5000, G9545, Sigma-Aldrich), the secondary antibody (1 of HRP- couplings:1000,Cell
Signaling Technology,Danvers,MA,USA);5×loading buffer(sangon);Film (Kodak,
USA);Automatic punching machine (MultiLine 400, USA).
11.2 experimental procedures
Cell abandons supernatant, and precooling PBS is washed one time;RIPA lysates (containing 1%PMSF) are added, crack 15min on ice;With thin
For born of the same parents' scraper by under cell scraper, 12,000rpm centrifuge 15min;It is total protein, -80 DEG C of preservations to take supernatant.BCA method protein quantifications;
SDS-PAGE electrophoresis;Transferring film;Closing;Primary antibody;Wash film;Secondary antibody;Wash film;Colour developing and tabletting.
12, chromatin immune is co-precipitated instrument and reagent used in 12.1 experiments
Dilution buffer:0.01%SDS, 1.1%Triton X-100,1.2mM EDTA, 16.7mM Tris-HCl, pH
8.1,167mM NaCl;SDS lysis buffers, 1%SDS, 10mM EDTA, 50mM Tris, pH 8.1;Less salt cleaning solution:
0.1%SDS, 1%Triton X-100,2mM EDTA, 20mM Tris-HCl, pH 8.1,150mM NaCl;High salt wash liquid:
0.1%SDS, 1%Triton X-100,2mM EDTA, 20mM Tris-HCl, pH 8.1,500mM NaCl;LiCl cleaning solutions:
0.25M LiCl, 1%IGEPAL-CA630,1% deoxycholic acids (sodium salt), 1mM EDTA, 10mM Tris, pH 8.1;TE is buffered
Liquid:24ml of 10mM Tris-HCl,1mM EDTA,pH 8.0.
12.2 experimental procedures
A. it is crosslinked:
10cm culture dishes cell (about 2 × 107It is a), estimate nutrient solution volume, calculating formaldehyde volume, (10ml culture solutions are added
The formaldehyde of 270 μ l 37%, final concentration of that formaldehyde directly 1%) is added in culture solution, room temperature fixes 10min.Addition 10 × sweet
Propylhomoserin (1.25M) room temperature terminates reaction 5min.Culture solution is blotted, ice precooling PBS is added and washes cell 3 times (operating on ice).It is added
PBS 1ml containing protease inhibitors cocktail, cell is scraped off, and is sucked in EP pipes.4 degree, 1000rpm centrifuges 5min
Collect cell fragment.1ml is added and contains the SDS lysates of protease inhibitors cocktail lytic cell 10min on ice.
B. ultrasonic:After cell fully cracks, with EP pipes, 200 μ l/ manage (about 4 × 10 for packing6A cell), ice-water bath
Ultrasound.4 degree of centrifugation 10min of 12000rpm, take supernatant to be pre-chilled in EP pipes in ice.Supernatant can freeze in -80 degree.
C.IP:
Prepare the dilution buffer containing protease inhibitors cocktail, the dilute of 9 times of cell pyrolysis liquid volumes is added
Release buffer solution.The protein A/G agarose of 60 μ l are added, after 4 degree are shaken 1 hour, 3000rpm centrifuges 1min, takes supernatant,
Remove agarose beads.The supernatant after 10% preclear is drawn as Input, 4 degree of preservations, remaining supernatant addition antibody 4
Degree rocks overnight.The protein A/G agarose of 60 μ l are added, 4 degree are rocked 1~2h.Centrifugation, abandons supernatant.Pearl is washed, every time
It rocks after washing 3~5min, 3000rpm centrifuges 1min and collects pearl.Eluted dna/albumen composition.
D. DNA is purified:
DNA/ albumen uncoupling:The 8 μ l 5M NaCl of (including Input) addition in 200 μ l eluents, 65 degree of incubations 4~
5h is stayed overnight.1 μ l RNase A are added and are incubated 30min in 37 degree.4 μ l 0.5M EDTA (pH 8.0), 8 μ l 1M are added
Tris-HCl (pH 6.5), 1 μ l (10mg/ml) Proteinase K, 45 degree of 1~2h of incubation.With DNA Purification Kits DNA.It is pure
The DNA product of change is detected with regular-PCR.
13, enzyme-linked immunosorbent assay
1) prepare:Kit is taken out from refrigerator, room temperature rewarming balances 30 minutes.
2) match liquid:20 times of concentrated cleaning solutions are diluted to former cleaning solution again with distilled water.
3) add standard items and sample to be tested:Sufficient amount of enzyme mark coating plate is taken, is fixed on frame, standard is respectively set
Sample wells, sample to be tested hole and blank control wells, record each hole site, and 50 μ L of standard items are added in standard sample wells;Sample to be tested
10 μ L of sample to be tested are added in Kong Zhongxian, then add 40 μ L of Sample dilution (i.e. 5 times of Sample Dilution);Blank control wells are not added with.
4) it incubates:37 DEG C of water-baths or insulating box incubate 30min.
5) board-washing:Liquid is discarded, is patted dry on blotting paper, cleaning solution is filled it up with per hole, 1min is stood, gets rid of cleaning solution, is absorbed water
It is patted dry on paper, so repeats board-washing 4 times (can also board-washing machine by specification be used to operate board-washing).
6) enzyme mark working solution:50 μ L of enzyme mark working solution are added per hole, blank control wells are not added with.
7) it incubates:Repeat 4 operation.
8) board-washing:Repeat 5 operation.
9) it develops the color:50 μ L of color developing agent A liquid are first added per hole, add 50 μ L of color developing agent B liquid, 37 DEG C are protected from light colour developing 15min.
10) it terminates:It takes out ELISA Plate, 50 μ L of terminate liquid is added per hole, terminate reaction (color turns yellow by blue is vertical).
11) it measures:It is returned to zero with blank well, after termination in 15 minutes, with the light absorption value (OD in each hole of 450nm wavelength measurements
Value).
12) it calculates:According to the concentration of standard items and corresponding OD values, the linear regression equation of standard curve is calculated, then
According to the OD values of sample, corresponding sample concentration is calculated on regression equation, can also be calculated using various application software.
Ultimate density is that practical measurement concentration is multiplied by extension rate.
14, statistical analysis
All experiments are at least in triplicate.Data are with mean+SD (mean ± standard deviation) table
Show.For gene relative expression analysis, the average value of control group is defined as 1.Student ' s t-test are used for two groups of data
It is for statistical analysis.One-way ANOVA are used for for statistical analysis to multi-group data.P<0.05 thinks poor with conspicuousness
It is different.
Two, experimental result
1.TNF- α promote to decompose by activating NF- κ B and MAPK signal paths to inhibit the anabolism of cartilage cell
Metabolism
Several proinflammatory inflammation factors in meniscus shakiness (DMM) OA rat pathogenic processes of operation induction are had detected first
The expression of (IL-1 β, TNF-α, IL-6, IL-10, IL-17A, IL-22) changes.The results show that the IL-1 β in OA generating processes
It is maximum (Fig. 1, A) with the expression ascensional range of TNF-α.
It is separately cultured Primary chondrocyte from rat knee joints and hip joint, and gives TNF-α (10ng/ml) stimulation, knot
Fruit shows that TNF-α can significantly inhibit the expression of sox-9, col2a1 and aggrecan in RNA and protein level, promotes simultaneously
The expression (Fig. 1, B, D, I) of MMP-3, MMP-13, Adamts-5 and COX-2.And the effect of TNF-α present time and dosage according to
Rely (Fig. 1, E, F).Meanwhile TNF-α stimulation cartilage cell can remarkably promote NF- κ B and MAPK signal path downstream molecules
The phosphorylation (Fig. 1, C) of (JNK, p38, Erk, p65, c-Jun and ATF2), and then cause p-p65, p-c-Jun and p-ATF2
Enter core (Fig. 1, J).When giving NF- κ B and MAPK signal pathway inhibitors (BAY11-7082, SP600125, SB203580 respectively
And PD98059) processing after, TNF-α is equal to the anabolic inhibiting effect of cartilage cell and to the facilitation of catabolism
It is apparent to weaken (Fig. 1, G, H).
The expression of 2.TNF- α negative regulations miR-145
Whether TNF-α plays an important role in meniscus shakiness (DMM) OA rat pathogenic processes to probe into operation induction,
As a result one week joint cavity injection TNF-α secretion inhibitor (CC-5013) after surgery shows that CC-5013 can be significantly inhibited
The secretion of TNF-α, and the generation (Fig. 2, A) of C2C (Type Ⅱ collagen catabolite) can be effectively inhibited.This result prompts
Unstable (DMM) the OA rats morbidity of meniscus of induction of performing the operation needs to rely on the generation of TNF-α.
Next, utilizing the microRNA regulated and controled by TNF-α in array screening cartilage cell.It is thin that TNF-α handles cartilage
After born of the same parents 24 hours, 15 microRNA are raised, and 18 by downward (Fig. 2, B).It is maximum to pick out 3 lower modulations
MicroRNA (miR-92a, miR-23b, miR-145) is verified with qRT-PCR, as a result shows miR-23b and miR-145
Significantly to inhibit (Fig. 2, C, D) by TNF-α.Since miR-145 expresses higher in cartilage cell, miR-145 is selected to do
Further research.
Further study show that miR-145 is horizontal (Fig. 2, E) in low expression in patient's OA cartilaginous tissue, and similarly, hand
MiR-145 is also in that low expression is horizontal in the OA rat knee joints cartilaginous tissues of art induction, and after giving CC-5013 processing,
MiR-145 expressions are restored (Fig. 2, H).Most it is interesting that the OA in patient's OA cartilaginous tissue and operation induction is big
MiR-145 is in apparent negative correlativing relation (Fig. 2, F, G) with TNF-α in mouse knee cartilage tissue.
In order to illustrate the specific mechanism that TNF-α negative regulation miR-145 is relied on, with NF- κ B and mapk pathway inhibitor point
Cartilage cell is managed in other places, and compared with other groups, BAY11-7082 can most effectively inhibit TNF-α to make the negative regulation of miR-145
With (Fig. 2, I).In order to verify result above, respectively p65, traf2 and tradd strike subtracting in cartilage cell, as a result be shown
P65 expressions are suppressed 80% or so (Fig. 2, J), in addition, p65, traf2 and tradd's strikes to subtract and can obviously inhibit
P65 phosphorylations (Fig. 2, K) caused by TNF-α, and inhibiting effect (Fig. 2, L) of the TNF-α to miR-145 can be blocked.The above knot
Fruit shows TNF-α by tradd-traf2-p65 axis come the expression of negative regulation miR-145.
3.miR-145 the generation of cartilage matrix degrading proteinase caused by negative regulation TNF-α
It, will in order to probe into whether miR-145 has regulating and controlling effect to the generation of cartilage matrix degrading proteinase caused by TNF-α
MiR-145 analogs, inhibitor and its negative control are transfected into cartilage cell, transfect final concentration of 20nM.As a result transfection is shown
After miR-145 analogs, miR-145 expressions significantly improve in cartilage cell, on the contrary, miR-145 inhibitor then can be bright
The aobvious expression (Fig. 3, A) for inhibiting miR-145.In order to exclude transfection miR-145 analogs, inhibitor influence cartilage cell is survived
As a result possibility shows miR-145 analogs, inhibitor using streaming and TUNEL dyeing detection Transfected cells existing states
Transfect into cartilage cell does not have an any influence (Fig. 3, B, C, D) to cell growth state, and to sox-9, col2a1 and
The no any influence of expression of aggrecan.It is interesting that MMP- caused by TNF-α can be significantly inhibited by being overexpressed miR-145
3, the up-regulation of MMP-13, Adamts-4 and Adamts-5 inhibits miR-145 that can then promote MMP-3, MMP- caused by TNF-α
13, the generation (Fig. 3, E, F, G, H) of Adamts-4 and Adamts-5.Therefore, miR-145 can be with soft caused by negative regulation TNF-α
The generation of bone matrix degradation protease is to inhibit the degradation of cartilage cell epimatrix.
4.miR-145 is directly targeted in MKK4
The generation of cartilage matrix degrading proteinase is specific caused by order to further probe into miR-145 negative regulation TNF-α
Mechanism passes through the possible target genes of Bioinformatics Prediction miR-145.TargetScan database displayings MKK43 '-UTR region
Contain highly conserved miR-145 binding sites (Fig. 4, H).
Next this prediction result is verified.First, miR-145 analogs and inhibitor are transfected respectively thin into cartilage
As a result born of the same parents show that expression of the MKK4 in RNA and protein level can be significantly inhibited by being overexpressed miR-145, on the contrary, inhibiting miR-
145 expression (Fig. 4, A, B) that can raise MKK4.And miR-145 to the inhibiting effect of MKK4 in dose-dependant (Fig. 4,
C).Similarly, miR-145 is overexpressed plasmid transfection into cartilage cell, MKK4 expressions are obviously suppressed, on the contrary, transfection
MiR-145, which strikes, to be subtracted plasmid then and can raise the expression of MKK4 (Fig. 4, D, E).
In order to which definitely for miR-145 to the targeting of MKK4, we further pay close attention to endogenous in cartilage cell
MiR-145 shows that TNF-α can obviously lower miR- in cartilage cell to the regulating and controlling effect of MKK4mRNA in view of result before
145 expression, and miR-145 can inhibit its translation in conjunction with 3 ' UTR regions of MKK4mRNA, therefore speculate TNF-α stimulation
MKK4 levels can be regulated and controled by lowering miR-145.The results show that really may be used within 6 hours with TNF-α stimulation cartilage cell later
With from RNA and protein level up-regulation MKK4 expression, after exogenous transfection miR-145 analogs, TNF-α is to the upper of MKK4 respectively
Tune effect is then suppressed (Fig. 4, F, G) completely.
In order to further verify targetings of the miR-145 to MKK4, construct respectively wild type (WT) MKK4 3'UTR and
Saltant type (MT) MKK4 3'UTR luciferase reporter gene carriers.Respectively by WT MKK4 3'UTR and MT MKK4 3'UTR
Luciferase reporter gene carrier is total in human osteosarcoma cell line SW1353 with miR-145 analogs and negative control respectively
Transfection.Luciferase result shows that miR-145 analogs can significantly inhibit MKK4WT 3'UTR Dual-Luciferase report carriers
Activity, but do not work (Fig. 4, I) to MT MKK4 3'UTR Dual-Luciferase report carriers.The above result shows that miR-145 can
Direct target MKK4 inhibits its expression in post-transcriptional level by being incorporated into the 3'UTR seed regions of MKK4.
In order to assess effects of MKK4 during OA occurrence and development, MKK4 is had detected first in rat different tissues
Expression specificity.The results show that compared to bone tissue, synovial tissue and tendon tissue, articular cartilage height expression MKK4 (Fig. 4,
J).In addition, it has been found that MKK4 and p-MKK4 horizontal significantly raised (Fig. 4, K, M) in patient's OA cartilaginous tissue;Similarly, exempt from
Also significantly raised (the figure of MKK4 and p-MKK4 expressions in the rat OA model cartilaginous tissues of epidemic disease group result display operation induction
4, L).
5.miR-145 inhibits the phosphorylation of JNK and p38 caused by TNF-α
MKK4 is one of MKK family members, and when cell is stimulated by inflammatory factor, phosphorylation occurs for MKK4, and then directly
Phosphorylation simultaneously activates JNK and p38, and the JNK and p38 of phosphorylation distinguish further p-c-Jun and ATF2, p-c-Jun and p-
ATF2 enters the transcription of core regulation and control series of genes.Cartilage matrix degrading proteinase caused by order to illustrate miR-145 regulation and control TNF-α
The specific molecular mechanism generated is overexpressed in cartilage cell, strikes and subtract miR-145 respectively, and subsequent TNF-α stimulates different time
(0,15,30,60,90,120 minute), Westernblot detection NF- κ B and MAPK access key signals molecule (p-JNK,
Total-JNK, p-Erk, Erk, p-p38, p38, p-p65, p65, I κ B α, p-c-Jun, p-ATF2 and p-MKK4).As a result it shows
Show, the phosphorylation of JNK, p38 caused by TNF-α can be significantly inhibited by being overexpressed miR-145, and the phosphorylation level of Erk, p65
It does not substantially change;Subtract the phosphorylation (Fig. 5, A, B) that miR-145 then promotes JNK, p38 caused by TNF-α on the contrary, striking.Equally
Ground, further investigation revealed that, being overexpressed miR-145 can obviously inhibit p-c-Jun's and p-ATF2 to enter core, strike and subtract miR-
145 promote p-c-Jun's and p-ATF2 to enter core (Fig. 5, C).The above result shows that miR-145 mainly passes through in cartilage cell
The phosphorylation of JNK, p38 is inhibited to carry out the generation of cartilage matrix degrading proteinase caused by negative regulation TNF-α.
With inhibitor (SP600125+SB203580) the Combined Treatment cartilage cell of JNK and p38.Subtract as previously mentioned, striking
MiR-145 is to the no any influence of the expression of sox-9, col2a1 and aggrecan, but SP600125+SB203580 is blocked completely
Inhibiting effect (Fig. 5, D) of the TNF-α to sox-9, col2a1 and aggrecan;It strikes and subtracts miR-145 and can cooperate with promotion TNF-α
The up-regulation of caused MMP-3, MMP-13 and Adamts-5, but SP600125+SB203580 processing can block miR-145 completely
Effect (Fig. 5, E).Therefore, miR-145 in cartilage cell mainly by inhibit JNK, p38 phosphorylation come negative regulation TNF-
The generation of cartilage matrix degrading proteinase caused by α inhibits JNK and p38 signal paths miR-145 can be blocked to TNF- completely
α causes the negative regulation effect that cartilage matrix is degraded.
It can inhibit the generation of cartilage matrix degrading proteinase caused by TNF-α 6. striking and subtracting MKK4
In order to further probe into works of the MKK4 played in the cartilage matrix degrading proteinase generation process that TNF-α mediates
With and the miR-145 negative regulation effects of cartilage matrix degradation that TNF-α is mediated whether need to rely on MKK4, design is simultaneously
3 specific siRNAs (#1, i.e. si-MKK4-366 for MKK4 are synthesized;#2, i.e. si-MKK4-701;#3, i.e. si-
MKK4-973).It 3 siRNA are transfected into cartilage cell, can be substantially reduced the expression of MKK4, and siRNA#1 strikes
Decreasing effect rate highest, up to 70% -90% (Fig. 6, A, B, C).Most subtract MKK4 and its upstream signaling molecule HGK it is interesting that striking
With obviously inhibit TNF-α mediate sox-9 and col2a1 downward, and significantly inhibit MMP-3, MMP-13 caused by TNF-α,
The generation (Fig. 6, D) of Adamts-5 and COX-2.
In order to further verify the above experimental result, it will be packaged into slow virus for the shRNA of MKK4, with postoperative infection cartilage
As a result cell shows that the slow virus with sh-MKK4 can significantly strike the expression (Fig. 6, E) of low MKK4.Striking low MKK4 can
The up-regulation of MMP-3, MMP-13, Adamts-5 caused by significantly inhibit TNF-α stimulation different time (6,12,24 hours), but
There is no any influence (Fig. 6, F) to the rna level of sox-9, col2a1 and aggrecan.Result is consistent above, utilizes slow disease
Poison strikes the downward for subtracting sox-9 and col2a1 that MKK4 can partly inhibit TNF-α to mediate, and can significantly inhibit TNF-α and cause
MMP-3, MMP-13, Adamts-5 and COX-2 generation, while can obviously inhibit the phosphorylation of MKK4, c-Jun and ATF2
(Fig. 6, G).It moreover has been found that strike subtract MKK4 can block miR-145 inhibitor to MMP-3, MMP-13 caused by TNF-α completely and
The synergistic effect (Fig. 6, H) of Adamts-5 up-regulations.Above the experimental results showed that the cartilage matrix that TNF-α mediates drops in miR-145
The negative regulation of solution acts on the MKK4 that needs to rely on.
It can inhibit the generation of cartilage matrix degrading proteinase caused by TNF-α 7. striking and subtracting HGK
HGK (Map4k4) belongs to one of MAPK kinase kinases (MAPKKKs) family member, can specific phosphorus
It is acidified and activates MKK4, to activate MKK4 downstream signaling molecules.JNK pathway inhibitors (SP600125) handle cartilage cell can
To significantly inhibit the phosphorylation (Fig. 7, A) of the JNK and c-Jun of TNF-α mediation, p38 pathway inhibitors (SB203580) processing is soft
Osteocyte then can obviously inhibit the phosphorylation (Fig. 7, B) of the p38 and ATF2 of TNF-α mediation, SP600125+SB203580 joints
Stimulation cartilage cell can effectively inhibit the phosphorylation of JNK, p38, MKK4, c-Jun and ATF2, and in dose-dependant (Fig. 7,
D).In view of HGK pecific phosphorylations and activate MKK4, MKK4 and then specific activation JNK and p38, we predict to strike subtract HGK or
MKK4 may have the effect of similar SP600125+SB203580 processing.It is consistent with expected results, it strikes and subtracts HGK or MKK4
To effectively inhibit the phosphorylation of JNK and p38, and then inhibit the phosphorylation (Fig. 7, C) of c-Jun and ATF2.In order to further bright
Effects of the true HGK during cartilage matrix degrading proteinase that TNF-α mediates generates, has designed and synthesized 3 for HGK
Specific siRNA (#1, i.e. si-HGK-491;#2, i.e. si-HGK-2990;#3, i.e. si-HGK-3230).3 siRNA are transfected
Into cartilage cell, it can be substantially reduced the expression of HGK, and siRNA#3 strikes decreasing effect rate highest, up to 85% (Fig. 7, E,
F).Col2a1 and aggrecan caused by TNF-α stimulation different time (6,12,24 hours) can be significantly inhibited by striking low HGK
The up-regulation of MMP-3 and Adamts-5 caused by downward and TNF-α stimulation different time (6,12,24 hours), but to sox-9
(Fig. 7, G) is not influenced with the rna level of MMP-13.
8. being overexpressed the generation of cartilage matrix degrading proteinase caused by MKK4 aggravation TNF-α
According to the above experimental result, thus it is speculated that cartilage caused by TNF-α may be aggravated by being overexpressed MKK4 in cartilage cell
The generation of matrix degrading protease enzyme.It is overexpressed slow virus it is assumed that constructing MKK4 in order to verify this, infection cartilage cell 72 is small
The RNA and protein level of Shi Hou, MKK4 are significantly raised (Fig. 8, A, B).TNF-α stimulation can significantly be aggravated not by being overexpressed MKK4
The up-regulation of MMP-3, MMP-13, Adamts-5 caused by same time (6,12,24 hours), but to sox-9, col2a1 and
The rna level of aggrecan does not have any influence (Fig. 8, C).It can be in albumen it is interesting that being overexpressed MKK4 using slow virus
Level promote TNF-α mediate sox-9 and col2a1 downward, and can significantly aggravate MMP-3, MMP-13 caused by TNF-α,
The generation of Adamts-5 and COX-2, while the phosphorylation (Fig. 8, D) of MKK4, c-Jun and ATF2 can be obviously promoted.In addition, hair
Now being overexpressed MKK4 can block miR-145 analogs to raise MMP-3, MMP-13 and Adamts-5 caused by TNF-α completely
Inhibiting effect, and inhibiting effect (Fig. 8, E) of the TNF-α to sox-9 and col2a1 can be aggravated.Above the experimental results showed that,
The generation of cartilage matrix degrading proteinase caused by TNF-α can be aggravated by being overexpressed MKK4.
9.MiR-145 can alleviate the regression of unstable (DMM) OA cartilage of rats of meniscus of operation induction
The above in vitro results show that miR-145 can be by MKK4-JNK/p38-c-Jun/ATF2 axis come negative regulation TNF-α
The expression of the cartilage matrix degrading proteinase of mediation, in order to which further whether clear miR-145 also plays same work(in vivo
Can, we construct unstable (DMM) rat OA models of meniscus of operation induction, experiment be divided into sham-operation group (n=6-8) and
DMM groups (n=6-8), the latter week DMM groups rat joint cavity of performing the operation inject respectively DMSO, JNK pathway inhibitor (SP60012),
P38 inhibitor (SB203580), PBS, miR-145 agonist (miR-145Agomir), miR-145 inhibitor (miR-
145Antagomir), Len-sh-MKK4 slow virus and Len-MKK4 slow virus (Fig. 9, A).Knee is taken after injecting 2 times, 8 weeks weekly
Joint carries out histologic analysis, and green coloration result shows SP60012, SB203580, miR-145Agomir and Len- to sarranine soon
Sh-MKK4 slow virus can significantly inhibit the regression of OA cartilage of rats, on the contrary, miR-145Antagomir and Len-MKK4
Slow virus then aggravates the regression (Fig. 9, B, C, D) of OA cartilage of rats.
Three, it discusses
The generation of OA is fundamentally due to improper mechanical stress and pro-inflammatory cytokine (IL-1 β and TNF-α performance
Most important effect) Inflammatory Pathway in cartilage cell (mainly NF- κ B and MAPK) is activated, make in MMPs and ADAMTS transcriptions
It adjusts, to break the unbalance of cartilage cell's anabolism and catabolism.Result of study before is consistent, our research
The result shows that IL-1 β and TNF-α are played the part of in the meniscus shakiness rat OA model pathogenic processes for the operation induction that we build
Most important proinflammatory cytokine.IL-1 β are related with cartilage destruction, and TNF-α is then responsible for driving cascade of response of inflammation.Initial TNF-
α the important function of OA generating processes be taken seriously be due to patient OA cartilage cell in the expression of TNF-α receptor p55 be apparently higher than
Normal chondrocyte, therefore patient OA cartilage cell shows more sensitive to the stimulation of TNF-α, is situated between to exacerbate TNF-α
The cartilage matrix degradation led, in addition, OA cartilages generate more Tumor necrosis factor convertases (TACE) compared with normal cartilage.
However to the miRNA of TNF-α response expression, there is presently no reports in cartilage cell.In our current research, I
Primarily determine that the stimulation of in cartilage cell TNF-α can significantly lower the expression of miR-145, and in the pathology mistake of OA
It is substantially reduced with the secretion miR-145 expressions of TNF-α in journey, in addition, TNF-α secretion inhibitor C C-5013 can be bright
Aobvious cartilage matrix degradation during inhibiting OA, most what is interesting is CC-5013 can partly restore the expression of miR-145, with
Above the result shows that there are negative correlativing relations with TNF-α by miR-145 in OA generating processes.Further study show that in cartilage cell
Middle overexpression miR-145 can significantly inhibit the generation of MMP-3, MMP-13 and ADAMTS-5 of TNF-α induction, on the contrary, inhibiting
MiR-145 is then obviously promoted the up-regulation of MMP-3, MMP-13 and ADAMTS-5 of TNF-α induction.We have found that MKK4 is miR-
145 target gene, and miR-145 inhibits the phosphorylation of MKK4 by the protein level of negative regulation MKK4, in turn
The phosphorylation and p-c-Jun/p-ATF-2 for inhibiting JNK/p38 enter core, the transcription of final regulation and control cartilage degeneration related gene.
It is surprisingly found that the promoter region of the MMP-13 and Adamts-5 binding site containing AP-1, and strike subtract MKK4 can
To abolish the facilitation for the inflammatory reaction that miR-145 inhibitor mediates TNF-α completely, similarly, it is then complete to be overexpressed MKK4
The full inhibiting effect for abolishing the inflammatory reaction that miR-145 agonists mediate TNF-α.To it is presumed that miR-145 negative regulations
The cartilage degeneration of regulation and control TNF-α induction is realized by MKK4-JNK/p38-c-Jun/ATF-2 axis.Experiment in vivo is into one
Step confirms result above, JNK pathway inhibitors (SP60012), p38 inhibitor (SB203580), miR-145 agonists
(Agomir-145) and MKK4 strikes and slows down viral (Len-sh-MKK4) and can significantly inhibit the regression of OA cartilage of rats, phase
Instead, it is soft then to aggravate OA rat articulars for miR-145 inhibitor (Antagomir-145) and MKK4 overexpression slow virus (Len-MKK4)
The regression of bone.
Our research finds that TNF-α stimulates the expression that can significantly lower miR-145, and in articular cartilage for the first time
The level of miR-145 is negatively correlated with the secretion of TNF-α in OA development processes.It blocks NF- κ B signals accesses or strikes that subtract p65 equal
Can with the downward of miR-145 caused by partial rescue TNF-α, this result shows that TNF-α to the effect of the negative regulation of miR-145 according to
Rely in classical NF- κ B signal accesses.It is surprising that IL-1 β stimulation to the expression of miR-145 in cartilage cell simultaneously
It does not influence, this prompts other signal paths that may simultaneously participate in the expression regulation to miR-145.
MMP-3, MMP-13 and ADAMTS-5 transcription and transcription of TNF-α induction can be significantly inhibited by being overexpressed miR-145
Horizontal raising afterwards.On the contrary, inhibiting the miR-145 then obviously productions of MMP-3, MMP-13 and ADAMTS-5 of the induction of aggravation TNF-α
It is raw.Subtract miR-145 in addition, being overexpressed or striking and there is no any influence to the growth conditions of cartilage cell.Therefore, miR-145 by
It is likely to become soft caused by significantly more efficient alleviation OA in the generation for the stromatin enzyme that can widely inhibit TNF-α induction
The therapy target of bone regression.Curiously, it is overexpressed or strikes and subtract miR-145 and do not influence TNF-α induction lower Sox-9, Col2a1
With the mRNA level in-site of Aggrecan, however be overexpressed miR-145 but can on protein level partial rescue TNF-α to Sox-
The inhibiting effect of 9 and Col2a1, this may be attributed to overexpression miR-145 and inhibit stromatin enzyme to Sox-9 and Col2a1
Degradation.These results prompt us, and there may be c-Jun's and ATF2 for the promoter region of MMP-3, MMP-13 and ADAMTS-5
Binding site, thus can by miR-145 by MKK4-JNK/p38-c-Jun/ATF-2 axis direct regulations and controls, and Sox-9,
Col2a1 and Aggrecan due to promoter region lacks the binding site of c-Jun and ATF2 cannot by miR-145 direct regulations and controls its
Transcriptional level.
MAPK and NF- κ B signal accesses participate in the expression of MMPs and ADAMTS in regulation and control cartilage cell by wide coverage,
Some researches show that three main map kinases (JNK, p38, ERK) and NF- κ B, and main work is played in cartilage degeneration
With.Since inflammatory reaction is acted in Cascaded amplification, this prompts us to be in the protein kinase of Inflammatory Pathway upstream, especially joins
Important inflammatory cascade driving effect may be played in the pathologic process of OA with the molecules upstream of NF- κ B and MAPK signal paths.
Some previous are studies have shown that celecoxib --- and cox 2 inhibitor is by blocking NF- κ B and JNK to reduce in cartilage cell
The generation of MMP-1, MMP-3, and hyaluronan is by inhibiting p38 come negative regulation MMP-1 and MMP-13 in cartilage cell
Expression.MKK4 and MKK7 can activate JNK, MKK4 that can activate p38 simultaneously.It is soft to demonstrate MKK4 participation regulation and control for the first time herein
The expression of MMP-3, MMP-13 and ADAMTS-5 in osteocyte, and the 3 ' areas UTR of MKK4 are containing there are one highly conserved miR-145
Binding site.Our result confirms that MKK4 is the target gene of miR-145, and MKK4 compares in patient's OA damaged cartilage tissue
Expression higher in the cartilaginous tissue in undamaged region, in addition, compared to sham-operation group, MKK4 and p-MKK4 are in DMM models
Higher is expressed in group cartilaginous tissue.The expression for subtracting MMP-3, MMP-13 and ADAMTS-5 that MKK4 significantly inhibits TNF-α induction is struck,
It is overexpressed the generation of MMP-3, MMP-13 and ADAMTS-5 that MKK4 then promotes TNF-α to induce.It is effectively blocked in addition, striking and subtracting MKK4
Synergistic effect of the miR-145 inhibitor to TNF-α induction MMP-3, MMP-13 and ADAMTS-5 up-regulation.On the contrary, being overexpressed MKK4
The inhibiting effect that can then block miR-145 to raise stromatin enzyme caused by TNF-α completely, the above result shows that miR-
The inhibiting effect for the cartilage degradation that 145 pairs of TNF-α mediate is that MKK4 is mediated.MKK4 can be activated by a variety of MKKKs, such as
ASK, MEKK and TAK, the MKK4 and then pecific phosphorylation JNK and p38 of phosphorylation.Our result indicate that in cartilage cell
Middle TNF-α can be struck with strong activation JNK and p38 and subtracted MKK4 or its molecules upstream HGK and can then significantly inhibit the phosphorus of JNK and p38
Acidification.
In conclusion our result of study show miR-145 be first be found in cartilage cell to TNF-
α is in the miRNA of response low expression, is overexpressed the generation for the stromatin enzyme that miR-145 then inhibits TNF-α to mediate in turn,
And it is realized by adjusting MKK4-JNK/p38-c-Jun/ATF-2 axis.Our result of study is found that miR-145
With the new mechanism in MKK4 cartilage matrix degradations caused by OA and the target spot of potential treatment OA may be used as.
Sequence table
<110>Shanghai Inst. of Life Science, CAS
<120>Applications of the miR-145 and MKK4 in treating or preventing cartilage damage, regression and osteoarthritis
<130> 170147
<160> 68
<170> PatentIn version 3.3
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gaaacacacg agacgctgaa 20
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atccactcag gcatcgacat 20
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gcgtcctaaa cagagacctg a 21
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ctgcttctcg ttgctctgtg 20
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gaaacgcaac tgaacgatga 20
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ggcagauaau ggcaguuaat t 21
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ggagaagcuu caggaccaut t 21
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augguccuga agcuucucct t 21
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ccaagaagaa aguggcaaut t 21
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auugccacuu ucuucuuggt t 21
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ccggccgccc cgactctaga gctgtgcaag ctcttctc 38
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gatccggcag ataatggcag ttaattcaag agattaactg ccattatctg ccttttttg 59
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aattcaaaaa aggcagataa tggcagttaa tctcttgaat taactgccat tatctgccg 59
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ttaagcttat ggcggctccg agcc 24
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cgggatcctc agtcgacata catggga 27
Claims (10)
1. raising the expression of miR-145 or the expression of active reagent and/or downward MKK4 or active reagent preparing inhibition
Cartilage damage or Bones and joints are alleviated in cartilage degeneration caused by the degradation of cartilage cell epimatrix, relief from osteoarthritis or treatment
Application in scorching drug.
2. application as described in claim 1, which is characterized in that the reagent is nucleic acid molecules, carbohydrate, lipid or small
Molecular compound;
Preferably, the expression vector that the reagent of the expression of the up-regulation miR-145 is miR-145;It is described to lower what MKK4 was expressed
Reagent is the carrier for knocking out MKK4.
3. application as claimed in claim 1 or 2, which is characterized in that the osteoarthritis is that spontaneous bone caused by aging closes
The osteoarthritis or sections inner side meniscus for the osteoarthritis, tears of anterior cruciate ligament induction that section is scorching, joint instability induces are cut
It removes and the osteoarthritis of the cross-section induction of medial collateral ligament.
4. the reagent for detecting miR-145 expressions and/or the reagent for detecting MKK4 expressions are soft in preparation monitoring
Application in the kit of the degradation of osteocyte epimatrix, cartilage damage or osteoarthritic condition progress.
5. application as claimed in claim 4, which is characterized in that the reagent for detecting MKK4 expressions includes detection
The reagent used during the reagent and detection MKK4 protein contents that are used in MKK4mRNA horizontal process.
6. application as described in claim 4 or 5, which is characterized in that the kit contains:
The horizontal reagent of the mRNA is detected using Northern hybrid methods, RT-PCR or RNA-seq;And/or
The reagent of protein content is detected using Western blot or ELISA.
7. a kind of detection kit, which is characterized in that the detection kit contains the examination for being useful for detection miR-145 expressions
Agent and/or reagent for detecting MKK4 expressions.
8. cartilage degeneration or treatment caused by a kind of degradation inhibiting cartilage cell epimatrix, relief from osteoarthritis are alleviated soft
The method of bone injury or osteoarthritis, which is characterized in that the method includes the combinations of either one or two following step:
(a) expression of miR-145 in object or the step of activity are raised;With
(b) expression of MKK4 in object or the step of activity are lowered.
9. method as claimed in claim 8, which is characterized in that the method includes giving object:Raise the expression of miR-145
Or expression or the active reagent of active reagent and/or downward MKK4.
10. a kind of diagnosis osteoarthritis or the method for monitoring osteoarthritic condition development, which is characterized in that the method includes inspections
Survey the expression step of miR-145 and/or MKK4 in object body fluid;
Preferably, the detection MKK4 expressions include detection MKK4mRNA levels and detection MKK4 albumen;
Preferably, the level of the mRNA is detected using Northern hybrid methods, RT-PCR or RNA-seq;Using Western
The content of blot or ELISA detection albumen;
Preferably, the body fluid is peripheral blood.
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111662972A (en) * | 2020-04-28 | 2020-09-15 | 上海交通大学医学院附属新华医院 | TRAF3 for preventing and treating arthritis by inhibiting IL-17 signal |
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2017
- 2017-03-28 CN CN201710191833.4A patent/CN108653738A/en active Pending
Non-Patent Citations (2)
Title |
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KOHEI NISHITANI ETAL: "PGE2 Inhibits MMP Expression by Suppressing MKK4-JNK MAP Kinase-c-JUN Pathway via EP4 in Human Articular Chondrocytes", 《JOURNAL OF CELLULAR BIOCHEMISTRY》 * |
张元民: "MiR-145在原发性膝关节骨性关节炎软骨中的表达及其意义", 《中国博士学位论文全文数据库医药卫生科技辑》 * |
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CN111662972A (en) * | 2020-04-28 | 2020-09-15 | 上海交通大学医学院附属新华医院 | TRAF3 for preventing and treating arthritis by inhibiting IL-17 signal |
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