CN110951735B - Long-chain non-coding RNA lnc-PMIF, small interfering RNA thereof and application thereof - Google Patents
Long-chain non-coding RNA lnc-PMIF, small interfering RNA thereof and application thereof Download PDFInfo
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Abstract
The invention discloses a long-chain non-coding RNA lnc-PMIF and a small interfering RNA of the lnc-PMIF. In addition, the invention also discloses application of the long-chain non-coding RNA lnc-PMIF in preparation or screening of products for diagnosing human skeletal system diseases, and application of small interfering RNA in preparation of medicines or pharmaceutical compositions for treating human skeletal system diseases. According to the invention, the function of lnc-PMIF is verified by designing a small interfering RNA sequence, and the lnc-PMIF is proved to be capable of inhibiting osteoblast migration through HuR, and the lnc-PMIF sequence can be used for diagnosing bone system diseases or serving as a therapeutic target; the designed small interfering RNA sequence can be used for preparing medicines and medicinal compositions for treating diseases of the skeletal system, including human osteoporosis, femoral head necrosis, joint degeneration, scoliosis and other diseases of the skeletal system.
Description
Technical Field
The invention belongs to the technical field of molecular biology and medicine, and particularly relates to a long-chain non-coding RNA lnc-PMIF, a small interfering RNA thereof and application thereof.
Background
Non-coding RNA (Non-coding RNA) refers to RNA that does not encode a protein. The RNA includes microRNA (miRNA), long non-coding RNA (lncRNA for short), and the like. The common feature of these RNAs is that they are all transcribed from the genome, but do not encode proteins, which enable their respective biological functions at the RNA level. Wherein the lncRNA is eukaryotic endogenous RNA with the length of more than 200nt, is widely distributed in eukaryotic organisms, and plays an important role in regulation and control in various physiological and pathological processes such as cell differentiation, organ development, disease occurrence and the like. While osteoblasts are the main functional cells for bone formation. As people age, bone development also changes dynamically, with osteoblast-directed bone formation and osteoclast-directed bone resorption constituting the dynamic balance of bone remodeling. When osteoblasts are abnormally differentiated, corresponding bone metabolic diseases can be caused, such as hyperosteogeny, osteoporosis, femoral head necrosis, joint degeneration, scoliosis and the like.
The 1ncRNA can play an important role in cell differentiation, ontogeny and diseases, for example, lnc-DC can regulate nerve dendritic cell differentiation, lnc RNA-DIGIT can regulate embryo inner embryo layer formation, lnc RNA-CLMAT1 can induce colorectal cancer liver transfer and the like; the research is mainly focused on the fields of nerves, development, tumors and the like, and the research on the involvement of lncRNA in bone metabolic diseases such as osteoporosis and the like is less reported.
DF Lee et al of West Neishan medical school of New York university found that the expression of lncRNA H19 was reduced in iPSC-derived osteoblasts of Li-Fremen syndrome (LFS) patients, and further analyzed that the expression of H19 in osteoblasts was restored to promote the differentiation of osteoblasts and simultaneously inhibit the tumorigenic capacity of osteosarcoma. The lncRNA is suggested to play a role in osteoblast differentiation, and the lncRNA can be used as a medicine for treating bone system diseases such as osteoporosis. However, the study was limited to osteoblasts derived from iPSC of Li-Fremen syndrome patients, and no study was made on osteoblasts under normal non-pathological conditions (e.g., aging, menopause, long-term bed rest, etc.) (Lee DF, et al. modeling facial cancer with induced plotent cells, cell.2015Apr 9; 161(2): 240-254).
Disclosure of Invention
The technical problem to be solved by the present invention is to provide a long non-coding RNA lnc-PMIF, which addresses the above-mentioned deficiencies of the prior art. According to the invention, through lncRNA high-throughput sequencing, cDNA terminal Rapid Amplification of CDNA Ends (RACE) and clone sequencing, an lncRNA is discovered and identified, named lnc-PMIF, a primer of the lnc-PMIF is designed, the expression of the lnc-PMIF in bone tissues of an aged mouse osteoporosis model and an ovariectomized osteoporosis mouse model is detected, and the correlation between the expression of the lnc-PMIF and the occurrence of osteoporosis is proved. And then the function of lnc-PMIF is verified by designing a small interfering RNA sequence, and the lnc-PMIF is proved to be capable of inhibiting osteoblast migration through HuR.
In order to solve the technical problems, the invention adopts the technical scheme that: a long non-coding RNA lnc-PMIF, which is characterized in that the cDNA sequence of the long non-coding RNA is shown as SEQ ID NO: 1 or the sequence of the cDNA of the long-chain non-coding RNA is the same as that of the cDNA shown in SEQ ID NO: 1 is fully complementary to an equal length of nucleotide sequence.
The long non-coding RNA lnc-PMIF is characterized in that the primer sequence for detecting the long non-coding RNA is as follows:
lnc-PMIF-1-Forward, the nucleotide sequence of which is shown in SEQ ID NO: 2;
lnc-PMIF-1-Reverse, the nucleotide sequence of which is shown in SEQ ID NO: 3;
lnc-PMIF-1-Probe, the nucleotide sequence of which is shown in SEQ ID NO: 4;
lnc-PMIF-2-Forward, the nucleotide sequence of which is shown in SEQ ID NO: 5;
lnc-PMIF-2-Reverse, the nucleotide sequence of which is shown in SEQ ID NO: 6.
in addition, the invention also provides application of the long-chain non-coding RNA lnc-PMIF in preparation of products for diagnosing human skeletal system diseases or screening of products for diagnosing human skeletal system diseases.
The use as described above, wherein the human skeletal system disease comprises osteoporosis, femoral head necrosis, joint degeneration or scoliosis.
The use as described above, wherein the product comprises a formulation, a chip, a reagent or a kit.
Further, the invention provides a small interfering RNA of the long non-coding RNA lnc-PMIF, which is characterized in that the sense strand sequence of the small interfering RNA is shown as SEQ ID NO: 7, the antisense strand sequence of the small interfering RNA is shown as SEQ ID NO: 8.
furthermore, the invention provides an application of the small interfering RNA of the long-chain non-coding RNA lnc-PMIF in preparing a medicine or a pharmaceutical composition for treating human skeletal system diseases.
The use as described above, wherein the human skeletal system disease comprises osteoporosis, femoral head necrosis, joint degeneration or scoliosis.
Compared with the prior art, the invention has the following advantages:
1. according to the invention, an lncRNA is discovered and identified through lncRNA high-throughput sequencing, cDNA terminal rapid amplification technology (RACE) and clone sequencing, is named lnc-PMIF, a primer of the lnc-PMIF is designed, the expression of the lnc-PMIF in bone tissues of an aged mouse osteoporosis model and an ovary-removed osteoporosis mouse model is detected, and the correlation between the expression and the occurrence of osteoporosis is proved.
2. According to the invention, the function of lnc-PMIF is verified by designing a small interfering RNA sequence, and the lnc-PMIF is proved to be capable of inhibiting osteoblast migration through HuR, and the lnc-PMIF sequence can be used for diagnosing bone system diseases or serving as a therapeutic target; the designed small interfering RNA sequence can be used for preparing medicines and medicinal compositions for treating diseases of the skeletal system, including human osteoporosis, femoral head necrosis, joint degeneration, scoliosis and other diseases of the skeletal system.
The technical solution of the present invention is further described in detail with reference to the accompanying drawings and embodiments.
Drawings
FIG. 1 is a graph of heatmap obtained by detecting the expression of lncRNA in bone-forming cells of an aged osteoporosis mouse model and an ovariectomized osteoporosis mouse model by using the Illumina sequencing technology platform in example 1 of the present invention.
FIG. 2 is an electrophoresis chart of the products of RACE experiments performed in example 2 of the present invention by using RACE technology to extract RNA from MC3T3-E1 cell line derived from mouse.
FIG. 3 is a graph showing the results of detecting the expression level of long non-coding RNA lnc-PMIF in bone-forming cells of an aged osteoporosis mouse model and an ovariectomized osteoporosis mouse model by using the qPCR technique in example 3 of the present invention.
FIG. 4 is a graph showing the results of the expression level of lnc-PMIF in osteoblasts of the control group of interference sequences and the group of transfected interference sequences transfected with small interference RNA sequence of lnc-PMIF according to example 4 of the present invention.
FIG. 5 is a graph showing the results of expression levels of cell migration-associated genes Actin and Mmp-2 in the interference sequence control group and the transfection interference sequence group into which the small interference RNA sequence of lnc-PMIF is transfected in example 5 of the present invention.
FIG. 6 is a graph showing the results of the scratch test and the Transwell test for the control group of interference sequences and the group of small interference RNA sequences transfected with lnc-PMIF according to example 6 of the present invention.
FIG. 7 is a graph showing the results of RNA pulldown-MS and RIP experiments using lnc-PMIF and HuR in example 7 of the present invention.
FIG. 8 is a graph showing the results of bone formation in mice transfected with C57/BL6 by tail vein injection using the small interfering RNA sequence of lnc-PMIF according to example 8 of the present invention.
FIG. 9 is a diagram showing the results of bone microstructure of C57/BL6 mice transfected by tail vein injection using the small interfering RNA sequence of lnc-PMIF in example 9 of the present invention.
Detailed Description
Embodiments of the present invention are illustrated below by specific examples, and unless otherwise indicated, the experimental methods disclosed in the present invention are all performed by conventional techniques in the art.
Example 1
Detecting a result heatmap picture of the expression of lnc RNA in bone forming cells of an aged osteoporosis mouse model and an ovariectomized osteoporosis mouse model by using an Illumina sequencing technology platform; indicating the expression difference of lncRNA in bone forming cells during the aging process.
(1) Establishing an old osteoporosis mouse model:
c57BL/6 experimental mice were selected and bred to 6 months of age (control group) and 18 months of age (aged group).
(2) Mice were sacrificed, femurs and tibias were collected, bone marrow was isolated and placed in osteoblast conditioned medium in 5% CO2Culturing at 37 deg.C.
(3) Extracting total RNA from cells by using a Trizol method, and detecting the purity of a sample by using a spectrophotometer (A260/A280);
(4) agilent 2100RNA Nano 6000Assay Kit (Agilent Technologies, CA, USA) to test the integrity and concentration of RNA samples;
(5) starting with 3. mu.g of total RNA per sample, Ribo-ZeroTMRemoving ribosome RNA (rRNA) in a sample by GoldKits, and respectively selecting different index labels according to the operation instruction of NEB Next Ultra direct RNA library Prep Kit for Illumina (NEB, Ispawich, USA) to construct an lnc RNA library;
(6) based on an Illumina sequencing technology platform, a double-end sequencing (PE150) method is utilized to construct a strand specificity library (the insert fragment is 350bp) from which ribosome RNA is removed for sequencing, lncRNA sequencing data is obtained, and each sample generates clean data not lower than 12 Gb.
The results are shown in FIG. 1. As can be seen from the figure, the expression level of lnc-RNA in osteoblasts of old osteoporosis mice is obviously different from that of osteoblasts of young mice, and the lnc-RNA is remarkably up-regulated, which indicates that the expression level of lnc-RNA in bone tissues is related to osteoporosis.
Example 2
Obtaining the sequence information of the lnc-PMIF full-length cDNA:
this example provides a method for obtaining a full-length cDNA sequence of long non-coding RNA lnc-PMIF, comprising the following steps:
step one, extracting total RNA from an MC3T3-E1 cell line derived from a mouse by adopting a Trizol method, removing ribosomal RNA (rRNA) in a sample, and constructing chain specificity library sequencing by utilizing a double-end sequencing method based on an Illumina sequencing technology platform to obtain lnc RNA sequence data;
step two, designing 3 'RACE and 3' RACE primers according to lnc RNA sequence information obtained by sequencing as follows;
TABLE 1 RACE primers
Step three, using RACE kit 3' -Full RACE Core Set with PrimeScriptTMRTase (Takara) and 5' -Full RACE Kit with TAP (Takara) were subjected to 3' RACE and 5' RACE, respectively, to obtain RACE products;
step four, subjecting the RACE products to agarose gel electrophoresis (see figure 2, proving that lnc-PMIF exists in bone forming cells), cutting gel and recovering cDNA bands with different sizes;
and step five, connecting the recovered cDNA to a sequencing vector pGEM-T by a T-A cloning method for sequencing to obtain a full-length cDNA sequence of the long-chain non-coding RNA lnc-PMIF, wherein the full-length cDNA sequence is shown as SEQ ID No: 1, lnc-PMIF was 1.5kb in length.
Example 3
The detection by utilizing the qPCR technology finds that lnc-PMIF is highly expressed in bone forming cell samples of an aged osteoporosis mouse model and an Ovariectomized (OVX) osteoporosis mouse model.
(1) Specific primers of lnc-PMIF are designed and prepared, and the sequences are shown in Table 2;
(2) establishing an old osteoporosis mouse model:
c57BL/6 experimental mice were selected, raised to 6 months of age (control group), 18 months of age (aged group), sacrificed, and femoral and bone marrow cell samples were collected.
(3) Establishing an OVX osteoporosis mouse model:
selecting C57BL/6 experimental female mice, shaving waist after anesthesia to be an operation area, opening 0.5 cm on two sides of dorsal midline respectively, finding ovary and uterine horn with forceps, dissociating ovary, removing, suturing, sterilizing, and removing a piece of adipose tissue around the ovary with the same size as the ovary in the control group. During the experiment the animals were free to eat, drink water, and after 3 months the mice were sacrificed and femoral and bone marrow cell samples were collected.
(4) Extracting RNA of a mouse bone tissue sample:
bone tissue samples were taken, bone marrow cells were aseptically flushed out using a 27G1/2 syringe, centrifuged at 2000rpm for 5 minutes, the supernatant was discarded, the cells were cultured in osteoblast conditioned medium, half a liquid change every 5 days, and the cells were harvested after 28 days. The cells were transferred to a 1.5mL EP tube, and 1mL TRIzol (Invitrogen) was added, followed by shaking and mixing. Centrifuge at 12000g for 20min at 4 ℃. The supernatant was removed and transferred to a new 1.5mL EP tube. Add 200. mu.L of chloroform to 1mL of TRIzol, shake the mixture up and down by hand for 15s, and leave the mixture at room temperature for 2-3 min. Centrifuge at 12000g for 15min at 4 ℃. The EP tube was removed, the sample was divided into three layers, and the upper aqueous phase was transferred to a new 1.5mL EP tube. Add 4 ℃ pre-chilled isopropanol (500. mu.L/1 mL TRIzol), gently pipette and mix, and precipitate RNA. Standing in a refrigerator at-20 deg.C for 30min to precipitate RNA. Centrifuge at 12000g for 10min at 4 ℃. The supernatant was removed, 1mL 75% ethanol was slowly added along the tube wall, and mixed by gentle pipetting. Centrifuge at 7500g for 10min at 4 ℃. The supernatant was removed and the pellet was dried at room temperature for 10-20min, but not completely. Add 50. mu.l RNase-free water, blow and mix well, dissolve RNA. (can be stored in a refrigerator at-80 ℃) and the absorbance values of RNA at 260nm and 280nm are measured by an ultraviolet spectrophotometer. The purity of the obtained RNA was determined based on the A260/A280 ratio, and the A260/A280 ratio of the pure RNA was about 2.0 (in general experiments, the ratio was preferably controlled to be 1.7 or more and 2.1 or less). Electrophoresis, results observed on a gel imager: the ratio of 28S bands to 18S bands should be close to 2:1, with the 5S bands unlighted.
(5) Detecting the expression change of lnc-PMIF in bone tissue samples of aged osteoporosis mice by utilizing a qPCR technology:
the extracted RNA was first reverse transcribed into cDNA using the TAKARA reverse transcription kit. The reverse transcription conditions were: 15min at 37 ℃; 15s at 85 ℃. Taking cDNA of each group as a template, taking GAPDH as an internal reference, and detecting the expression quantity of the lnc-PMIF gene in osteoblasts of aged osteoporosis mice by adopting qPCR. The qPCR reaction conditions were: denaturation at 95 ℃ for 30 s; 10s at 95 ℃; 60 ℃ for 30s, 44 cycles. The primer sequences of lnc-PMIF and GAPDH used were as follows:
TABLE 2 qPCR primer sequences
The results are shown in fig. 3 (values are expressed as "mean ± sd", significance between the two groups was tested by students't,. P <0.05,. P <0.001), and it can be seen that lnc-PMIF is highly expressed in osteoblasts of aged osteoporotic mice, indicating that the expression level of lnc-PMIF in bone tissue is related to osteoporosis.
Example 4
The RNA interference sequence of lnc-PMIF can effectively inhibit the expression of lnc-PMIF in preosteoblasts MC3T 3-E1.
(1) Preparation of small interfering RNA sequence of lnc-PMIF:
synthesizing an oligonucleotide single strand and a complementary double strand according to the nucleotide sequence of lnc-PMIF, purifying by high performance liquid chromatography, carrying out front-and-back 2 ' -4 ' site thiophosphoryl skeleton modification and 3' end cholesterol labeling, and finally obtaining the small RNA interference sequence si-lnc-PMIF of the lnc-PMIF. Next, a negative control interfering sequence was synthesized. The synthesized oligonucleotides were diluted to 20. mu.M stock using RNase free water, aliquoted and frozen at-80 ℃. The small interfering RNA sequence of lnc-PMIF is as follows:
TABLE 3 Small interfering RNA sequences of lnc-PMIF
(2) Transfection of preadipocytes MC3T3-E1 with the small interfering RNA sequence of lnc-PMIF:
grouping experiments: transfection scrambling sequence group: transfecting lnc-PMIF siRNA; interfering sequence control group: siRNA-NC was transfected. Each well was prepared by taking preosteoblasts MC3T3-E1 at 1X 106Cell number per well 2mL of α -MEM cell culture medium containing 10% FBS, 100 μ g/mL streptomycin, 100 μ g/mL penicillin 10% FBS, 1% L-glutamine in 6-well plates; cell confluence reaches 70-90% within 24 hours; adding 20pmol siRNA 5ul into 250 ul of alpha-MEM serum-free medium, and mixing the mixture softly and evenly; diluting 5. mu.L lipofectamin 2000 reagent with 250. mu.L serum-free alpha-MEM, mixing gently, and standing at room temperature for 5 minutes; mixing the diluted siRNA and lipofectamin 2000 reagent, gently mixing uniformly, and standing for 20 minutes at room temperature to form an siRNA/lipofectamin compound; mu.L of the complex was added to the wells of the plate containing the cells, supplemented with 1.5mL of α -MEM serum-free medium, and the plate was gently shaken back and forth. Exposing the cells to CO2After incubation for 6 hours at 37 ℃ in the incubator, the medium was changed and incubation continued. After 48h, additional detection steps after transfection were performed.
(3) RNA extraction from MC3T3-E1 cells after transfection:
TRIzol (Invitrogen) was added to each well of the plate containing the transfected cells, and after the cells were sufficiently lysed, they were transferred to a new 1.5mL EP tube, and the extraction steps were the same as above.
(4) The expression level of lnc-PMIF was measured by qPCR (same procedure as above), and the primer sequences of lnc-PMIF and GAPDH used were as shown in Table 2.
The results are shown in figure 4 (values are expressed as "mean ± standard deviation", significance between the two groups was tested using students't test,. times.p < 0.001). As can be seen in the figure, the expression level of lnc-PMIF is significantly reduced after transfection with the small interfering RNA sequence. The small interfering RNA sequence of lnc-PMIF designed and prepared by the invention can effectively inhibit the expression of lnc-PMIF in preosteoblasts MC3T 3-E1.
Example 5
The small interfering RNA sequence of lnc-PMIF can effectively promote the expression level of cell migration related genes Actin and Mmp-2 in preosteoblasts.
Transfection and RNA extraction reference example 4 was followed by taking cDNA of each group as a template and GAPDH as an internal reference, and qPCR was used to detect the expression of cell migration-associated genes Actin and Mmp-2. The sequence of the primer of the Actin and Mmp-2 is as follows:
TABLE 4 primer sequences for genes associated with cell migration
The results are shown in fig. 5 (values are expressed as "mean ± standard deviation", significance between the two groups is tested by students't test,. P <0.05,. P <0.001), and it can be seen from the figure that, in the preosteoblasts, when the expression level of lnc-PMIF is decreased, the expression level of the genes related to cell migration is increased, which indicates that the small interfering RNA sequence of the present invention can effectively promote the expression of the genes Actin and Mmp-2 related to cell migration, and can promote osteoblast migration.
Example 6
The small interfering RNA sequence of lnc-PMIF can effectively promote the migration of preosteoblasts.
(1) Scratch test:
grouping experiments: transfection scrambling sequence group: transfecting lnc-PMIF siRNA; interfering sequence control group: siRNA-NC was transfected. Transfection reference example 4. Collecting the treated preosteoblasts MC3T3-E1 at 5 × 105The number of cells/well was seeded in 6-well plates (straight lines drawn behind the wells in advance with a marker pen), each set was prepared with 3 multiple wells, and after overnight the plates were scratched with a tip perpendicular to the transverse line behind the ruler. Washed 3 times with PBS, the scraped cells were removed and serum-free medium was added. Put in 37 ℃ 5% CO2And (5) an incubator for culture. Photographs were taken at 0 and 18 hours and the width of the scratch was calculated.
(2) Transwell experiment:
siRNA-treated preosteoblasts MC3T3-E1 were taken, the cells were carefully washed with PBS to remove serum completely, the medium was changed to serum-free medium, and cell starvation was performed for 18 h. Digesting the starved cells, adding a serum-free culture medium, uniformly blowing, centrifuging at 800rpm for 5 min; and (4) discarding the supernatant, adding the cell precipitate into a serum-free culture medium, blowing, beating, uniformly mixing and counting. The Transwell chamber was taken out and placed in a 24-well plate, 700. mu.l of the lower chamber medium (. alpha. -MEM containing 10% FBS) was added, and 200. mu.l of the cell suspension (0.6X 10) was added to the chamber5) And the other group of lower chambers is added with serum-free culture medium. The Transwell chamber is placed at the corresponding position of the 24-hole plate, so that bubbles at the bottom of the chamber are prevented, and the experimental result is influenced. After incubation for 12 hours at 37 ℃, the cells were respectively taken out for subsequent 0.1% crystal violet staining observation.
The results are shown in FIG. 6, and it can be seen from the figure that, in the preosteoblasts, when the expression level of lnc-PMIF is reduced, the cell migration capacity is improved, the distance and the number of osteoblast migration can be effectively increased by transfecting the small interfering RNA sequence of lnc-PMIF, which indicates that the small interfering RNA sequence of lnc-PMIF can inhibit osteoblast migration, and suggests that lnc-PMIF can be used as a target for the action of drugs for diagnosing and treating bone system related diseases such as osteoporosis.
Example 7
lnc-PMIF can specifically bind HuR.
(1) Bioinformatics verification
The binding score of lnc-PMIF to HuR was predicted to be 0.90 by the RPISeq predictive analysis tool (http:// pridb. gdcb. iastate. edu/RPISeq /); the binding sites were predicted by catRAPID online prediction website (http:// service. tartaglalab. com/page/catapid _ group) as follows:
TABLE 5 binding sites of lnc-PMIF to HuR predicted by catRAPID
(2)RNA Pulldown：
The lnc-PMIF gene was cloned into pGEM-T (Promega), labeled with Biotin (Biotin RNA labeling Mix, Roche) as a probe, and the protein interacting with lnc-PMIF was extracted from a cell lysate of MC3T3-E1 cells using an RNA probe, and Western Blot detection was performed using HuR antibody (abcam, ab200342, 1:1000) and Lamin A/C antibody (Boshide, BA1227, 1: 400).
(3) And (RIP): MC3T3-E1 cells were treated with a protein lysate (100mM of KCl,5mM of MgCl. sub.C.) supplemented with protease inhibitors (Byunnan Co.) and RNase inhibitors (Takara Co.)2,10mM of HEPES[pH 7.0]0.5% NP-40, and 1mM of dithiothreitol) for 30 minutes on ice and centrifuged at 12,000g for 15 minutes. The supernatant was mixed with Protein A/G magnetic beads and incubated with HuR antibody (abcam, ab200342, 1:1000) and IgG Protein (Bycy, Inc.) at 4 ℃ overnight, respectively. The Protein A/G magnetic bead complex was separated by a magnetic stand. RNA was extracted by the conventional Trizol method and qPCR detection was performed (the primer sequences are shown in tables 1 and 3).
(4) qPCR detects expression of Actin (with GAPDH as an internal control): grouping experiments: transfection scrambling sequence group: transfecting lnc-PMIF siRNA; interfering sequence control group: siRNA-NC was transfected.
The results are shown in fig. 7 (values are expressed as "mean ± standard deviation", significance between the two groups using students't test,. P <0.05,. P <0.01,. P <0.001), demonstrating the regulatory mechanism of lnc-PMIF on osteoblast migration, i.e. inhibition of osteoblast migration by binding to HuR. The small interfering RNA sequence of lnc-PMIF can be specifically combined with HuR and lnc-PMIF can promote the expression of Actin.
Example 8
The small interfering RNA sequence of lnc-PMIF can effectively promote bone formation.
(1) Establishing an old osteoporosis mouse model:
c57BL/6 experimental mice were selected and fed to 18 months of age.
(2) Randomly dividing the aged osteoporosis mice into two groups, respectively injecting interference sequence contrast and interference sequence into tail vein, wherein the dosage is 10mg/kg body weight), injecting for 1 time in 2 weeks, and continuously for 4 times; sacrificed two weeks after the last injection; injecting calcein solution into each mouse at a dose of 20mg/kg body weight on the last-to-last day and the last-to-last day before sacrifice respectively; mice were sacrificed and femurs were collected and preserved in 10% formalin.
(3) The femurs were cryosectioned and bone-forming lines were photographed under a fluorescence microscope, see figure 8 (values are expressed as "mean ± standard deviation", significance between the two groups was tested by students't,. P < 0.05).
As can be seen from FIG. 8, the lnc-PMIF small interfering RNA sequence designed by the invention can effectively promote bone formation.
Example 9
The lnc-PMIF interference sequence can effectively improve the bone microstructure.
(1) Establishing an old osteoporosis mouse model:
c57BL/6 experimental mice were selected and fed to 18 months of age.
(2) Randomly dividing the aged osteoporosis mice into two groups, respectively injecting interference sequence contrast and interference sequence into tail vein, wherein the dosage is 10mg/kg body weight), injecting for 1 time in 2 weeks, and continuously for 4 times; sacrificed two weeks after the last injection; injecting calcein solution into each mouse at a dose of 20mg/kg body weight on the last-to-last day and the last-to-last day before sacrifice respectively; mice were sacrificed and femurs were collected and preserved in 10% formalin.
(3) Femurs were subjected to micro-CT scanning and imaged and their bone volume/tissue volume (BV/TV) indices were analyzed as shown in figure 9 (values are expressed as "mean ± standard deviation", significance between the two groups was tested using students't test,. P < 0.01).
As can be seen from FIG. 9, the small interfering RNA sequence of lnc-PMIF designed by the invention can effectively improve the bone microstructure.
According to the invention, through lncRNA high-throughput sequencing, cDNA terminal Rapid Amplification of CDNA Ends (RACE) and clone sequencing, an lncRNA is discovered and identified, named lnc-PMIF, a primer of the lnc-PMIF is designed, the expression of the lnc-PMIF in bone tissues of an aged mouse osteoporosis model and an ovariectomized osteoporosis mouse model is detected, and the correlation between the expression of the lnc-PMIF and the occurrence of osteoporosis is proved. And then the function of lnc-PMIF is verified by designing a small interfering RNA sequence, and the lnc-PMIF is proved to be capable of inhibiting osteoblast migration through HuR. The lnc-PMIF sequence can be used for diagnosing skeletal system diseases or used as a therapeutic target; the designed small interfering RNA sequence can be used for preparing medicines and medicinal compositions for treating diseases of the skeletal system, including human osteoporosis, femoral head necrosis, joint degeneration, scoliosis and other diseases of the skeletal system.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, changes and equivalent structural changes made to the above embodiment according to the technical spirit of the present invention still fall within the protection scope of the technical solution of the present invention.
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Claims (8)
1. A long non-coding RNA lnc-PMIF, which is characterized in that the cDNA sequence of the long non-coding RNA is shown as SEQ ID NO: 1 or the sequence of the cDNA of the long-chain non-coding RNA is the same as that of the cDNA shown in SEQ ID NO: 1 is fully complementary to an equal length of nucleotide sequence.
2. The long-chain non-coding RNAlnc-PMIF of claim 1, wherein the primer sequences for detecting the long-chain non-coding RNA are as follows:
lnc-PMIF-1-Forward, the nucleotide sequence of which is shown in SEQ ID NO: 2;
lnc-PMIF-1-Reverse, the nucleotide sequence of which is shown in SEQ ID NO: 3;
lnc-PMIF-1-Probe, the nucleotide sequence of which is shown in SEQ ID NO: 4;
lnc-PMIF-2-Forward, the nucleotide sequence of which is shown in SEQ ID NO: 5;
lnc-PMIF-2-Reverse, the nucleotide sequence of which is shown in SEQ ID NO: 6.
3. use of the long chain non-coding RNAlnc-PMIF of claim 1 in the preparation of a product for diagnosing skeletal system diseases in humans or in screening products for diagnosing skeletal system diseases in humans.
4. Use according to claim 3, wherein the human skeletal system disease comprises osteoporosis, femoral head necrosis, joint degeneration or scoliosis.
5. The use of claim 3, wherein the product comprises a formulation, chip, reagent or kit.
6. The long non-RNAlnc-PMIF-encoding small interfering RNA of claim 1, wherein the sense strand of the small interfering RNA has the sequence set forth in SEQ ID NO: 7, the antisense strand sequence of the small interfering RNA is shown as SEQ ID NO: 8.
7. use of the small interfering RNA of the long non-coding RNA lnc-PMIF according to claim 6 for the preparation of a medicament or pharmaceutical composition for the treatment of a disease of the human skeletal system.
8. Use according to claim 7, wherein the human skeletal system disease comprises osteoporosis, femoral head necrosis, joint degeneration or scoliosis.
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