CN114702566A

CN114702566A - Small peptide coded by long-chain non-coding RNA LINC01234 and application thereof

Info

Publication number: CN114702566A
Application number: CN202210318574.8A
Authority: CN
Inventors: 余露山; 唐春媛; 周蓥; 孙文; 刘宇茜; 曾苏
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2022-03-29
Filing date: 2022-03-29
Publication date: 2022-07-05
Anticipated expiration: 2042-03-29
Also published as: CN114702566B

Abstract

The invention provides a small peptide coded by a long-chain non-coding RNA LINC01234 and application thereof. The LINC01234ORF region contained in the long non-coding RNA LINC01234 can encode and express a novel human small peptide MBOP, and the small peptide is highly expressed in colorectal cancer and cervical cancer and is mainly positioned in cytoplasm. The small peptide MBOP can regulate and control the cell migration and proliferation process of colorectal cancer, so the small peptide MBOP can be used as a novel molecular typing marker and a drug target for diagnosing and treating colorectal cancer and cervical cancer.

Description

Small peptide coded by long-chain non-coding RNA LINC01234 and application thereof

Technical Field

The invention belongs to the field of tumor molecular biology and the field of medicine, and particularly relates to a small peptide MBOP (MEK-binding on peptide) coded by a long-chain non-coding RNA LINC01234, a method for generating an antibody of the small peptide, and application of the small peptide.

Background

The development of high throughput sequencing technology has brought researchers to notice the existence of non-coding RNA (ncRNA) and the various regulatory roles these ncRNA play in cell migration, proliferation, development and immunity. In recent years, with the continuous update of technologies such as ribosome sequencing and mass spectrometry, researchers found that Open Reading Frames (ORFs) encoding small peptides existed in ncrnas that were originally thought to have no coding function. Long non-coding RNA (lncRNA) is ncRNA with the length of more than 200 nucleotides, and is a kind of functional RNA molecules which are identified to have strong potential for coding small peptides in recent years. Small peptides encoded by LncRNA have been reported to play important regulatory roles in the development of various neoplastic diseases such as colorectal Cancer, triple negative breast Cancer and liver Cancer (Cancer Lett.2021Jan 28; 497: 89-99.). Therefore, the invention combines the encoding capacity prediction database and in vivo and in vitro biological experiments to search lncRNA with ORF, and the small peptide coded by the ORF is characterized in expression level, subcellular localization and research on biological function in tumor cells, so as to provide a new marker (biomar) for clinical diagnosis of tumor diseases and a new drug target for treatment.

Disclosure of Invention

Aiming at the current situation that the molecular typing analysis of tumor diseases is not deep enough, one object of the invention is to provide a small peptide encoded by a long-chain non-coding RNA LINC 01234.

The nucleotide sequence of an open reading frame LINC01234ORF of the long-chain non-coding RNA LINC01234 is shown as SEQ ID No. 1, and the amino acid sequence of the small peptide MBOP generated by the coding region is shown as SEQ ID No. 2.

Based on the amino acid sequencing result of human colorectal cancer tissues, the amino acid sequence detected in the cancer tissues of colorectal cancer is shown as SEQ ID No. 3, and the amino acid sequence detected in the corresponding paracarcinoma tissues is shown as SEQ ID No. 4.

Based on the 68-82 amino acid (aa) fragment of SEQ ID No. 2 as an antigen, the antibody for detecting the MBOP content is generated through an immune reaction.

The overexpression plasmid pORF-FLAG of the nucleotide sequence LINC01234ORF of the small peptide is coded. The construction method of the plasmid pORF-FLAG for over-expressing the small peptide MBOP comprises the following steps: analyzing the restriction site distribution of the sequence SEQ ID No. 1 and the plasmid vector pcDNA3.1+, and screening out restriction enzymes BamH I and EcoR I; adding a FLAG label to the tail end of a target gene SEQ ID No. 1, designing a primer according to the restriction enzyme cutting site of endonuclease, and synthesizing a target fragment suitable for plasmid construction; and (3) carrying out enzyme digestion, purification, connection and transformation on the target fragment and the pcDNA3.1+ vector by using restriction enzyme to obtain an overexpression plasmid for overexpression of MBOP.

The invention also aims to provide application of the small peptide as a marker for diagnosing and treating colorectal cancer and cervical cancer.

Still another object of the present invention is to provide the use of said small peptide in the preparation of a medicament targeting MBOP against colorectal and cervical cancers.

The expression of the small peptide MBOP in the colorectal cancer and cervical cancer cell lines is higher than that in normal colon epithelial cells.

The expression of the small peptide MBOP in the colorectal cancer tissues is higher than that in paired paracancerous tissues.

The small peptide MBOP of the invention is positioned as cytoplasm in subcellular location in colorectal cancer cells.

The small peptide MBOP can promote the migration and proliferation functions of colorectal cancer cells HCT116 and HCT 15.

The invention has the following advantages:

the invention develops a new tumor marker and a drug target, in particular to a small peptide SEQ ID No. 2 coded by a nucleotide sequence of SEQ ID No. 1. The small peptide is highly expressed in colorectal cancer cell lines and hardly expressed in normal colon epithelium; the small peptide is highly expressed in cervical cancer cells; the expression of the small peptide in the colorectal cancer tissue is higher than that in the corresponding paracancerous normal tissue. The small peptide can promote the migration and proliferation capacity of the colorectal cancer cell lines HCT116 and HCT 15. The small peptide has a cancer promotion effect in colorectal cancer cells. These results indicate that the small peptide MBOP can be used as a marker for diagnosing and treating colorectal cancer and cervical cancer; the compound has the potential of being used as a drug target of colorectal cancer and cervical cancer and preparing MBOP-targeted drugs (such as compound inhibitors and proteolytic targeting chimeras).

Drawings

Figure 1 is a schematic representation of the localization of the small peptide MBOP on its template strand LINC 01234.

FIG. 2 is a diagram of the naturally occurring MBOP fragments in a pair of colorectal cancer tissues in an amino acid sequencing experiment. 3 segments can be detected in cancer tissues, and the coverage rate is higher and reaches 82.35%; 2 segments were detectable in the para-carcinoma tissue with a coverage of 61.18%.

FIG. 3 Western blot experiment proves the effectiveness of antibody anti-MBOP targeting small peptide MBOP.

FIG. 4 Western blot experiment proves that the small peptide MBOP is highly expressed in the colorectal cancer tissue.

FIG. 5 Western blot experiment demonstrating that the expression of the small peptide MBOP in the colorectal cancer cell lines HCT116, HCT15 and HT29 is higher than that in normal colon epithelial cell FHC; the small peptide MBOP is highly expressed in the cervical cancer cell Hela; the expression level of MBOP in the colorectal cancer cell lines and the cervical cancer cells was higher than that in the gastric cancer cell lines MKN45 and HGC27 and the kidney embryo cells 293T.

FIG. 6 Western blot experiment shows that the small peptide MBOP exists in colon part of female BALB/c Nude mouse, which indicates that the sequence of the small peptide MBOP has certain conservation.

Figure 7 subcellular localization of the small peptide MBOP is cytoplasmic. After nuclear isolation of HCT116 and HCT15 cells, (A) LINC01234ORF and (B) MBOP were found to be distributed mainly in the cytoplasm.

Figure 8 immunofluorescence experiments demonstrated that subcellular localization of the small peptide MBOP is cytoplasmic.

Figure 9 small peptide MBOP without membrane anchor sequence. Via database (a) transmurane hidden Markov model (TMHMM) server 2.0; (B) SignalP-5.0server and (C) ProtScale analysis revealed that the small peptide MBOP had no membrane anchor sequence.

FIG. 10 Transwell experiments demonstrated the ability of the small peptide MBOP to promote migration of the colorectal cancer cell lines HCT116 and HCT 15.

FIG. 11 scratch test demonstrates the ability of the small peptide MBOP to promote migration of the colorectal cancer cell lines HCT116 and HCT 15.

FIG. 12 cell cycle experiments demonstrate the ability of the small peptide MBOP to promote its proliferation by increasing the ratio of G2/M phases of the colorectal cancer cell lines HCT116 and HCT 15.

FIG. 13 clonogenic experiments demonstrate the ability of the small peptide MBOP to promote proliferation of the colorectal cancer cell lines HCT116 and HCT 15.

FIG. 14 stable over-expressed cell lines co-GFP, ORF-GFP and ORFm-GFP of HCT116 maintained over-expressed in nude mice. (A) The ORF-GFP group of cells produced the small peptide MBOP in vivo. (B) Both the cells of the group overexpressing small peptide ORF-GFP and the promoter mutated ORFm-GFP group produced significant overexpression of the nucleotide sequence LINC01234ORF in nude mice.

FIG. 15 xenograft tumor model experiments demonstrated the ability of the small peptide MBOP to promote proliferation of the colorectal cancer cell line HCT 116. The ORF-GFP group showed a marked increase in tumor proliferation (FIG. 15A, B). The body weights of three groups of nude mice all decreased, but there was no significant difference (fig. 15C). Immunohistochemistry confirmed that MBOP expression was higher in tumors in the ORF-GFP group than in the co-GFP and ORFm-GFP groups (FIG. 15D).

Detailed Description

The invention is further explained by the accompanying drawings and examples. The following examples are illustrative of the invention and are not intended to limit the invention in any way.

Example 1

The coding region LINC01234ORF is present in the human long non-coding RNA LINC01234 and can code a small peptide MBOP.

1. Method of producing a composite material

1.1 analysis of the potential of the Long non-coding RNA LINC01234 to encode Small peptides

lncRNA associated with tumor disease was analyzed using the online databases lncRNAdisease2.0(http:// www.rnanut.net/lncRNAdisease /) and NCBI (https:// www.ncbi.nlm.nih.gov /), and then the long-chain non-coding RNA LINC01234 was locked by the coding capacity prediction databases RegRNA2.0(http:// regrna2.mbc. nctu. edu. tw /), CPC (http:// CPC. cbi. pku. edu. cn /) and CPAT (http:// lilab. research.bcm. edu/CPAT/index. php). A high coding potential region exists in LINC01234 by utilizing POSTAR3(http:// pop tar. ncrnalab. org) and GWIPS. viz (https:// GWIPS. ucc. ie/index. html). The nucleotide sequence of an open reading frame LINC01234ORF of LINC01234 is shown in SEQ ID No. 1, and the amino acid sequence of a small peptide MBOP generated by the coding region is shown in SEQ ID No. 2.

1.2 Western blot

Unfreezing the subpackaged RIPA lysate (strong) on ice, and adding protease inhibitor PMSF (final concentration is 1mmol/L), Leupepptin (final concentration is 0.5 mu g/mL) and Pepstatin (final concentration is 0.7 mu g/mL) before use. After media was aspirated, cells were gently rinsed with 1mL of pre-cooled 1 XPBS buffer, and 1 XPBS buffer was aspirated. 1mL of 1 XPBS buffer was added, the cells were suspended with a cell scraper, transferred to a 1.5mL RNase-free EP tube with a pipette, centrifuged at 13400 g for 10min at 4 ℃ and the supernatant was aspirated. 50-100 mul of lysis solution was added to each well. And (3) blowing the cell suspension by using a pipettor for about 10 times, then moving the EP tube into a rotating disc of a chromatography cabinet at 4 ℃, and cracking for about 30-60 min. Then, the mixture was centrifuged at 13400 g for 10min at 4 ℃ to obtain a supernatant as a sample of total cellular protein, which was transferred to a new 1.5mL RNase-free EP tube. Sucking a part of protein sample, adding 1/4 volume of 5 Xprotein loading buffer solution, mixing, denaturing in 100 deg.C water bath for 10min, and performing electrophoresis or freezing at-80 deg.C.

Preparing 15% SDS-PAGE gel, sucking 10 μ L of the above denatured protein samples, adding into gel lane, performing constant voltage 80V electrophoresis for 25min, and performing constant voltage 130V electrophoresis for 70 min. The proteins were then transferred to 0.22. mu.M PVDF membrane at a constant current of 200mA for 45 min. After membrane transfer, blocking was performed at room temperature for 2h using 5% skim milk buffer. After washing the membrane with TBST buffer, the membrane was incubated overnight at 4 ℃ with primary antibody dilution. After washing the membrane with TBST buffer, the membrane was incubated with a secondary antibody dilution for 2h at room temperature. After washing the film with TBST buffer, exposure imaging was performed.

1.3 amino acid sequencing analysis of the expression of a small peptide MBOP in colorectal cancer tissues

Taking a pair of colorectal cancer tissues, and extracting, quantifying and denaturing tissue proteins. After separating the protein samples by 15% SDS-PAGE gel electrophoresis, the portion of the bromophenol blue front to 15kDa was excised from the colorectal cancer tissue and the para-cancer tissue, respectively. And respectively carrying out cracking and reduction, enzyme digestion and mass spectrometry on the two gel samples. The chromatographic system consisted of a trapping column (75 μm × 2cm, nanoscale, C18, 3 μm,

) And an analytical column (50 μm × 15cm, nanoscale, C18, 2 μm,

) And (4) forming. Data acquisition was performed by combining an FTMS (Fourier transform ion cyclotron resonance Mass Analyzer) analyzer equipped with a Nanospray Flex ionization source of Thermo LTQ-Orbitrap Velos Pro and an ion trap analyzer equipped with Thermo LTQ-Orbitrap Elite.

2. Results of the experiment

2.1 database predicted presence of a long non-coding RNA LINC01234 in the colorectal cancer cell line HCT116 of an open reading frame LINC01234ORF, which encodes the sequence of the small peptide MBOP (FIG. 1).

2.2 amino acid sequencing of human colorectal cancer tissue and alignment with the sequence of the small peptide MBOP found naturally, and the sequence coverage in cancer tissue was higher than that in paracancerous tissue (FIG. 2).

Example 2

High expression of small peptide MBOP coded by human long-chain non-coding RNA LINC01234 in colorectal cancer

1. Method of producing a composite material

1.1 antibody preparation

Epitope property prediction analysis is carried out on the full-length amino acid sequence of the small peptide MBOP, and the result of amino acid sequencing is utilized to determine 68-82aa (PSDHASVWGNEDQPR) of the MBOP sequence as the antigen sequence for generating the MBOP antibody.

Firstly, a 68-82aa sequence is synthesized, then a KLH label is coupled, and 3 experimental-grade white rabbits are immunized. After sacrifice of the big ear white rabbits, the antibody anti-MBOP was affinity purified.

1.2 construction of overexpression plasmids

Adding a FLAG tag at the end of the predicted SEQ ID No. 1 sequence, designing primers according to the digestion sequences of restriction enzymes BamH I and EcoR I, synthesizing a target fragment linearly connected with the SEQ ID No. 1 and the FLAG tag, carrying out water bath digestion on the synthesized target fragment and pcDNA3.1+ vector plasmid for 4h at 37 ℃ by using the restriction enzymes BamH I and the EcoR I, purifying, connecting with T4 ligase at 16 ℃ for 12h, and transforming to DH5 alpha competent cells to obtain pORF-FLAG plasmid. The promoter mutation operation is to mutate the first promoter ATG of SEQ ID No. 1 sequence to ATT, and the rest steps are the steps of the construction of the pORF-FLAG plasmid, so that the promoter mutant plasmid pORFmut-FLAG can be obtained.

1.3 plasmid transfection

The day before transfection, cells were seeded into 6-well plates to achieve a cell confluence of about 40% to 60%. The transfection system was prepared according to the ratio of plasmid/jetPrime Buffer/jetPrime ═ 2 ug/200. mu.L/5. mu.L, pcDNA3.1+, pORF-FLAG and pORFmut-FLAG plasmids were transfected into cells, and the cells were harvested after 48 h.

1.4Western blot

And (3) detecting the effectiveness of the antibody anti-MBOP of the targeting small peptide MBOP by using a Western blot experiment, and comparing the expression conditions of the small peptide MBOP in the colorectal cancer. And (3) detecting the expression of the small peptide MBOP in the sample by using an anti-MBOP antibody and a goat anti-rabbit secondary antibody.

2. Results

2.1 validation of the effectiveness of the antibody anti-MBOP targeting the small peptide MBOP. Cell protein samples were collected 48h after transfection of plasmids pcDNA3.1+, pORF-FLAG and pORFmut-FLAG in colorectal cancer cell lines HCT116 and HCT15 for validation of antibodies targeting small peptide MBOP. The anti-MBOP antibody is found to generate a band at the same position with the antibody targeting the tag protein FLAG, which indicates that the anti-MBOP antibody targeting the small peptide is effective (figure 3).

2.2 the small peptide MBOP is highly expressed in colorectal cancer tissues. A protein sample of 9 colorectal cancer tissues is extracted and subjected to protein content detection of a small peptide MBOP, and the expression of the MBOP in 6 cancer tissues is found to be higher than that of corresponding paracarcinoma tissues, which indicates that the MBOP is highly expressed in the colorectal cancer tissues (figure 4).

2.3 the small peptide MBOP is highly expressed in colorectal cancer cell lines. Taking colorectal cancer cell lines HCT116, HCT15, HT29, SW48, SW620 and RKO, a normal colon epithelial cell line FHC, a gastric cancer cell line MKN45 and HGC27, a cervical cancer cell line Hela and a kidney embryo cell line 293T to carry out content detection on the small peptide MBOP, and finding that the expression of the small peptide in the HCT116, the HCT15 and the HT29 is higher than that in the FHC; the expression of the small peptide MBOP in the colorectal cancer cell lines HCT116, HCT15, HT29, SW48, SW620 and RKO was higher than in the gastric cancer cell lines, cervical cancer cell lines and kidney germ cells (fig. 5).

2.4 in each organ of a female BALB/c Nude mouse, the expression of the small peptide MBOP exists only in the colon part, which indicates that the small peptide MBOP has certain sequence conservation and certain tissue specificity (figure 6).

Example 3

Subcellular localization of the small peptide MBOP encoded by the human long-chain non-coding RNA LINC01234 to the cytoplasm

1. Method of producing a composite material

1.1 plasmid and nucleus separation experiments

Paris separation kit according to nucleus^TMKit's instructions, Cell Fraction Buffer, Cell precipitation Buffer were placed on ice to pre-cool and proteins in the cytoplasm and nucleus were extracted according to the following procedure: each preparation was 1X 10⁶HCT116 and HCT15 cells, which were pelleted on ice. The Cell pellet was gently resuspended using 300-500. mu.L of ice Cell Fraction Buffer and allowed to stand on ice for 5-10 min. Centrifuging for 1-5 min at 4 ℃ under the condition of 500 g. Carefully pipette the supernatant into another RNase-free centrifuge tube using a 200. mu.L pipette, and the supernatant is the cytoplasmic fraction protein. And (3) washing the sediment part which is a Cell nucleus part once again by using ice Cell Fraction Buffer to remove cytoplasm of the Cell nucleus part, adding 300-500 mu L of ice Cell precipitation Buffer, and violently blowing and beating the Cell sediment to obtain the Cell nucleus protein sample. Protein samples were stored at-80 ℃.

1.2Western blot experiment

And detecting the subcellular localization condition of the small peptide MBOP by using a Western blot experiment. GAPDH is used as cytoplasmic internal reference protein, H3 is used as nuclear internal reference protein, and anti-MBOP antibody is used to detect the distribution of the small peptide MBOP in the natural state in the protein sample separated from the nucleus.

1.3 detection of mRNA expression level of Gene

1.3.1 Total RNA extraction from cells

According to the instructions of the AxyPrep total RNA preparation kit, the cell RNA is extracted according to the following steps: abandoning the culture medium, adding 300 mu L of lysate Buffer R-I into each hole, slowly rotating the cell plate to ensure that the lysate can uniformly cover all adherent cells, blowing and beating for about 10 times by using a pipettor, and completely transferring into a 1.5mL RNase-free centrifuge tube in the reagent kit. After sucking with a sterile syringe equipped with a No. 21-25 pillow about 10 times, 110. mu.L of Buffer R-II was added, vortexed for about 30s, and centrifuged at 12000 g at 4 ℃ for 5 min. In this procedure, 1.5mL RNase-free EP tube was prepared, 200. mu.L isopropanol was added, and the spin column in the kit was inserted into a 2mL receiver tube. Sucking the centrifuged supernatant into an EP tube containing 200. mu.L of isopropanol, blowing, mixing uniformly, transferring all the mixture to a centrifugal column, and centrifuging at 6000 g for 1min at 4 ℃. The column was removed and the liquid in the receiving tube was discarded, and the column was replaced in the receiving tube. Next, the spin column was rinsed with 500. mu.L of Buffer W1A, 700. mu.L of Buffer W2, and 700. mu.L of Buffer W2 in this order, and centrifuged at 12000 g for 1min each at 4 ℃. After the final rinse, the spin column was placed in a receiving tube and centrifuged at 12000 g for 1min at 4 ℃ to remove the rinse sufficiently. And transferring the centrifugal column into a 1.5mL RNase-free centrifugal tube in a reagent box, opening a cover in a fume hood, placing for about 2min, adding 50-100 mu L Buffer TE according to the amount of the cells, standing for 1min at room temperature, centrifuging for 1min at 12000 g at 4 ℃ to obtain a total RNA sample of the cells, and storing at-80 ℃.

1.3.2 tissue Total RNA extraction

Before the experiment, scissors, tweezers and steel balls are wrapped by tinfoil paper, are placed in a 120 ℃ oven for high-temperature baking for 4 hours, and are naturally cooled. According to the Tiangen RNAsim Total RNA kit Total RNA was extracted from the tissue samples. A tissue sample with a size of half a granule is cut and placed in a 2mL centrifuge tube of RNase-free, and a steel ball is added, and then 1mL of lysis solution RZ is added. The centrifuge tube was added to a pre-cooled tissue grinder cannula base and tissue grinding was performed at 60Hz for 180 s. After the sample tube was left at room temperature for 5min, it was centrifuged at 13400 g for 5min at 4 ℃ and the supernatant was pipetted into a new 1.5mL RNase-free centrifuge. Then 200. mu.L of chloroform was added, vigorously shaken at room temperature for 20s, and allowed to stand for 3 min. After centrifugation at 13400 g for 10min at 4 ℃ the sample was divided into three layers, the RNA sample was mainly in the first colorless aqueous phase, this layer was carefully pipetted (typically 400. mu.L) into a new 1.5mL RNase-free centrifuge, 200. mu.L of absolute ethanol was added, after pipetting the whole system into an adsorption column CR3, and centrifugation at 13400 g for 0.5min at 4 ℃ to discard the waste. After rinsing once with 500. mu.L deproteinized solution RD, it was centrifuged at 13400 g for 0.5min at 4 ℃. Then, 500. mu.L of the rinsing solution RW was added thereto, and the mixture was allowed to stand at room temperature for 2min and centrifuged at 13400 g at 4 ℃ for 0.5 min. After repeating the RW rinse step, another 4 ℃ centrifuge at 13400 g was performed for 2min in an effort to remove residual rinse. The column was transferred to a new 1.5mL EP tube and placed in a fume hood for about 2 min. Finally, the appropriate amount of RNase-free ddH is added according to the amount of the tissue₂And O, standing at room temperature for 2min, and centrifuging at 13400 g for 2min at 4 ℃ to obtain a final tissue RNA sample. RNA samples were stored at-80 ℃.

1.3.3 reverse transcription

Prior to reverse transcription, detection and calculation of the concentration of the RNA sample is required. When the Nanodrop 2000 is used for concentration measurement in an RNA mode, the values of A260/A280 are distributed in the range of 2.00-2.08, and the values of A260/A230 are all larger than 1.5, which indicates that the RNA sample has high quality and is free from DNA, protein and reagent pollution.

Reverse transcription system was prepared on ice according to the reverse transcription reagent instructions, a volume corresponding to 500ng RNA sample was pipetted into a 200. mu.L EP tube and RNase-free ddH was used₂Adding O to make up to 8 μ L, and adding 2 μ L

II qRT SuperMix^aAnd the mixture is blown and beaten uniformly by a liquid shifter and centrifuged. The EP tube was transferred to a 50 ℃ water bath and after 15min of reaction, it was transferred to 85 ℃ and after 5s of reaction immediately transferred to ice. Absorb 40. mu.L of RNase-free ddH₂O was added to the EP tube so that the final concentration became 10 ng/. mu.L. The cDNA samples can be stored at-20 ℃.

1.3.4 RT-qPCR

The following reaction system was prepared on ice:

the PCR amplification procedure was as follows:

data processing:

gene quantification was calculated using Microsoft Excel: GAPDH, U6 was selected as the reference gene, the expression level of the control group was 1, and the relative expression level of the objective gene in the experimental group was 2^-△△Ct。

1.4 construction of Stable transformants

According to the construction method of the overexpression plasmid, the target fragment is inserted into pCDH-CMV-MCS-EF1-copGFP-T2A-Puro plasmid with green fluorescent protein to respectively obtain ORF-GFP plasmid for overexpression small peptide and ORFm-GFP plasmid for overexpression promoter mutation sequence, and the empty vector is marked as co-GFP.

Lentiviral packaging was performed using the 293T cell line. 293T cells from 3 cell culture dishes of 6cm should be plated one day in advance to achieve approximately 70% -80% of the degree of cell fusion for the next day.

The packaging system is as follows:

the preparation process comprises the following steps: several sterilized EP tubes were prepared, 3 of them were each added 400. mu.L of Opti-MEM blank medium and 30. mu.LL Lipofectamine 2000, and mixing uniformly; then, 3 EP tubes were added to 400. mu.L each of the Opti-MEM blank medium and two packaging vector plasmids, and 5. mu.g of co-GFP, ORF-GFP and ORFm-GFP plasmids, and mixed well. After standing for 5min, Lipofectamine 2000 diluted with Opti-MEM was added one-to-one to the plasmid diluted with Opti-MEM, mixed well and allowed to stand for 20 min. Taking out 293T cells from the advanced plating, removing the culture medium, adding 3mL of blank culture medium, gently adding the transfection system for packaging lentivirus into the 293T cells, placing at 37 ℃ and 5% CO₂Culturing for 6h in a cell culture box, taking out cells, replacing the culture medium with a complete culture medium, and then returning to the cell culture box for culturing for 42 h.

Cells that required the construction of stably overexpressing cell lines were seeded into 6-well plates 24h prior to transfection with virus fluid to achieve approximately 20% confluence the following day. The 293T cells transfected with lentivirus for 48h were removed, the medium was aspirated with a 5mL syringe (without needle), the virus fluid was filtered through a disposable filter (0.45 μm), and an amount of polybrene (final concentration 8 μ g/mL) was added. The media in the 6-well plates was discarded and 1mL of virus solution containing co-GFP, ORF-GFP and ORFm-GFP plasmids was added to each of the 3 wells of the plates. After 24h, the cell status was observed under a microscope and 1mL of complete medium was added. After another 24h, the whole medium was aspirated and replaced with complete medium, at which time green fluorescence in the cells should be observed under a microscope. In the later stage, the cells are seeded into a 96-well plate by a stepwise dilution method, and then the cells are cultured in a culture medium containing puromycin with a certain concentration, so that a monoclonal cell line is selected.

1.5 immunofluorescence

One drop of culture medium was added dropwise to each of 3 wells of the 12-well plate, and the cell slide was held with tweezers, and the cell slide was carefully placed on the culture medium drop in the 12-well plate, so that it was adsorbed and fixed. The 12-well plate was returned to the cell incubator and incubated for 15 min. The stably over-expressed cell lines co-GFP, ORF-GFP and ORFm-GFP were digested, counted and diluted. The 12-well plate was removed, and 2 drops of the cell suspension were aspirated with a 200. mu.L pipette tip and dropped into the center of the cell slide. And (3) putting the 12-hole plate back to the cell incubator, incubating for 6-8 h, attaching the cells to a cell slide, adding 1mL of complete culture medium, and putting the cell slide back to the cell incubator to incubate overnight.

The next day 12 well plates were removed, the whole medium was aspirated off, and the cell slide was gently rinsed 3 times with 1 × PBS. Then, 500. mu.L of 4% fixed formaldehyde was added to each well in the dark, and the wells were incubated for 1 hour in the dark. After gently rinsing the cell slide with 1 XPBS for 3 times, 500. mu.L of PBS containing 0.5% (v/v) Triton X-100 was added to each well and the cell slide was gently rinsed with 1 XPBS for 3 times after incubating at room temperature for 20 min. PBS blocking solution containing 10% (v/v) goat serum is prepared, added to the cell slide, blocked for 1h at room temperature, and the cell slide is rinsed gently with 1 × PBS for 3 times. anti-MBOP primary anti-dilution solution is prepared according to the set dilution ratio and added on a cell slide for overnight at 4 ℃. The next day, the whole operation needs to be protected from light. The 1 XPBS solution was pre-warmed, primary dilutions were recovered and cells were gently rinsed 3 times with pre-warmed 1 XPBS solution. Fluorescent secondary antibody was added to the PBS blocking solution in proportion and applied to a cell slide, and after incubation at room temperature for 1h, the cells were gently rinsed 3 times with pre-warmed 1 XPBS solution. After staining the nuclei with hochests for 20min, the cells were gently rinsed 3 times with pre-warmed 1 x PBS solution. Finally, a drop of the anti-fluorescence quencher was dropped onto the slide, the cell slide was picked up with the syringe needle, the forceps were carefully lifted, and the lower edge was placed against a piece of absorbent paper to remove excess liquid, and the cell slide was covered with the quencher with the cell side down, taking care to avoid air bubbles. And then 3-4 drops of transparent nail polish are dripped to the edge position sealing piece of the cell climbing piece. Preferably, the fluorescence photography is performed within half a month after the preparation is completed.

1.6 analysis of the Membrane localization potential of the Small peptide MBOP

The cell membrane anchoring potential of MBOP was analyzed using a transmurane hidden Markov model (TMHMM) server 2.0, SignalP-5.0server and ProtScale.

2. Results

2.1 RT-qPCR and Western blot experiments demonstrated that the nucleotide sequence LINC01234ORF and the small peptide MBOP are mainly distributed in the cytoplasm. The experimental result shows that the cytoplasm and nucleus parts of HCT116 and HCT15 cells are well separated by the plasmid-nucleus separation experiment, the content of the nucleotide sequence LINC01234ORF and the small peptide MBOP in the cytoplasm is obviously higher, and the small peptide MBOP is mainly distributed in the cytoplasm (figure 7).

2.2 immunofluorescence experiments demonstrated that the small peptide MBOP is distributed predominantly in the cytoplasm (FIG. 8).

2.3 database prediction analysis of the small peptide MBOP membrane-free anchor sequence (FIG. 9), the small peptide MBOP is therefore predominantly localized in the cytoplasm.

Example 4

Small peptide MBOP (small peptide) encoded by human long-chain non-coding RNA LINC01234 for promoting migration and proliferation of colorectal cancer cells

1. Method of producing a composite material

1.1Transwell experiment

Complete medium of 15% fetal bovine serum (v/v) was prepared, and this medium was added to the lower chamber of the transwell plate in an amount of 600. mu.L per well, the upper chamber of the transwell was put into the well to which the medium was added, air bubbles were removed, and it was left to stand in a cell incubator for about 20 min. The transfected cells were then digested for 48h, counted and resuspended in serum-free medium. Cells were counted under a microscope using a hemocytometer according to 2X 10 for each transwell chamber⁴The density of each cell was calculated to obtain the required cell suspension. One sterile EP tube of 1.5mL each was pipetted to aspirate 2 times the required amount of cell suspension and replenished to 400 μ L with serum-free medium and pipetted evenly. The transwell plates were removed and 200. mu.L of each cell suspension was pipetted and carefully added to the upper chamber of the transwell, after carefully shaking up, the plates were returned to the cell incubator and the incubation continued. After 48h incubation, the transwell plates were removed, the lower chamber medium was aspirated off, 600 μ L of 4% fixed formaldehyde solution was added, and the plates were fixed in the dark at room temperature for 30 min. Then the fixed formaldehyde solution is sucked away and replaced by PBS solution, and the solution is rinsed once. The PBS solution was aspirated off, 600 μ L of crystal violet solution was added, after incubation for 20min at room temperature, the crystal violet solution was recovered and the transwell was rinsed multiple times with PBS solution until there was no excess crystal violet solution. The non-migrated cells on the top surface of the upper chamber of the transwell were wiped off with a small cotton swab and allowed to air dry. The transwell cell was observed under a microscope and recorded by taking a photograph in the bright field.

1.2 scratch test

Transfected cells were digested for 48h, counted and resuspended in serum-free medium. Cells were counted under microscope using a hemocytometerCounting, calculating 3 × 10 of each well⁵The volume of cell suspension required for each cell. A new 6-hole plate is taken, parallel straight lines are drawn on a bottom plate of the plate by paint pens, and generally, three parallel lines can be drawn on one hole. Adding the calculated cell suspension into the line-drawn 6-well plate, supplementing liquid to 2mL of each well, fully shaking the cell suspension in the 6-well plate, and then putting the cell suspension back to the cell culture box for culture. The next day, cells were observed under a microscope when the degree of cell fusion reached about 95%, and a 200 μ L pipette tip was used to draw a straight line in the 6-well plate, which should be perpendicular to the parallel line of the paint stroke of the previous day. And abandoning the culture medium, rinsing the cells by using PBS (phosphate buffer solution) to wash off the scraped cells in the line drawing process, changing the cells into the culture medium containing 1 percent fetal calf serum (v/v), immediately taking the cells under a microscope to take a picture in a bright field, and taking pictures in the bright field after 24 hours and 48 hours.

1.3 cell cycle experiments

The transfected cells for 48h were centrifuged at 1000 rpm for 5min at 4 ℃. The supernatant was carefully pipetted off and approximately 50. mu.L of medium could be retained to avoid aspiration of the cell pellet during the pipetting process. The cell pellet was resuspended in 1mL PBS and centrifuged at 1000 rpm at 4 ℃ for 5 min. The supernatant was carefully pipetted off, and about 50. mu.L of PBS could be retained. 1mL of 70% ethanol solution was added, the cells were gently resuspended, and the sample tube was inserted on a rotating disk in a 4 ℃ chromatography cabinet, fixed for at least 12 hours, and then centrifuged at 1000 rpm at 4 ℃ for 10 min. The supernatant was aspirated off, and approximately 50. mu.L of ethanol was retained to avoid aspiration of the cells. The cell pellet was resuspended in 1mL PBS and centrifuged at 1000 rpm for 5min at 4 ℃. In the centrifugation process, a propidium iodide staining working solution is prepared according to the number of samples required by the experiment, and the staining working solution required by each sample to be tested comprises 500. mu.L staining buffer solution, 25. mu.L propidium iodide staining solution (20X) and 10. mu.L RNase A (50X). After the supernatant of the above-mentioned centrifuged sample was aspirated, 450. mu.L of propidium iodide staining working solution was transferred by a pipette, gently resuspended, and the cell sample was stored in a 37 ℃ water bath in the dark.

Before loading, a 40-mesh screen is needed to process the cell suspension, the cell liquid is added into a flow-type loading tube, and a flow cytometer is arranged to record the percentage of G1 phase, S phase and G2/M phase of cells in pcDNA3.1+, pORF-FLAG and pORFmut-FLAG groups respectively.

1.4 clone formation experiments

The stably over-expressed cell lines co-GFP, ORF-GFP and ORFm-GFP were digested and the cells were resuspended in complete medium containing 15% fetal bovine serum (v/v), counted in a hemocytometer and diluted to a cell concentration of 1000 cells/2 mL by stepwise dilution. Cells of the stably overexpressed cell line were seeded into 6-well plates at 1000 cells per well, the cells were thoroughly shaken and placed in a cell culture incubator for further culture, and the medium used was complete medium containing 15% fetal bovine serum (v/v). After 10-14 days, the 6-well plate was removed, the medium in the well was aspirated away, and the cell plate was rinsed with 1mL PBS. Then 600. mu.L of 4% fixed formaldehyde solution was added and fixed in the dark at room temperature for 30 min. The fixed formaldehyde solution was aspirated away, 600 μ L of crystal violet solution was added, and after incubation at room temperature for 20min, the crystal violet solution was recovered and the cell plates were rinsed with PBS solution. After the plate is naturally dried, the recorded data is shot by a microscope camera.

1.5 xenograft tumor model experiment

1.5.1 first graft

Three stable over-expressed cells, co-GFP, ORF-GFP and ORFm-GFP, of HCT116 cells were amplified, digested and the cells collected. The cell pellet was washed once more with PBS, and after centrifugation, the cells were resuspended in a small amount of ice PBS. After aspirating about 20. mu.L of cell suspension and diluting appropriately, the cell suspension was counted under a microscope using a hemocytometer, and the cell suspension concentration was adjusted to 8X 10 with ice PBS⁶Cells/150. mu.L. mu.L of the cell suspension was inoculated into the subcutaneous site under the axilla of 4-week female BALB/cNude nude mice using a 1-mL medical syringe, and after about 2 weeks, the nude mice developed visible tumor masses subcutaneously. After about another 1 month, the tumor mass in the nude mice grew large enough to provide enough tumor mass for secondary tumor inoculation.

1.5.2 Secondary Functions

Before the experiment, scissors, tweezers and steel balls are wrapped by tinfoil paper and then placed in a 120 ℃ oven for high-temperature baking for 4 hours, and then the mixture is naturally cooled, so that ice physiological saline and a cell cuvette are prepared. After the nude mice are sacrificed, the tumor is stripped and the tumor mass is removedThe surrounding tissue is cut off, and the bean curd residue-like tissue inside the tumor mass is also cut off. A small piece was taken out of the middle part, RNA and protein were extracted, and expression level detection of LINC01234ORF and MBOP was performed. Shearing most of the remaining tissue to about 1mm³The tissue pieces, in volume, were placed in ice saline for future use. A small opening is cut on the ventral side of a 4-week female BALB/c Nude mouse by a pair of scissors, a tissue block is pushed into the small opening, and a straight forceps is used for pushing the tissue block to the armpit position from the ventral side so as to prevent the tissue block from falling out of the small opening in the movement process of the Nude mouse. Several days after the tissue mass was tumorigenic, the state of the nude mice and the state of the tissue mass in vivo were closely followed.

1.5.3 collecting data

On day 9 after the second inoculation, the body weight of the nude mice, as well as the size of the nude mice tumor, began to be recorded for the first time. Tumor volume of nude mice was determined as V1/2 × a × b²Where a is the length of the tumor and b is the width of the tumor. Continuously recording to the eighth time according to the frequency of once every three days, wherein the tumor in nude mice does not exceed 2000 mm³In case of volume, nude mice were sacrificed humanely. The tumor is stripped and the tissue of the surface and core of the tumor is removed. The remaining tumor parenchyma part is subjected to RNA extraction, protein extraction, immunohistochemical pretreatment and cryopreservation at the temperature of minus 80 ℃.

2. Results

2.1 through transwell experiments, the small peptide MBOP is proved to have the function of promoting migration. The principle of the Transwell experiment is to use the difference in serum concentration in the culture medium to induce cell migration from the top surface of the Transwell chamber to the bottom surface of the Transwell chamber. The experimental result shows that the number of the cells migrated in the small peptide MBOP overexpression group is higher than that in the pcDNA3.1+ group transfected with the empty vector and the promoter mutant plasmid pORFmut-FLAG group, which indicates that the small peptide MBOP has the function of promoting the cell migration (FIG. 10).

2.2 the scratch test proves that the small peptide MBOP has the function of promoting migration. The principle of the scratch experiment is to artificially make space intervals between adherent cells and induce the cells to migrate from a dense place to a cell-free place. The experimental result shows that the number of the cells migrated in the small peptide MBOP overexpression group is higher than that in the pcDNA3.1+ group transfected with the empty vector and the promoter mutant plasmid pORFmut-FLAG group, which indicates that the small peptide MBOP has the function of promoting the cell migration (FIG. 11).

2.3 through cell cycle experiments, the small peptide MBOP has the function of promoting proliferation (FIG. 12). The amount of the ratio of the cell cycle G2/M phase is considered as an index of the activity degree of cell proliferation, and the ratio of the G2/M phase of the cell in the small peptide MBOP overexpression group is higher than that in the pcDNA3.1+ group transfected with an empty vector and that in the promoter mutant plasmid pORFmut-FLAG group, which indicates that the small peptide MBOP has the function of promoting cell proliferation (FIG. 12).

2.4 through clone formation experiments, the small peptide MBOP has the function of promoting proliferation. The ORF-GFP group stably overexpressing the small peptide MBOP formed a higher number of cell clones than the co-GFP of the control group and the ORFm-GFP of the promoter mutant group, and thus the small peptide MBOP had a function of promoting cell proliferation (FIG. 13).

2.5 the small peptide MBOP has the function of promoting proliferation proved by a xenograft tumor model experiment. The LINC01234ORF and MBOP in the tumor mass were examined after the first tumor inoculation, demonstrating that the stably overexpressing cell line was able to maintain significant overexpression in vivo (fig. 14). After secondary tumor inoculation is carried out on the tumor mass after verification, the tumor mass volume proliferation of the small peptide MBOP overexpression group is found to be remarkably higher than that of the control group and the promoter mutation group, and the small peptide MBOP is proved to have the function of promoting proliferation (figure 15).

Sequence listing

<110> Zhejiang university

<120> small peptide coded by long-chain non-coding RNA LINC01234 and application

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 258

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 1

atgaaagaga gagggagaga aaggaaggaa gagaaggagg gagaatggaa ggatgtatgg 60

atttggatgt atgggttcca ttccttctac cctggcaaaa gcttactcat ccttcagtgt 120

ccatccaaaa tggcatctct tcttgggcac catctccaga gtctcctgca agcaggtagc 180

tacatcccac aaaacaacca cccatctgac catgcaagtg tgtgggggaa tgaagaccag 240

cccaggaatc tgagatga 258

<210> 2

<211> 85

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 2

Met Lys Glu Arg Gly Arg Glu Arg Lys Glu Glu Lys Glu Gly Glu Trp

1 5 10 15

Lys Asp Val Trp Ile Trp Met Tyr Gly Phe His Ser Phe Tyr Pro Gly

20 25 30

Lys Ser Leu Leu Ile Leu Gln Cys Pro Ser Lys Met Ala Ser Leu Leu

35 40 45

Gly His His Leu Gln Ser Leu Leu Gln Ala Gly Ser Tyr Ile Pro Gln

50 55 60

Asn Asn His Pro Ser Asp His Ala Ser Val Trp Gly Asn Glu Asp Gln

65 70 75 80

Pro Arg Asn Leu Arg

85

<210> 3

<211> 70

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 3

Glu Gly Glu Trp Lys Asp Val Trp Ile Trp Met Tyr Gly Phe His Ser

1 5 10 15

Phe Tyr Pro Gly Lys Ser Leu Leu Ile Leu Gln Cys Pro Ser Lys Met

20 25 30

Ala Ser Leu Leu Gly His His Leu Gln Ser Leu Leu Gln Ala Gly Ser

35 40 45

Tyr Ile Pro Gln Asn Asn His Pro Ser Asp His Ala Ser Val Trp Gly

50 55 60

Asn Glu Asp Gln Pro Arg

65 70

<210> 4

<211> 52

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 4

Ser Leu Leu Ile Leu Gln Cys Pro Ser Lys Met Ala Ser Leu Leu Gly

1 5 10 15

His His Leu Gln Ser Leu Leu Gln Ala Gly Ser Tyr Ile Pro Gln Asn

20 25 30

Asn His Pro Ser Asp His Ala Ser Val Trp Gly Asn Glu Asp Gln Pro

35 40 45

Arg Asn Leu Arg

50

Claims

1. A small protein peptide encoded by the long non-coding RNA LINC01234, characterized in that: the nucleotide sequence of the protein small peptide is shown as SEQ ID No. 1 and is named as LINC01234 ORF; the amino acid sequence is shown as SEQ ID No. 2 and is named as MBOP.

2. Overexpression plasmid pORF-FLAG containing the nucleotide sequence LINC01234ORF of claim 1, encoding a small peptide.

3. The method for constructing over-expression plasmid pORF-FLAG according to claim 2, comprising the steps of: analyzing the restriction site distribution of the sequence SEQ ID No. 1 and the plasmid vector pcDNA3.1+, and screening out restriction enzymes BamH I and EcoR I; adding a FLAG label to the tail end of a target gene SEQ ID No. 1, designing a primer according to the restriction enzyme cutting site of endonuclease, and synthesizing a target fragment suitable for plasmid construction; and (3) carrying out enzyme digestion, purification, connection and transformation on the target fragment and the pcDNA3.1+ vector by using restriction enzyme to obtain an overexpression plasmid for overexpression of MBOP.

4. The use of the small peptide according to claim 1 as a marker for the diagnosis and treatment of colorectal cancer and cervical cancer.

5. The use of the small peptide of claim 1 as a tumor marker in the preparation of MBOP-targeted drugs against colorectal cancer and cervical cancer.