CN111110693B - Application of agent for down-regulating expression of circular gene in preparation of medicine for preventing and/or treating pulmonary fibrosis and medicine - Google Patents

Application of agent for down-regulating expression of circular gene in preparation of medicine for preventing and/or treating pulmonary fibrosis and medicine Download PDF

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CN111110693B
CN111110693B CN202010001372.1A CN202010001372A CN111110693B CN 111110693 B CN111110693 B CN 111110693B CN 202010001372 A CN202010001372 A CN 202010001372A CN 111110693 B CN111110693 B CN 111110693B
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CN111110693A (en
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宋晓冬
吕长俊
张瑾锦
刘博�
曹国红
刘伟丽
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Binzhou Medical College
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Abstract

The invention relates to application of a reagent for down-regulating expression of cyclic has _ circ _0023858 in preparation of a medicament for preventing and/or treating pulmonary fibrosis and a medicament for down-regulating expression of cyclic has _ circ _0023858, belonging to the technical field of medicament preparation. The medicine prepared by the reagent for down-regulating the expression of the cyclic has _ circ _0023858 can realize prevention and/or treatment of pulmonary fibrosis, and particularly prevents or treats pulmonary fibrosis by regulating a Hippo signal pathway through miR-324-5p adsorption; can inhibit differentiation, activation, proliferation and migration of lung fibroblast.

Description

Application of agent for down-regulating expression of circular gene in preparation of medicine for preventing and/or treating pulmonary fibrosis and medicine
Technical Field
The invention relates to the technical field of medicine preparation, in particular to application of a reagent for down-regulating expression of cyclic has _ circ _0023858 in preparation of a medicine for preventing and/or treating pulmonary fibrosis and a medicine for down-regulating expression of cyclic has _ circ _ 0023858.
Background
Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive, fibrotic, substantive lung disease of unknown etiology, lacking effective intervention. In recent years, the incidence of idiopathic pulmonary fibrosis is increasing year by year due to haze caused by environmental pollution, and the treatment thereof still belongs to a worldwide problem. The disease has no medicine with definite curative effect, and the lung transplantation is the only important means for treating the terminal pulmonary fibrosis, but the cost is high, the donor source is difficult, and the application is limited. Until now, no clinically applied molecular marker can reveal the molecular mechanism of pulmonary fibrosis diseases, so that susceptibility analysis, diagnosis, especially early diagnosis, treatment and prognosis of pulmonary fibrosis are extremely difficult.
Disclosure of Invention
The invention aims to provide application of an agent for down-regulating the expression of cyclic has _ circ _0023858 in preparing a medicament for preventing and/or treating pulmonary fibrosis and a medicament for down-regulating the expression of cyclic has _ circ _ 0023858. The reagent for down-regulating the expression of the cyclic has _ circ _0023858 can realize prevention and/or treatment of pulmonary fibrosis, and particularly prevents or treats pulmonary fibrosis by regulating a Hippo signaling pathway through adsorbing miR-324-5 p; can inhibit differentiation, activation, proliferation and migration of lung fibroblast.
The invention provides application of an agent for down-regulating the expression of cyclic has _ circ _0023858 in preparing a medicament for preventing and/or treating pulmonary fibrosis, wherein the nucleotide sequence of the cyclic has _ circ _0023858 is shown as SEQ ID NO. 1.
The invention also provides application of an agent for down-regulating the expression of the cyclic has _ circ _0023858 in preparing a medicament for preventing or treating pulmonary fibrosis by regulating a Hippo signal pathway through adsorbing miR-324-5p, wherein the nucleotide sequence of the cyclic has _ circ _0023858 is shown in SEQ ID No. 1.
The invention also provides application of a reagent for down-regulating the expression of the cyclic has _ circ _0023858 in preparing a medicament for preventing or treating pulmonary fibrosis by regulating the target gene YAP1 through adsorbing miR-324-5p and further regulating a Hippo signal pathway, wherein the nucleotide sequence of the cyclic has _ circ _0023858 is shown in SEQ ID No. 1.
The invention also provides application of a reagent for reducing the expression of the cyclic has _ circ _0023858 in preparing a medicament for preventing or treating pulmonary fibrosis by regulating the target gene AJUBA through adsorbing miR-324-5p and further regulating the Hippo signal pathway, wherein the nucleotide sequence of the cyclic has _ circ _0023858 is shown in SEQ ID No. 1.
The invention also provides application of a reagent for down-regulating the expression of the circular has _ circ _0023858 in preparing a medicament for inhibiting the differentiation of lung fibroblasts, wherein the nucleotide sequence of the circular has _ circ _0023858 is shown as SEQ ID No. 1.
The invention also provides application of an agent for down-regulating the expression of the circular has _ circ _0023858 in preparing a medicament for inhibiting the activation of lung fibroblasts, wherein the nucleotide sequence of the circular has _ circ _0023858 is shown as SEQ ID No. 1.
The invention also provides application of an agent for down-regulating the expression of the circular has _ circ _0023858 in preparing a medicament for inhibiting the proliferation of lung fibroblasts, wherein the nucleotide sequence of the circular has _ circ _0023858 is shown as SEQ ID No. 1.
The invention also provides application of an agent for down-regulating the expression of the circular has _ circ _0023858 in preparing a medicament for inhibiting the migration of lung fibroblasts, wherein the nucleotide sequence of the circular has _ circ _0023858 is shown as SEQ ID No. 1.
The invention also provides a medicament for down-regulating the expression of the circular has _ circ _0023858, wherein the nucleotide sequence of the circular has _ circ _0023858 is shown as SEQ ID NO.1, the medicament comprises an interfering double-stranded RNA, the interfering double-stranded RNA is synthesized according to the sequence of nucleotides shown as SEQ ID NO.2, and the interfering double-stranded RNA has the following structure:
Figure DEST_PATH_IMAGE002
wherein each "|" represents base pairing.
The invention provides application of an agent for down-regulating expression of cyclic has _ circ _0023858 in preparation of a medicament for preventing and/or treating pulmonary fibrosis. The reagent for down-regulating the expression of the cyclic has _ circ _0023858 can realize prevention and/or treatment of pulmonary fibrosis, and particularly prevents or treats pulmonary fibrosis by regulating a Hippo signaling pathway through adsorbing miR-324-5 p; can inhibit differentiation, activation, proliferation and migration of lung fibroblast.
Drawings
FIG. 1 is a graph of the change in expression of has _ circ _0023858 in a fibrosis model and an IPF blood sample, provided by the present invention;
FIG. 2 is a graph showing the effect of siRNA interfering fragment of has _ circ _0023858 on myofibroblast activation-associated protein expression;
FIG. 3 is a graph showing the results of siRNA interference fragments of has _ circ _0023858 on the proliferation and migration ability of myofibroblasts;
FIG. 4 is a diagram of the expression change of miR-324-5p provided by the invention;
FIG. 5 is a graph showing the effect of mics and inhibitors of miR-324-5p provided by the invention on the expression of myofibroblast activation-associated protein;
FIG. 6 is a graph showing the results of migration ability of miR-324-5p myofibroblasts provided by the invention;
fig. 7 is a technical route provided by the present invention.
Detailed Description
The invention provides application of an agent for down-regulating the expression of cyclic has _ circ _0023858 in preparing a medicament for preventing and/or treating pulmonary fibrosis, wherein the nucleotide sequence of the cyclic has _ circ _0023858 is shown as SEQ ID NO. 1. The nucleotide sequence of the non-coding RNA (has _ circ _0023858) provided by the invention is shown as follows (SEQ ID NO. 1):
TGTCTTCATTGGCTGCTCTGGCATGGAGCTGATATCACACACGTAACAACGAGAGGTTGGACAGCATCTCACATAGCTGCAATCAGGGGTCAGGATGCTTGTGTACAGGCTCTTATAATGAATGGAGCAAATCTGACAGCCCAGGATGACCGGGGATGCACTCCTTTACATCTTGCTGCAACTCATGGACATTCTTTCACTTTACAAATAATGCTCCGAAGTGGAGTGGATCCCAGTGTGACTGATAAGAGAGAATGGAGACCTGTGCATTATGCAGCTTTTCATGGGCGGCTTGGCTGCTTGCAACTTCTTGTTAAATGGGGTTGTAGCATAGAAGATGTGGACTACAATGGAAACCTTCCAGTTCACTTAGCAGCCATGGAAGGCCACCTTCACTGTTTCAAATTCCTAGTCAGTAGAATGAGCAGTGCGACGCAAGTTTTAAAAGCTTTCAATGATAATGGAGAAAATGTACTGGATTTGGCCCAGAGGTTCTTCAAGCAGAACATTTTACAGTTTATCCAGGGGGCTGAGTATGAAGGAAAAGACCTAGAGGATCAGGAAACTTTAGCATTTCCAGGTCATGTGGCTGCCTTTAAGGGTGATTTGGGGATGCTTAAGAAATTAGTGGAAGATGGAGTAATCAATATTAATGAGCGTGCTGATAATGGATCAACTCCTATGCATAAAGCTGCTGGACAAGGCCACATAGAGTGTTTGCAGTGGTTAATTAAAATGGGAGCAGACAGTAATATTACCAACAAAGCAGGGGAGAGACCCAGTGATGTGGCAAAGAG。
the invention also provides application of an agent for down-regulating the expression of the cyclic has _ circ _0023858 in preparing a medicament for preventing or treating pulmonary fibrosis by regulating a Hippo signal pathway through adsorbing miR-324-5p, wherein the nucleotide sequence of the cyclic has _ circ _0023858 is shown in SEQ ID No. 1. The invention annotates and clarifies the structure and the function of a genome from the different angle of protein coding genes by a genetic information transmission mode and an expression regulation network mediated by non-coding genes, and can provide a new strategy and scheme for gene therapy of IPF.
The invention also provides application of a reagent for down-regulating the expression of the cyclic has _ circ _0023858 in preparing a medicament for preventing or treating pulmonary fibrosis by regulating the target gene YAP1 through adsorbing miR-324-5p and further regulating a Hippo signal pathway, wherein the nucleotide sequence of the cyclic has _ circ _0023858 is shown in SEQ ID No. 1.
The invention also provides application of a reagent for reducing the expression of the cyclic has _ circ _0023858 in preparing a medicament for preventing or treating pulmonary fibrosis by regulating the target gene AJUBA through adsorbing miR-324-5p and further regulating the Hippo signal pathway, wherein the nucleotide sequence of the cyclic has _ circ _0023858 is shown in SEQ ID No. 1.
FIG. 7 is a flow chart of the technical scheme provided by the present invention, which first identifies the nature of circRNA-0023858 and defines the clinical value of circRNA-0023858. And (3) knocking down and increasing the expression of circRNA-0023858 by using siRNA and an overexpression vector respectively, and detecting the activation of fibroblasts and the change of a function related index under the intervention of circRNA-0023858. Detecting the intracellular localization of the circRNA-0023858 by using in situ hybridization to determine the regulation and control mode of the cells; the combination of circRNA-0023858 and miR-324-5p is determined, and the combination relation of the circRNA-0023858 and miR-324-5p is further determined by applying RNA pull down, Ago RIP and dual-luciferase reporter gene experiments. The influence of miR-324 on the activation and function of myofibroblasts is further clarified, and the miR-324-5p regulates and controls a Hippo signal path through the target genes YAP1 and AJUBA respectively, so that the mechanism of regulating and controlling IPF by circRNA-0023858 is researched, and the aim of preventing and treating pulmonary fibrosis is fulfilled.
The invention also provides application of a reagent for down-regulating the expression of the circular has _ circ _0023858 in preparing a medicament for inhibiting the differentiation of lung fibroblasts, wherein the nucleotide sequence of the circular has _ circ _0023858 is shown as SEQ ID No. 1.
The invention also provides application of an agent for down-regulating the expression of the circular has _ circ _0023858 in preparing a medicament for inhibiting the activation of lung fibroblasts, wherein the nucleotide sequence of the circular has _ circ _0023858 is shown as SEQ ID No. 1.
The invention also provides application of an agent for down-regulating the expression of the circular has _ circ _0023858 in preparing a medicament for inhibiting the proliferation of lung fibroblasts, wherein the nucleotide sequence of the circular has _ circ _0023858 is shown as SEQ ID No. 1.
The invention also provides application of an agent for down-regulating the expression of the circular has _ circ _0023858 in preparing a medicament for inhibiting the migration of lung fibroblasts, wherein the nucleotide sequence of the circular has _ circ _0023858 is shown as SEQ ID No. 1.
The invention also provides a medicament for down-regulating the expression of the circular has _ circ _0023858, wherein the nucleotide sequence of the circular has _ circ _0023858 is shown as SEQ ID NO.1, the medicament comprises an interfering double-stranded RNA, the interfering double-stranded RNA is synthesized according to the sequence of nucleotides shown as SEQ ID NO.2, and the interfering double-stranded RNA has the following structure:
Figure DEST_PATH_IMAGE004
wherein each "|" represents base pairing.
The invention obtains the interference double-stranded RNA (interference sequence) aiming at the has _ circ _0023858 gene, and specifically aims at a target gene with a nucleotide sequence shown as SEQ ID NO. 2: 5'-GTGGCAAAGAGTGTCTTCA-3', 19 base pairs, and the first and second strands are each designed with two overhanging bases dT at the 3 ' end. According to the invention, the specific interference sequence aiming at has _ circ _0023858 is designed and transfected into cells to play a role in down-regulating the expression of has _ circ _0023858, so that the aim of preventing and treating pulmonary fibrosis is fulfilled.
The application of the agent for downregulating the expression of cyclic ha _ circ _0023858 in the preparation of the medicament for preventing and/or treating pulmonary fibrosis and the medicament for downregulating the expression of cyclic ha _ circ _0023858 are further described in detail with reference to the following specific examples, and the technical scheme of the invention includes but is not limited to the following examples.
The specific experimental scheme of the invention is carried out according to the following specific operations:
preparation of commonly used reagents
(1) DEPC water: sucking 1mL of DEPC stock solution by using a 1mL pipette, adding into 1L of triple distilled water, uniformly stirring by using a magnetic stirrer, subpackaging in 500 mL glass bottles, sterilizing by high pressure and moist heat, and storing at 4 ℃ for later use.
(2) 0.01M PBS: and (3) adding all 1 bottle of PBS solid crystals into 2000 mL of triple distilled water, uniformly stirring by using a magnetic stirrer, subpackaging in 500 mL glass bottles, and storing at room temperature for later use.
(3) 10% F12(1 ×) medium: according to F12: fetal bovine serum: the double antibody (9: 1: 0.1) was prepared into F12 medium containing 10% fetal bovine serum and stored at 4 ℃ for further use.
(4) 10% EMEM (1 ×) medium: according to EMEM: fetal bovine serum: the double antibody (9: 1: 0.1) was prepared in F12 medium containing 5% fetal bovine serum and stored at 4 ℃ for further use.
Method
Cell resuscitation
(1) And (3) opening a water bath kettle in advance for preheating, taking out the cells from the liquid nitrogen when the temperature reaches 37 ℃, quickly putting the cells into the preheated water bath kettle, rotating the cells in time, quickly melting the cells, and quickly taking out the cells when only small crushed ice blocks are left.
(2) Wiping the cryopreserved tube with 75% ethanol in a sterile workbench, transferring the cryopreserved tube to a 15 mL EP tube, adding 2 mL of fresh culture medium, gently blowing and mixing the cell suspension, 950rpm for 4min, centrifuging the tube, removing the supernatant, adding 1mL of fresh F12(1 x) or EMEM (1 x) culture medium containing 5% fetal bovine serum, and gently blowing and dispersing the mixed cell sediment.
(3) At room temperature, 950rpm, 4min, centrifuging, discarding the supernatant, adding 1mL of fresh medium, and mixing by pipetting.
(4) 3 mL of fresh F12 (1X) or EMEM (1X) medium containing 10% fetal bovine serum was placed in a 25 cm format2The culture bottle is taken out of the new culture bottle, 3 mL of new culture medium is added, 1mL of cell suspension is added, and the uniformly mixed cell suspension is transferred into the culture bottle.
(5) Slowly shaking to uniformly distribute the cells in the culture flask, standing for 3-4 min, and observing the cell distribution condition.
(6)37 ℃,5% CO2Culturing in a constant temperature incubator for 24h, and allowing the cells to adhere to the wall.
Cell passage
(1) From the incubator, the flask was removed and the medium was discarded.
(2) Cells were washed once with 1 × PBS.
(3) Adding pancreatin 700 μ L, incubating and digesting in a constant temperature incubator at 37 deg.C for 1 min, and adding 1.4 mL of new culture medium to neutralize the pancreatin.
(4) The pipette is blown and uniformly mixed and transferred into a 15 mL EP tube.
(5) 950rpm, 4min, two new flasks were removed at room temperature and 3 mL of fresh medium containing 10% fetal bovine serum was added.
(6) After centrifugation, the 15 mL EP tube was removed, the supernatant removed, the cell pellet retained, 3 mL fresh medium added, and the cell suspension was blown up and mixed. (slow action, prevention of cell damage due to excessive mechanical force)
(7) Adding 1.5 mL of each into a new culture bottle, shaking gently to mix uniformly to make the cells uniformly distributed at the bottom of the culture bottle, and standing for 4min to settle the cells.
(8) 5% CO in a 37 ℃ constant temperature incubator2And (5) culturing. (before the glass bottle is put in, the bottle cap is loosened to ensure the circulation of the air in the bottle and the normal growth of cells; before the plastic bottle is added, the bottle cap is screwed up.)
Cell cryopreservation
(1) - (4) same as above.
(5) 950rpm, 4min, centrifugation at room temperature, and discarding the supernatant, leaving the cell pellet.
(6) Preparing ice-making freezing liquid: 1000. mu.L of fetal bovine serum + 100. mu.L of DMSO (which is toxic to cells at normal temperature and has protective effect on cells at low temperature).
(7) The frozen solution was added to a 1.5 mL EP tube, the suspension cells were blown up, and transferred to a cryopreservation tube.
(8) Freezing and storing procedures: 4 ℃, 4min → -20 ℃, 30 min → -80 ℃ overnight → transfer to liquid nitrogen.
Cell plate
When the cells grow to cover more than 70% of the bottom of the flask, plating can be performed by first discarding the old medium, adding 1mL of PBS to wash the cell surface by gentle shaking, then adding 1mL of 0.25% trypsin solution to digest, standing at 37 ℃ for 40s, and immediately adding 1.5 mL of fresh medium to stop the digestion. Cells were transferred to 15 mL EP tubes at 1000 rpm for 4min, centrifuged, and six well plates were removed during this process, with 1mL of fresh medium added to each well. To be centrifugedRemoving the supernatant, adding 2 mL of fresh culture medium containing 5% fetal calf serum, gently blowing, mixing, continuously adding 4 mL of fresh culture medium containing 5% fetal calf serum, gently blowing, mixing cell suspension, adding 1mL of cell suspension into each six-well plate, gently shaking to uniformly distribute cells at the bottom of the six-well plate, 37 ℃, and 5% CO2Culturing for 8 h in a constant temperature incubator, and carrying out subsequent experiments when the wall adheres to the incubator.
circRNA smart silence transfection
(1)37 ℃,5% CO2Culturing in a constant temperature incubator for 24h, and allowing the cells to adhere to the wall. The cell grouping includes: normal group, model group + NC group, model group + Smart group.
(2) And (3) transfection process: mu.L of circRNA smart silicone stock was diluted with 240. mu.L of 1 XriboFECTTM CP Buffer and incubated at room temperature for 3 min; then 24. mu.L of riboFECTTM CP Reagent was added, incubated at room temperature for 12 min until a complex was formed, and the mixture was added to 3726. mu.L of medium and added to the cells uniformly.
(3) After 6 h of transfection, TGF-beta 1 was added to a final concentration of 5 ng/mL to stimulate RNA extraction for 72h or protein extraction for 72 h.
Cellular RNA extraction
(1) Collecting cells treated at different stimulation times, adding 1mL of Trizol into the cells respectively, then fully blowing the uniformly mixed cells by a pipette, and standing the cells for 5min at room temperature to ensure that the Trizol fully cracks the cells to release RNA in the cells;
(2) adding 1mL of trichloromethane into each tube, shaking vigorously for 20s, and standing at room temperature for 5 min; centrifuging at 4 ℃ and 12000 rpm for 15 min;
(3) putting 400 mu L of supernatant into new 1.5 mL of EP, adding 500 mu L of isopropanol into each tube, slightly reversing the mixture up and down, uniformly mixing, standing for 10 min, removing slightly less fat-soluble impurities, and simultaneously increasing the polarity of the solution to be beneficial to RNA precipitation;
(4) centrifuging at 4 deg.C and 10000 rpm for 10 min, and removing supernatant; adding 1mL of 75% glacial ethanol into each tube, slightly reversing the mixture from top to bottom, and uniformly mixing to remove impurities with larger polarity;
(5) centrifuging at 4 deg.C and 7400 rpm for 5min, removing supernatant, and air drying at room temperature for 7 min;
(6) finally, adding 20 mu L of RNase-free water into each tube, fully and uniformly mixing RNA precipitates, and standing at 60 ℃ for 3 min to fully dissolve the RNA;
(7) detecting RNA concentration on a machine, marking and recording, and storing at-80 ℃ for later use.
Reverse transcription of circRNA
The extracted cellular RNA, quantified at 1ug, was reverse transcribed according to the following procedure:
step 1: 2.0. mu.L 5 XgDNA Eraser Buffer, 1.0. mu.L gDNA Eraser and Total RNA plus RNase Free dH2O is 10 mu L in total, 42 ℃ and 2 min, and after the reaction is finished, the mixture is immediately put into a refrigerator for ice breeding.
Step2:4.0 μL 5× Prime Scrit Buffer,1.0 μL Prime Scrit RTEnzyme MixI,1.0 μLRT Primer Mix,4.0 μLRNase Free dH2O, 20 μ L in total, 37 ℃, 15 min → 85 ℃, 5s → 4 ℃, 2 min, and storing the sample at-20 ℃ after the reverse transcription is finished.
qRT-PCR detection
The reverse transcription of the received cDNA, sample loading and amplification detection are carried out according to the following processes: 7.2 μ L of LDEPC water, 0.4 μ L of has _ circ _0023858 forward primer (5 'CTGGACAAGGCCACATAGAGT 3', SEQ ID NO. 3), 0.4 μ L of has _ circ _0023858 reverse primer (5 'CAGAGCAGCCAATGAAGACAC 3', SEQ ID NO. 4), 10 μ L of LSYBR®Premix Ex TaqTM(Tli RNaseH Plus). The program after sample adding is set as follows: 95 ℃, 30s → 95 ℃, 5s → 60 ℃, 20s, addition of SYBR green fluorescence, 20. mu.L reaction, 45 cycles. 2.9 Western blot immunoblot analysis
Cell protein extraction
(1) The cells were harvested, the old medium was discarded, washed once with PBS, and pancreatin was added in 0.5mL, digested for 30s, and neutralized with pancreatin in 0.5mL of fresh medium.
(2) It was transferred into a new 1.5 mL EP tube, 950rpm, 4min, and centrifuged at room temperature.
(3) The cells were lysed, and a cell lysate (RIPA lysate: PMSF =100: 1) was prepared, and 70 μ L of the lysate was added to each 1.5 mL EP tube, followed by lysis on ice for 27 min. Centrifuging at 12000 rpm at 4 deg.C, discarding precipitate, collecting supernatant, mixing in new 1.5 mL EP tube, and storing at-80 deg.C.
Detecting the protein concentration of a sample
(1) PBS was added to the periphery of the 96-well plate and the humidity was kept in equilibrium.
(2) A standard curve was drawn by adding 20, 18, 16, 14, 12, 8, 4, 0. mu.LPBS and 0, 2, 4, 6, 8, 12, 16, 20. mu.L of the standard protein solution in sequence. (Standard protein solution 5 mg/mL, which was diluted 10-fold with PBS for use.)
(3) And (3) detecting the OD value of the sample, restarting one line, sequentially and respectively adding 16 mu L of PBS into each hole, and then sequentially adding 4 mu L of sample protein.
(4) Add 200. mu.L of BCA working solution (Cu solution: B solution =1: 50)
(5) Placing into a 37 deg.C incubator, and incubating for 36 min.
(6) And detecting OD values of the standard protein solution and the sample protein solution by using an enzyme-labeling instrument, drawing a standard curve, and calculating the concentration of the sample protein.
Western blot immunoblot analysis
(1) And (4) washing the glass plate (the glass plate must be washed clean, otherwise, the glue running quality is easily influenced).
(2) Fixing the glass plate on a fixing frame after drying, selecting glue preparation concentration when preparing glue, wherein 10% glue and 6% glue can run target protein with protein molecular weight (20 KD-80 KD) and (50 KD-150 KD), the glue contains two kinds including separation glue and concentrated glue
(3) Buffer solution system
Western blot is commonly used in a buffer solution system in 3, and comprises an electrophoresis buffer solution, a membrane transfer buffer solution and a TBST buffer solution.
(4) Boiling samples: taking out the sample from-80 ℃, adding 1/4 protein solution volume of protein loading buffer solution after melting, mixing uniformly, boiling the sample at 95 ℃ for 10 min.
(5) Loading: the sample loading is generally 20-50 ug. Firstly, the glass plate is taken down from the fixed frame, the gel leaked outside the glass plate is washed by using double distilled water, then the glass plate is arranged on the fixed electrophoresis frame, and the electrophoresis buffer solution is poured until the glass plate is submerged. The comb was slowly pulled out to expose the lane. 5 mul of Marker is added, and then the sample solution with the corresponding volume is added according to the sample loading quantity.
(6) Electrophoresis: constant voltage electrophoresis, 85V, 30 min (to concentrate the protein for aggregation), 100V, 120 min (to separate the protein).
(7) Film transfer: and taking out the glass plate, slightly opening the glass plate thin plate, and sequentially cutting the strips of the corresponding target proteins by using a rubber cutting plate according to the Marker specification.
Treatment of the sandwich plate: add the transmembrane buffer, immerse the sandwich plate in it, put the sequence: negative pole → sponge → three layers of filter paper → glue → film → three layers of filter paper → sponge → positive pole (three layers of filter paper must drive up the air bubble, otherwise affect the film transfer efficiency).
Film treatment: methanol 2 min → distilled water 5min → transfer buffer 10 min.
Film transferring conditions: constant flow to membrane, 200 mA, 120 min (determined according to the molecular weight of the target protein), and low-temperature membrane transfer.
(8) Primary membrane washing: after the membrane is turned off, the power is turned off, the membrane is taken out and put into a dish, 1 × TBST buffer solution, 5min × 3, and the membrane is quickly washed (quickly shaken on a shaker).
(9) And (3) sealing: after washing the membrane, adding the sealing liquid, slowly shaking at room temperature, and sealing for 150 min.
(10) And (3) secondary membrane washing: the blocking solution was discarded, washed three times with 1 × TBST 4min and shaken quickly on a shaker.
(11) Sealing a first antibody: transferring the membrane into an antibody box with the right side facing upwards, adding 5mL of corresponding specific protein antibody diluent into the antibody box, combining with corresponding protein on the membrane, sealing primary antibody, and keeping the temperature at 4 ℃ for more than 8 hours.
(12) And (3) incubation: the antibody cassette was placed at 37 ℃ and incubated for 32 min, then the primary antibody was recovered and the membrane was transferred to a dish.
(13) And (3) washing the membrane for the third time: the membrane was washed rapidly on a shaker with 1 × TBST for 8 min three times in sequence.
(14) Sealing secondary antibody: adding 7 mL of secondary antibody diluent into the antibody box, then sequentially transferring the membrane into the antibody box, facing upwards, keeping the temperature at room temperature, slowly shaking on a shaking table for 70 min, and recovering the secondary antibody diluent.
(15) Washing the membrane for four times: the membrane was transferred to a dish, washed with 1 × TBST 12 min × 3 and shaken quickly on a shaker.
(16) And (3) computer detection: and (3) preparing a chemiluminescence reagent (liquid A: liquid B =1:1), exposing on a western blot detection exposure device when the chemiluminescence reagent is used in the preparation, and observing the expression condition of the target protein in the sample.
RTCA real-time label-free cell proliferation assay
(1) Firstly, a detection table of an RTCA cell real-time dynamic analyzer is placed in a constant temperature incubator at 37 ℃.
(2) Adding 50 mu L of complete culture medium into a hole of an E-Plate detection Plate in an ultra-clean workbench, enabling the culture medium to cover the bottom of the Plate, paying attention to no bubbles, vertically putting a proliferation Plate into a detector, displaying OK through a message, namely successfully connecting, detecting a base line, and taking out and placing the Plate in the ultra-clean workbench after the test is completed.
(3) Digesting the transfected cells with 0.5mL of pancreatin without EDTA, stopping digestion with 1mL of complete medium, collecting the cells, centrifuging at 950rpm for 4min, discarding the supernatant, adding 1mL of serum-containing medium, suspending the cells, and blowing and mixing the cells uniformly.
(4) Pipette 10. mu.L of cell suspension into counting plate, count and adjust the concentration to 8X 103And (4) sucking 100 mu L of the adjusted cell suspension, adding the cell suspension into an E-Plate proliferation Plate, patting the cell suspension for 10 times in a front-back left-right mode, uniformly mixing, standing in an ultra-clean workbench for 20min, and waiting for the cells to sink to the bottom of the hole.
(5) And vertically placing the E-Plate proliferation Plate added with the cell suspension into the detector again, displaying OK through the message, setting various parameters, and detecting the real-time dynamic state of cell proliferation within 72 hours to obtain a cell proliferation curve.
RTCA real-time label-free cell migration assay
(1) Assembling a CIM Plate detection Plate: in an ultra-clean workbench, a CIM Plate transfer Plate is placed in a corresponding groove of a CIM clamp, and a blue mark point is positioned at the upper right of the clamp and aligned with the clamp;
(2) sucking 165 mu L of serum-containing culture medium, placing the serum-containing culture medium in a lower chamber of the transfer plate, and preventing air bubbles from being generated so that the culture medium in a lower chamber hole forms a convex meniscus;
(3) place the upper chamber on the lower chamber with the blue marks aligned and press down quickly with the hand without generating air bubbles.
(4) And (3) placing 30 mu L of serum-free culture medium in an upper chamber, and patting the medium for 10 times all around to uniformly distribute the medium on the bottom layer.
(5) Placing the assembled CIM Plate migration Plate into a 5% CO solution at 37 deg.C2Balancing for 1h in a cell incubator;
(6) and vertically placing the migration detection plate on the detection platform, displaying OK through the message, namely successfully connecting, setting various parameters, detecting a base line, taking out after the test is finished, and placing the detection plate in an ultra-clean workbench.
(7) Digesting the transfected cells with 0.5mL of pancreatin without EDTA, stopping digestion with 1mL of complete medium, collecting the cells, centrifuging at 950rpm for 4min, discarding the supernatant, adding 1mL of serum-containing medium, suspending the cells, and blowing and mixing the cells uniformly.
(8) Pipette 10. mu.L of cell suspension into counting plate, count cells and adjust the concentration to 2.0X 104one/mL.
(9) Sucking 100 mu L of the adjusted cell suspension into an upper chamber hole of a CIM Plate detection Plate, and lightly beating the cell suspension for 5 times in front, back, left and right to ensure that the cell suspension is uniformly distributed;
(10) standing in an ultraclean workbench at room temperature for 20min until the cells sink to the bottom of the hole;
(11) and vertically placing the CIM Plate migration detection Plate into the detector again, starting Step2, and detecting the real-time dynamic state of cell migration within 72h to obtain a cell migration curve.
Example 1
Expression profiles of has _ circ _0023858 in fibrosis models and IPF blood samples
Experimental methods
Clinical human blood sample of material
According to idiopathic pulmonary fibrosis diagnosis and treatment guideline american thoracic association/european respiratory association idiopathic pulmonary fibrosis (ATS/ERS/JRS/ALAT) issued by the respiratory society of the chinese medical society, the diagnosis and treatment evidence-following guideline 2015, patients with pulmonary fibrosis meeting the diagnosis criteria were selected as study subjects, blood samples were collected, and controls were selected in parallel according to gender, age, and age. All patients signed informed consent.
A549 alveolar epithelial cells and MRC-5 fibroblast strains are purchased from an ATCC cell bank;
EMEM medium, F12 medium, Fetal Bovine Serum (FBS) were purchased from Hyclone, USA; TGF-. beta.1 was purchased from invitrogen; riboFECTTM CP transfection reagent (RiboBiotech, Inc., Guangzhou, China) SYBR Green PCR Master Mix (TAKARA, Dalian, China); total RNA extraction reagent (Trizol) (TAKARA Baozhitian Co., Ltd.).
Second, the method
1. Cell culture and grouping: a549 cells in F12 (1X) culture solution containing 10% fetal calf serum, and MRC-5 cells in EMEM culture solution containing 10% fetal calf serum at 37 deg.C and 5% CO2Culturing in an incubator with saturated humidity. Selecting cells in logarithmic growth phase, preparing single cell suspension, and inoculating the single cell suspension to a six-hole culture plate. And (3) setting a blank control group and a TGF-beta 1 stimulation group, and culturing for 24 hours by using a serum-free culture medium when the cells grow to 70-80% fusion, so that the cells grow synchronously. TGF-beta 1 stimulation group administered with 5nM final concentration TGF-beta 1 stimulation 72h completed the cell conversion to myofibroblasts.
qRT-PCR validation analysis of changes in expression of has _ circ _0023858
(1) After total RNA extraction, RNA concentration and quality were checked using a NanoDrop 2000 spectrophotometer and RNA integrity was assessed by standard denaturing agarose gel electrophoresis. Reverse transcription was performed using the M-MLV reverse transcriptase kit according to the manufacturer's instructions. qRT-PCR was performed using Rotor Gene 3000 and SYBR Green kit.
(2) RT Synthesis of cDNA (reaction solution prepared on ice)
The extracted cellular RNA, quantified at 1. mu.g, was reverse transcribed according to the following procedure:
step 1: 2.0. mu.L 5 XgDNA Eraser Buffer, 1.0. mu.L gDNA Eraser and Total RNA plus RNase Free dH2O is 10 mu L in total, 42 ℃ and 2 min, and after the reaction is finished, the mixture is immediately put into a refrigerator for ice breeding.
Step2:4.0 μL 5× Prime Scrit Buffer,1.0 μL Prime Scrit RTEnzyme MixI,1.0 μLRT Primer Mix,4.0 μLRNase Free dH2O, 20 μ L in total, 37 ℃, 15 min → 85 ℃, 5s → 4 ℃, 2 min, and storing the sample at-20 ℃ after the reverse transcription is finished.
(3) PCR reaction system (reaction mixture prepared on ice)
Double distilled water 7.2 μ L
cDNA as template 2. mu.L
has _ circ _0023858 upstream primer (SEQ ID NO. 3) 0.4. mu.L
0.4 μ L of has _ circ _0023858 downstream primer (SEQ ID NO. 4)
SYBR 10μL
The final volume was 20. mu.L.
Mixing the above mixed solution, pre-denaturing at 95 deg.C for 30s, denaturing at 95 deg.C for 5s, annealing at 60 deg.C for 20s,
extension at 72 ℃ for 40sec, 40 cycles, final extension at 72 ℃ for 7min, storage at 4 ℃. GAPDH was used as an internal control.
Results of three
1. has _ circ _0023858 is up-regulated in TGF-beta 1 stimulation of alveolar epithelial cell A549 and fibroblast MRC-5 (see A in FIG. 1, A is expression change in alveolar epithelial cell A549 and fibroblast MRC-5), indicating that has _ circ _0023858 is closely related to the occurrence of pulmonary fibrosis.
2. Blood samples of clinical IPF patients were collected and blood samples of corresponding parallel control groups (normal persons) were selected according to age and gender for further verification of the expression trend of has _ circ _ 0023858. The qRT-PCR data showed that has _ circ _0023858 was significantly upregulated in IPF patients compared to normal, indicating high expression in IPF patient blood (see B in fig. 1, B is the amount of expression in IPF patient peripheral blood mononuclear cells).
Example 2
Effect of siRNA interference fragment specific to has _ circ _0023858 on myofibroblast transdifferentiation
The experimental method comprises the following steps: 1. cell culture and grouping: a549 and mrc-5 cells in logarithmic growth phase were selected, made into single cell suspension with 0.25% trypsin and seeded in 6-well plates. Setting a blank control group, a TGF-beta 1 stimulating group, a TGF-beta 1+ transfection group and a TGF-beta 1+ transfection control group; when the cells grow to 70-80% fusion, transfecting the siRNA interference fragment and the control siRNA into the TGF-beta 1+ transfection group and the TGF-beta 1+ transfection control group respectively by using riboFECTTM CP transfection reagents, wherein the final concentration is 50nM, the blank control group is cultured by a serum-free culture medium, other groups are stimulated by 5nM TGF-beta 1, and the cells are collected after 72h of culture.
Western blot detection of expression Change in the marker protein (same experimental protocol)
The effect of the siRNA interfering fragment of has _ circ _0023858 on myofibroblast activation-associated protein expression results are shown in fig. 2, and the results were analyzed: further detecting the change of the expression quantity of the alpha-SMA and other pulmonary fibrosis related proteins by a Western blot experiment. These proteins are intercellular mesenchymal markers, and in the process of pulmonary fibrosis, namely under the influence of TGF-beta 1, the fibrotic cells are transformed into myofibroblasts, so that the expression level of the proteins is increased. The results show that in alveolar epithelial cells A549, the TGF-beta 1 stimulation group has up-regulated expression of alpha-SMA, collagen I, collagen III and vimtein compared with the normal control group, and after the transfection of has _ circ _0023858 smart silence, the expression of alpha-SMA, collagen III and vimtein is down-regulated (A in figure 2, which is the result of the expression change of pulmonary fibrosis related proteins in A549 cells given different treatments); in fibroblast MRC-5, the expression of collagen I and alpha-SMA is up-regulated in TGF-beta 1 stimulated group compared with normal control group, and is down-regulated after the transfection of has _ circuit _0023858 smart site (B in figure 2, the result of the expression change of pulmonary fibrosis related protein in MRC-5 given different treated cells).
Example 3
Effect of siRNA interfering fragment specific to has _ circ _0023858 on myofibroblast proliferation and migration
The experimental method is the same as the experimental scheme:
the results of the siRNA interference fragment of has _ circ _0023858 on the proliferation and migration ability of myofibroblasts are shown in FIG. 3, and the results are analyzed as follows: in order to further research the functions of has _ circuit _0023858, the RTCA real-time monitoring technology is adopted to detect the influence of has _ circuit _0023858 on the proliferation and migration of cells, and the detection result shows that the proliferation and migration speed of the fibrotic cells are accelerated after TGF-beta 1 stimulation and are slowed down after ha _ circuit _0023858 smart site transfection compared with a normal group; it was shown that has _ circ _0023858 can promote cell proliferation (a in fig. 3, as a result of siRNA interfering fragment of has _ circ _0023858 on myofibroblast proliferation potency) and migration (B in fig. 3, as a result of siRNA interfering fragment of has _ circ _0023858 on myofibroblast migration potency).
Example 4
Molecular mechanism of has _ circ _0023858 in regulating function of myofibroblasts
And (4) analyzing results: the TGF-beta 1 is used for stimulating alveolar epithelial cells A549 to establish a pulmonary fibrosis cell model, the expression trend of miR-324-5p is verified from the cell level, and qRT-PCR data show that miR-324-5p is expressed and reduced at 12 h, 24h, 48 h and 72h compared with a normal group in the TGF-beta 1 treatment group, wherein the reduction is most obvious at 72h and is opposite to the expression of has _ circ _0023858 (figure 4, an expression change graph of miR-324-5 p).
The experimental method is the same as the experimental scheme (western blot analysis)
And (4) analyzing results: compared with a blank control group, the TGF-beta 1 stimulation group has the advantages that the expression levels of alpha-SMA, vim and collagen are obviously up-regulated, and the TGF-beta 1 can induce the cells to be transformed into myofibroblasts; compared with a TGF-beta 1 stimulation group, the alpha-SMA and vim expression levels of the A549/miR-324-5 p-mici group and the mrc-5/miR-324-5 p-mici group are obviously reduced. The expression of the group A549/miR-324-5p inhibitor is opposite (figure 5, a graph of the effect of the mimic and inhibitor of miR-324-5p on the expression of the protein related to the activation of myofibroblasts), wherein A is the expression change of the protein related to pulmonary fibrosis in the cells A549 cells treated by the miR-324-5p-mimic, B is the expression change of the protein related to pulmonary fibrosis in the cells MRC-5 cells treated by the miR-324-5p-mimic, and C is the expression change of the protein related to pulmonary fibrosis in the cells A549 cells treated by the miR-324-5 p-inhibitor).
FIG. 6 is a result graph of migration ability of miR-324-5p myofibroblasts, wherein A is a result graph of mimic of miR-324-5p on migration ability of myofibroblasts, and B is a result graph of inhibitor of miR-324-5p on migration ability of myofibroblasts.
And (4) analyzing results: the influence of miR-324-5p on cell migration is detected by adopting an RTCA real-time monitoring technology, and the detection result shows that the migration speed of the fibrotic cells is accelerated after TGF-beta 1 stimulation compared with a normal group, and the migration speed of the fibrotic cells is accelerated after miR-324-5p inhibitor transfection; after miR-324-5p imic is transfected, the migration speed of the fibrotic cells is accelerated.
The results show that a regulation network of has _ circ _0023858-miR-324-5p may exist in pulmonary fibrosis, and the reduction of the expression of has _ circ _0023858 can inhibit the activation and migration of myofibroblasts, and can be used as a molecular and drug target for pulmonary fibrosis treatment. Therefore, a specific interference sequence aiming at has _ circ _0023858 is designed and transfected into cells to play a role in down-regulating the expression of has _ circ _0023858, so that the aim of preventing and treating pulmonary fibrosis can be fulfilled.
The invention utilizes siRNA and an overexpression vector to knock down and increase the expression of circRNA-0023858 respectively, and detects the activation of fibroblast and the change of function related indexes under the intervention of circRNA-0023858. Detecting the intracellular localization of the circRNA-0023858 by using in situ hybridization to determine the regulation and control mode of the cells; the combination of circRNA-0023858 and miR-324-5p is determined, and the combination relation of the circRNA-0023858 and miR-324-5p is further determined by applying RNA pull down, Ago RIP and dual-luciferase reporter gene experiments. The influence of miR-324 on the activation and function of myofibroblasts is further clarified, and the miR-324-5p regulates and controls a Hippo signal path through the target genes YAP1 and AJUBA respectively, so that the mechanism of regulating and controlling IPF by circRNA-0023858 is researched, and the aim of preventing and treating pulmonary fibrosis is fulfilled.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Binzhou medical college
<120> application of reagent for down-regulating expression of circular gene in preparation of medicine for preventing and/or treating pulmonary fibrosis and medicine
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aatggagcaa atctgacagc ccaggatgac cggggatgca ctcctttaca tcttgctgca 180
actcatggac attctttcac tttacaaata atgctccgaa gtggagtgga tcccagtgtg 240
actgataaga gagaatggag acctgtgcat tatgcagctt ttcatgggcg gcttggctgc 300
ttgcaacttc ttgttaaatg gggttgtagc atagaagatg tggactacaa tggaaacctt 360
ccagttcact tagcagccat ggaaggccac cttcactgtt tcaaattcct agtcagtaga 420
atgagcagtg cgacgcaagt tttaaaagct ttcaatgata atggagaaaa tgtactggat 480
ttggcccaga ggttcttcaa gcagaacatt ttacagttta tccagggggc tgagtatgaa 540
ggaaaagacc tagaggatca ggaaacttta gcatttccag gtcatgtggc tgcctttaag 600
ggtgatttgg ggatgcttaa gaaattagtg gaagatggag taatcaatat taatgagcgt 660
gctgataatg gatcaactcc tatgcataaa gctgctggac aaggccacat agagtgtttg 720
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Claims (9)

1. A medicament for down-regulating expression of circular has _ circ _0023858, the medicament comprising an interfering double-stranded RNA, the nucleotide sequence of the circular has _ circ _0023858 being shown in SEQ ID No.1, the interfering double-stranded RNA being synthesized according to the sequence of nucleotides shown in SEQ ID No.2, the interfering double-stranded RNA having the following structure:
Figure FDA0002881832870000011
wherein each "|" represents base pairing.
2. Use of a medicament according to claim 1 for the preparation of a medicament for the prevention and/or treatment of pulmonary fibrosis.
3. The use of the medicament of claim 1 in the preparation of a medicament for preventing or treating pulmonary fibrosis by regulating a Hippo signaling pathway through the adsorption of miR-324-5 p.
4. The application of the medicine of claim 1 in preparing the medicine for preventing or treating pulmonary fibrosis by regulating the target gene YAP1 and further regulating a Hippo signal pathway through adsorbing miR-324-5 p.
5. The use of the medicament of claim 1 in the preparation of a medicament for preventing or treating pulmonary fibrosis by regulating a target gene AJUBA through adsorption of miR-324-5p and further regulating a Hippo signal pathway.
6. Use of the medicament of claim 1 for the preparation of a medicament for inhibiting differentiation of lung fibroblasts.
7. Use of a medicament according to claim 1 for the preparation of a medicament for inhibiting the activation of lung fibroblasts.
8. Use of a medicament according to claim 1 for the preparation of a medicament for inhibiting proliferation of lung fibroblasts.
9. Use of the medicament of claim 1 for the preparation of a medicament for inhibiting migration of lung fibroblasts.
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