CN112410432A - Application of LINC02474 as colorectal cancer diagnosis marker and treatment target - Google Patents
Application of LINC02474 as colorectal cancer diagnosis marker and treatment target Download PDFInfo
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Abstract
The invention belongs to the technical field of biological medicines, and relates to a colorectal cancer diagnosis marker, a colorectal cancer treatment target and application thereof. The invention firstly provides that LINC02474 can be used as a colorectal cancer diagnosis biomarker and a treatment target point, and provides a new idea for diagnosis and treatment of colorectal cancer. The study shows that LINC02474 is significantly up-regulated in tumor tissues of colorectal cancer patients compared with normal control, and the biomarker provided by the invention has higher accuracy. The result of the invention shows that the interference of LINC02474 can inhibit the migration and invasion capacity of colorectal cancer cells, shows that LINC02474 plays a role in promoting oncogene in the occurrence and development of colorectal cancer, provides a new idea for the clinical treatment of colorectal cancer, and is expected to obtain a diagnosis and treatment scheme which is more acceptable for patients and feasible in clinic.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and relates to a colorectal cancer diagnosis marker, a colorectal cancer treatment target and application thereof.
Background
The long-chain non-coding RNA is a highly conserved long-chain non-coding RNA molecule which does not code protein per se and has a transcript length of more than 200nt, and can be expressed in a tissue-specific or development stage-specific manner. Involved in the regulation of genes in many aspects (epigenetic regulation, transcriptional regulation, post-transcriptional regulation, etc.). In recent years, long non-coding RNAs have attracted interest to biomedical researchers due to their wide range of biological functions, and their functions have been analyzed.
The biological functions of long-chain non-coding RNA are mainly divided into the following 3 types: (1) the role of long non-coding RNA in transcriptional regulation; (2) long-chain non-coding RNAs regulate different aspects of mRNA processing at the post-transcriptional level; (3) long non-coding RNAs affect a variety of epigenetic modifications including histone and DNA methylation, histone acetylation, and the like.
In recent years, the association of long non-coding RNAs with cancer has become an important issue in the field of cancer research, and hundreds of long non-coding RNAs have been shown to be abnormally expressed in various human cancers including colorectal cancer. Long non-coding RNAs have been found to be involved in cell proliferation, apoptosis, epithelial-mesenchymal transition (EMT), invasion and metastasis of cancer cells, and thus, long non-coding RNAs are useful as biomarkers for disease diagnosis.
At present, the clinical diagnosis of colorectal cancer mainly comprises rectoscopy and imaging examination, but the rectoscopy is invasive examination and has bleeding and infection risks. The problems of radiation dose accumulation, high cost and the like exist in the imaging examination and judgment of lymph node metastasis. Therefore, the discovery of early colorectal cancer diagnosis markers can reduce the use of rectoscopy and PET-CT, and make early colorectal cancer diagnosis more accurate and economical.
Disclosure of Invention
The invention provides a novel colorectal cancer diagnosis marker, a novel colorectal cancer diagnosis target and application thereof, aiming at the problems in the traditional colorectal cancer diagnosis and treatment.
In order to achieve the purpose, the invention is realized by adopting the following technical scheme:
according to the invention, colorectal cancer data set data is downloaded from a TCGA (TCGA), differential expression genes in a colorectal cancer tissue sample and a tissue sample beside cancer are analyzed by using a bioinformatics method, and LINC02474 with obvious differential expression is selected as a research target. In addition, 80 cases of colorectal cancer and paracancer normal tissues are selected from a tissue specimen library, tissue RNA is extracted and is reversely transcribed into cDNA, the expression level of LINC02474 in the tissue specimen is verified through qRT-PCR, and the expression of LINC02474 in the colorectal cancer tissues is found to be obviously up-regulated; LINC02474 can promote colorectal cancer invasion and metastasis.
A long-chain non-coding RNA LINC02474 is derived from human chromosome 1, and has deoxyribonucleotide sequence of
>NR_149071.1 Homo sapiens long intergenic non-protein coding RNA 2474 (LINC02474), long non-coding RNA
CCTCCTGCTCTTTGCTCCGTGAGAAAGATCCACCTACGACCTCAGGTCCTCAGACCGACTAGCCCAAGAAACATCTCACCAATTTCAAATCTGACCTTTGCAAGAGGGTCCGAGACATTTGCATCATCATTCAGTGAGACCTGTAAACACAGCATCTGCCTTTGACCACATCCATCTGGAAGAACCTGAGAGATAATCCATTTTATGAAATTTTCCCTACCCTGAAATGGGAGAATGAATCTAATTTGAAGCACTGAGAAGGATAAGGCATCCATTTGAAAAGGACTCCTATATTGCAACATGAATTCTGCTAAAATTGAAGCAAGAACAAACATCAAATTTATGATGAAGTTTGGGTAGAAGAACGATGAAATTAGTGACGCTTTATAAAAAGTTTATTGGGACAATACCCCAAATGAATCAGCAGTTTAAAAATGGATA are provided. The position of the long non-coding RNA LINC02474 in the chromosome is shown in FIG. 1.
The invention provides application of the long-chain non-coding RNA LINC02474 as a colorectal cancer molecular biological marker. Detecting an expression level of LINC02474 in a sample from a suspected colorectal cancer patient, wherein a higher expression level of LINC02474 is associated with a high likelihood that the subject has colorectal cancer and a poor prognosis.
The invention provides application of the long-chain non-coding RNA LINC02474 in preparation of a colorectal cancer diagnostic reagent or kit. The detection method of the LINC02474 molecular marker comprises the steps of total RNA extraction of a sample, reverse transcription of RNA and real-time fluorescence quantitative PCR amplification. The method comprises the following specific steps: total RNA extraction using feijast 2000 kit, second step: RNA was reverse transcribed into cDNA using prime-Script, GAPDH as an internal control, and all qRT-PCR reactions were performed using SYBR Green PCR Master Mix.
Application of substances for detecting high and low expression level of RNA LINC02474 in preparation of colorectal cancer diagnostic reagent or kit.
The primer for specifically amplifying the RNA LINC02474 has the sequence,
forward TGCAAGAGGGTCCGAGACAT and reverse ATGTGGTCAAAGGCAGATGCT.
A diagnostic kit for colorectal cancer, comprising the following primers: forward TGCAAGAGGGTCCGAGACAT, reverse ATGTGGTCAAAGGCAGATGCT; internal reference primers: forward ACCCACTCCTCCACCTTTGAC and reverse TGTTGCTGTAGCCAAATTCGTT.
Use of a specific small interfering RNA for down-regulating the expression level of RNA LINC02474 in the preparation of a medicament for the treatment of colorectal cancer
si-LINC02474#1：
Forward GGGUCCGAGACAUUUGCAUTT, reverse AUGCAAAUGUCUCGGACCCTT; si-LINC02474# 2:
forward GGUAGAAGAACGAUGAAAUTT and reverse AUUUCAUCGUUCUUCUACCTT.
Application of specific small interfering RNA for down-regulating RNA LINC02474 expression level in preparing medicine for treating colorectal cancer, and the acting object is DLD-1 cell strain.
The first purpose of the invention is to provide a potential diagnosis biomarker and a treatment target of colorectal cancer, which is LINC 02474.
A second object of the invention is to provide a primer pair, comprising a forward primer and a backward primer, which specifically recognizes LINC 02474. The nucleotide sequences of the primers are shown in Table 1.
A third object of the invention is to disclose the expression profile of LINC02474 in tumor tissue of a colorectal cancer patient.
A fourth object of the invention is to disclose the function of LINC02474 in colorectal cancer cells.
The invention provides application of long-chain non-coding RNA LINC02474 serving as a new potential colorectal cancer molecular diagnostic marker, and relates to discovery, detection and application of the LINC02474 molecular marker, and design and synthesis of a detection primer specifically used for real-time fluorescence quantitative PCR and small interfering RNA (siRNA) interfering with LINC02474 in vitro. LINC02474 expression was significantly upregulated in tissue samples from colorectal cancer patients compared to normal controls. In vitro experiments show that LINC02474 can promote invasion and migration of colorectal cancer cells. The research result of the invention shows that LINC02474 can be used as a diagnosis marker of colorectal cancer patients, has poor compliance for colorectal microscopic screening by target people, is invasive, and aims at the current situation that most of clinically diagnosed colorectal cancer patients are in middle and late stages and the early diagnosis rate is low overall, so that an ideal early molecular early warning signal is found in the process of colorectal cancer lesion, and early screening and diagnosis of cases are performed in a targeted manner, so as to relieve the pain of the patients, avoid over-medical treatment and save medical resources.
Compared with the prior art, the invention has the advantages and positive effects that:
1. the invention firstly provides that LINC02474 can be used as a colorectal cancer diagnosis biomarker and a treatment target point, and provides a new idea for diagnosis and treatment of colorectal cancer.
2. The study shows that LINC02474 is significantly up-regulated in tumor tissues of colorectal cancer patients compared with normal control, and the biomarker provided by the invention has higher accuracy.
3. The result of the invention shows that the interference of LINC02474 can inhibit the migration and invasion capacity of colorectal cancer cells, shows that LINC02474 plays a role in promoting oncogene in the occurrence and development of colorectal cancer, provides a new idea for the clinical treatment of colorectal cancer, and is expected to obtain a diagnosis and treatment scheme which is more acceptable for patients and feasible in clinic.
Drawings
Figure 1 is a schematic representation of the location of LINC02474 on a chromosome.
FIG. 2 is a graph showing the results of electrophoresis obtained after qRT-PCR using LINC02474 specific primers.
FIG. 3 is a graph showing the results of tissue expression level measurement in a patient with colorectal cancer using a LINC 02474-specific primer. Represents a p value < 0.05. Wherein, FIG. 3A is a qRT-PCR result graph; figure 3B is a characteristic curve of the subject's performance.
FIG. 4 is a qRT-PCR validation graph of the transfection knockdown efficiency of LINC02474 small interfering RNA (siRNA) in DLD-1 cell line. si-NC stands for negative control group; si-LINC02474# 1, si-LINC02474# 2 and si-LINC02474# 3 represent groups of siRNAs transfected with three targeted LINC02474 reverse splice sites, respectively.
FIG. 5 is a graph showing the results of the changes of migration and invasion capabilities of colorectal cancer cell line DLD-1 after the expression of LINC02474 is interfered by siRNA.
Detailed Description
In order that the above objects, features and advantages of the present invention may be more clearly understood, the present invention will be further described with reference to specific embodiments. It should be noted that the embodiments and features of the embodiments of the present application may be combined with each other without conflict.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced in other ways than those specifically described herein, and thus the present invention is not limited to the specific embodiments of the present disclosure.
Example 1
This example provides the use of LINC02474 in the diagnosis of colorectal cancer, based on studies of its expression in colorectal cancer and its regulatory role in colorectal cancer biological function.
The corresponding deoxyribonucleotide sequence of LINC02474 is shown below and is derived from human chromosome 1, in the positions shown in figure 1.
>NR_149071.1 Homo sapiens long intergenic non-protein coding RNA 2474 (LINC02474), long non-coding RNA
CCTCCTGCTCTTTGCTCCGTGAGAAAGATCCACCTACGACCTCAGGTCCTCAGACCGACTAGCCCAAGAAACATCTCACCAATTTCAAATCTGACCTTTGCAAGAGGGTCCGAGACATTTGCATCATCATTCAGTGAGACCTGTAAACACAGCATCTGCCTTTGACCACATCCATCTGGAAGAACCTGAGAGATAATCCATTTTATGAAATTTTCCCTACCCTGAAATGGGAGAATGAATCTAATTTGAAGCACTGAGAAGGATAAGGCATCCATTTGAAAAGGACTCCTATATTGCAACATGAATTCTGCTAAAATTGAAGCAAGAACAAACATCAAATTTATGATGAAGTTTGGGTAGAAGAACGATGAAATTAGTGACGCTTTATAAAAAGTTTATTGGGACAATACCCCAAATGAATCAGCAGTTTAAAAATGGATA。
In this example, 80 cases of colorectal cancer and paracancer normal tissues were selected from tissue samples, total tissue RNA was extracted from Trizol (Invitrogen, Carlsbad, ca. usa), and reverse transcription was performed to obtain cDNA, and the expression level of LINC02474 in the tissue samples was verified by qRT-PCR, and it was found that LINC02474 expression was significantly up-regulated in colorectal cancer tissues. As shown in fig. 3.
The differences in the expression of LINC02474 in colorectal cancer tissue samples were verified by methods employing real-time fluorescent quantitative PCR.
Extracting total RNA in a colorectal cancer cell line DLD-1, wherein the capacity of each part of RNA is less than 1ug, adding 4ul of 5XPrimeScript Buffer (for real time), 1ul of PrimeScript RT Enzyme Mix I and 1ul of Oligo dT Primer, complementing the system to 20ul by RNase-free water, reacting at 37 ℃ for 30min, keeping at 85 ℃ for 5s, reversing the RNA into cDNA, performing qRT-PCR by using forward and reverse primers, and amplifying a transcript LINC 02474.
1. Experimental materials and methods
And (3) collecting clinical samples: collecting fresh tissues of colorectal cancer and corresponding paracarcinoma, and collecting samples according to the following standards: (1) the pathological examination confirms the diagnosis of colorectal cancer; (2) uncomplicated with other malignancies or diseases; (3) before operation, the patient does not receive any treatment such as radiotherapy, chemotherapy and the like; (4) washing residual blood on the surface of the postoperative tissue with precooled PBS, subpackaging on ice, and quickly freezing in liquid nitrogen for 30min in an ultra-low temperature refrigerator at-80 ℃.
Cell lines and cell culture: the human colorectal cancer cell line DLD-1 was purchased from Shanghai Life sciences research institute of Chinese academy of sciences. Colorectal cancer cell line DLD-1 was cultured in DMEM medium containing 10% fetal bovine serum, mixed with 100U/ml penicillin streptomycin and 5% CO at 37 ℃2Culturing the cells in the environment ofAnd (4) cells.
RNA extraction and real-time fluorescent quantitative PCR (qRT-PCR): extracting total RNA of the tissue by Trizol; total RNA of the cells is extracted by adopting an RNA extraction kit fast 2000. The reverse transcription Kit adopts PrimeScriptTM RT reagent Kit; the real-time fluorescent quantitative PCR kit adopts TB Green TM Premix Ex TaqTM II; performing PCR reaction by using a CFX96 real-time fluorescent quantitative PCR instrument; GAPDH is used as an internal reference; by using 2-ΔΔCtCalculating the relative expression quantity of RNA; the primer is synthesized by Jinan Boshang biotechnology, Inc.; the primer sequences are shown in Table 1.
TABLE 1 primer sequences for qRT-PCR
Transfection: specific small interfering rna (sirna) targeting LINC02474 reverse splice site and negative control (si-NC) were synthesized from the gimar gene (shanghai); 80umol of siRNA was transferred into colorectal cancer cells DLD-1 using Lipofectamine 3000; the siRNA sequences are shown in Table 2. the interference efficiency is shown in FIG. 4.
TABLE 2 siRNA sequences
The 24-well wells were placed in a 24-well plate. Cells were trypsinized, digestion was stopped in medium containing 10% fetal bovine serum, and cells were counted, 8000 cells/well were removed for the corresponding number of cells, centrifuged at 800rpm for 5 minutes at room temperature, the supernatant was discarded, and resuspended in serum-free medium. 8000 cells/200 ul were seeded in 24-well wells. The lower chamber was filled with medium containing 20% fetal bovine serum. 37 ℃ and 5% CO2After culturing for 24h, the supernatant and the lower chamber medium were discarded, the cells that did not transfer in the upper chamber were wiped off with a cotton swab, washed twice with PBS, and fixed with 4% paraformaldehyde for 1 h. After fixation, the fixation solution is discarded, PBS is used for washing twice, and the chamber is dyed for 2-4h by using Giemsa dye solution. The cell was washed twice with PBS, the cell well membrane was cut off and fixed with neutral gum. The pictures were taken using a Zeiss microscope. Counting the cells。
The 24-well cell pool was placed in a 24-well plate. Adding 60ul matrigel into the upper chamber, placing at 37 deg.C and 5% CO2 for 1-2h, and allowing the gel to solidify into jelly. Cells were trypsinized, digestion was stopped with medium containing 10% fetal bovine serum, and cell counting was performed, the corresponding number of cells was taken out at 10000 cells/well, centrifuged at 800rpm at room temperature for 5 minutes, the supernatant was discarded, and the cells were resuspended in serum-free medium. 10000 cells/200 ul were seeded into a 24-well cell pool. The lower chamber was filled with medium containing 20% fetal bovine serum. After culturing at 37 ℃ in an environment of 5% CO2 for 48h, the supernatant and the culture medium in the lower chamber were discarded, the uninfected cells in the upper chamber were wiped off with a cotton swab, washed twice with PBS, and fixed with 4% paraformaldehyde for 1 h. After fixation, the fixation solution is discarded, PBS is used for washing twice, and the chamber is dyed for 2-4h by using Giemsa dye solution. The cell was washed twice with PBS, the cell well membrane was cut off and fixed with neutral gum. The pictures were taken using a Zeiss microscope. The number of cells was counted.
Statistical analysis: the results were statistically analyzed using SPSS23.0 software. Plots were made using GraphPad prism7.0 software. Statistical analysis was performed using either the t-test or Wilcoxon signed rank sum test as appropriate. Data were from three independent experiments. Data are presented as means ± sd, all P values are two-sided, P <0.05 is considered statistically significant.
2. The experimental results are as follows:
as shown in fig. 1, LINC02474 is derived from chromosome 1, the location of which is shown in fig. 1.
As shown in FIG. 2, specific primers were designed based on the sequence of LINC02474, and the cDNA extracted from DLD-1 cells was amplified using the primers, and the results showed that the specific primers were able to amplify the product.
As shown in fig. 3A, qRT-PCR results showed that LINC02474 expression was significantly up-regulated in colorectal cancer tissues compared to normal tissues. As shown in fig. 3B, the area under the receiver operating characteristic curve (ROC) was 0.6118, which is better in diagnostic performance. Represents a p value < 0.05.
As shown in FIG. 4, the expression level of LINC02474 in DLD-1 cell line was significantly down-regulated after transfection with siRNA to LINC 02474.
As shown in FIG. 5, after transfection of siRNA of LINC02474, migration invasion ability of DLD-1 of colorectal cancer cell line was significantly inhibited.
The above results indicate that LINC02474 is up-regulated in colorectal cancer tissue; in vitro experiments show that LINC02474 can promote migration and invasion capacity of colorectal cancer cell DLD-1, and show that LINC02474 plays a cancer promotion role in colorectal cancer; LINC02474 is expected to become a new colorectal cancer diagnosis biomarker and a new colorectal cancer treatment target.
The above description is only a preferred embodiment of the present invention, and not intended to limit the present invention in other forms, and any person skilled in the art may apply the above modifications or changes to the equivalent embodiments with equivalent changes, without departing from the technical spirit of the present invention, and any simple modification, equivalent change and change made to the above embodiments according to the technical spirit of the present invention still belong to the protection scope of the technical spirit of the present invention.
SEQUENCE LISTING
<110> secondary Hospital of Shandong university
<120> application of LINC02474 as colorectal cancer diagnosis marker and treatment target
<130> 1
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 441
<212> DNA
<213> Artificial sequence
<400> 1
cctcctgctc tttgctccgt gagaaagatc cacctacgac ctcaggtcct cagaccgact 60
agcccaagaa acatctcacc aatttcaaat ctgacctttg caagagggtc cgagacattt 120
gcatcatcat tcagtgagac ctgtaaacac agcatctgcc tttgaccaca tccatctgga 180
agaacctgag agataatcca ttttatgaaa ttttccctac cctgaaatgg gagaatgaat 240
ctaatttgaa gcactgagaa ggataaggca tccatttgaa aaggactcct atattgcaac 300
atgaattctg ctaaaattga agcaagaaca aacatcaaat ttatgatgaa gtttgggtag 360
aagaacgatg aaattagtga cgctttataa aaagtttatt gggacaatac cccaaatgaa 420
tcagcagttt aaaaatggat a 441
<210> 2
<211> 41
<212> DNA
<213> Artificial sequence
<400> 2
tgcaagaggg tccgagacat atgtggtcaa aggcagatgc t 41
<210> 3
<211> 43
<212> DNA
<213> Artificial sequence
<400> 3
acccactcct ccacctttga ctgttgctgt agccaaattc gtt 43
<210> 4
<211> 84
<212> DNA
<213> Artificial sequence
<400> 4
ggguccgaga cauuugcaut taugcaaaug ucucggaccc ttgguagaag aacgaugaaa 60
uttauuucau cguucuucua cctt 84
Claims (8)
1. A long-chain non-coding RNA LINC02474 which is derived from human chromosome 1, and the deoxyribonucleotide sequence is shown in a sequence table SEQ ID NO: 1 is shown.
2. Use of the long non-coding RNA LINC02474 according to claim 1 as molecular biological marker for colorectal cancer.
3. Use of the long non-coding RNA LINC02474 according to claim 1 for the preparation of a diagnostic reagent or kit for colorectal cancer.
4. Application of substances for detecting high and low expression level of RNA LINC02474 in preparation of colorectal cancer diagnostic reagent or kit.
5. The primer for specifically amplifying RNA LINC02474 is characterized in that the primer sequence is,
forward TGCAAGAGGGTCCGAGACAT and reverse ATGTGGTCAAAGGCAGATGCT.
6. A diagnostic kit for colorectal cancer, comprising the following primers: forward TGCAAGAGGGTCCGAGACAT, reverse ATGTGGTCAAAGGCAGATGCT; internal reference primers: forward ACCCACTCCTCCACCTTTGAC and reverse TGTTGCTGTAGCCAAATTCGTT.
7. Use of a specific small interfering RNA for down-regulating the expression level of RNA LINC02474 in the preparation of a medicament for the treatment of colorectal cancer, characterized in that the sequence of said specific small interfering RNA is
si-LINC02474#1：
Forward GGGUCCGAGACAUUUGCAUTT, reverse AUGCAAAUGUCUCGGACCCTT; si-LINC02474# 2:
forward GGUAGAAGAACGAUGAAAUTT and reverse AUUUCAUCGUUCUUCUACCTT.
8. Use of a specific small interfering RNA for the down-regulation of the expression level of the RNA LINC02474 according to claim 7, characterized in that the subject is the DLD-1 cell line.
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