CN115851914A - Application of LOC107985869 in preparation of ophthalmoplegia specific auxiliary diagnostic reagent - Google Patents
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Abstract
The invention belongs to the technical field of biomedical detection, and particularly relates to application of LncRNA-LOC107985869 in preparation of an Ophthalmoplegia (ONP) specific auxiliary diagnostic reagent, and an ONP LncRNA-LOC107985869 diagnostic kit, which can be used for assisting diagnosis and disease judgment of ONP according to the expression level of LncRNA-LOC107985869 in an individual extraocular muscle. The kit disclosed by the invention is used for the first time for assisting the diagnosis of ONP by detecting the content of LOC107985869 in extraocular muscles by using a quantitative PCR method, has the characteristics of low cost, strong operability and convenience in use, and enables LncRNA-LOC107985869 to be expected to become a biological marker for scientifically judging the occurrence of ONP and the prognosis of a patient.
Description
Technical Field
The invention belongs to the technical field of biomedical detection, and particularly relates to application of LncRNA-LOC107985869 in preparation of an ophthalmoplegia specific auxiliary diagnostic reagent, and an ophthalmoplegia LncRNA-LOC107985869 diagnostic kit, which can be used for assisting diagnosis and disease condition judgment of ophthalmoplegia according to the LncRNA-LOC107985869 expression level in an individual extraocular muscle.
Background
Oculomotor Nerve Palsy (ONP), also known as third cranial nerve palsy, is a common cause of non-common strabismus and is classified into congenital and acquired. The etiology of acquired ONP includes vascular etiology, such as aneurysm, as well as idiopathic etiology, trauma, and tumor. The oculomotor nerve is the cranial nerve which is most important for controlling eyeball motion, is divided into an upper branch and a lower branch after entering an orbit, and simultaneously innervates four muscles of six extraocular muscles, including the superior rectus muscle, the internal rectus muscle, the inferior rectus muscle and the inferior oblique muscle, and also innervates the superior palpebral muscle and the sphincter pupillae muscle. Different locations and different causes of ONP can lead to different external ocularis and paralysis in different degrees, and the clinical manifestations are complex and different, and the symptoms are different. In addition, many cases have difficulty finding the primary disease, resulting in difficulty in clinical diagnosis. To date, there are no biological indicators for ONP-specific aided diagnosis.
Surgical treatment is the main treatment of ONP, including neurosurgery and ophthalmic surgery. Among them, the ophthalmic surgery is mainly performed for correcting strabismus. Different surgical protocols are available depending on the amount and degree of paralysis of the external muscles, but the efficacy and long-term efficacy of surgery in such patients is often unsatisfactory. Even the most experienced strabismus surgeon, treatment of ONP is a significant challenge. Therefore, there is a need to find biomarkers for ONP to make scientific judgments about the occurrence of ONP and the prognosis of patients.
Long non-coding RNA (LncRNA) is a type of non-coding RNA with a length of more than 200 nucleotides, and has not been considered to have any biological function. In recent years, the structure and role of LncRNA in transcriptome of various species have been revealed based on the development of sequencing technology. Studies have shown that LncRNA plays an important role in regulating gene expression, and its dysfunction may be involved in the pathophysiological processes of various diseases, such as cancer and autoimmune diseases. In addition, lncRNA has been found to be associated with the pathogenesis and prognosis of a variety of neuroparalytic diseases, including progressive supranuclear palsy, diabetic peripheral neuropathy, and thyrotoxic periodic paralysis, among others.
LOC107985869 (NCBI Reference Sequence: XR _ 001739409.1) belongs to a member of the LncRNA family, encodes a gene that maps to chromosome 2p22.2 and is ubiquitously expressed in various tissue cells of humans, particularly liver, prostate, uterus, and skeletal muscle. At present, there is no report on LncRNA-LOC107985869 as a biological marker for ONP-assisted diagnosis. When LncRNA-LOC107985869 is used as a biological marker for ONP-assisted diagnosis, surgical sampling is not necessary, and minimally invasive muscle biopsy is also possible.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a new application of LncRNA-LOC107985869, which can be used as a specific auxiliary diagnostic reagent for the auxiliary diagnosis of ONP, and the sequence of the LncRNA-LOC107985869 is shown as SEQ ID NO 1.
The invention also aims to provide an LncRNA-LOC107985869 detection kit for ONP specificity auxiliary diagnosis, which comprises an RNA extraction system, an RNA reverse transcription system and a PCR reaction system, wherein the PCR reaction system contains a primer sequence for specifically amplifying a gene sequence SEQ ID NO. 1, and the primer sequence is a specific RT-qPCR upstream and downstream primer of LOC 107985869.
Further, the RNA extraction system in the detection kit comprises a total RNA extraction reagent, and the RNA reverse transcription reaction system comprises reverse transcriptase, a reverse transcription system buffer solution and an RNase inhibitor;
further, the PCR reaction system comprises an amplification system and a primer system, wherein the amplification system is prepared by PerfectStart TM Green qPCR Supermix reagent composition; the primer system comprises an RNA reverse transcription random primer and a primer of LOC107985869 specific RT-qPCR;
further, the RNA reverse transcription random primer is a quantitative PCR primer sequence of GAPDH and/or Beta-actin,
GAPDH quantitative PCR primer sequence, wherein the upstream primer sequence is shown as SEQ ID NO. 2, and the downstream primer sequence is shown as SEQ ID NO. 3;
the Beta-actin quantitative PCR primer sequence has the upstream primer sequence shown as SEQ ID No. 4 and the downstream primer sequence shown as SEQ ID No. 5.
The upstream primer of the LOC107985869 specific RT-qPCR is shown as SEQ ID NO. 6, and the downstream primer is shown as SEQ ID NO. 7.
The invention relates to an antisense nucleotide for treating ONP, which is an antisense nucleotide of LncRNA-LOC107985869, and the sequence of the antisense nucleotide is shown as SEQ ID NO: shown in fig. 8.
Application of antisense nucleotide of LncRNA-LOC107985869 in preparing medicine for treating ONP.
In summary, the advantages and positive effects of the invention are: the kit disclosed by the invention is used for assisting the diagnosis of ONP by detecting the content of LOC107985869 in extraocular muscles by utilizing a quantitative PCR method for the first time, has the characteristics of low cost, strong operability and convenience in use, and ensures that LncRNA-LOC107985869 is expected to become a molecular marker for scientifically judging the occurrence of ONP and the prognosis of a patient. When LncRNA-LOC107985869 is used as a biological marker for ONP-assisted diagnosis, surgical sampling is not necessary, and minimally invasive muscle biopsy is also possible.
Drawings
FIG. 1 is a diagram of differentially expressed LncRNA
FIG. 2 shows the results of quantitative PCR of LOC107985869
FIG. 3 is a LOC107985869 ROC curve analysis
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following examples, and the apparatuses and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.
Example 1: correlation verification of LncRNA-LOC107985869 and ONP
The first step is as follows: sample preparation: internal rectus muscle samples from ophthalmic ONP post-surgery (experimental group, n = 4) and from patients with constant external strabismus (control group, n = 4), RNA was extracted using TRIzol (Invitrogen) reagent and stored at-80 ℃ for future use.
The second step is that: differential expression LncRNA screening:
the LncRNA associated with ONP generation was analyzed using high-throughput RNA sequencing technology.
The analysis comprises the following specific steps: ribosomal RNA (rRNA) was removed from the extracted Total RNA, a strand-specific RNA-seq library was constructed using TruSeq Stranded Total RNAwith Ribo-Zero Gold (Illumina), followed by high throughput RNA sequencing using Illumina NovaSeq 6000System (Illumina). The constructed transcripts are merged by Stringtie software, and are subjected to one-to-one comparison by cuffmatch software, so that RNAs with more than 200 nucleotides and 2 exons are screened out, and are compared in databases of CPC, CNCI, pfam and PLEK, so that RNAs with coding capacity are removed. Subsequently, lncrnas differentially expressed in the experimental group and the control group were screened by DESeq data packet in R language (fig. 1).
The third step: experimental results of sequencing were verified using quantitative PCR (figure 2): and then determining lncRNA with P less than 0.05 and the difference multiple of more than 2 times as the differential expression LncRNA according to a t-test unitary unpaired algorithm by combining GO and KEGG signal path analysis. In combination with Receiver Operating Characteristic (ROC) curve analysis (FIG. 3), the results showed that LncRNA-LOC107985869 expression in ONP patients and control groups was more different (P values were smallest) and the area under the ROC curve (AUC) was largest, suggesting that LncRNA-LOC107985869 has a greater correlation with ONP.
Example 2: preparation of the kit of the invention
The sequence of LncRNA-LOC107985869 is shown in SEQ ID NO:1, the specific quantitative PCR upstream and downstream primers and the quantitative PCR upstream and downstream primers of internal reference GAPDH and/or Beta-actin are designed by Shanghai Europe and Yi biomedicine science and technology limited company, are synthesized by Beijing Ongskaceae New industry Biotechnology limited company, and have the purity of PAGE grade, and the synthesized primers adopt DEPC ddH 2 O dissolved at a total concentration of 10. Mu.M.
Preparing a kit comprising the following components:
(a) Extraction system
1) Trizol reagent,1 tube, 2000. Mu.L/tube;
2) Chloroform, 1 tube, 500 μ L/tube;
3) Absolute ethyl alcohol, 1 tube, 8000 mul/tube;
4)DEPC ddH 2 o,1 tube, 1000. Mu.L/tube;
5)ddH 2 o,1 tube, 2000. Mu.L/tube;
6) Isopropanol, 8000 μ Ι _/tube;
(b) Reverse transcription system
1) 5 × TransScript All-in-one Supermix for qPCR,1 tube, 200 μ l/tube;
2) 5 × TransScript All-in-one No-RT Control Supermix for qPCR,1 tube, 20 μ l/tube;
3) gDNARemover,1 tube, 50. Mu.l/tube;
4) DEPC ddH 2 0,1 tubes, 1000 μ l/tube;
(c) PCR system
1)2×PerfectStart TM Green qPCR Supermix,1 tube, 1000. Mu.l/tube;
2) Passive Reference Dye (50X), 1 tube, 40. Mu.l/tube;
3) Upstream primer of LOC107985869 specific RT-qPCR, 1 tube, 10 μ M,100 μ L/tube;
LOC107985869 specific RT-qPCR downstream primer, 1 tube, 10 μ M,100 μ L/tube;
GAPDH quantitative PCR upstream primer, 1 tube, 10. Mu.M, 100. Mu.L/tube;
GAPDH quantitative PCR downstream primer, 1 tube, 10. Mu.M, 100. Mu.L/tube;
and/or, beta-actin quantitative PCR upstream primer, 1 tube, 10 MuM, 100 MuL/tube;
beta-actin quantitative PCR downstream primer, 1 tube, 10 MuM, 100 MuL/tube;
4) Nucleic-free water,1 tube, 1000. Mu.l/tube.
RNA quality detection:
the concentration of RNA was determined at 260nm and 280nm absorbance using Nanodrop 2000 (Thermo Scientific); the ratio of A260/A280 of the RNA solution is the RNA purity, and the ratio range is 1.8 to 2.1; agarose gel electrophoresis was used to check RNA integrity.
Reverse transcription of RNA to obtain cDNA sample:
the RNA to be detected is reversely transcribed into cDNA by utilizing a TransScript All-in-One First-Strand cDNAsynthesis SuperMIX for qPCR kit. The reverse transcription system is as follows: total RNA, 0.5. Mu.g; 5 × TransScript All-in-one Super Mix for qPCR,2 μ l; gDNARemover, 0.5. Mu.l; DEPC ddH 2 O was added to 10. Mu.l. Reaction procedure: 15min at 42 ℃, 5s at 85 ℃. After completion of reverse transcription, 90. Mu.l DEPC ddH was added 2 O is stored in a refrigerator at-20 ℃ for later use.
Real-time fluorescent quantitative PCR:
using PerfectStart TM Green qPCR Supermix kit in
Reactions were performed on a model 480 II fluorescent quantitative PC R instrument (Roche). The method comprises the following steps: 2 x PerfectStart TM Green qPCR SuperMix,5 μ Ι; upstream primer, 0.2. Mu.l; downstream primer, 0.2. Mu.l; cDNA, 1. Mu.l; nucleic-free water, 3.6. Mu.l.
PCR conditions were as follows:
30s at 94 ℃;94 ℃ for 5s,60 ℃ for 30s,45 cycles. Detecting product specificity by using a melting curve after circulation is finished: the temperature was slowly raised from 60 ℃ to 97 ℃ and fluorescence signals were collected 5 times per ℃ C.
And (3) data analysis:
the gene expression value is calculated by a Delta Ct method, and the amplification efficiency of the target gene and the reference gene is assumed to be close to 100% and the relative deviation is not more than 1Ct; Δ Ct = mean Ct of target gene-mean Ct of reference gene; wherein the reference gene is selected for GAPDH and/or Beta-actin; the Δ Ct range was determined for healthy and ONP patients, respectively.
And (5) judging a result:
calculating the delta Ct of the sample to be detected, analyzing whether the delta Ct is in the normal range of the healthy person, and if the delta Ct of the sample to be detected is in the delta Ct range of the healthy person or is smaller than the delta Ct range, considering that the sample to be detected is ONP negative; and if the delta Ct of the sample to be detected is larger than the range of the delta Ct of the healthy person, the sample to be detected is considered to be positive ONP. The Δ Ct ranges for healthy persons are shown in table 1.
Table 1: delta Ct range in healthy humans
| LOC107985869Ct mean-GAPDH Ct mean | |
| Internal rectus muscle sample | ＞7.8 |
According to the results, simultaneously comparing with the results of actual clinical pathology analysis and diagnosis, 26 unknown clinical samples are respectively selected, and the accuracy rate of the method based on LOC107985869 detection is evaluated, wherein the results are shown in table 2. The results indicate that LncRNA-LOC107985869 can be used as a biomarker for ONP diagnosis.
Table 2: method detection accuracy based on LOC107985869
| LOC107985869Ct mean-GAPDH Ct mean | |
| Internal rectus muscle sample | 76.9％ |
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (7)
1. The LncRNA-LOC107985869 with the sequence shown in SEQ ID NO:1 is applied to the preparation of a specific auxiliary diagnostic reagent for the paralysis of the oculomotor nerve.
2. An ONP LncRNA-LOC107985869 detection kit comprises an RNA extraction system, an RNA reverse transcription system and a PCR reaction system, wherein the PCR reaction system contains a primer sequence for specifically amplifying a gene sequence of SEQ ID NO. 1, and the primer sequence is a RT-qPCR upstream primer SEQ ID NO:6 and downstream primer SEQ ID NO:7.
3. the test kit according to claim 2, wherein the RNA extraction system comprises a total RNA extraction reagent, and the RNA reverse transcription reaction system comprises a reverse transcriptase, a reverse transcription system buffer and an RNase inhibitor.
4. The detection kit of claim 2, wherein the PCR reaction system comprises an amplification system and a primer system, and the amplification system is characterized by PerfectStart TM Green qPCR Supermix reagent composition; the primer system comprises RNA reverse transcription random primers and primers of LOC107985869 specific RT-qPCR.
5. The detection kit of claim 2, wherein the RNA reverse transcription random primer is a quantitative PCR primer sequence of GAPDH and/or Beta-actin, the GAPDH quantitative PCR primer sequence is shown as an upstream primer sequence in SEQ ID NO. 2, and a downstream primer sequence is shown as SEQ ID NO. 3; the Beta-actin quantitative PCR primer sequence has the upstream primer sequence shown as SEQ ID No. 4 and the downstream primer sequence shown as SEQ ID No. 5.
6. An antisense nucleotide for treating ONP, the antisense nucleotide is an antisense nucleotide of LncRNA-LOC107985869, the sequence of the antisense nucleotide is shown in SEQ ID NO: shown in fig. 8.
7. The use of an antisense nucleotide as claimed in claim 6 in the preparation of a medicament for the treatment of ONP.
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