CN113151478A

CN113151478A - Application of long-chain non-coding RNA LINC00892 as bladder cancer molecular marker

Info

Publication number: CN113151478A
Application number: CN202110509165.1A
Authority: CN
Inventors: 金红蕾; 任书纬; 沈丽萍; 黄海山
Original assignee: Wenzhou Medical University
Current assignee: Wenzhou Medical University
Priority date: 2021-05-11
Filing date: 2021-05-11
Publication date: 2021-07-23

Abstract

The invention discloses application of a long-chain non-coding RNA LINC00892 as a bladder cancer molecular marker and application in preparation of a bladder cancer treatment drug, and finds that expression of a new long-chain non-coding RNA LINC00892 in bladder cancer clinical samples and human bladder cancer cell lines has a significant down-regulation trend for the first time, can inhibit migration, infiltration and metastasis of bladder cancer, has great potential as a new target for prognosis diagnosis of bladder cancer patients, and provides a theoretical basis for research and development of a new bladder cancer metastasis-resistant drug or a new technology taking LINC00892 as a target.

Description

Application of long-chain non-coding RNA LINC00892 as bladder cancer molecular marker

Technical Field

The invention relates to the technical field of molecular biology and tumor prevention and treatment, in particular to application of a long-chain non-coding RNA LINC00892 as a bladder cancer molecular marker.

Background

Malignant tumor is a disease which endangers human health, and chemotherapy is one of the main means for treating malignant tumor at present. However, malignant tumor metastasis is often the leading cause of tumor chemotherapy failure. In addition, because the traditional anti-tumor drugs have poor selectivity, the traditional anti-tumor drugs have strong killing effect on normal cells and tissues while killing tumors, cause adverse reaction after treatment of patients, and are not beneficial to treatment and recovery of the patients to a great extent. Therefore, the research on the mechanism of malignant tumor metastasis and the search for and development of drugs for reducing or eliminating malignant tumor metastasis are the key and difficult points in the current tumor prevention and treatment research field.

Bladder cancer is a common urogenital malignancy. According to Journal survey of CA Cancer Journal for Clinicians, 549393 new cases of bladder Cancer, deaths 199922 were identified worldwide in 2018, with the incidence being sixth and ninth among male patients. In China, bladder cancer is one of the most common tumors in the urogenital system, new cases of bladder cancer in men are 62100, women are 18400, the mortality rate in men is 25100, and women are 7800.

The pathological types of bladder tumors can be divided into two types according to the parts: non-muscle-invasive tumors and muscle-invasive tumors. Although 70% of these are diagnosed with non-muscle-invasive tumors, the rate of recurrence is high in this population (50-70%), whereas 15-20% of recurrent bladder cancers progress to muscle-invasive disease. The 5-year survival rate for patients with bladder cancer that develop regional or distant metastases is only 45% and 6%, and nearly 80% of those with lymph node metastases emerge in the first 5 years after diagnosis.

In recent years, radical cystectomy for invasive bladder cancer remains the standard therapy in many parts of the world, but almost half of these patients develop metastases within two years after cystectomy and subsequently die from the disease. In the last two decades, cisplatin-based combination chemotherapy regimens such as CMV (cisplatin, methotrexate, and vinblastine) or M-VAC (methotrexate, vinblastine, doxorubicin, and cisplatin) have been used primarily in patients with advanced bladder cancer. However, the overall prognosis remains poor and the adverse effects caused by these combination chemotherapies are very severe. Therefore, there is an urgent need to develop new molecular target drugs for bladder cancer.

Only 1.5% of the nucleic acid sequences in 30 hundred million base pairs of the human genome are used for protein coding, with 98.5% of the genome being non-protein-coding. In recent years, with the rapid development of high-throughput deep sequencing technology, a large amount of long-chain non-coding rna (lncrna) is continuously discovered in eukaryotes, and a large number of LINCRNA are identified in 2012. The lncRNA is a non-coding RNA (ncRNA) molecule with the length of more than 200nt, rich biological functions and conserved higher-level structure, can directly act with molecules such as protein, micro RNA (microRNA) and the like, and regulates and controls the expression of a target gene at an epigenetic level, a transcription level and a post-transcription level. However, the mechanism of action of LNCRNA is closely related to its localization, and in order to precisely study the mechanism of action of LNCRNA, it is necessary to clarify its localization, which is more diverse than the localization of MRNA (ribosomes which are very specifically localized in the cytoplasm), since some LNCRNAs can occupy chromatin, subduclear domains, nucleoplasm or cytoplasm. In the nucleus, LNCRNA is used as a guide molecule to guide the protein complex to be positioned to a specific site and regulate the expression of a target gene; can also be used as a scaffold molecule, and simultaneously recruits different protein complexes to regulate the expression of target genes; also as a protein decoy molecule, adsorbs the target protein, repressing its action on the target gene. In cytoplasm, LNCRNA acts as a decoy molecule for miRNA, and adsorbs miRNA in a target-mimicking manner, inhibiting its further action on the target, and regulating gene expression. Although a great deal of LNCRNA is continuously excavated in the organism, the research still stays in the surface layer stage, and a long way is left for completely revealing the biological function of the 'dark substance' in the gene.

Due to the limitations of traditional therapies for treating bladder cancer, as well as the enormous adverse effects on the patient's body, and the heavy economic and work burden on the patient's family, medical personnel and society. Therefore, the treatment of bladder cancer faces an unprecedented severe situation, the alleviation of the situation is an urgent task for researchers, and the research and development of new drugs targeting the inhibition of metastasis and the application of corresponding new treatment measures are important ways for recognizing the situation, so that the identification of corresponding new molecular targets for inhibiting the metastasis of bladder cancer becomes one of the hot spots in the research of bladder cancer prevention and treatment.

In recent years, LNCRNA has been increasingly studied in bladder cancer and plays an important biological role, but no effective therapeutic target has been found so far.

Disclosure of Invention

The invention provides application of a long-chain non-coding RNA LINC00892 as a bladder cancer molecular marker and application in preparation of a bladder cancer treatment drug, and finds that expression of a novel long-chain non-coding RNA LINC00892 in bladder cancer clinical samples and human bladder cancer cell lines has a remarkable down-regulation trend for the first time, can inhibit migration, infiltration and metastasis of bladder cancer, has great potential as a new target for prognosis diagnosis of bladder cancer patients, and provides a theoretical basis for research and development of a new bladder cancer metastasis resistant drug or a new technology taking the LINC00892 and related intermediate signal molecules as targets.

The specific technical scheme is as follows:

the invention provides application of a long-chain non-coding RNA LINC00892 as a bladder cancer molecular marker, wherein the nucleotide sequence of the long-chain non-coding RNA LINC00892 is shown as SEQ ID No. 1.

The LINC00892 gene is positioned in human Xq26.3, and is determined to be non-coding RNA between long-chain genes according to the encoded protein ability of UCSC website and CPAT database, and is LNCRNA with unknown function at present.

The invention utilizes a bioinformatics tool to analyze the relationship between LINC00892 and the prognosis of a bladder cancer patient, and discovers for the first time that the expression of the long-chain non-coding RNA LINC00892 in a bladder cancer clinical sample and a human bladder cancer cell line has a significant down-regulation trend, and the expression level of LINC00892 is positively correlated with the life cycle of the bladder cancer patient; the long-chain non-coding RNA LINC00892 has great potential as a new target for prognosis diagnosis of bladder cancer patients.

Further, the detection reagent of the long-chain non-coding RNA LINC00892 is used for preparing an in vitro diagnosis product for early diagnosis and prognosis diagnosis of bladder cancer.

Further, the in vitro diagnosis product comprises a kit, a gene chip and a solid support.

Further, the kit is a fluorescent quantitative PCR detection kit.

Further, the gene chip is an lncRNA chip, comprising: a solid phase carrier and oligonucleotide probes orderly fixed on the solid phase carrier;

the oligonucleotide probe specifically corresponds to a part or all of the sequence of the long-chain non-coding RNA LINC 00892.

The invention also provides application of the long-chain non-coding RNA LINC00892 in preparation of a medicament for treating bladder cancer, wherein the nucleotide sequence of the long-chain non-coding RNA LINC00892 is shown as SEQ ID No. 1.

Further, the drug for treating bladder cancer is a drug for inhibiting invasion and/or migration of bladder cancer cells.

Further, the drug includes nucleic acid molecules, lipids, small molecule chemical drugs, antibody drugs, polypeptides, interfering lentiviruses.

Compared with the prior art, the invention has the following beneficial effects:

the invention discovers novel targets which are differentially expressed and closely related to the prognosis of patients through deep mining of the TCGA database of the bladder cancer: long non-coding RNA (LINC 00892); the low expression of the LINC00892 in bladder cancer is found for the first time, and in vivo and in vitro experiments prove that the over-expression LINC00892 can inhibit the migration, infiltration and metastasis of bladder cancer, thereby providing a theoretical basis for researching and developing new bladder cancer metastasis resistant drugs or new technologies taking LINC00892 and related intermediate signal molecules as targets.

Drawings

FIG. 1 is a bioinformatics analysis of the relative expression of LINC00892 in TCGA database 19 versus a clinical sample of fully paired bladder cancer;

wherein an asterisk indicates that there is a significant difference (p < 0.05); n is normal tissue; t: tumor tissue.

Fig. 2 is a bioinformatics analysis that LINC00892 expression positively correlated with survival of bladder cancer patients (n 403) in the TCGA database.

FIG. 3 is a graph showing the relative expression level of LINC00892 in 27 pairs of fully-paired human bladder cancer clinical samples (bladder cancer patients treated radically- -bladder resection samples, normal bladder epithelial tissue can be obtained) detected by fluorescence quantitative PCR technology;

wherein an asterisk indicates that there is a significant difference (p < 0.05); n: normal tissue; t: tumor tissue.

FIG. 4 is a graph showing the detection of the expression level of LINC00892 in bladder cancer cell lines RT4, RT112, J82, TCCSUP, UMUC3, T24T and UROTSA cells, a normal urothelial cell line, using fluorescence quantitative PCR;

where asterisks indicate significant differences (p < 0.05).

FIG. 5 shows the fluorescent quantitative PCR technology for detecting the relative expression of LINC00892 in 24 cases of clinical non-metastatic bladder cancer and 14 cases of clinical metastatic bladder cancer (bladder cancer patients- -bladder resection samples, normal bladder epithelial tissue can be obtained);

where asterisks indicate significant differences (p < 0.05).

FIG. 6 is a graph showing the expression of LINC00892 in stable cell lines of T24T and J82 cells overexpressing LINC00892 constructed by artificial lipofection using fluorescent quantitative PCR;

wherein Vector represents a blank plasmid control; asterisks indicate significant differences (p < 0.05).

FIG. 7 shows that overexpression of LINC00892 significantly inhibited bladder cancer cell invasion in vitro;

wherein A is a graph of the results of detecting the invasive ability of T24T (Vector, LINC00892) cells using a BD BioCoail matrigel Invasion Chamber; b is a statistical histogram of the A picture results; c is a graph of the results of measuring the invasive potential of J82(Vector, LINC00892) cells using a BD BioCoail matrigel Invasion Chamber; d is a statistical histogram of the results of the C picture.

FIG. 8 shows that overexpression of LINC00892 significantly inhibited metastatic potential of bladder cancer cells in vivo;

wherein, A is a result graph of selecting representative lung tissues and performing HE staining analysis; b is a histogram of the number of lung metastases; c isThe result of analyzing the genomic DNA expression level of T24T cells in the lung tissue of the nude mice by using fluorescent quantitative PCR; d is 2.5 x 10⁶Injecting the individual cells into the tail vein of the nude mouse, taking a picric acid fixed picture of the lung after 8 weeks of injection, and counting the metastasis to obtain a metastasis statistical chart; asterisks indicate significant differences (p)<0.05)。

Detailed Description

The present invention will be further described with reference to the following specific examples, which are only illustrative of the present invention, but the scope of the present invention is not limited thereto.

Example 1 bioinformatic analysis LINC00892 expression Down-Regulation in the TCGA database

RNAseq V2 for bladder cancer (BLCA) in TCGA data (https:// TCGA-data. nci. nih. gov /) and clinical data were downloaded, and samples with both RNAseq and miRNASeq information were screened for 424 cases, including 405 tumor tissues and 19 paracarcinoma tissues. The detection platform of the Sequencing data was IlluminaHiSeq 2000RNA Sequencing platform, a sample (n 19) with cancer-paracanced tissue was selected as an analysis object, and differential analysis was performed.

The specific difference analysis method comprises the following steps: genes expressed in low amounts in the data were knocked out (genes whose expression values were 90% or more and 0 were filtered). Lnc data were preprocessed using R-package edgeR (Version: 3.4, http:// www.bioconductor.org/pac kages/release/bioc/html/edgeR. html), respectively, raw count was normalized to log-CPM values, linear modeling was performed, and the mean variance relationship was adjusted using precision weights calculated from the voom function. Differential expression analysis was performed on lncRNA data Tumor VS Normal using the paired T-test method provided in limma package, respectively. And (3) obtaining corresponding P.value values after T test of all genes, and performing multiple test correction by using a Benjamini and Hochb erg method to obtain the corrected p value, namely adj.P.value. The lncRNA differential expression thresholds in this example are all adj.p. value <0.05 and | log2FC | > 2.

The results are shown in fig. 1, LINC00892 was significantly down-regulated in cancer tissue by about 3.5-fold.

In addition, the relationship between the expression level of LINC00892 and the survival of bladder cancer patients in the TCGA database (403 total cases, 19 cases with normal tissue samples) was analyzed, and it was found that the survival of the patients with high expression of LINC00892 was significantly better than that of the patients with low expression of LINC00892 (p <0.05) (fig. 2).

Example 2LINC00892 expression in clinical bladder cancer matched pairs and human bladder cancer cell lines is significantly downregulated

The specific steps of the tissue/cell total RNA extraction method, LncRNA reverse transcription method and the method for detecting LncRNA expression level by fluorescent quantitative PCR referred to in this example are as follows:

1) tissue/cell total RNA extraction

SuperScript was used as purchased by Invitrogen corporation of America^TMIV, extracting by using a reverse transcription kit, wherein the extraction steps are as follows:

a. removing clinical samples of bladder cancer from a freezer at-80 deg.C, each sample about 30-50ug in an EP tube, and placing on ice; about 1X 10 cells⁷Within one.

b. 1000ul of trizol was added and mixed well, and the large tissue was cut into pieces with scissors.

c. Mixing completely, standing at room temperature for 5-10 min. The tissue needs to be homogenised using a homogeniser at maximum speed of rotation, usually for 2min, the whole process being carried out on ice.

d. Add 140. mu.L chloroform (chloroform), shake vigorously for 1min, and stand at room temperature for 2-3 min.

e. The samples were centrifuged at 12000g at 4 ℃ for 15min and after centrifugation the supernatant was slowly pipetted into a new de-enzymed EP tube.

f. 1.5 volumes of 100% absolute ethanol (generally 525. mu.L) were added and mixed up and down several times to prepare a sample for the next step.

g. Placing RNeasy Mini spin column in 2ml centrifuge tube, sucking the above sample 700ul into centrifugal column, centrifuging at room temperature for 30s, rotating at 10000rpm, and discarding filtrate.

h. And 7, repeating the step until the sample is filtered completely.

i. Adding 700 mu LBuffer RWT into a centrifugal column, rotating at 10000rpm, centrifuging at room temperature for 30s, and discarding filtrate;

j. adding 500 mu L Buffer RPE into the centrifugal column, rotating at 8000g, centrifuging at room temperature for 30s, and removing the filtrate;

k. and adding 500 mu L of Buffer RPE into the centrifugal column again, centrifuging at 8000g of rotating speed for 1min, then centrifuging at 10000rpm for 1min so as to effectively enrich RNA, and discarding the filtrate.

RNeasy Mini spin column was placed in a new 2ml centrifuge tube and centrifuged at 1000g for 2min at room temperature.

m. the column was placed in a new 1.5ml enzyme-removed EP tube, RNase-free water 30-50. mu.L (directly on the membrane) was added, centrifuged at 10000rpm at room temperature for 1min, and the filtrate was collected.

And n, sucking the filtrate to a centrifugal column membrane, repeating the step 13, and collecting the filtrate.

o. the concentration was measured using Biodrop, and the sealing film was sealed and stored at-80 ℃.

2) LncRNA reverse transcription

After total RNA of the required tissue and cells is extracted according to the method, reverse transcription is carried out by using a reverse transcription kit, and the steps are as follows: the RT reaction system was prepared as follows and operated on ice.

Shaking the above system, mixing well, dropping, placing into PCR instrument, and setting program at 65 deg.C for 5min → placing on ice for at least 1 min.

Shaking and uniformly mixing the system, then dotting and separating the system, and putting the system into a PCR instrument for setting the program as follows, wherein the temperature is 23 ℃ for 10min and the temperature is 50-55 ℃; 10min, 80 ℃ 10 min. Sealing with sealing film, and storing at-80 deg.C or performing fluorescence quantitative experiment.

3) Fluorescent quantitative PCR detection of LncRNA expression

After obtaining cDNA from desired tissues and cells, PCR was performed using the MiScript SYBR Green PCR Kit available from Qiagen, USA, and the PCR reaction system was prepared as follows:

and (3) fully mixing the reaction system, adding the mixture into a 384-well plate, setting 3 multiple wells for each sample, centrifuging the 384-well plate for a short time, and placing the 384-well plate into a Q6 fluorescent quantitative PCR instrument for detection. Q-PCR reaction, pre-denaturation at 95 ℃, 15min later cycle conditions as follows, amplification for 40 cycles, denaturation at 95 ℃, 15 seconds, annealing at 55 ℃, 30 seconds, extension at 72 ℃ and 30 seconds. The main primer sequences are shown above, GAPDH was purchased from shanghai sony as an internal control.

The main primer sequences are as follows:

LINC00892-F 5’-AAAGACTACTGGGCTGGAAGTCA-3’；

LINC00892-R 5’-CTCATGGGCTCGTTCTTCTCTTAC-3’。

1. significant downregulation of LINC00892 expression in clinical bladder cancer paired samples

Tissue specimens were collected from 27 patients undergoing total bladder resection in urology surgery at the hospital affiliated to the university of medical science in Wenzhou. The clinical samples of bladder cancer are mostly male patients and the bladder is completely cut, and the tissues are pathologically diagnosed as bladder urothelial cancer. Tumor tissue was collected from each specimen, while paracancerous normal tissue was excised approximately 3cm from the periphery of the tumor as a control, and each sample was frozen in a liquid nitrogen tank after tissue isolation. All patient data includes name, gender, age, pathological diagnosis, etc.

Tissue samples of the 27 patients and matched paracancer normal bladder epithelial tissue samples thereof are taken, and the relative expression level of LINC00892 is detected by fluorescent quantitative PCR.

The results are shown in fig. 3, with low expression of LINC00892 in bladder cancer tissue; the expression level of LINC00892 was significantly down-regulated by about 5-fold in bladder cancer tissue compared to paracancerous normal bladder tissue.

2. LINC00892 expression in human bladder cancer cell line

Taking human bladder cancer cell lines UROTSA, RT4, RT112, J82, TCCSUP, UMUC3 and T24T, and detecting the relative expression level of LINC00892 on the bladder cancer cell line level by using fluorescent quantitative PCR.

The results are shown in fig. 4, and the expression level of LINC00892 in bladder cancer cell lines was significantly down-regulated compared to the normal urothelial cell line UROTsa control group.

Example 3LINC00892 expression levels in clinically metastatic bladder cancer samples were significantly downregulated compared to non-metastatic bladder cancer samples

Tissue specimens from 38 patients who underwent total cystectomy in urology surgery at the university of medical science, Wenzhou (24 non-metastatic bladder cancer and 14 metastatic bladder cancer) were collected. The clinical samples of bladder cancer are mostly male patients and the bladder is completely cut, and the tissues are pathologically diagnosed as bladder urothelial cancer. Tumor tissue was collected from each specimen, while paracancerous normal tissue was excised approximately 3cm from the periphery of the tumor as a control, and each sample was frozen in a liquid nitrogen tank after tissue isolation. All patient data includes name, gender, age, pathological diagnosis, etc.

Cancer tissue samples of the above cases were collected, and the relative expression level of LINC00892 was determined by fluorescent quantitative PCR (see example 2 for details).

The results are shown in fig. 5, with LINC00892 being less expressed in metastatic bladder cancer compared to non-metastatic bladder cancer; its expression level was significantly down-regulated by about 2-fold.

Example 4 overexpression of LINC00892 inhibits the invasive and migratory Capacity of bladder cancer cells

On the basis of example 2, two cell lines T24T and J82 with relatively low LINC00892 expression level are selected, an artificial liposome transfection method is adopted to transfect an overexpression plasmid of LINC00892 into T24T and J82 cells, and a corresponding stable cell line is successfully constructed through 3-week G418 screening (figure 6).

1. LINC00892 can remarkably inhibit invasion capability of bladder cancer cells in vitro

To further investigate the role of LINC00892 in bladder cancer cell invasion, we performed migration/infiltration experiments, photographing to record cell distribution 24h after chamber, and analyzing by counting the penetration of cells through Matrigel.

The migration/infiltration experiment comprises the following specific steps:

a. incubating the small chamber with the gel: taking out the small chamber with the gel from the temperature of minus 20 ℃, placing the small chamber in a 24-hole plate, standing the small chamber at room temperature for 10 minutes, adding 400 mu L of serum-free DMEM medium into the upper chamber, adding 700 mu L of serum-free DMEM medium into the lower chamber, and placing the small chamber in a 37 ℃ incubator for incubation for about 2 hours; the cells without the gel were incubated for 0.5 h.

b. Digesting the cells: after the cells were digested, the cells were resuspended in 0.1% serum DMEM/F-12 or 0.1% serum MEM medium and counted in a well.

c. Plate paving: the chamber was discarded and 400. mu.L of cell suspension (containing 3X 10 cells) was added to the upper chamber⁴Cells), 700. mu.L of 5% serum DMEM/F-12 or 10% FBS MEM medium was added to the lower chamber, and the mixture was left to stand in an incubator at 37 ℃ for 24 hours;

d.4% paraformaldehyde fixation: the upper chamber medium was discarded, washed twice with PBS, placed in a clean 24-well plate, 400. mu.L of the upper chamber medium was added, 700. mu.L of 4% paraformaldehyde was added to the lower chamber medium, and the mixture was fixed at room temperature for 5 minutes;

e.100% methanol pass through: after fixation, washing twice with PBS, placing the small chamber in a clean 24-pore plate, adding 400 μ L of 100% methanol into the upper chamber, adding 700 μ L of 100% methanol into the lower chamber, and allowing permeation at room temperature for 20 min;

f. giemsa staining: washing with PBS twice, placing the small chamber in a clean 24-hole plate, adding 400 μ L of filtered Giemsa working solution into the upper chamber, adding 700 μ L of Giemsa working solution into the lower chamber, and dyeing for 15-30 min at room temperature in dark place;

g. after dyeing is finished, washing the chamber twice by PBS, and then dipping the PBS by a cotton swab to wipe the bottom of the chamber;

h. images were taken, the number of cells passing through the chamber was counted and statistically analyzed, and a picture representative of the statistical analysis was taken.

The results are shown in fig. 7, where overexpression of LINC00892 significantly inhibited the invasive potential of bladder cancer cells compared to control cells.

2. In nude mice, LINC00892 inhibits the metastatic potential of bladder cancer cells

To further explore whether the in vivo animal level has the same function, we constructed an in vivo nude mouse lung metastasis model.

The construction method of the transfer model comprises the following steps:

(1) sterilizing the injection part of the tail vein of the nude mouse by using 75% alcohol, fully and uniformly mixing the cells before inoculating the cells, sucking 100ul of cell suspension by using a 1ml sterile syringe, and injecting the cell suspension into the tail vein of the nude mouse, wherein each cell is injected into 10 nude mice;

(2) normally breeding nude mice for 6-8 weeks, randomly killing 5 mice per group, fixing lungs with picric acid for 36h, photographing and counting the number of metastasis foci of lung tissues on the surface, and extracting genomic DNA in the lungs of the nude mice to determine the intrapulmonary metastasis condition;

(3) after the number of lung metastases was counted by photographing, lung tissues were used for paraffin embedding and HE staining for analyzing the details of metastases.

The T24T cells and the stable transfected cell strain thereof over-expressing LINC00892 are used as a study object, BALB/c nude mice of 4 weeks old are randomly divided into 2 groups, and the tail veins of the 2 groups of nude mice are respectively injected with 2.5 multiplied by 10⁶And (3) killing the nude mice after 8 weeks of cell injection, taking lung tissues, fixing the lung tissues by picric acid for 36 hours, selecting representative lung tissues for display, and performing HE (high-grade hematoxylin) staining analysis (figure 8A), wherein the statistical data are shown in figure 8B.

For the reliability of data, the expression quantity of the human genome DNA in the lung tissue of the nude mouse is analyzed by fluorescent quantitative PCR, and the result shows that the content of the human genome in the lung tissue of the nude mouse is remarkably reduced by over-expressing LINC00892 (FIG. 8C). The stereomicroscope takes pictures and counts the metastases, as shown in the metastasis statistics table (fig. 8D). From the above preliminary results, it can be seen that LINC00892 specifically inhibits the metastatic potential of bladder cancer cells without significantly affecting their growth potential.

Sequence listing

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Claims

1. The application of the long-chain non-coding RNA LINC00892 as a bladder cancer molecular marker is characterized in that the nucleotide sequence of the long-chain non-coding RNA LINC00892 is shown as SEQ ID No. 1.

2. The use of claim 1, wherein the detection reagent for the long-chain non-coding RNA LINC00892 is used for the preparation of an in vitro diagnostic product for the early and prognostic diagnosis of bladder cancer.

3. The use of claim 2, wherein the in vitro diagnostic product comprises a kit, a gene chip, a solid support.

4. The use of claim 3, wherein the kit is a fluorescent quantitative PCR assay kit.

5. The use of claim 3, wherein said gene chip is a lncRNA chip comprising: a solid phase carrier and oligonucleotide probes orderly fixed on the solid phase carrier;

6. The application of the long-chain non-coding RNA LINC00892 in preparing a medicament for treating bladder cancer is characterized in that the nucleotide sequence of the long-chain non-coding RNA LINC00892 is shown as SEQ ID No. 1.

7. The use of claim 6, wherein the medicament for treating bladder cancer is a medicament that inhibits the invasion and/or migration of bladder cancer cells.

8. The use of claim 6, wherein the drug comprises a nucleic acid molecule, a lipid, a small molecule chemical, an antibody drug, a polypeptide, an interfering lentivirus.