CN116287179A - Identification, application and detection method of non-coding RNA FPASL of molecular marker for diagnosis of hypertrophic scar - Google Patents
Identification, application and detection method of non-coding RNA FPASL of molecular marker for diagnosis of hypertrophic scar Download PDFInfo
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Abstract
The identification, application and detection method of the marker non-coding RNA FPASL for the diagnosis of the hypertrophic scar molecules comprises the following steps: collecting normal skin tissue samples of hypertrophic scars and paraspinal scars during the operation of a patient with hypertrophic scars; culturing primary fibroblasts derived from a hypertrophic scar and normal skin beside the scar from a tissue sample; detecting expression of non-coding RNA FPASL in the two sets of tissue samples and primary fibroblasts; predicting specificity and sensitivity of non-coding RNA FPASL diagnosis proliferation scar through ROC curve; analyzing non-coding RNA FPASL non-coding property by adopting bioinformatics; the long-chain non-coding RNA FPASL expression is interfered by using a locked nucleotide technology (LNA), the non-coding RNA FPASL is overexpressed by using a slow virus, and the proliferation of the fibroblast is detected by using CCK 8. The invention can obviously reduce non-coding RNA FPASL in the hypertrophic scar, has high specificity and high sensitivity, and provides powerful technical support for diagnosing the hypertrophic scar.
Description
Technical Field
The invention belongs to the field of molecular diagnosis, and in particular relates to an identification, application and detection method of a molecular marker non-coding RNA for diagnosing a hypertrophic scar in the hypertrophic scar.
Background
Hypertrophic scars are a common post-injury complication of skin that can occur after the skin has been damaged by burns, surgery, vaccination, etc., and are difficult to heal or resolve spontaneously. The hypertrophic scar accounts for about 30% to 50% after surgery or trauma, and the prevalence after burn is even higher. These hypertrophic scars can cause symptoms such as pain, itching, etc., and in severe cases can result in limited activity due to lack of elasticity or contracture, which can in part cause long-term physical dysfunction and psychological stress to the patient. Thus, the hypertrophic scar following injury and its attendant aesthetic and functional sequelae remain major challenges for hypertrophic scars. The mechanism of action of the hypertrophic scars is still unclear, and the prevention and treatment of the hypertrophic scars are hindered to a certain extent. Local silica gel application, compression or massage therapy, triamcinolone (TAC), intralesional injection of corticosteroids or 5-fluorouracil (5-FU), laser ablation and surgery are the most common options for preventing or treating hypertrophic scars. However, many of these therapies lack evidence of effectiveness and safety and appear to have a high recurrence rate. Currently, there is no ideal molecular marker for assessing the progression of hypertrophic scars and the risk of recurrence in the clinic. Therefore, finding new highly specific, highly sensitive biomarkers would help to diagnose and prevent hypertrophic scars, and develop potential therapeutic targets to achieve efficient, safe hypertrophic scar healing and recovery with low recurrence rate.
Disclosure of Invention
The invention aims to provide application of a long-chain non-coding RNA FPASL of a novel molecular diagnostic marker of a hypertrophic scar, and secondly, the invention provides a kit for identifying and detecting the expression quantity of the marker non-coding RNA FPASL in a hypertrophic scar tissue and primary fibroblasts derived from the hypertrophic scar, and an identification and detection method.
Currently, with respect to non-coding human genomes, most human genomes are transcribed, but only about 2% of human genomes contain protein-encoding genes, and thus there is increasing interest in the role of non-coding regions of human genomes. Using the latest sequencing techniques, various regulatory non-coding RNAs have been identified. These non-coding RNAs (ncrnas) include small ncrnas less than 200 nucleotides (nts) in length, such as miRNA and piRNA, and long non-coding RNAs (lncRNA) greater than 200nts in length. Although the expression levels of most lncRNA are low compared to messenger RNA (mRNA), many lncRNA play a central role in the regulation of cellular homeostasis and gene expression. With the advancement of next generation sequencing technology, recent studies have demonstrated the diversity of lncRNA functions. This diversity may be due to differences in their mechanism of action, spatiotemporal expression and/or abundance, all of which may vary with the particular cell type or tissue. lncRNA FPASL is a non-coding RNA newly discovered and identified in this application, and expression is significantly reduced in hypertrophic scars. Currently, the important role that lncRNA FPASL plays in the normal skin and in the development of hypertrophic scarring is still under further investigation. Whether lncRNA FPASL can be used as a novel hypertrophic scar marker for preventing and treating hypertrophic scar has not been reported yet. Thus, the present application demonstrates for the first time the feasibility of lncRNA FPASL as a novel hypertrophic scar marker for diagnosing scarring and development in hypertrophic scar patients.
The specific technical scheme of the application is as follows:
the identification, application and detection method of the marker non-coding RNA FPASL for the diagnosis of the hypertrophic scar molecules specifically comprises the following steps:
(1) Collecting normal skin tissue samples of hypertrophic scars and paraspinal scars during the operation of a patient with hypertrophic scars;
(2) Culturing primary fibroblasts derived from a hypertrophic scar and normal skin beside the scar from a tissue sample;
(3) Detecting expression of long non-coding RNA FPASL in the two sets of tissue samples and primary fibroblasts;
(4) Predicting specificity and sensitivity of the long-chain non-coding RNA FPASL to diagnose the hypertrophic scar through the ROC curve;
(5) Analyzing the non-coding property of the long-chain non-coding RNA FPASL by adopting bioinformatics;
(6) The long non-coding RNA FPASL expression is interfered by using a locked nucleotide technology (LNA), the long non-coding RNA FPASL is overexpressed by using a lentivirus, and the proliferation of the fibroblast is detected by using CCK 8.
The preparation for identifying and detecting the diagnosis of the patient with the hypertrophic scar is a real-time fluorescent quantitative PCR detection kit, and the kit comprises a specific primer for detecting the expression of non-coding RNA FPASL, wherein the nucleic acid sequences of the specific primer and a specific primer of an internal reference gene GAPDH are shown in a sequence table.
The sequence of the lncRNA FPASL is shown as SEQ ID NO. 1;
the sequence of the upstream primer of the FPASL primer sequence is shown as SEQ ID NO. 2, and the sequence of the downstream primer is shown as SEQ ID NO. 3;
the sequence of the upstream primer of the GAPDH primer sequence is shown as SEQ ID NO. 4, and the sequence of the downstream primer is shown as SEQ ID NO. 5.
The kit also contains all reagents for extracting RNA from tissues and cells and performing reverse transcription and real-time fluorescent quantitative PCR.
The identification and detection method is used for drawing ROC curves on the expression quantity of the long-chain non-coding RNA FPASL of the detected novel molecular diagnostic marker of the hypertrophic scar, and evaluating the predictive value of the long-chain non-coding RNA FPASL.
The identification and detection method adopts bioinformatics to analyze the non-coding and open reading frame of the long-chain non-coding RNA FPASL.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention discovers that the long-chain non-coding RNA FPASL is obviously reduced in the hypertrophic scar through real-time fluorescence quantitative PCR and ROC curve analysis, has high specificity and high sensitivity, and provides powerful technical support for diagnosing the hypertrophic scar.
2. The invention predicts the non-coding property of the long-chain non-coding RNA FPASL by adopting bioinformatics, and discovers that the long-chain non-coding RNA can regulate and control the proliferation of fibroblasts, thereby providing a new theoretical basis for treating the hypertrophic scar and being beneficial to reducing the occurrence and recurrence risk of the hypertrophic scar.
Drawings
FIG. 1 is a schematic representation of the differences in expression of long non-coding RNA FPASL in normal skin tissue beside a hypertrophic scar. Ordinate: the amount of FPASL expressed; abscissa: divided into two groups, HS: hypertrophic scar tissue, NS: normal skin tissue beside scar; * Representing a statistically significant difference in the reduction of the expression level of FPASL in the HS group compared to the NS group, with p less than 0.05.
FIG. 2 is a schematic representation of the differences in expression of long non-coding RNA FPASL in primary fibroblasts derived from hypertrophic scar and paracetamol normal skin tissue. Ordinate: the amount of FPASL expressed; abscissa: the HSFBs are divided into two groups: primary fibroblasts derived from hypertrophic scar tissue, NSFBs: primary fibroblasts derived from scar normal skin tissue; * Representing a statistically significant difference in the reduction of FPASL expression in the HSFBs group compared to the NSFBs group, with p less than 0.05.
FIG. 3 is a graph showing the predictive value (ROC curve) of long-chain non-coding RNA FPASL on hypertrophic scar. Ordinate: sensitivity represents Sensitivity; abscissa: 1-Specificity represents Specificity; AUC represents area under the curve; CI represents a confidence interval; the area under the ROC curve is between 0.5 and 1. The closer the AUC is to 1, the better the diagnostic effect. The AUC is lower in accuracy when 0.5-0.7, has certain accuracy when 0.7-0.9, and has higher accuracy when more than 0.9.
FIG. 4 is a schematic diagram of the potential of the bioinformatics website CAPT, CPC, CNCI to analyze long non-coding RNA FPASL encoded proteins;
FIG. 5 is a schematic diagram of the number of open reading frames of NCBI ORF finder analysis of long non-coding RNA FPASL;
FIG. 6 is a schematic representation of CCK8 detection of fibroblast viability following interference or overexpression of long non-coding RNA FPASL. Ordinate: cell Viability (450 nm) represents Cell Viability, which is the absorbance value of the Cell measured at a wavelength of 450 nm; abscissa: representative of the time of treatment (days); CTL represents the blank, OE-nc+lna-NC represents the empty vector, OE-fpasl+lna-NC represents the over-expressed FPASL, OE-nc+lna-FPASL represents the interfering FPASL.
The sequence table is a nucleic acid sequence of long-chain non-coding RNA FPASL; nucleic acid sequences of long non-coding RNA FPASL and reference gene GAPDH specific primers.
Detailed Description
The technical scheme of the invention is described in detail below with reference to the accompanying drawings and the specific embodiments.
1 Material
1.1 materials and the reagent green streptomycin (Solarbio, china); PBS (Hyclone, china); gentamicin (biotupped, beijing); collagen type I protein (Solarbio, china); fetal Bovine Serum (FBS) (BI, israel); DMEM-F12 medium (Hyclone, china); edU kit (libo, guangzhou); CCK8 kit (APExBIO, usa); TBST buffer (double helix, shanghai); cDNA (complementary DNA) kit, RT-qPCR (real-time fluorescent quantitative PCR) kit (Japan, takara Co.); lipo2000 (Thermo Fisher, usa); primers were synthesized by Shanghai Biotechnology Co., ltd; lentiviruses were constructed by Tianjin Seisakusho as Sta; LNA Gapmers were synthesized by Kaijer, germany. 1.2 instruments and apparatus: negative pressure aspirator (ROCKER 300, china); a hand-held vortex oscillator (Kylin-bell, china); precision electronic balances (Sartorius, germany); micropipettes (Eppendorf, germany); double temperature water bath shaking table (HLD, jiangsu); ultraviolet ultra-clean bench (antai, china); desk top centrifuge (Eppendorf, germany); hand-held portable centrifuges (SCILOGEX, usa); biological safety cabinets (su jing antai, china); ice maker (BILON, china); a homogenizer (FLUKO, germany); biotek microplate reader (deluxe, beijing); 6-well plates, 96-well plates (corning, usa); horizontal shaking table (Kylin-bell, china); gradient PCR instrument (Eppendorf, germany); fluorescent quantitative PCR apparatus (analytik jena, germany); LUNA cytometer (tokyo, beijing); gel imager (BIO-RAD, USA).
2 method
2.1 selection of clinical specimens
The present study obtained 15 skin specimens from the surgery of patients with hypertrophic scar tissue and included normal adjacent skin tissue surrounding the hypertrophic scar. Patients had undergone surgery at the university of Ningxia medical university general hospital from 4 months 2019 to 12 months 2020.
2.2 Primary culture of human fibroblasts
The hypertrophic scar and normal tissue beside the scar are obtained in the operation process, and are put into PBS which is precooled at 4 ℃ and contains 1% of green streptomycin for transportation back to a laboratory; cutting the tissue as small as possible into pieces of approximately 1mm3 with a scalpel or other sharp instrument; washing with PBS containing diabody and gentamicin for 30min under vigorous shaking for 3 times; placed in a 15ml centrifuge tube containing 0.125% TE-digestive enzyme, and refrigerated overnight at 4 ℃; after overnight (the next day), the epidermis was removed and the remaining tissue was minced with a scalpel as much as possible (minced meat-like); the minced tissue was digested in dishes with CoA collagenase (collagen type I protein) added, and incubated overnight at 37 ℃; centrifuging at 1000r for 10min, and discarding supernatant; observing the climbing-out condition of primary cells in the culture process, and washing twice by PBS after 2-3 days, and changing the liquid; the cell morphology and the growth condition of the cells are observed regularly, liquid exchange is carried out in time, when the cell density reaches 80% -90%, trypsin is used for digestion and then passaging, and the cells are passaged for 3-5 times and are directly used for experiments. .
2.4 RT-qPCR (real-time quantitative fluorescent PCR) detection of long-chain non-coding RNA FPASL expression
Total RNA (ribonucleic acid) of primary fibroblasts was extracted according to the instructions of Takara RNA kit, and reverse transcription was performed according to the instructions of reverse transcription kit. RT-qPCR (Real-time quantitative fluorescent PCR) the expression level of the long-chain non-coding RNA FPASL was detected and analyzed by a Yes PCR instrument using Takara Real-Time PCR Master Mix (Real-time PCR mixture labeled with green fluorescent dye) kit. GAPDH was used as a control. The experimental results were calculated according to the following formula: detection of the relative expression level of the Gene of interest=2 －△△Ct Wherein, the method comprises the steps of, wherein, ΔΔct= [ CtGI (test sample) -CtGAPDH (test sample)]- [ CtGI (calibration sample) -CtGAPDH (calibration sample)]. GI refers to the target gene, ct refers to the fluorescence signal intensity in the reaction system, and Ct refers to the fluorescence signal intensity in the reaction systemPositive samples refer to all samples selected to represent 1-fold the amount of gene expression of the subject.
2.5 CCK8 cell viability assay
Inoculating 5000 fibroblasts per well in a 96-well plate, 5 wells per experimental well, at least 3 wells per control well and blank well; for the corresponding time after transfection, 10 μl of CCK8 solution was added to each well and incubated for 2 hours at 37 ℃; taking out the 96-well plate, and lightly shaking the plate on a shaking table for 1min in a dark place; detecting an OD value with the wavelength of 450nm by using an enzyme-labeled instrument; the OD values of the six wells were averaged or cell viability (%) = [ (As-Ab)/(Ac-Ab) ]×100 was calculated by the formula (as=experimental well absorbance; ab=blank well absorbance; ac=control well absorbance).
3. Statistical treatment
The results were analyzed and processed analytically using prism5.0 statistical software, expressed as Mean ± standard deviation (Mean ± SD), with a Student's t test for comparison between the two sample averages, an One-way ANOVA test for comparison between multiple sample averages, a Student-Newan-Keuls test for comparison between groups, and p.ltoreq.0.05.
4.1 real-time fluorescent quantitative PCR detection of expression of Long non-coding RNA FPASL in hypertrophic scar tissue
The real-time fluorescent quantitative PCR detects the expression of the long-chain non-coding RNA FPASL in the hypertrophic scar tissue, and the result shows that: the expression of long non-coding RNA FPASL was significantly reduced in the hypertrophic scar tissue compared to the paracetamol normal skin tissue (P < 0.05). See fig. 1.
4.2 real-time fluorescent quantitative PCR detection of expression of Long non-coding RNA FPASL in Primary fibroblast cells of tissue origin
Real-time fluorescent quantitative PCR detects expression of long-chain non-coding RNA FPASL in primary fibroblasts derived from hypertrophic scar tissue and periscar adjacent normal skin tissue, showing that: long-chain non-coding RNA FPASL expression was significantly reduced in primary fibroblasts (HSFBs) derived from hypertrophic scar tissue compared to primary fibroblasts (NSFBs) derived from adjacent normal skin tissue next to scar (P < 0.01). See fig. 2.
4.3 predictive value of long non-coding RNA FPASL
The area under the curve (AUC) of the area under the curve of the long-chain non-coding RNA FPASL predicted the hypertrophic scar was 0.789, and the sensitivity and the specificity were 80.00% and 73,30%, respectively, as shown in FIG. 3.
4.4 potential of bioinformatics analysis websites to analyze long non-coding RNA FPASL encoded proteins
The long-chain Non-Coding RNA FPASL was analyzed for its protein-encoding ability using Coding Potential Assessment Tool (CPAT), contrastive Predictive Coding (CPC) and Coding-Non-Coding Index (CNCI), respectively, and the results showed that: all three websites showed very low protein-encoding capacity of long non-coding RNA FPASL. See table 4.
4.5 NCBI ORF finder analysis of the number of open reading frames of long non-coding RNA FPASL
Open Reading Frame (ORF) finder analyses the number of open reading frames of the long non-coding RNA FPASL and searches for homologous protein sequences by Swiss-Prot database, the results show that: the number of open reading frames of the long non-coding RNA FPASL is less than 75nts, and a total of 6 open reading frames are found, and the protein sequence without homology is searched through the Swiss-Prot database, so that the fact that the long non-coding RNA FPASL cannot code the protein is further explained. See table 5.
4.6 CCK8 detection interference and fibroblast viability after overexpression of long-chain non-coding RNA FPASL
The activity of the fibroblast after interference and overexpression of the long-chain non-coding RNA FPASL is detected by a CCK8 method, the activity of the fibroblast after interference (OE-NC+LNA-FPASL) of the long-chain non-coding RNA FPASL is found to be increased, and the activity of the fibroblast after overexpression of (OE-FPASL+LNA-NC) of the long-chain non-coding RNA FPASL is found to be reduced, so that the activity of the fibroblast can be regulated and controlled by the long-chain non-coding RNA FPASL. See fig. 6.
In conclusion, the invention collects the hyperplastic scar and normal skin tissues beside the hyperplastic scar of a clinical hyperplastic scar patient, extracts primary fibroblasts, discovers and identifies a new long-chain non-coding RNA FPASL, and finally determines the diagnostic value of the long-chain non-coding RNA FPASL, thereby providing theoretical basis for treating and preventing recurrence of the hyperplastic scar, and having important significance.
Sequence listing
NINGXIA MEDICAL University
Identification, application and detection method of non-coding RNA FPASL of molecular marker for diagnosis of hypertrophic scar
lncRNA FPASL sequences
TGGAGCACGAGGACACTGACAGGGACGAACGGCTCGCCAATGCAAAGGAGGCCCCTCGGGGAGACGGCAGGCAGCAAAAGGCTTCCTTGGCCGGGGGTCTGGGCGTTTGGGGAGGGGAGGTGCTCAGAGATAGGGCCGAGGATGGAGCTCCCAGGTGGGTTGGAGGCGGGGGTGGCTGGGCTCGAGGACGCTGTTGGAGTCCGGGGTCTGGAAGAGGCGATCCCCGGGAAAGCCAGCTTCAGAGCCGGGAAGGGTCGGCAGGGGACCTACTGCAAGAGCCACCAGGTTGGGGTAGGGAGGGGGCCCAGACCTGGGCCGGGAGGGATGCTGAATCCCCCGACCTAGCTCTGGGGCTTTGCAAACCAGCATCACTGCAGCATTTTGTGGCTCAAGGAATGGGGCAGGGCGGCCAGACCAGCTGTGGCTGGTGCGTGCGTGAGGCCCTGAGGGATGCGGCCAATGCTATTTTGCTGTTTTGTACTTACTCAGTTTCATTTTGGCTCTGTCGCCACTAGGCGAAAGAAATCGAAGTGCTAACATACCACGGGGAGCAATGATTATTACTCAATGTGGCCCCGCCCCAGCAGCGGGAGGTGGCATCTGTTTGACCTTGGAGACTTGGACGGTATTTAGGGAGATTACATGAAAAAAAGACTCACCCCACAATGCTTGCAGTCAGCTACAAGCTGCCTTGTCCTTCATACCTTTTAGCCTTTATTTCCTGAGTCTATCTTGTCTGTTTCTAATGTAGTCTCCTGATTTAGGGCAAGAGAATATTTCTCACTCTTGGTCTGCCCACTACCCACAATCCTTGTCCGAGGATTTACTCGGCAAACTTAGCAGGCAAAACCCCTTGTCAACTTAACACTAAAAAATACCGTCTCTTGGAAGGCAGTCTGCTTTGATAAACCTAATGGAATAAAAAGTGAGTTTGCTATCTGGCTTTAAAGAACATAACCCAGCTGGGCATGGTGGCTCACACCTGTAATCCCAACACTTTGGGAGACCGAGGCAGGAGGATCGCTTAAGCCCAGGCGTTCAAGACCAGCCTGGGCAACATAGTATAGTAAAACCCAGTCTCTGCAAAAAAAAAAAAAAA
FPASL primer sequences
An upstream primer:
TACCGTCTCTTGGAAGGCAGTCTG
a downstream primer:
GCCTCGGTCTCCCAAAGTGTTG
GAPDH primer sequences
An upstream primer:
ATTCCACCCATGGCAAATTCC
a downstream primer:
GACTCCACGACGTACTCAGC
Claims (5)
1. the identification, application and detection method of the marker non-coding RNA FPASL for the diagnosis of the hypertrophic scar molecules is characterized by comprising the following steps:
(1) Collecting normal skin tissue samples of hypertrophic scars and paraspinal scars during the operation of a patient with hypertrophic scars;
(2) Culturing primary fibroblasts derived from a hypertrophic scar and normal skin beside the scar from a tissue sample;
(3) Detecting expression of non-coding RNA FPASL in the two sets of tissue samples and primary fibroblasts;
(4) Predicting specificity and sensitivity of non-coding RNA FPASL diagnosis proliferation scar through ROC curve;
(5) Analyzing the non-coding property of the long-chain non-coding RNA FPASL by adopting bioinformatics;
(6) The expression of non-coding RNA FPASL is interfered by using a locked nucleotide technology (LNA), and the proliferation of fibroblast cells is detected by using lentivirus to overexpress long-chain non-coding RNA FPASL and CCK 8.
2. The identification, application and detection method of non-coding RNA FPASL for the markers for the diagnosis of hypertrophic scar molecules according to claim 1, wherein the preparation for identifying and detecting the diagnosis of hypertrophic scar patients is a real-time fluorescent quantitative PCR detection kit, and the kit comprises specific primers for detecting the expression of non-coding RNA FPASL.
3. The method for identifying, using and detecting non-coding RNA FPASL as claimed in claim 2, wherein said kit further comprises all reagents for extracting RNA from tissues and cells and performing reverse transcription and real-time fluorescent quantitative PCR.
4. The identification, application and detection method of the non-coding RNA FPASL of the markers for the diagnosis of the hypertrophic scar molecules according to claim 1, wherein the identification and detection method is characterized in that ROC curve drawing is carried out on the expression quantity of the long-chain non-coding RNA FPASL of the novel markers for the diagnosis of the hypertrophic scar obtained by detection, and the predictive value of the non-coding RNA FPASL is evaluated.
5. The method for identifying, applying and detecting the marker non-coding RNA FPASL for the diagnosis of the hypertrophic scar molecules according to claim 1, wherein the identification and detection method adopts bioinformatics to analyze the non-coding and open reading frames of the long-chain non-coding RNA FPASL.
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