CN112063706B - Application of long-chain non-coding RNA LINC01679 in diagnosis and treatment of primary pterygium - Google Patents

Abstract

According to the invention, the gene expression profile changes of human primary pterygium tissues and adjacent normal tissues are compared through high-throughput sequencing, the abnormal high expression of long-chain non-coding RNA LINC01679 and LOC102724238 are found, the surface of the long-chain non-coding RNA LINC01679 and LOC102724238 is mostly concentrated in a cytoplasmic region, cytological experiments prove that the high-expression LINC01679 and LOC102724238 promote the proliferation, migration and invasion of cells, and the expression of the LINC01679 and LOC102724238 is inhibited by using specific inhibitors to reverse the phenotype of the cells, so that the LINC01679 and LOC102724238 have a promoting effect on the generation and development of the human primary pterygium and are potential treatment targets.

Description

Application of long-chain non-coding RNA LINC01679 in diagnosis and treatment of primary pterygium

The invention belongs to the field of biomedicine, and particularly relates to the application field of long-chain non-coding RNA to primary pterygium.

Background

Pterygium (Pterygium) is a common ocular disease occurring only in humans, and epidemiological investigations by Wu et al in southern china have shown that Pterygium occurs in 33.01% of people over the age of 50. At present, the specific pathogenesis of pterygium is still unclear, an effective treatment method is also lacked except for surgery, and the recurrence rate of simple pterygium excision is as high as more than 30%, which brings great pain to patients. Therefore, intensive research on the pathogenesis of pterygium, more effective prevention of pterygium occurrence and reduction of postoperative recurrence rate is a key problem to be solved urgently by clinicians and basic research.

In recent years, with the intensive study on the pathogenesis of pterygium, the characteristics of the tumor-like characteristics of pterygium, such as local infiltrates, high recurrence rate, etc., have been confirmed by more and more basic and clinical studies. Epidemiological studies have shown that pterygium occurrence is closely related to developmental and environmental factors. The present opinion is intended to suggest that pterygium is a fibrovascular abnormally proliferative disease associated with ultraviolet irradiation, possibly closely related to abnormal proliferation and apoptosis of cells, and is pathologically represented by a large amount of hyperplastic blood vessels and degenerative fibers contained in pterygium tissues, accompanied by abnormal proliferation of a large amount of bulbar conjunctival epithelial cells. Recent tumor molecular function studies further prove that important molecular signaling pathways such as TGF-beta, Notch and growth factors (PDGF and EGF) play a crucial role in regulating the progress of pterygium. However, studies on the development of long non-coding RNAs in primary pterygium have been relatively blank.

High-Throughput Sequencing, also known as Deep Sequencing or Next-Generation Sequencing (NGS), is the first choice for studying gene expression and identifying novel RNA species. The main advantage of this is that it enables the production of larger amounts of data at lower cost compared to the first generation sequencing methods; compared with the traditional DNA microarray, the high-throughput sequencing method has the advantages of lower background noise, larger detection dynamic range and capability of identifying and quantifying rare transcripts without specific probes. Most importantly, high throughput sequencing directly reveals sequence identity, which is crucial for the analysis of unknown genes and new transcript isoforms. High throughput sequencing technologies can be used for genome sequencing and resequencing, transcriptome analysis (RNA-Seq), DNA protein interaction (ChIP-Seq), and epigenome sequencing. Transcriptome (transcriptome) broadly refers to the collection of all transcripts in a cell under a certain physiological condition, including messenger RNA, ribosomal RNA, transfer RNA, and non-coding RNA. Transcriptome-directed studies are crucial for elucidating the function of the genome and for revealing the molecular composition of cells and tissues as well as for understanding development and disease. The main objectives of transcriptomics studies are: cataloguing transcripts from all species, including mRNA, non-coding RNA, and small RNA; determining the transcription structure of the gene from the aspects of initiation sites, cutting modes, other post-transcriptional modifications and the like; and quantifying changes in transcript expression levels during development and under different circumstances.

The research results of the human genome project and the subsequent DNA element project show that the gene sequence of the coding protein only accounts for 1-3% of the human genome sequence, while the vast majority of the transcribable sequences in the human genome are non-coding RNAs (non-coding RNAs) including small fragments such as microRNAs and long-chain non-coding RNAs (lncRNAs). Many studies in recent years have shown that IncRNA is involved in a series of important cell regulatory processes. For example, long non-coding RNAs are involved in regulating many key regulatory processes such as transcription of target genes, genetic imprinting, and nuclear trafficking, and as well as other epigenetic forms including dna methylation, genetic imprinting, and chromatin remodeling, disease association continues to be a focus of attention.

LncRNA is non-coding RNA with the transcript length exceeding 200 nucleic acids, and Tsai finds that long-chain non-coding RNA is helpful for early diagnosis of swelling and pain in 2011, and prognosis judgment and can be used as a target gene for molecular whip-direction treatment, so that the LncRNA has a good clinical application value. And a large number of reports prove that the long-chain non-coding RNA has close relation with the formation and development of various tumors, such as lung cancer, liver cancer, breast cancer and the like. The Chinese academy of sciences classifies long non-coding RNAs according to their different characteristics, for example, intragenic, intergenic, sense and antisense according to the location and background of the genome; the expression level is classified into a transcription level, a post-transcription level, a translation level, and the like according to a mechanism for exerting a function; according to the penis searching mechanism, the method can be divided into a signal prototype, a smart prototype, a guide prototype and a molecular scaffold prototype; the DNA sequence is classified into cis-acting and trans-acting according to the mode of action on the DNA sequence. The LncRNA realizes the regulation and control of gene level mainly through different signal transduction pathways such as epigenetic level, genetic imprinting, transcriptional activation, post-transcriptional regulation, protein function regulation and the like. Currently, in non-coding RNA research, mirnas have achieved significant results; thus, long non-coding RNAs may also represent an important source of molecules. The related lncRNA and the effect thereof in the human primary pterygium are not discovered at present, and the application firstly proves that the lncRNA in the human primary pterygium can be used as a diagnostic marker and a therapeutic target.

Based on the insufficient research on the relationship between the long-chain non-coding RNA and the human primary pterygium in the prior art, whether the influence of the LINC01679 and the LOC102724238 on the generation and development of the human primary pterygium is discussed from various aspects of cell viability, migration and invasion capacity and the like of LINC01679 and LOC102724238 on the primary epithelial cells (hPECs) of the human primary pterygium.

Disclosure of Invention

One aspect of the present invention is to provide a diagnostic marker for invasive hyperplasia of conjunctival epithelium; in a specific embodiment, the invasive hyperplasia is pterygium, preferably a primary pterygium; in another specific embodiment, the marker is a non-coding RNA, and further, the non-coding RNA is a long non-coding RNA; further, the long non-coding RNA is highly expressed in human primary pterygium tissue; further, the long non-coding RNA is selected from LINC01679, LOC102724238, LOC101928725 and LINC00880, and preferably, the long non-coding RNA is LINC01679 and LOC 102724238. In a specific embodiment, the nucleotide sequences of LINC01679 and LOC102724238 are respectively shown in SEQ ID NO. 1 and NO. 2, or the nucleotide sequences of LINC01679 and LOC102724238 are respectively shown in SEQ ID NO. 1 and NO. 2, have 99% homology and are derived from human nucleic acid sequences.

Another aspect of the invention is the use of an agent for detecting long non-coding RNA in the preparation of a reagent for diagnosing or treating conjunctival invasive hyperplasia. In a specific embodiment, the invasive hyperplasia is pterygium, preferably a primary pterygium; in another specific embodiment, the long non-coding RNA is highly expressed in human primary pterygium tissue; further, the long non-coding RNA is selected from LINC01679, LOC102724238, LOC101928725 and LINC00880, and preferably, the long non-coding RNA is LINC01679 and LOC 102724238; in another specific embodiment, the detection reagent is a detection reagent known to those skilled in the art, and can be selected from, but not limited to, primers, probes, and antibodies.

Another aspect of the invention is the use of an inhibitor of long non-coding RNA for the manufacture of a medicament for the treatment of tumors. In a specific embodiment, the invasive hyperplasia is pterygium, preferably a primary pterygium; in another specific embodiment, the long non-coding RNA is highly expressed in human primary pterygium tissue; further, the long non-coding RNA is selected from LINC01679, LOC102724238, LOC101928725 and LINC00880, and preferably, the long non-coding RNA is LINC01679 and LOC 102724238; in another specific embodiment, the inhibitor is an agent known to those skilled in the art to inhibit long non-coding RNA, and may be selected from, but not limited to, small compounds, interfering RNA, antibodies, and the like. In a specific embodiment, the inhibitor is siRNA, and further, the sequences of the siRNA against LINC01679 and LOC102724238 are shown in the table of example 2 below.

Drawings

FIG. 1 high throughput sequencing scatter plot (A), Gene heatmap (B).

Figure 2qRT-PCR demonstrated LINC01679, LOC102724238, LOC101928725, LINC00880 expression in primary pterygium tissue, siRNA knockdown efficiency for LINC01679, LOC102724238 and LINC01679, LOC102724238 distribution in cells.

FIG. 3 the effect of LINC01679, LOC102724238 on the proliferative capacity of hPECs.

(A) - (B) proliferation of hPECs at 24, 48 and 72 hours after transfection of the highly efficient small interfering RNAs targeting LINC01679(si-1, si-2) and LOC102724238(si-3, si-4). Negative Control transfected cells were used as a Control.

FIG. 4 influence of LINC01679, LOC102724238 on migration ability of hPECs.

(A) - (B) healing of the scrapings of hPECs after 24 hours, 48 hours after transfection of the highly efficient small interfering RNAs targeting LINC01679(si-1, si-2) and LOC102724238(si-3, si-4). Negative Control transfected cells were used as a Control.

FIG. 5 the effect of LINC01679, LOC102724238 on the invasive capacity of hPECs.

(A) - (B) cases of hPECs cell invasion after transfection of the highly efficient small interfering RNAs targeting LINC01679(si-1, si-2) and LOC102724238(si-3, si-4). Negative Control transfected cells were used as a Control. 24 hours after the transfection was completed, hPECs cells were incubated in a transwell chamber plated with a primer for 16 hours, and finally fixed, stained, and photographed.

Detailed Description

Material method

1. hPECs cells were cultured from human primary pterygium head tissue. 1) In the conventional pterygiectomy, head tissue was taken out, and the tissue pieces were placed in DMEM-F12 medium containing 10% fetal bovine serum and kept on ice, and primary cell culture was started within two hours. 2) Tissue pieces were first cut into small pieces 2-3mm in diameter and washed three times with Hank's solution. 3) The tissue mass was submerged in 0.8U of Dispase/Collagenase IV solution (Roche, USA) at 37 deg.C in 5% CO₂The digestion was continued for 5 minutes with 0.25% pancreatin after 3 hours. 4) The digestate was collected, centrifuged at 1000rpm for 5 minutes, and cultured in DMEM-F12 medium containing 10% fetal bovine serum in basic suspension.

2. Cell culture: cell recovery, cell passage and cell freezing are all operation techniques well known to those skilled in the art.

3. Cell proliferation assay (CCK-8 method for cell viability)

1) Inoculating cells:taking hPECs in logarithmic growth phase, carrying out cocyte passage, processing adherent cells into uniform cell suspension, counting by using a cell counting plate, and adjusting the cell concentration to 2 x 10 by using complete culture medium⁴hPECs cells were seeded in 96-well plates at a volume of 100. mu.L cell suspension per well. The 96-well plate was placed in a 37 ℃ incubator overnight. 2) The 450nm excitation wavelength was selected, the absorbance of each well was measured using an enzyme linked immunosorbent assay, and the results were recorded. Cell viability was expressed by calculating the ratio of absorbance of the treated samples to the control samples, with three replicates per group set.

4. Scratch healing experiments:

1) inoculating cells: taking hPECs (human hematopoietic progenitor cells) in logarithmic growth phase, carrying out cocyte passage to obtain adherent cells, processing the adherent cells into dispersed and uniform single cell suspension, counting and adjusting the concentration of the cell suspension, and counting the number of the cells in each hole to 4 multiplied by 10⁵hPECs cells were plated in 6-well plates. The 6-well plate was placed in an incubator to continue the culture. 2) After the cells reached 100% confluence, the monolayer was wounded by scratching the surface as evenly and straightly as possible with a sterilized 200 μ L pipette tip, resulting in a scratch. Rinsing with PBS buffer solution for three times, adding serum-free culture medium, and placing in a constant temperature incubator for continuous culture. 3) Samples were taken at 0h, 24h and 48h and photographed to observe the healing of the scratch.

5. Transwell invasion test

1) The Matrigel base was gel frozen overnight and the tip pre-cooled with an eppendorf tube at 4 ℃. 2) And (4) icing, and placing Matrigel substrate glue, a serum-free culture medium, a gun head and an eppendorf tube. The transwell cells were placed into the wells of a 24-well plate, numbered at the cell edges. The thawed Matrigel was mixed by vortexing, diluted with serum-free medium at a ratio of 1:40, whipped several times, mixed well and placed on ice. Each well was 100. mu.L of serum-free medium, 2.5. mu.L of substrate gel, and 1 well was prepared. 100. mu.l of diluted Matrigel primer was added to the transwell chamber, avoiding the addition to the side walls and not generating bubbles. Placing in an incubator for 1-2 h. 3) Discarding the culture medium of the cells to be treated (fusion degree 70-80%), passaging with the cells, treating the adherent cells into single cell suspension, centrifuging, discarding pancreatin, and removing trypsinThe serum medium was resuspended. The cell concentration was adjusted to 1.25X 10 by counting⁵one/mL. 4) After the matrix of the transwell chamber Matrigel in 1) was coated, excess liquid (serum-free medium) was aspirated off immediately, 200. mu.L of cell suspension (containing 2.5X 10 cells)⁴Individual cells) were added to the chamber to prevent the basement membrane from drying. Adding 600 μ L complete culture medium into the lower chamber to ensure that no air bubbles are attached to the permeation support membrane/polycarbonate membrane, and placing in an incubator for 12-16 h. 5) The 24-well plate with the transwell chamber was removed, 2 wells were selected in the 24-well plate, 1ml PBS buffer was added, the medium in the chamber was poured out, placed in PBS buffer, 1ml PBS was added to the upper chamber, and washing was repeated 2 times without sticking to the membrane. 6) Add 600. mu.L of 4% paraformaldehyde into 24 wells without adding in the upper chamber, and let stand for 5-20 min. The step as in 5) was washed twice with PBS buffer. 7) Add 500. mu.L crystal violet dye to 24-well plate and stain for 5 min. The upper and lower chambers were washed 2-3 times with PBS buffer, the upper cells of the membrane were wiped off 2-3 times with a moist cotton swab, and the overall situation was observed under a microscope. 8) After drying, the film was photographed by a microscope and three pictures were taken for subsequent statistics.

6. RNA extraction and reverse transcription experiments were performed according to the molecular cloning laboratory Manual published by Cold spring harbor.

7. Transcriptome sequencing analysis

1) Quality control and quantification of RNA: RNA degradation and contamination were monitored on a 1% agarose gel, RNA purity, concentration and integrity were measured, and subsequent experiments were performed after the samples were qualified. 2) Library preparation for lncRNA sequencing: the extracted total RNA is first labeled with a biotin-labeled specific probe (Ribo-Zero)^TMrRNA Removal Kit) to remove ribosomal rRNA. After purification, the RNA is fragmented at a temperature and under ionic conditions. Is then used

Random primers and reverse transcriptase in the Stranded kit synthesize one strand of cDNA, and then DNA polymerase I and RNaseH are used to synthesize double-Stranded cDNA. During cDNA double-strand synthesis, the RNA template is removed and dTTP is replaced by dUTP. The involvement of dUTP prevents the double strand cDNA from being amplified in subsequent processing because polymerase does not extendThe method spans the dUTP site on the template. The double stranded cDNA product is then ligated with an "A" base and an adaptor. The ligation product will be amplified and purified to obtain the final cDNA library. 3) Data filtering and gene assembly: after sequencing, the Reads are aligned to the ribosome database by using short Reads alignment tool SOAP, 5 mismatches are allowed at most, Reads on the aligned ribosome are removed, and the retained data is called clean Reads for subsequent analysis. After aligning clean reads to the reference genome using the alignment software HISAT, assembly was performed with StringTie to obtain all transcript sequences in each sample. 4) And (3) data analysis: these transcripts were compared with known mRNA and lncRNA using cuffmatch (one of the tools of Cufflinks) to obtain information on their positional relationship to each other. For the assembly results of individual transcripts, we require that each transcript meet the following requirements: FPKM>＝0.5，Coverage>1，Length>200. The low expressed genes were not assembled sufficiently in a single repeat due to insufficient sequencing depth, we pooled the multiple assembly results with Cuffmerge to obtain complete transcripts, which were used as final results for subsequent analysis. All subsequent analyses are based on these high quality data to be analyzed. 5) And (3) coding capability prediction: after obtaining new transcripts, we used three prediction software, CPC, txcdredict, CNCI, and a database, pfam, to predict the coding capacity of transcripts to distinguish mRNA from lncRNA. All three prediction software scored the coding capacity of transcripts and then set a scoring threshold to distinguish lncRNA from mRNA. pfam is protein database, and if the transcript can be compared with the upper pfam, the transcript is considered mRNA, otherwise the transcript is lncRNA. In the four judgment methods, at least three judgment methods are consistent, and only if the transcript is determined to be mRNA or lncRNA, the transcript is determined to be mRNA or lncRNA. The scoring thresholds for the three types of software are as follows: CPC _ threshold is 0, transcripts greater than 0 are mRNA, less than 0 are lncRNA; CNCI _ threshold is 0, transcripts greater than 0 are mRNA, less than 0 are lncRNA; txcdpredict _ threshold of 500, transcripts greater than 500 are mrnas and less than 500 are lncrnas. 6) Analysis of gene differential expression: we calculated FPKM (fragments per kilo-base per millions reads) to evaluate gene transcription expression levels, completion genesQ-value (FDR adjusted p-value) or p-value due to differential expression analysis<A gene of 0.05 was defined as a differentially expressed gene as a candidate gene for further analysis. 7) Functional analysis of differentially expressed genes: the Differentially Expressed Genes (DEG) were subjected to a heat map analysis at http:// www.heatmapper.ca/expression/. GO and KEGG enrichment analyses of up-and down-regulated genes were made by the DAVID website (https:// DAVID. ncifcrf. gov /). The GO enrichment analysis is respectively carried out in the aspects of biological process, molecular function, cell composition and the like.

8. Statistical analysis:

all experiments were repeated three or more times. The measurement data conforming to normal distribution in the data result are all expressed by mean +/-standard deviation, and significant difference analysis is carried out by using t test or variance analysis. Statistical analysis and mapping were performed using GraphPad Prism 7 data processing software with a P value <0.05 as a criterion for significant differences.

Example 1 alteration of expression of Long noncoding RNA of Primary pterygium

We used high throughput sequencing technology to detect primary pterygium lncRNA expression. lncRNA with a p-value less than 0.05 was defined as Differentially Expressed Genes (DEGs). We found that the total expression levels of 2280 lncRNA in the primary pterygium tissue (experimental group) were statistically significantly changed compared to the expression levels of lncRNA in the normal conjunctival tissue (control group), as shown in the scattergram (A) and the Genethermograph (B) of FIG. 1, and that 1327 lncRNA were up-regulated and 953 lncRNA were down-regulated in the 2280 lncRNA. The 2280 lncRNA with different expression levels screened by the inventor comprises 1867 confirmed lncRNA and 413 potential lncRNA. We picked 1327 lncrnas that were upregulated and further studied to investigate the relationship of these lncrnas whose expression was upregulated to the development of primary pterygium. In the sequencing result, lncRNA with the expression quantity up-regulated in the primary pterygium tissue (experimental group) is potentially lncRNA for promoting the progress and invasion of pterygium compared with the normal conjunctival tissue (control group). Since lncRNA has not been fully studied as a target for pterygium therapy, has potential research and therapeutic value, and we have long focused on the specific role and mechanism of lncRNA in the development of primary pterygium, we next investigated whether these upregulations of expression potentially promote progress of pterygium and affect lncRNA on proliferation, migration, and invasion of hPECs cells.

First, we verified the lncRNA sequencing results on primary pterygium tissues using real-time fluorescent quantitative PCR. As shown in fig. 2(a), LINC01679, LOC102724238, LOC101928725 and LINC00880 are the lncrnas that are most significantly upregulated in the sequencing results compared to normal conjunctival tissue. The real-time fluorescent quantitative PCR verification shows that LINC01679 and LOC102724238 are most remarkably upregulated in pterygium tissues, and the upregulation times reach 1.7-24.8 times. It is shown that LINC01679 and LOC102724238 have a strong possibility of playing a certain role in the progress process of the primary pterygium, such as increasing the proliferation, migration and invasion capacity of hPECs. Therefore, we designed a series of experiments against LINC01679 and LOC102724238 to verify our hypothesis.

Example 2 distribution of LINC01679 LOC102724238 in hPECs

Firstly, RNA of nucleus and cytoplasm of hPECs is extracted respectively, reverse transcription is carried out on the RNA to cDNA, and distribution of LINC01679 and LOC102724238 in hPECs is verified by a real-time fluorescence quantitative PCR method. As shown in fig. 2(C), LINC01679 and LOC102724238 are both distributed mainly in the cytoplasm.

Example 3, LINC01679, LOC102724238 Effect on hPECs cell proliferation, migration and invasion

First, we designed two small interfering RNAs (sirnas) for LINC01679 and LOC102724238, and verified the knockdown efficiency of the small interfering RNA in hPECs cells for LINC00205 by using a real-time fluorescence quantitative PCR method. The sequences of the siRNAs are shown in the following table:

the experimental results are shown in FIG. 2(B), and the knocking-down efficiency of LINC01679-homo-461(si-1) and LINC01679-homo-930(si-2) in hPECs cells is 60% and 52% respectively after 24 hours of transfection; LOC102724238-homo-1997(si-3) and LOC102724238-homo-1325(si-4) have knockdown efficiencies of 41% and 86%, respectively, on LOC102724238 in hPECs. All four sirnas were used in subsequent experiments. Since LINC01679 and LOC102724238 are up-regulated in primary pterygium tissue compared to normal conjunctival tissue, we hypothesize that LINC01679 and LOC102724238 can promote the proliferation, migration, and invasion capacity of hPECs cells. To verify this hypothesis, we examined the changes in the proliferation, migration and invasion abilities of hPECs after knocking down the expression levels of LINC01679 and LOC102724238 in hPECs. As shown in fig. 3, 4 and 5, in the hPECs cells, the proliferation capacity, migration capacity and invasion capacity of the hPECs cells are significantly reduced compared with the control group in the LINC01679 knock-down group and the LOC102724238 knock-down group, respectively, and the reduction degree is proportional to the knocking-down efficiency. The above experimental results can preliminarily confirm that LINC01679 and LOC102724238 can mediate the increase of the proliferation, migration and invasion capacities of hPECs.

The gene expression profile of the primary pterygium and the expression condition of the long-chain non-coding RNA are comprehensively analyzed by using a high-throughput sequencing technology, and the effects of LINC01679 and LOC102724238 in the generation, development and regulation of the primary pterygium are preliminarily verified by combining the omics analysis results, so that certain revelation is provided for subsequent research.

The above examples are only the best mode to represent the research idea of the present invention, and any technical solutions that can be obviously obtained by those skilled in the art from the prior art disclosures fall within the invention to be deprotected in the present application.

Sequence listing

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gacgcgtgcg cagatctcga tcccaaactc cctcctgcca gaatctggac ccgaatccac 420

ccattgcccg ttttctgctg ccgctggaga gaatctctga ggtccccagg agagcctgcc 480

tgcacggaag agatgcctcc tcagtatggc cgcccccgga gaggagcgat taagtgcaga 540

cctccatgtt gctcttgagc ctgagcggct tcagggagcc atgtttgtta ctggcgggcg 600

ccgacctcac tgagcatgtg cagccctggc cgggcggcct caaagttctg acatcacagg 660

gcggttcctg aagtggacgt agttgtaaga gctagttatt ttagacaatg cctctgggat 720

cagggactct aatctggaaa taggtagtag gagaggtcgg tgatgcagtc tctggatcag 780

agacctgagc tatatggggt tagagagggg ccctgggcag gcgagtctct ggggagtgtg 840

gtgagaatcc ttgtgtaaga tgctgggagg aggtggggtc agggctgggg tccgtgggcc 900

gacgggttgg gggatggcca gcgtcaggga tcagtagtag agattctatg tgccctgatc 960

gccagtggag gttttaaata cagagtattc atgagtttag caatgttatt cgcctctcat 1020

tatttaaata atttgaaatt ttccccaata actagtgtct tagaagtcat gaaaatttca 1080

gaaaatgaca ggtctcctga gttcgtgatg ggagttgggc agcagtcgca tacgagcacc 1140

tggagagtcc ttgccagttc tttggggatg gggagctctt aagactgccc tgagaccgcc 1200

ctttgacctc attatggtcc tttctagatc aaatgctgtt ttccatgact gtctctgttc 1260

ttcccatacg cgaatggcag gcatccagat ctccaagaat agaggattag gagagactcc 1320

accacctatg tcctcacagt taattatcga tttgtgtcag ttgcccattt tctcacttcc 1380

tgtttgtgtg tcaaggagta ttaataaatc tttgcctatt taaagatatt agccttggat 1440

tttcaaatat tgcatatttt gacatgtaaa aatttgttag ttttattttt tcaaccgatc 1500

tgacctgtat agttgggctt gaggaagctt cctcattcta catgttacta tggattttct 1560

aatattaaca cagggttgta ttttttccat ataactttct attgaatttc tttttccata 1620

tgataggtgg tgaaggttta gcccagtaaa gcagagaggt taagaggtta gattgggggc 1680

tctggagcca gacctatgta gatctgagtc ctggctctgg cacttgaaag ccgtgtgacc 1740

ttggttaaga tacttagtcc ctctctgcca aatggagaaa taagggcacc taccccatag 1800

ggtagttgtg tgattacaca agttaataca cttcaatcag tagcaagaaa gtgaatgtca 1860

agctgtattt gttcaggcag ccatatggta gccccacgtc cttagtaaac tgaggtattg 1920

aagtatcttc tttttctttc agaaaatatt tgtcaagcac cttctgtgtt ctaagaactg 1980

ttctagagct ttgcatgcta tggaggtcta gaaattcaca ttatggtggc aggtgacaga 2040

caatacaaag agataaagca atttcatata gtgataagct agggaggagt gctaaaagga 2100

gccaaggtgt agtggtgact ggaaggtgac cagggaaacc actgtagggt ttctaagtga 2160

tgagatctgt gacaaatccc tgtggtcagc agcaggcatt ggatatgtaa ctttattctg 2220

tcccattgat cttgtagtca accctggtct gttctgtatt gttttaatta cataa 2275

<210> 3

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cccaggacau cacacacaat t 21

<210> 4

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

uuguguguga uguccugggt t 21

<210> 5

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ccacauggag uuuauccaat t 21

<210> 6

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

uuggauaaac uccauguggt t 21

<210> 7

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gcuauggagg ucuagaaaut t 21

<210> 8

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

auuucuagac cuccauagct t 21

<210> 9

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ccuauguccu cacaguuaat t 21

<210> 10

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

uuaacuguga ggacauaggt t 21

<210> 11

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

uucuccgaac gugucacgut t 21

<210> 12

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

acgugacacg uucggagaat t 21

Claims (3)

1. Use of a reagent for specifically detecting a long non-coding RNA (long non-coding RNA) for the preparation of a reagent for diagnosing primary pterygium in a human, wherein the long non-coding RNA is LINC 01679.

2. The use as claimed in claim 1, wherein the long non-coding RNA has the sequence of SEQ ID NO. 1 or an RNA sequence having 99% homology with SEQ ID NO. 1 and derived from a human.

3. The application of the inhibitor of the long-chain non-coding RNA in preparing the medicine for treating the primary pterygium of a human is characterized in that the long-chain non-coding RNA is LINC01679, the inhibitor is interfering RNA, and the sequence of the interfering RNA is shown as any one of SEQ ID NO. 3-6.

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