CN111411155A

CN111411155A - Application of lncRNA IGF L2-AS 1 AS colon cancer diagnosis marker

Info

Publication number: CN111411155A
Application number: CN202010205269.9A
Authority: CN
Inventors: 李小俊; 毕焕京; 闫飞; 李倩
Original assignee: Shaanxi Fan Chang Biotechnology Co ltd
Current assignee: Xi'an Aorui Jingchuang Biotechnology Co.,Ltd.
Priority date: 2020-03-20
Filing date: 2020-03-20
Publication date: 2020-07-14
Anticipated expiration: 2040-03-20
Also published as: CN111411155B

Abstract

The invention discloses an application of lncRNA IGF L-AS 1 AS a colon cancer diagnosis marker, belonging to the field of tumor molecular biology.A lncRNA IGF L2-AS 1 provided by the invention shows high-level expression in colon cancer and plays an important role in the processes of malignant transformation of colon cancer cells and formation of tumors.

Description

Application of lncRNA IGF L2-AS 1 AS colon cancer diagnosis marker

Technical Field

The invention belongs to the field of tumor molecular biology, and particularly relates to application of lncRNA IGF L2-AS 1 AS a colon cancer diagnosis marker.

Background

Worldwide, the incidence of colorectal cancer is third in the malignancy, and mortality is second, and is considered a major threat to public health. Due to poor eating patterns and living habits, the incidence and mortality of colon cancer presents a significantly rising trend and is one of the most common gastrointestinal malignancies. In China, about 17 ten thousand new cases of colon cancer occur each year, and the patients are younger in disease onset age, which causes great harm to the health of people in China. Colorectal carcinogenesis is a multistep biological process involving the dysregulation of multiple oncogenes and tumor suppressor genes. Despite the relatively deep understanding of the disease colorectal cancer, the continuous progress of diagnostic techniques and the increasing efficiency of therapeutic measures, the overall survival rate of colorectal cancer patients remains relatively low. At present, with the increasing incidence and poor prognosis of colorectal cancer, there is an urgent need to understand the specific molecular mechanisms involved in the pathogenesis of colorectal cancer more deeply, since this may facilitate the development of more effective therapeutic strategies, ultimately improving the prognosis of the patient.

To date, with improvements in whole genome and transcriptome sequencing technologies, it has been found that the whole genome of most mammals can be transcribed, but most of the transcripts have limited or no protein-encoding capacity at all, and these genes are referred to as ncrnas. IncRNAs are non-coding RNAs of length greater than 200 nucleotides that are transcribed by RNA polymerase II (RNAPII), and which have few or no Open Reading Frames (ORFs). A great deal of research results show that lncRNA plays an irreplaceable role in various basic biological processes such as embryonic development, epigenetic silencing, transcription and translation control, various biological behaviors of cells, tumorigenesis and the like. In addition, extensive research has provided strong evidence that lncRNA plays a functional role in a range of human diseases, including various types of cancer, such as breast, pancreatic, lung, gastric, and the like. And a plurality of lncRNA are also found to participate in carcinogenesis and cancer suppression processes in the process of generating and developing tumors, for example, lncRNA MVIH can be used AS an oncogene to promote the proliferation and invasion of non-small cell lung cancer cells, and on the contrary, lncRNA KCNK15-AS1 is proved to play a role in protecting the non-small cell lung cancer cells AS a cancer suppressor. Recent studies have shown that lncrnas also play a role as modulators in the development and progression of colorectal cancer, suggesting their potential as new therapeutic targets. Research on the molecular mechanisms and biological functions of lncRNA in the development and progression of colorectal cancer is still in the stage of inception, and although researchers have had limited success to date in elucidating specific molecular mechanisms, these limited studies are sufficient to demonstrate that lncRNA is a potential biological target for colorectal cancer diagnosis and treatment.

Therefore, the invention provides a breakthrough point for treating the colon cancer by detecting the expression and the function of lncRNA IGF L2-AS 1 in the colon cancer.

Disclosure of Invention

The invention aims to provide application of lncRNA IGF L2-AS 1 AS a colon cancer diagnosis marker.

The technical scheme adopted by the invention is as follows:

application of lncRNA IGF L2-AS 1 AS colon cancer diagnosis marker.

Furthermore, the nucleotide sequence of lncRNA IGF L2-AS 1 is shown in SEQ ID NO. 1.

Application of a reagent for detecting lncRNA IGF L2-AS 1 in preparation of a colon cancer diagnosis detection reagent or kit.

Further, the detection reagent or the kit is used for detecting the expression level of lncRNA IGF L2-AS 1 in the biological sample.

Further, the detection reagent or the kit comprises a PCR primer with detection specificity to lncRNA IGF L2-AS 1.

Further, PCR primers with detection specificity for lncRNA IGF L2-AS 1 were AS follows:

an upstream primer: 5'-TGCTGAGCAC AGACATGTGA-3', respectively;

a downstream primer: 5'-GTGCTGCAGA ATCAACGACC-3' are provided.

The sequences of lncRNA IGF L-AS 1 gene targets are shown in SEQ ID NO: 1, and the interfering RNAs comprise at least one of siRNA-1, siRNA-2 and siRNA-3.

Further, the sequence of siRNA-1 is: 5'-AAAAUUGGUGAGAAGUUCCUU-3' are provided.

Further, the sequence of siRNA-2 is: 5'-UAAAAUUGGUGAGAAGUUCCU-3' are provided.

Further, the sequence of siRNA-3 is: 5'-ACUUCUUCAUUCUUGUUGGCU-3' are provided.

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

artificially knocking down lncRNA IGF L2-AS 1 in colon cancer cells can obviously inhibit the proliferation, migration, invasion capacity and malignancy of SW620 cells, shows that lncRNA IGF L2-AS 1 plays an important role in the colon cancer morbidity process, and can be used AS a new molecular marker and a drug target for diagnosing and treating colon cancer.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 is a Real-time PCR assay of the expression of IncRNA IGF L2-AS 1 in colon cancer and paracarcinoma tissues;

FIG. 2 shows the expression of IncRNA IGF L2-AS 1 in SW620 cell line with knockdown of IncRNA IGF L2-AS 1;

FIG. 3 shows the CCK-8 proliferation of SW620 cell line knocking down IncRNA IGF L2-AS 1;

FIG. 4 shows the results of cell migration of SW620 cell line with knockdown of IncRNA IGF L2-AS 1;

FIG. 5 shows the results of cell invasion experiments on SW620 cell line with knockdown of IncRNA IGF L2-AS 1.

Detailed Description

The invention utilizes fluorescent quantitative PCR experiment to research that the expression quantity of lncRNA IGF L2-AS 1 in colon cancer is obviously higher than that of tissues beside cancer, extracts total RNA of the colon cancer and tissues beside cancer, carries out reverse transcription to synthesize cDNA, designs lncRNA IGF L2-AS 1PCR primer to carry out PCR amplification, and clones for the first time to obtain lncRNA IGF L2-AS 1 gene.

By designing siRNA, SW620 colon cancer cell line with low lncRNA IGF L2-AS 1 is obtained, and the influence of lncRNA IGF L2-AS 1 on the proliferation, migration, invasion and malignancy of SW620 cells is researched, so that the effect of lncRNA IGF L2-AS 1 in the colon cancer pathogenesis process is discussed, and the application of the lncRNA IGF L-AS 1 AS a new tumor marker in colon cancer clinical diagnosis, treatment and drug development is discussed.

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

In the following embodiments, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

Example 1 Real-time PCR detection of IncRNA IGF L2-AS 1

Extracting total RNA:

adding 1m L from colon cancer tissue sample and paracarcinoma tissue sample

Putting the Reagent at room temperature (15-30 ℃) to completely separate the nucleoprotein compound, adding 0.2m L chloroform after 5min, covering a tube cover, holding by a hand and violently oscillating for 15s, placing at room temperature for 2-3 min, centrifuging for 15min at 12000 g at 2-8 ℃, transferring the upper colorless aqueous phase to another 1.5m L Eppendorf tube, adding 0.5m L isopropanol, placing at room temperature for 10min, centrifuging for 10min at 12000 g at 2-8 ℃, discarding the supernatant, washing the RNA precipitate with 1m L75% ethanol, mixing by a vortex mixer, centrifuging for 5min at 7500g at 2-8 ℃, discarding the supernatant, drying for 5-10 min by air, and then using ddH without RNase₂And dissolving the RNA by using O, repeatedly blowing and sucking by using a microsyringe, and incubating for 10min at 55-60 ℃ to promote the RNA to be dissolved. Measuring RNA concentration at 260nm, and maintaining the extracted RNA at-70 deg.CAnd (4) storing.

Designing a primer:

primers were designed by the Primer design tool of NCBI (Primer B L AST) based on the transcript sequence of lncRNA IGF L2-AS 1.

An upstream primer: 5'-TGCTGAGCAC AGACATGTGA-3', respectively; as shown in SEQ ID NO: 2, respectively.

A downstream primer: 5'-GTGCTGCAGA ATCAACGACC-3', respectively; as shown in SEQ ID NO: 3, respectively.

Reverse transcription reaction:

taking the extracted total RNA (1 mu g) as a template, adding the following reaction system:

Buffer 4μL，

RT Enzyme Mix 1. mu. L, Oligo dT Primer (50. mu.M) 1. mu. L, Random6mers (100. mu.M) 1. mu. L, as RNase-free ddH₂O made up the reaction volume to 20. mu. L.

The mixture was incubated at 37 ℃ for 15min and 85 ℃ for 5s to obtain cDNA. The cDNA can be used for IncRNA Real-time PCR detection.

Real-time PCR detection:

according to Takara Japan

Optimum concentration of primers recommended by Premix Ex TaqTM (Tli RNaseH Plus) kit (10. mu.M), Real-time PCR specific primers for IncRNA IGF L2-AS 1 were dissolved in deionized water, and a reaction system was established in which SYBR Premix Ex TaqTM (2 ×) 25. mu. L Reference Dye (50 ×) 1. mu. L Forward Primer (10. mu.M) 1. mu. L Reverse Primer (10. mu.M) 1. mu. L4. mu. L, and ddH was sterilized₂O18 mu L, pre-denatured at 95 ℃ for 20s, denatured at 95 ℃ for 10s, annealed at 60 ℃ for 20s, and extended at 70 ℃ for 10s, and the Ct value obtained was calculated according to the method recommended by ABI PRISM 7300Sequence Detection System user Bulletin #2, Applied biosystems.

The results confirm that the expression of lncRNA IGF L2-AS 1 in colon cancer tissues is obviously higher than that in paracarcinoma tissues (FIG. 1), and suggest that lncRNA IGF L2-AS 1 may be involved in regulating the development process of colon cancer.

Example 2 knock-down of cancer cells lncRNA IGF L2-AS 1

The SW620 cell line was selected for the lncRNA IGF L2-AS 1 knock-down experiment, and the related gene expression and cell migration ability after knock-down were tested.

Knock-down of lncRNA IGF L2-AS 1:

the knock-down of lncRNA IGF L2-AS 1 is realized by using a siRNA method, which comprises the following steps of paving cells on a six-well plate, and starving for 30min by using Opti-MEM when the plate grows over 50% on the next day;

dissolving 2.5nmol siRNA/NC with 125 μ L DEPC water, wherein the amount of each well is 10 μ L, taking two tubes with the reference number of A, B, adding 100 μ L Opti-MEM into each tube, adding 10 μ L lipofectamine 3000 into tube A, adding siRNA or NC 10 μ L into tube B, respectively mixing, standing at room temperature for 5min, fully mixing two tubes AB, standing for 15min, adding into a six-well plate, and transfecting for 6-8 h.

The sequences of the siRNAs used in the research of the invention are as follows:

5’-AAGGAACTTCTCACCAATTTTAA-3’

siRNA-1: 5'-AAAAUUGGUGAGAAGUUCCUU-3', as shown in SEQ ID NO: 4 is shown in the specification;

5’-AGGAACTTCTCACCAATTTTAAA-3’

siRNA-2: 5'-UAAAAUUGGUGAGAAGUUCCU-3', as shown in SEQ ID NO: 5 is shown in the specification;

5’-AGCCAACAAGAATGAAGAAGTGG-3’

siRNA-3: 5'-ACUUCUUCAUUCUUGUUGGCU-3', as shown in SEQ ID NO: and 6.

FIG. 2 shows that the expression level of lncRNA IGF L2-AS 1 in SW620 cell line is reduced after the three siRNAs are transferred into the cell line, and the subsequent experiments are carried out by using siRNA-1 and siRNA-2.

Example 3: CCK-8 experiment

At 1 × 10⁵Cell number/well SW620 cells were seeded in 12-well plates at 37 ℃ with 5% CO₂Culturing, removing original culture medium when the cells grow to 70% confluence the next day, adding cells,after 24h pancreatin digestion, 3000 cells/well were plated in 96-well plates, 5 wells per group. Adding 10 mul CCK-8 into each well after culturing for 1-7 days, culturing for 1h, adjusting to zero with blank wells, measuring the OD value with a full-automatic enzyme standard reading instrument, wherein the wavelength is 450 nm.

As shown in FIG. 3, the cell into which siRNA was transferred had a decreased proliferative capacity as compared with the tumor cells that had not been knocked down.

Example 4: testing of the migratory Capacity of tumor cells

The tumor cells are subjected to a scratching experiment by the following specific method:

transfected cells grew 80-90% of the plate and two perpendicular scratches were made with a suitable size tip. Culturing in serum-free culture solution, and observing healing conditions of the scratch (migration of cells to the middle) after 0h and 48 h;

as a result, as shown in FIG. 4, the migration ability of the cells into which siRNA was transferred was decreased as compared with that of the tumor cells that were not knocked down.

Example 5: transwell invasion test

The matrigel and DMEM medium were mixed as 1: 8 preparing a matrigel-DMEM mixed solution, and placing on ice for later use. Transwell cells were placed in a 24-well plate with 500. mu.l DMEM medium in the lower chamber, 60. mu.l matrigel-DMEM mixture was added to each cell, and 5% CO was added at 37 ℃₂Culturing in a cell culture box for 5 h.

The cells were prepared in DMEM medium to 5 × 10⁵One cell suspension per ml, 100. mu.l of cell suspension per chamber, i.e.5 5 × 10 per well⁴Each cell was set with 3 wells. And (3) putting the 24-well plate into a cell culture box, taking out the chamber after 12h, dyeing and fixing the chamber, taking a picture under a microscope for counting, and observing the number of the cells entering the lower chamber to prove the invasion condition of the cells.

As shown in FIG. 5, the siRNA-transfected cells were less invasive than the non-knocked-down tumor cells.

The results show that the knocking-down of lncRNA IGF L2-AS 1 can obviously inhibit the proliferation, migration and invasion capabilities of colon cancer cells SW620, so that lncRNA IGF L2-AS 1 expressed at low level in colon cancer participates in a plurality of processes for regulating and controlling tumorigenesis, has the potential of promoting colon canceration, and provides a new molecular marker and a drug target for diagnosis and treatment of colon cancer.

While the foregoing description shows and describes several preferred embodiments of the invention, it is to be understood, as noted above, that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

The application of lncRNA IGF L2-AS 1 AS a colon cancer diagnosis marker.
2. The use of claim 1, wherein the incrna IGF L2-AS 1 has the nucleotide sequence shown in SEQ ID No. 1.
3. Application of a reagent for detecting lncRNA IGF L2-AS 1 in preparation of a colon cancer diagnosis detection reagent or kit.
4. The use of claim 3, wherein the detection reagent or kit is used for detecting the expression level of IncRNA IGF L2-AS 1 in a biological sample.
5. The use according to claim 4, wherein the detection reagent or kit comprises PCR primers with detection specificity for IncRNAIGF L2-AS 1.
6. The use of claim 5, wherein the PCR primers with detection specificity for IncRNA IGF L2-AS 1 are AS follows:

an upstream primer: 5'-TGCTGAGCAC AGACATGTGA-3', respectively;

a downstream primer: 5'-GTGCTGCAGA ATCAACGACC-3' are provided.
7. A group of interfering RNAs aiming at lncRNA IGF L2-AS 1 gene targets is characterized in that the sequences of the lncRNA IGF L2-AS 1 gene targets are shown in SEQ ID NO. 1, and the interfering RNAs comprise at least one of siRNA-1, siRNA-2 and siRNA-3.
8. The interfering RNA of claim 7, wherein the sequence of siRNA-1 is: 5'-AAAAUUGGUGAGAAGUUCCUU-3' are provided.
9. The interfering RNA of claim 7, wherein the sequence of siRNA-2 is: 5'-UAAAAUUGGUGAGAAGUUCCU-3' are provided.
10. The interfering RNA of claim 7, wherein the sequence of siRNA-3 is: 5'-ACUUCUUCAUUCUUGUUGGCU-3' are provided.