CN108774642A - Purposes of the miR-214 as the biomarker of renal fibrosis in urine - Google Patents

Purposes of the miR-214 as the biomarker of renal fibrosis in urine Download PDF

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CN108774642A
CN108774642A CN201810768246.1A CN201810768246A CN108774642A CN 108774642 A CN108774642 A CN 108774642A CN 201810768246 A CN201810768246 A CN 201810768246A CN 108774642 A CN108774642 A CN 108774642A
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urine
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reverse transcription
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张爱华
白咪
贾占军
张玥
黄松明
于晓文
雷娟
郭燕
陈爽
杜阳
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Nanjing Childrens Hospital of Nanjing Medical University
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    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

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Abstract

New application the invention discloses miR-214 in urine as the biomarker of renal fibrosis, while proposing and a kind of early diagnosing the kit with dynamic monitoring renal fibrosis by detecting urine miR-214.Result of study shows that miR-214 is detected in the urine of chronic kidney disease infant has important clinical meaning, and important foundation is provided for the degree of clinical judgment renal fibrosis, can be with the progress of early intervention renal fibrosis.

Description

Purposes of the miR-214 as the biomarker of renal fibrosis in urine
Technical field
The present invention relates to the new opplication of miR-214, the biology more particularly to miR-214 in urine as renal fibrosis The new application of marker.
Background technology
Renal fibrosis is the common pathology base that a variety of chronic renal diseases (CKD) proceed to end stage renal failure (ESRD) Plinth, pathophysiological process include mainly：The intrinsic cellular damage of kidney including renal cells and stage, fiber Signal transfer stages, fiber formation stages and the kidney damage stage of formation.Wherein, injury of proximal cells and damage after The series of biochemical reactions of induction is to start and promote the principal element of renal interstitial fibrosis.
Albuminuria is the common feature of numerous chronic renal diseases, including Children Kidney Diseases.Which part infant is being controlled It can fully recover after treatment, but still with the presence of the lasting albuminuria of part infant, and it is now recognized that long-term High-grade Proteinuria is always between kidney Matter damage is with appearance.Find occur in albuminuria using the construction rat albumin overloading experiment of large dosage of bovine serum albumin(BSA) There is renal interstitial inflammation in very short time afterwards, and then kidney region fibrosis and kidney function damage occurs.Therefore this part is held Continuous High-grade Proteinuria infant can gradually appear kidney region fibrosis, go further to end-stage renal disease.Renal fibrosis diagnoses at present Renal needle biopsy this invasive detection methods are relied primarily on, since the technology of this technology itself requires, invasive and parent With the factors such as the feared state of mind existing for infant so as to the renal puncture of chronic kidney disease infant only in frequent recurrence or treatment It could be carried out when ineffective, the best opportunity that treatment renal fibrosis may be missed, delay kidney injury.
In recent years, Microrna (microRNA, miRNA) attracts attention in urine.MiRNA is raw in one kind , length be about 20-24 nucleotide tiny RNA, by with target gene mRNA complementations in conjunction with by regulate and control the expression of target gene, ginseng With a variety of pathophysiological processes in organism.MiRNAs can be stabilized in the body fluid of people, therefore can be used as and be examined substantially Disconnected marker.Studies have found that the expression of miRNA and kidney trouble are closely related in urine, as lupus nephritis, IgA nephrosis, acute kidney injury etc..But there has been no the reports in relation to miR-214 in urine as the biomarker of renal fibrosis Road.
Invention content
The present invention has been found surprisingly that in urine that miR-214 can especially be sent out as the biomarker of renal fibrosis The expression of miR-214 reduces in present chronic kidney disease infant urine, negatively correlated with the degree of albuminuria.
Present invention further proposes a kind of reagents for predicting renal fibrosis degree by detecting miR-214 in urine Box, the kit include miR-214 reverse transcriptions relevant primer, miR-214 PCR relevant primers, reverse transcription Mix reagents, reverse Record buffer solution, real-time quantitative PCR Mix reaction reagents, internal reference U6 reverse transcriptions relevant primer, U6 PCR relevant primers, reverse transcription The reagent and PCR Mix reaction reagents of Mix.Wherein, miR-214 and U6 reverse transcriptions and PCR relevant primers are purchased from the sharp rich life in Guangzhou Object Science and Technology Ltd..Reverse transcription Mix reagents are purchased from Takara companies of Japan, and real-time quantitative PCR Mix reaction reagents are purchased from south The bio tech ltd Jing Nuoweizan.
Specifically, the kit includes box body and box cover, wherein
The inner surface of the box cover, which is respectively arranged with, can rub magnetic sheet and bar code area, and the bar code area includes in kit Reagent preserves and configuration information, can rub magnetic sheet and can be used for experimenter's record experiment information conveniently；
The box body includes upper, middle and lower-ranking, wherein upper layer is identical with middle level, is divided into 8 subregions, each subregion Interior setting test tube hole is respectively used to place the Reagent Tube equipped with miR-214 reverse transcription relevant primers, miR-214 PCR phases is housed It closes the Reagent Tube of primer, the Reagent Tube equipped with reverse transcription Mix, the Reagent Tube equipped with RT Buffer, real-time quantitative PCR be housed The Reagent Tube of Mix reaction reagents, the Reagent Tube equipped with internal reference U6 reverse transcription relevant primers and the examination equipped with U6 PCR relevant primers Agent pipe and white board marker；
The lower layer of box body is separated into two cavitys, and drawable drawer is respectively arranged in two cavitys, can pull Drawer in fill refrigeration material respectively.
Preferably, the drawable drawer outside is provided with draw ring.
It is separated by partition between the middle level and lower layer of box body.
Advantageous effect
Compared with prior art, the present invention has the following advantages that and good effect：
(1) this present invention predicts the degree of renal fibrosis by simply detecting the expression quantity of miR-214 in urine, Renal needle biopsy this invasive detection methods are avoided, important foundation is provided for the degree of clinical judgment renal fibrosis, It can be with the progress of early intervention renal fibrosis；
(2) reagent cartridge configuration designed by is reasonable, easy to use, preservation easy to carry；
(2) it is entire to accumulate monitoring in real time when detecting the expression quantity of miR-214 by fluorescence signal for kit of the invention PCR processes, can it is more accurate, easily obtain monitoring result, and high degree of automation, pollution is effectively reduced, convenient for facing Bed monitoring is promoted.
Description of the drawings
Fig. 1 is tables of the miR-214 in the urine of urine examination normal child (normal) and Children with Nephrotic Syndrome (NS) It reaches；
Fig. 2 be miR-214 it is light, in, the expression in severe albuminuria Urine in Patients and with the correlation of albuminuria；
Fig. 3 be miR-214 it is light, in, the expression in severe albuminuria patient's renal needle biopsy tissue and and albuminuria Correlation；
Fig. 4 be Fibronectin (marker as fibrosis) it is light, in, severe albuminuria patient's renal needle biopsy Expression in tissue and the correlation with miR-214；
Fig. 5 is the structural schematic diagram for detecting the kit of miR-214 predictions renal fibrosis degree in urine.
Specific implementation mode
The present invention has unexpectedly discovered that miR-214 can especially be sent out as the biomarker of renal fibrosis in urine The expression of miR-214 reduces in present chronic kidney disease infant urine, negatively correlated with the degree of albuminuria.
In order to more easily detect the expression quantity of miR-214 in urine, the invention also provides one kind by detecting urine The kit of middle miR-214 predictions renal fibrosis degree, the kit includes miR-214 reverse transcriptions relevant primer, miR- 214 PCR relevant primers, reverse transcription Mix reagents, RT Buffer, real-time quantitative PCR Mix reaction reagents, internal reference U6 are reversed Record relevant primer, U6 PCR relevant primers, the reagent of reverse transcription Mix and PCR Mix reaction reagents.Wherein, miR-214 and U6 Reverse transcription and PCR relevant primers are purchased from the Guangzhou bio tech ltd Rui Bo.Reverse transcription Mix reagents are public purchased from Japan Takara Department, real-time quantitative PCR Mix reaction reagents are purchased from Nanjing Vazyme Biotechnology Co., Ltd..
Specifically, kit includes box body 1 and box cover 2, wherein the inner surface of box cover, which is respectively arranged with, can rub magnetic sheet 3 and bar code area 4, bar code area includes that seminal plasma fructose detection kit preserves and configuration information, can rub magnetic sheet can be used for experimenter with Written notes record experiment information.
Box body includes upper, middle and lower-ranking, wherein upper layer 5 and middle level 6 are identical, are divided into 8 subregions, in each subregion Test tube hole is set, is respectively used to place the Reagent Tube equipped with miR-214 reverse transcription relevant primers, miR-214 PCR correlations is housed The Reagent Tube of primer, the Reagent Tube equipped with RT Buffer, is equipped with real-time quantitative PCR at the Reagent Tube equipped with reverse transcription Mix The Reagent Tube of Mix reaction reagents, the Reagent Tube equipped with internal reference U6 reverse transcription relevant primers and the examination equipped with U6 PCR relevant primers Agent pipe and white board marker；
Box is separated by partition between the middle level and lower layer of box body, the lower layer 7 of body is separated into two cavitys, two cavitys It is inside respectively arranged with drawable drawer, fills refrigeration material respectively in drawable drawer, drawable drawer outside is set It is equipped with draw ring 8.Lower layer is the freeze space of entire kit, can be used for preserving the reagent that needs refrigerate, such as equipped with reverse Record the Reagent Tube of Mix and the Reagent Tube equipped with PCR Mix reaction reagents.
When in use, in the title or label symbol of the upper end labelled reagent pipe of each Reagent Tube conduct mark, detection The upper layer of box body is removed, each Reagent Tube can expose identification division, and so as to clearly differentiate each Reagent Tube, that removes is upper Layer can be further used for placing the Reagent Tube for having added sample.
In 52 chronic kidney disease infants of clinic collection, (any control was not done in different degrees of albuminuria, first visit for this research Treat) and 46 urine examination normal children random urine, by detect urine in miR-214 expression, the results are shown in Figure 1, send out The expression of miR-214 reduces in present chronic kidney disease infant urine, negatively correlated with the degree of albuminuria.
Specifically, the expression of miR-214 detects by the following method in urine：
(1) in urine micro RNA extraction：
1. the urine 3000rpm collected centrifuges 10min, supernatant 500ul is taken, Trizol500 μ l are added, be acutely vortexed shake 30s is swung, 5min is stored at room temperature；
2. 200 μ l isopropanols are added, mixing is overturned, the concussion 2min that is acutely vortexed is stored at room temperature 5min to liquid-transparent；
3.4 DEG C, 13000rpm, centrifuge 15min；
4. 500 μ l supernatants are transferred to new EP to manage, isometric 500 μ l chloroforms are added, overturn mixing, be acutely vortexed concussion 1min is stored at room temperature 5min；
5.4 DEG C, 13000rpm, centrifuge 15min；
6. supernatant is again transferred to new EP to manage, the isopropanol of 3/4 volume is added, overturns mixing, is stored at room temperature 10min；
7.4 DEG C, 13000rpm, 10min is centrifuged, precipitates RNA；
8. removing supernatant (suction is abandoned), 75% ethyl alcohol (DEPC water configures, -20 DEG C of precoolings) is washed, 7500rpm, and 5min is centrifuged；
9. abandoning supernatant, dry 10min, 10 μ lDEPC water dissolutions RNA are buckled on paper；
10. surveying concentration, prepare reverse transcription.
(2) reverse transcription step：
Using grads PCR instrument, 500ng mass total serum IgEs is taken to carry out reverse transcription, reaction system is 20 μ l.MiR-214 and U6 Reverse transcription respectively prepares RT reaction solutions by following component (reaction solution is prepared to carry out on ice).
1. reverse transcription reaction system of table
(3) it after soft mixing centrifugation, is put into grads PCR instrument and carries out reverse transcription reaction, condition is：37 DEG C of 15min, 85 DEG C 5sec, 4 DEG C of holdings.The short-term 4 DEG C of refrigerators of cDNA preserve, long-term -20 DEG C of storages.
Real-time quantitative PCR step：
Using Roche SYBR green PCR methods, reaction system is 20 μ l, is shown in Table 2.
Table 2.Real-time PCR reaction systems (25 μ l)
Mixing slightly centrifuges, and carries out PCR amplification in 7500 fluorescence quantitative PCR instruments of ABI PRISM, reaction condition is：95 DEG C, 10min；Then 95 DEG C, 15s and 60 DEG C, 1min is recycled 35 times.Mesh miR-214 is calculated using reference gene U6 Δ Δ Ct methods The relative quantity of expression.
Then have chosen the chronic kidney disease infant 18 for doing renal needle biopsy, male 11 (61%), female 7 (39%), average age 6 months 9 years old.CKD infants are divided into mild proteinuria group (mild, Urine proteins by the degree of albuminuria< 1.0g/24h) 6, moderate albuminuria group (moderate, Urine proteins 1-3g/24h) 6 and severe albuminuria group (severe, Urine proteins>3.0g/24h) 6.Normal group of Urine proteins (0-1g/24h) 6, renal tissue derive from tumor of kidney resection Tumour periphery nephridial tissue afterwards comes from attached Nanjing children's hospital Urology Surgery, and ratifies and obtain infant by Ethics Committee The informed consent of parent.Using the expression of miR-214 in the method detection biopsy nephridial tissue of fluorescence in situ hybridization, as a result such as Fig. 2 Shown in~Fig. 4, it is found that it is mainly expressed in renal tubular cell, be proportionate with albuminuria.To the fibrosis journey of these patients Degree carries out immunohistochemical staining (Fibronectin) and assesses, it is found that the expression of fibrosis and nephridial tissue miR-214 are in positive It closes.Therefore it is presumed that, in chronic kidney disease infant, High-grade Proteinuria causes miR-214 expression and accumulation in renal tubule to increase Add, is discharged and reduces in urine, and then aggravate renal fibrosis.
The above results illustrate that miR-214 is detected in the urine of chronic kidney disease infant has important clinical meaning, is The degree of clinical judgment renal fibrosis provides important foundation, can be with the progress of early intervention renal fibrosis.
The specific steps of fluorescence in situ hybridization：
1. tissue prepares：
The paraffin mass of the biopsy of selection is sliced (5 μ m-thick), 65 DEG C are toasted 1 hour, then 37 DEG C of incubators Overnight.Room temperature, it is spare.
2. preparation of reagents：
1) antigen retrieval buffers (0.01M trisodium citrate sodium citrate pH value of solution 6.0) --- configuration 500ml：It will 1.47g trisodium citrates are dissolved in 500ml ddH2In O, pH value of solution is adjusted to 6.0；
2) penetrating liquid Permeabilization buffer (1 × PBS/0.5%Triton X-100) --- configuration 50ml：10 × PBS of 5ml are diluted to 40ml., 250 μ l Triton X-100, mixing is then added；
3) 1 × PBS --- configuration 1L：10 × PBS of 100ml are added in 900ml ddH2O, mixing；
4) 4 × SSC --- configuration 1L：20 × SSC of 200ml (purchase) are added in 800ml ddH2O, mixing；
5) prehybridization solution Prehybridization buffer (4 × SSC of 3%BSA in) --- configuration 100ml：It weighs 3g BSA are added in 4 × SSC of 100ml, mixing；
6) hybridization solution Hybridization buffer (4 × SSC of 10%dextran sulfate in) --- configuration 10ml：20 × SSC of 2ml, 4ml 25%dextran sulfate are added to 4ml ddH2In O, mixing；
7) washing lotion I Washing buffer I (4 × SSC, 0.1%Tween-20) --- configuration 1L：By 200ml 20 × SSC and 1ml Tween-20 are added to 799ml ddH2In O, mixing；
8) washing lotion II Washing buffer II (2 × SSC) --- configuration 1L：20 × SSC of 100ml are added to 900ml ddH2In O, mixing；
9) washing lotion III Washing buffer III (1 × SSC) --- configuration 1L：20 × SSC of 50ml are added to 950ml ddH2In O, mixing.
3. dewaxing and rehydration
1) piece is dried --- paraffin piece is placed in 65 DEG C 30min or 47 DEG C overnight, keeps tissue not easily to fall off；
2) dewax --- paraffin piece is completely soaked in dimethylbenzene, is repeated 3 times, (dimethylbenzene has severe toxicity to ask to each 10min It is operated in draught cupboard, the safeguard procedures such as whole wearing gloves and mask)；
3) removal xylene --- paraffin piece is completely soaked in 100% alcohol, is repeated 3 times, each 3min；
4) rehydration --- paraffin piece is impregnated into graded ethanol, is followed successively by 90%, 80%, each 3min of 70% alcohol finally soaks ddH2In O.
5) antigen retrieval
A. paraffin piece is heated into 10min in 0.01M trisodium citrates (pH 6.0) solution boiled；
B. be sliced in the solution natural cooling 30min to room temperature；
C. paraffin section is soaked in ddH2It is cleaned 3 times in O, each 5min；
D. paraffin section is soaked in 1 × PBS；It steeps in ddH2In O；
4.RNAs probe in detecting
1) drawn a circle at 1mm around the organization edge using liquid wax crayon, in order to later hybridization and dying operation；
2) prehybridization solution prehybridization buffer are added dropwise to tissue, paraffin section is placed in wet box and is sealed 20min (covering is advisable) is closed, closure temperature is generally below the prediction Tm values (being usually 45-55 DEG C) of probe, is used in this experiment RT 22–25℃；
3) prehybridization simultaneously, preheating temperature hybridization solution hybridization buffer when using formal hybridization；
4) under the conditions of being protected from light, probe is added in hybridization buffer and (refers to reagent configuration) configuration and is hybridized Liquid；
5) prehybridization buffer are discarded, appropriate hybridization solution is added, 22-25 DEG C of hybridization 1h (pay attention to：Extend Hybridization time cannot improve signal strength, background signal can be made to enhance instead), hybrid process whole process is protected from light, and hybridization solution is complete Tissue is covered, prevents tissue from air-drying；
6) it is protected from light, under conditions of higher than 5 DEG C of hybridization temperature, (refers to reagent to match with washing lotion I washing buffer I Set) the every hole cell 3 times, each 5min of cleaning is shaken, to reduce background signal；
7) under conditions of step e, cell is cleaned 1 time with washing lotion II washing buffer II；
8) under conditions of step e, cell is cleaned 1 time with washing lotion III washing buffer III
9) it is protected from light, cell, room temperature 5min is cleaned with 1 × PBS.
5.DNA is dyed
1) it is protected from light, DAPI dyes 10min；
2) it is protected from light, at room temperature, cleaning cell 3 times, each 5min is shaken with 1 × PBS, then in laser after mountant mounting It makes film under Laser Scanning Confocal Microscope.
The specific steps of immunohistochemistry：
Paraffin section carries out anticreep piece processing after cutting：65 DEG C are toasted 1 hour, and then 37 DEG C of incubators are overnight.Second day, often Rule dewaxing (paraffin piece is completely soaked in dimethylbenzene, is repeated 3 times, each 10min), aquation (paraffin piece impregnate graded ethanol, according to Secondary is each 3min of 100%*3,90%, 80%, 70% alcohol)；Slice is immersed in antigen retrieval buffers, micro-wave oven height fire boils Afterwards, it is stored at room temperature 10min, then sets in fire and boils again, after natural cooling, is washed three times with 1 × PBS, each 5min；It is added dropwise 3% hydrogen peroxide, room temperature 20min (inactivating endogenous catalase), is then washed three times with 1 × PBS, each 5min；It is added dropwise Immunostaining confining liquid, room temperature 20min, gets rid of surplus liquid, without washing；Fibronectin antibody (1 is added dropwise：200 is dilute Release), it sets 4 DEG C of fixations in wet box and stays overnight；Second day 37 DEG C of incubator rewarming 45min, 1 × PBS are washed 5 times, each 5min；Examination is added dropwise Agent I (biotin labeling secondary antibody), 37 DEG C are incubated 20min, and 1 × PBS is washed 5 times, each 5min；Reagent II (horseradish peroxidating is added dropwise The streptomysin avidin working solution of object enzyme label), 37 DEG C are incubated 20min, and 1 × PBS is washed five times, each 5min；DAB colour developings 2- 5min grasps dye levels under microscope, and tap water fully rinses；Haematoxylin is redyed 2 minutes, and hydrochloric acid breaks up 1-2s, returns indigo plant 3min, tap water fully rinse；Conventional dehydration, transparent, mounting.

Claims (6)

1. purposes of the miR-214 as the biomarker of renal fibrosis in urine.
2. purposes according to claim 1, which is characterized in that the expression of miR-214 in chronic kidney disease infant urine It reduces, it is negatively correlated with the degree of albuminuria.
3. a kind of kit for predicting renal fibrosis degree by detecting miR-214 in urine, which is characterized in that the reagent Box includes miR-214 reverse transcriptions relevant primer, miR-214PCR relevant primers, reverse transcription Mix reagents, RT Buffer, reality When quantitative PCR Mix reaction reagents, internal reference U6 reverse transcriptions relevant primer, U6PCR relevant primers, reverse transcription Mix reagent and PCR Mix reaction reagents.
4. kit according to claim 3, which is characterized in that the kit includes box body and box cover, wherein
The inner surface of the box cover, which is respectively arranged with, can rub magnetic sheet and bar code area, and the bar code area includes seminal plasma fructose detection kit Preservation and configuration information；
The box body includes upper, middle and lower-ranking, wherein upper layer is identical with middle level, is divided into 8 subregions, is set in each subregion Test tube hole is set, is respectively used to place the Reagent Tube equipped with miR-214 reverse transcription relevant primers, miR-214PCR relevant primers is housed Reagent Tube, the Reagent Tube equipped with reverse transcription Mix, the Reagent Tube equipped with RT Buffer, equipped with real-time quantitative PCR Mix it is anti- Answer the Reagent Tube, the Reagent Tube equipped with internal reference U6 reverse transcription relevant primers and the Reagent Tube equipped with U6PCR relevant primers of reagent with And white board marker；
The lower layer of box body is separated into two cavitys, drawable drawer is respectively arranged in two cavitys, in drawable pumping Refrigeration material is filled in drawer respectively.
5. kit according to claim 3, which is characterized in that the drawable drawer outside is provided with draw ring.
6. kit according to claim 3, which is characterized in that be separated by partition between the middle level and lower layer of box body.
CN201810768246.1A 2018-07-13 2018-07-13 Purposes of the miR-214 as the biomarker of renal fibrosis in urine Pending CN108774642A (en)

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Cited By (1)

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CN114250287A (en) * 2021-07-27 2022-03-29 皖南医学院第一附属医院（皖南医学院弋矶山医院） Use of urine exosome cyclic RNA for predicting kidney fibrosis

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CN206359543U (en) * 2016-12-26 2017-07-28 上海新培晶医学检验所有限公司 A kind of kit for being used to detect the first-line drug expression of drug resistance genes amount for the treatment of malignant tumour
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Publication number Priority date Publication date Assignee Title
CN206359543U (en) * 2016-12-26 2017-07-28 上海新培晶医学检验所有限公司 A kind of kit for being used to detect the first-line drug expression of drug resistance genes amount for the treatment of malignant tumour
CN206418109U (en) * 2017-01-19 2017-08-18 广州精科医学检验所有限公司 The kit and Reagent Tube studied for immune group storehouse

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Title
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M.BAI等: "MiR-214 Expression in Kidney Tissue of CKD Patients and Its Relationship with Proteinuria", HONG KONG JOURNAL OF NEPHROLOGY, vol. 17, no. 2, pages 86, XP029301930, DOI: 10.1016/j.hkjn.2015.09.130 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114250287A (en) * 2021-07-27 2022-03-29 皖南医学院第一附属医院（皖南医学院弋矶山医院） Use of urine exosome cyclic RNA for predicting kidney fibrosis
CN115851900A (en) * 2021-07-27 2023-03-28 皖南医学院第一附属医院（皖南医学院弋矶山医院） Application of circRNA in preparation of kidney fibrosis diagnosis reagent and diagnosis equipment

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