CN114702566B

CN114702566B - Small peptide encoded by long-chain non-coding RNA LINC01234 and application thereof

Info

Publication number: CN114702566B
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: 2023-08-01
Anticipated expiration: 2042-03-29
Also published as: CN114702566A

Abstract

The invention provides a small peptide coded by long-chain non-coding RNA LINC01234 and application thereof. The region of the LINC01234ORF contained in long-chain non-coding RNA LINC01234 is capable of encoding and expressing a novel human small peptide MBOP, which is highly expressed in colorectal and cervical cancers and is predominantly localized in the cytoplasm. The small peptide MBOP can regulate the migration and proliferation process of colorectal cancer cells, so that the small peptide MBOP can be used as a novel molecular parting marker and a drug target for diagnosing and treating colorectal cancer and cervical cancer.

Description

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

Technical Field

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

Background

The continued development of high throughput sequencing technology has led researchers to notice the presence of non-coding RNAs (ncrnas), and the many modulations that these ncrnas exert in cell migration, proliferation, development, and immunity. In recent years, with the continuous updating of techniques such as ribosome sequencing and mass spectrometry sequencing, researchers have found that open reading frames (open reading frame, ORF) encoding small peptides exist in ncrnas that were originally thought to have no encoding function. Long non-coding RNAs (lncRNA) are ncrnas that are more than 200 nucleotides in length, and are a class of functional RNA molecules that have been identified in recent years as having a strong potential to encode small peptides. LncRNA encoded small peptides have been reported to play an important role in the development of a variety of 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 coding capacity prediction database and in-vivo and in-vitro biological experiments, searches for lncRNA with ORF, and performs characterization on expression level and subcellular localization of small peptide coded by the ORF and research on biological functions of the small peptide in tumor cells, so as to provide a new marker (biomarker) for diagnosis of clinical tumor diseases and provide 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 aim of the invention is to provide a small peptide encoded by long-chain non-coding RNA LINC01234.

The nucleotide sequence of the 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 a pair of human colorectal cancer tissues, the amino acid sequence detected in the colorectal cancer tissues is shown as SEQ ID No. 3, and the amino acid sequence detected in the corresponding paracancerous tissues is shown as SEQ ID No. 4.

Antibodies for detecting the MBOP content are produced by an immune reaction based on the 68-82 amino acid (aa) fragment of SEQ ID No. 2 as antigen.

The overexpression plasmid pORF-FLAG of the nucleotide sequence LINC01234ORF of the coding small peptide. The construction method of the plasmid pORF-FLAG for over-expressing the small peptide MBOP comprises the following steps: analyzing the distribution of enzyme cutting sites of the sequence SEQ ID No. 1 and the plasmid vector pcDNA3.1+, and screening restriction enzymes BamHI and EcoRI; adding a FLAG tag to the tail end of a target gene SEQ ID No. 1, designing a primer according to the 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 the over-expression plasmid for over-expressing the MBOP.

It is another object of the present invention to provide the use of said small peptides as markers for diagnosis and treatment of colorectal cancer and cervical cancer.

It is a further object of the invention to provide the use of said small peptides for the preparation of a medicament for the targeted MBOP against colorectal and cervical cancers.

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

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

The subcellular localization of the small peptide MBOP of the invention in colorectal cancer cells is cytoplasmic.

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

The invention has the following advantages:

the invention discloses a novel tumor marker and a drug target, in particular a small peptide SEQ ID No. 2 encoded by a nucleotide sequence of SEQ ID No. 1. The small peptide is highly expressed in colorectal cancer cell lines, but hardly expressed in normal colon epithelium; the small peptide is highly expressed in cervical cancer cells; the expression of the small peptide in colorectal cancer tissues is higher than that in corresponding paracancerous normal tissues. The small peptides are capable of promoting migration and proliferation of colorectal cancer cell lines HCT116 and HCT 15. The small peptide has a carcinomatous effect in colorectal cancer cells. These results indicate that the small peptide MBOP can be used as a marker for diagnosing colorectal cancer and cervical cancer; there is also the potential to be used as a drug target for colorectal and cervical cancer, for the preparation of MBOP-targeted drugs (e.g. compound inhibitors, proteolytically targeted chimeras).

Drawings

FIG. 1 is a schematic representation of the localization of small peptides MBOP on their template strand LINC01234.

FIG. 2 is a representation of a pair of naturally occurring MBOP fragments in colorectal cancer tissue in an amino acid sequencing experiment. 3 fragments can be detected in cancer tissues, so that the coverage rate is higher and reaches 82.35%; 2 fragments were detected in the paracancerous tissue with a coverage of 61.18%.

FIG. 3 Western blot experiments demonstrates the effectiveness of antibodies anti-MBOP targeting small peptides MBOP.

FIG. 4 shows that the small peptide MBOP is highly expressed in colorectal cancer tissue by Western blot experiments.

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

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

FIG. 7 subcellular localization of small peptide MBOP to the cytoplasm. After cytoplasmic nuclear separation of HCT116 and HCT15 cells, (a) LINC01234ORF and (B) MBOP were found to be predominantly distributed in the cytoplasm.

FIG. 8 immunofluorescence experiments demonstrate that subcellular localization of small peptide MBOP is cytoplasmic.

FIG. 9 small peptide MBOP has no membrane anchor sequence. Via database (a) transmembrane hidden Markov model (TMHMM) server 2.0; (B) SignalP-5.0server and (C) ProtScale analysis, small peptide MBOP was found to have no membrane anchor sequence.

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

Figure 11 scratch experiments demonstrate the ability of the small peptide MBOP to promote migration of colorectal cancer cell lines HCT116 and HCT 15.

FIG. 12 cell cycle experiments demonstrate the ability of the small peptide MBOP to promote proliferation of colorectal cancer cell lines HCT116 and HCT15 by increasing the ratio of G2/M phases.

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

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

Figure 15 xenograft tumor model experiments demonstrate the ability of the small peptide MBOP to promote proliferation of colorectal cancer cell line HCT 116. The ORF-GFP group showed a significant increase in tumor proliferation (FIGS. 15A, B). The body weights of all three groups of nude mice tended to decrease, but were not significantly different (fig. 15C). Immunohistochemical experiments demonstrated higher expression of MBOP in the tumors of the ORF-GFP group than in the co-GFP and ORFm-GFP groups (FIG. 15D).

Detailed Description

The invention is further described with reference to the drawings and examples. The following examples illustrate the invention, but do not limit it in any way.

Example 1

The coding region LINC01234ORF exists in human long-chain non-coding RNA LINC01234 and can code small peptide MBOP.

1. Method of

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

lncRNA associated with tumor disease was analyzed using the online databases LncRNAdata 2.0 (http:// www.rnanut.net/lncrnadisease /) and NCBI (https:// www.ncbi.nlm.nih.gov /), and then long-chain non-coding RNA LINC01234 was locked using the coding ability prediction database RegRNA2.0 (http:// regnna2. Mbc. Nctu. Edu /), CPC (http:// CPC. Cbi. Pku. Edu. Cn /) and CPAT (http:// lip. Research. Bcm. Edu/CPAT/index. Php) analyses. POSTAR3 (http:// POSTAR. Ncrnalab. Org) and GWIPS-viz (https:// GWIPS. Ucc. Ie/index. Html) are utilized to analyze to find that a region with high coding potential exists in LINC01234, the nucleotide sequence of the open reading frame LINC01234ORF of LINC01234 is shown as SEQ ID No. 1, and the amino acid sequence of a small peptide MBOP generated by the coding region is shown as SEQ ID No. 2.

1.2 Western blot

Thawing the split-charged RIPA lysate (strong) on ice, and adding protease inhibitor PMSF (final concentration 1 mmol/L), leupeptin (final concentration 0.5 μg/mL), pepstatin (final concentration 0.7 μg/mL) before use. After pipetting the medium, the cells were gently rinsed with 1mL of pre-chilled 1 XPBS buffer and the 1 XPBS buffer was pipetted. Then, 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 13 400g at 4℃for 10min, and the supernatant was aspirated. 50-100 mu L of lysate is added to each well. After the cell suspension was blown about 10 times with a pipette, the EP tube was transferred to a turntable of a chromatography cabinet at 4℃for about 30-60 min of lysis. And centrifuging at 4 ℃ for 10min at 13 g for 10min, sucking the supernatant to a new 1.5mL RNase-free EP tube, wherein part of the supernatant is the cell total protein sample. Absorbing a part of protein sample, adding 1/4 volume of 5 Xprotein loading buffer solution, mixing, denaturing in a water bath at 100deg.C for 10min, and performing electrophoresis or freezing at-80deg.C.

Preparing 15% SDS-PAGE gel, sucking 10 mu L of each denatured protein sample, adding into gel lanes, and continuing electrophoresis at constant pressure of 80V for 25min and constant pressure of 130V for 70min. The proteins were transferred to 0.22. Mu.M PVDF membrane with a constant current of 200mA for 45 min. After transfer, the membrane was blocked with 5% nonfat dry milk buffer for 2h at room temperature. After washing the membrane with TBST buffer, it was incubated overnight with primary anti-dilution at 4 ℃. After washing the membrane with TBST buffer, it was incubated with secondary antibody dilution for 2h at room temperature. After washing the membrane with TBST buffer, exposure imaging was performed.

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

And (3) taking a pair of colorectal cancer tissues, and extracting, quantifying and denaturing tissue proteins. After separation of the protein samples by 15% SDS-PAGE gel electrophoresis, the portions of bromophenol blue leading edge to 15kDa of colorectal cancer tissue and paracancestral tissue were excised, respectively. And respectively carrying out cleavage, reduction, enzyme digestion and mass spectrometry on the two glue samples. The chromatographic system consisted of a trapping column (75 μm x 2cm, nanoscale, C18,3 μm,) And analytical column (50 μm. Times.15 cm, nanoscale, C18,2 μm, & lt/EN & gt)>) Composition is prepared. Data acquisition was performed by a combination of 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 ellite.

2. Experimental results

2.1 Long non-coding RNA LINC01234 predicted by database there is an open reading frame LINC01234ORF in colorectal cancer cell line HCT116 that encodes the sequence of the small peptide MBOP (FIG. 1).

2.2 amino acid sequencing was performed on human colorectal cancer tissue using a pair and aligned with the sequence of small peptide MBOP, which was found to occur naturally and with higher sequence coverage in cancer tissue than in paracancerous tissue (fig. 2).

Example 2

Small peptide MBOP encoded by human long-chain non-coding RNA LINC01234 is highly expressed in colorectal cancer

1. Method of

1.1 antibody preparation

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

First 68-82aa sequence was synthesized, then KLH tag was conjugated, and then 3 experimental-grade white rabbits were immunized. After human tract killing of white rabbits, antibody anti-MBOP was affinity purified.

1.2 construction of the overexpression plasmid

Adding a FLAG tag at the tail end of the predicted SEQ ID No. 1 sequence, designing a primer according to the digestion sequences of restriction enzymes BamH I and EcoR I, synthesizing a target fragment of the linear ligation SEQ ID No. 1 and the FLAG tag, performing 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 EcoR I, purifying, performing water bath ligation for 12h at 16 ℃ by using T4 ligase, and transforming into DH5 alpha competent cells to obtain the pORF-FLAG plasmid. The promoter mutation operation is to mutate the first promoter ATG of SEQ ID No. 1 sequence into ATT, and the other steps are as the construction steps of the pORF-FLAG plasmid, thus obtaining the promoter mutation plasmid pORFut-FLAG.

1.3 plasmid transfection

The day before transfection, cells were seeded into 6-well plates to achieve a cell confluency of about 40% -60%. The transfection system was prepared in the ratio of plasmid/jetPrime Buffer/jetprime=2 ug/200. Mu.L/5. Mu.L, pcDNA3.1+, pORF-FLAG and pORFut-FLAG plasmids were transfected into cells, and the cells were harvested after 48 hours.

1.4Western blot

And detecting the effectiveness of an antibody anti-MBOP targeting the small peptide MBOP by using a Western blot experiment, and comparing the expression conditions of the small peptide MBOP in the colorectal cancer. And 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 effectiveness verification of antibodies anti-MBOP targeting small peptides MBOP. 48h after transfection of plasmids pcDNA3.1+, pORF-FLAG and pORFput-FLAG in colorectal cancer cell lines HCT116 and HCT15, cellular protein samples were collected for validation of antibodies targeting the small peptide MBOP. Experiments find that the anti-MBOP antibody can generate a strip at the same position as an 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 tissue. Protein samples of colorectal cancer tissues were extracted 9, and protein content detection of small peptide MBOP was performed, and found that MBOP was expressed higher in 6 cancer tissues than in corresponding paracancerous tissues, indicating that MBOP was highly expressed in colorectal cancer tissues (fig. 4).

2.3 small peptide MBOP was highly expressed in colorectal cancer cell lines. Taking colorectal cancer cell lines HCT116, HCT15, HT29, SW48, SW620 and RKO, normal colon epithelial cell line FHC, gastric cancer cell lines MKN45 and HGC27, cervical cancer cell line Hela and kidney embryo cell line 293T for detecting the content of small peptide MBOP, and finding that the expression of the small peptide in HCT116, HCT15 and HT29 is higher than that in FHC; colorectal cancer cell lines HCT116, HCT15, HT29, SW48, SW620 and RKO were higher in the expression of the small peptides MBOP than gastric cancer cell lines, cervical cancer cell lines and kidney blasts (fig. 5).

2.4 in each organ of a BALB/c Nude female mouse, only the colon part was expressed with small peptide MBOP, which showed that small peptide MBOP has a certain sequence conservation and its expression has a certain tissue specificity (FIG. 6).

Example 3

Subcellular localization of human long-chain non-coding RNA LINC 01234-encoded small peptide MBOP to cytoplasm

1. Method of

1.1 Mass Nuclear separation experiment

According to the plasma and nuclear separation kit PARIS ^TM Kit instructions Cell Fraction Buffer, cell Disruption Buffer were pre-chilled on ice and the proteins in the cytoplasm and nucleus were extracted as follows: each preparation 1×10 ⁶ HCT116 and HCT15 cells were pelleted and placed 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 at 4 deg.c and 500g for 1-5 min. The supernatant, the cytoplasmic fraction protein, was carefully pipetted into another RNase-free centrifuge tube using a 200. Mu.L pipette. The cell nucleus is taken as the sediment, and the cell nucleus can be removed by washing with ice Cell Fraction Buffer once again, and then 300-500 mu L of ice Cell Disruption Buffer is added, and then the cell sediment is blown off vigorously, thus obtaining the cell nucleus protein sample. Protein samples were stored at-80 ℃.

1.2Western blot experiments

And detecting subcellular localization of the small peptide MBOP by using a Western blot experiment. Using GAPDH as an internal reference protein of cytoplasm and H3 as an internal reference protein of nucleus, the distribution of small peptide MBOP in the natural state of the above protein sample isolated from the nucleus was detected using anti-MBOP antibody.

1.3 detection of mRNA expression level of Gene

1.3.1 Total RNA extraction from cells

Cellular RNAs were extracted according to the following steps according to the instructions of the AxyPrep total RNA preparation kit: the medium was discarded, 300. Mu.L of lysate Buffer R-I was added to each well, and the cell plate was slowly rotated so that the lysate could uniformly cover all adherent cells, and then the cells were beaten about 10 times with a pipette and transferred all to a 1.5mL RNase-free centrifuge tube in the kit. After about 10 times of blowing and sucking with a sterile syringe equipped with a No. 21-25 pillow, 110. Mu.L of Buffer R-II was added, and after vortexing for about 30s, centrifugation was performed at 4℃for 5min at 12 g. In this procedure, an EP tube of 1.5mL RNase-free was prepared, 200. Mu.L of isopropanol was added, and the spin column in the kit was inserted into a 2mL receiving tube. The centrifuged supernatant was aspirated into an EP tube containing 200. Mu.L of isopropanol, and after air-blow mixing, all was transferred to a centrifuge column and centrifuged at 6 g for 1min at 4 ℃. The column is removed and the liquid in the receiving tube is discarded and the column is replaced in the receiving tube. Next, the 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 12 g for 1min at 4 ℃. After the last rinsing, the column was again placed in a receiving tube and centrifuged at 12 g for 1min at 4℃to remove the rinsing liquid sufficiently. Transferring the centrifugal column into a 1.5mL RNase-free centrifuge tube in the kit, opening a cover in a fume hood, standing for 1min at room temperature after adding 50-100 mu L Buffer TE according to the cell quantity, centrifuging for 1min at 4 ℃ at 12 g to obtain a total RNA sample of the cells, and storing at-80 ℃.

1.3.2 Total RNA extraction from tissues

Before the experiment, scissors, tweezers and steel balls are wrapped by tinfoil paper, and are placed in a baking oven at 120 ℃ to be baked for 4 hours at high temperature, and then are naturally cooled. Total RNA in the tissue samples was extracted according to Tiangen RNAsimple Total RNA kit. The half grain size tissue sample is cut and placed in a 2mL centrifuge tube of RNase-free, one steel ball is added, and then 1mL of lysis solution RZ is added. The centrifuge tube was added to the pre-chilled tissue mill cannula mount and tissue milling was performed at a frequency of 60Hz for 180 s. After the sample tube was left at room temperature for 5min, it was centrifuged at 13 g for 5min at 4℃and the supernatant was aspirated into a new 1.5mL RNase-free core. 200. Mu.L of chloroform was added thereto, and the mixture was vigorously shaken at room temperature for 20s and allowed to stand for 3 minutes. After centrifugation at 13 g for 10min at 4℃and 13 g, the sample was divided into three layers, the RNA sample was mainly in the first colorless aqueous phase, this layer was carefully aspirated (typically 400. Mu.L) into a new 1.5mL RNase-free centrifuge, 200. Mu.L absolute ethanol was then added, the whole system was transferred to the column CR3 after pipetting, and centrifugation at 13 g for 0.5min at 4℃was performed to discard the waste liquid. After rinsing with 500. Mu.L of deproteinized solution RD once, centrifugation is performed at 13 g for 0.5min at 4 ℃. Then, 500. Mu.L of a rinse solution RW was added thereto, and the mixture was allowed to stand at room temperature for 2 minutes, followed by centrifugation at 13 g for 0.5 minutes at 4 ℃. After repeating the RW rinsing step once again, the temperature was again 4℃and 13 g was centrifuged for 2min to try to remove residual rinse. The column was transferred to a fresh 1.5mL EP tube and placed in a fume hood for about 2 minutes. Finally, adding an appropriate amount of RNase-free ddH according to the amount of the tissue ₂ O, standing for 2min at room temperature, and centrifuging at 4 ℃ for 2min at 13 g to obtain a final tissue RNA sample. RNA samples were stored at-80 ℃.

1.3.3 reverse transcription

Detection and calculation of the concentration of the RNA sample is required prior to reverse transcription. Concentration measurement is carried out by using Nanodrop 2000 under the RNA mode, the value distribution of A260/A280 is 2.00-2.08, and the values of A260/A230 are all more than 1.5, which indicates that the quality of the RNA sample is higher, and DNA, protein and reagent pollution is avoided.

A reverse transcription system was prepared on ice according to the reverse transcription reagent instructions, and a volume corresponding to 500ng of RNA sample was aspirated into 200. Mu.L of EP tube, and RNase-free ddH was used ₂ O is added to the mixture to be 8 mu L, and 2 mu L of the mixture is addedII qRT SuperMix ^a And the mixture was uniformly beaten by a pipette and centrifuged. The EP tube was transferred to a 50℃water bath, reacted for 15min, then transferred to 85℃and immediately transferred to ice after 5s of reaction. mu.L of RNase-free ddH was pipetted ₂ O was added to the EP tube to a final concentration of 10 ng/. Mu.L. The cDNA sample may be stored at-20deg.C.

1.3.4 RT-qPCR

The following reaction system was formulated on ice:

the PCR amplification procedure was as follows:

and (3) 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 target gene of the experimental group was 2 ^-△△Ct 。

1.4 construction of stable transgenic plants

According to the construction method of the over-expression plasmid, the target fragment is inserted into pCDH-CMV-MCS-EF 1-copGGFP-T2A-Puro plasmid with green fluorescent protein to respectively obtain ORF-GFP plasmid of over-expression small peptide and ORFm-GFP plasmid of over-expression promoter mutation sequence, and the empty vector is marked as co-GFP.

Lentiviral packaging was performed using 293T cell line. 293T cells of 3 cell culture dishes of 6cm should be planted one day in advance, so that the cell fusion degree of the next day reaches about 70% -80%.

The packaging system is as follows:

the specific preparation process is as follows: preparing a plurality of sterilized EP tubes, taking 3 of the EP tubes, adding 400 mu L of Opti-MEM blank culture medium and 30 mu L of Lipofectamine 2000 into each tube, and uniformly mixing; then, 3 EP tubes were taken, 400. Mu.L of Opti-MEM blank medium and two packaging vector plasmids, and 5. Mu.g of co-GFP, ORF-GFP and ORFm-GFP plasmids were added, and mixed. After standing for 5min, lipofectamine 2000 diluted with Opti-MEM was added one-to-one to the Opti-MEM diluted plasmid, mixed and then left for 20min. Taking out 293T cells of the advanced seed plates, sucking and removing the culture medium, adding 3mL of blank culture medium, gently adding the transfection system for packaging the lentivirus into the 293T cells, and placing at 37 ℃ and 5% CO ₂ Culturing in a cell culture box for 6h, taking out cells, changing the culture medium into a complete culture medium, and then placing the complete culture medium back into the cell culture box for culturing for 42h.

24 hours before transfection of the virus liquid, the cells required to construct stable overexpressing cell lines were seeded into 6-well plates to achieve a confluency of about 20% the next day. The 293T cells transfected with lentivirus were removed for 48h, the medium was aspirated with a 5mL syringe (no needle), the virus solution was filtered with a disposable filter head (0.45 μm), and a quantity of polybrene (final concentration 8. Mu.g/mL) was added as an auxiliary infectious agent. The medium in the 6-well plate was aspirated, and 1mL of virus solution containing co-GFP, ORF-GFP and ORFm-GFP plasmids was added to each of 3 wells in the plate. After 24h, the cell state was observed under a microscope and 1mL of complete medium was added. After a further 24 hours, the whole medium was aspirated and replaced by complete medium, at which time green fluorescence in the cells should be observed under the microscope. Later stage, the cells can be planted in a 96-well plate by a stepwise dilution method, and then the cells are cultured by a culture medium added with puromycin with a certain concentration, so that a monoclonal cell line is selected.

1.5 immunofluorescence

One drop of culture medium was dropped into each of 3 wells of the 12-well plate, and the cell climbing sheet was held by forceps, and carefully covered on the culture medium drop in the 12-well plate, so that it was adsorbed and immobilized. The 12-well plate was returned to the cell incubator and incubated for 15min. The stable overexpressing 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 pipetted with a 200. Mu.L pipette tip and added dropwise to the middle of the cell slide. The 12-hole plate is put back into a cell culture box for incubation for 6-8 h, after cells are attached to a cell climbing plate, 1mL of complete culture medium is added, and the cells are put back into the cell culture box for incubation overnight.

The following day the 12-well plate was removed, all media was aspirated, and the cell slide was gently rinsed 3 times with 1 XPBS. 500. Mu.L of 4% fixed formaldehyde was added to each well in the dark and incubated for 1h in the dark. After gently rinsing the cell slide 3 times with 1 XPBS, 500. Mu.L of PBS permeabilization solution containing 0.5% (v/v) Triton X-100 was added to each well, and after incubation for 20min at room temperature, the cell slide was gently rinsed 3 times with 1 XPBS. PBS blocking solution containing 10% (v/v) goat serum was prepared, and was added to the cell slide, blocked at room temperature for 1h, and the cell slide was gently rinsed 3 times with 1 XPBS. An anti-MBOP primary anti-dilution solution is prepared according to a given dilution ratio, and is added to a cell slide at 4 ℃ overnight. The following day, all operations need to be protected from light. The 1 XPBS solution was warmed, primary anti-dilution was recovered, and cells were gently rinsed 3 times with the warmed 1 XPBS solution. Fluorescent secondary antibodies were added to PBS blocking solution in proportion and onto cell slide plates, after incubation for 1h at room temperature, cells were gently rinsed 3 times with pre-warmed 1 x PBS solution. After 20min of nuclear staining with host, the cells were gently rinsed 3 times with pre-warmed 1 XPBS solution. Finally, a drop of anti-fluorescence quenching agent is dripped on the glass slide, the cell climbing sheet is picked up by a needle head of a syringe, the forceps are carefully clamped, the lower edge of the cell climbing sheet is abutted against the water absorbing paper to remove redundant liquid, the cell surface of the cell climbing sheet faces downwards, and the cell climbing sheet is covered on the quenching agent, so that air bubbles are carefully avoided. Then 3-4 drops of transparent nail polish are dripped to the edge position sealing piece of the cell climbing piece. Fluorescence photographing is preferably performed within half a month after completion of the tablet preparation.

1.6 analysis of Membrane localization potential of small peptides MBOP

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

2. Results

2.1 RT-qPCR and Western blot experiments prove that the nucleotide sequence LINC01234ORF and the small peptide MBOP are mainly distributed in cytoplasm. The experimental results show that the cytoplasmic and nuclear fractions of HCT116 and HCT15 cells were better isolated by the plasma-nuclear separation experiment, and the contents of the nucleotide sequence LINC01234ORF and the small peptide MBOP in the cytoplasm were significantly higher, indicating that the small peptide MBOP was mainly distributed in the cytoplasm (FIG. 7).

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

2.3 database predictive analysis of the small peptide MBOP membraneless anchor sequences (fig. 9), so the small peptide MBOP is predominantly localized in the cytoplasm.

Example 4

Small peptide MBOP encoded by human long-chain non-coding RNA LINC01234 promotes migration and proliferation of colorectal cancer cells

1. Method of

1.1Transwell experiments

A complete medium of 15% fetal bovine serum (v/v) was prepared, and this medium was added to the lower chamber of the transwell plate at 600. Mu.L per well, the upper chamber of the transwell was placed in the well to which the medium was added, the air bubbles were removed, and the mixture was allowed to stand in a cell incubator for about 20 minutes. Cells transfected for 48h were again digested, counted, and resuspended in serum-free medium. The cells were counted under a microscope using a hemocytometer, 2× for each transwell chamber10 ⁴ The density of individual cells, the cell suspension to be aspirated, is calculated. A1.5 mL sterile EP tube was taken, the required amount of 2-fold cell suspension was aspirated, and the solution was supplemented to 400. Mu.L with serum-free medium and was pipetted in a pipette for homogenization. The transwell plates were removed, 200. Mu.L of each cell suspension was aspirated, carefully placed in the transwell upper chamber, carefully shaken, and returned to the cell incubator for further culture. After 48h of incubation, the transwell plate was removed, the lower chamber medium was aspirated, 600. Mu.L of 4% fixative formaldehyde solution was added, and fixation was performed at room temperature in the absence of light for 30min. The fixed formaldehyde solution was then removed, replaced with PBS and rinsed once. The PBS solution was removed by pipetting, 600. Mu.L of crystal violet solution was added, after incubation for 20min at room temperature, the crystal violet solution was recovered and the transwell upper chamber was rinsed multiple times with PBS solution until no excess crystal violet solution was present. The non-migrated cells on the top surface of the transwell upper chamber were erased with a small cotton swab and naturally dried. The transwell cells were observed under a microscope and recorded in a bright field photograph.

1.2 scratch test

Cells transfected for 48h were digested, counted, and resuspended in serum-free medium. Cells were counted under a microscope using a hemocytometer to calculate 3×10 cells per well ⁵ Cell suspension volume required for individual cells. A new 6-hole plate is taken, a paint pen is used for drawing parallel straight lines on the bottom plate of the new 6-hole plate, and generally one hole can draw three parallel lines. The calculated cell suspension is added into a 6-well plate with a line, and the liquid is replenished to 2mL for each well, and the cell suspension in the 6-well plate is fully shaken and then placed into a cell culture box for culture. The next day, cells were observed under a microscope, at which point the cell confluency reached about 95%, and a 200 μl pipette tip was used to draw a straight line in the 6-well plate, which line would be perpendicular to the parallel lines of the paint strokes of the previous day. And discarding the culture medium, rinsing the cells with PBS solution to wash the scraped cells in the line drawing process, changing the scraped cells into the culture medium containing 1% fetal bovine serum (v/v), immediately taking the culture medium under a microscope to carry out bright field photographing, and carrying out bright field photographing after 24 hours and 48 hours respectively.

1.3 cell cycle experiments

The transfected cells were centrifuged at 1 000rpm at 4℃for 5min for 48 h. The supernatant was carefully pipetted away and about 50. Mu.L of medium was kept to avoid pipetting away the cell pellet during pipetting. The cell pellet was resuspended in 1mL of PBS and centrifuged at 1 000rpm for 5min at 4 ℃. The supernatant was carefully pipetted away and about 50. Mu.L of PBS was retained. 1mL of 70% ethanol solution was added, the cells were gently resuspended, and the sample tube was inserted on a turntable in a chromatography cabinet at 4℃and after fixing for at least 12 hours, centrifuged at 1 000rpm at 4℃for 10min. The supernatant was aspirated off, and about 50. Mu.L of ethanol was retained to avoid aspiration of cells. The cell pellet was resuspended in 1mL of PBS and centrifuged at 1 000rpm for 5min at 4 ℃. In the centrifugation, propidium iodide staining solution was prepared according to the number of samples required for the experiment, and the staining solution required for each sample to be measured included 500. Mu.L of staining buffer, 25. Mu.L of propidium iodide staining solution (20X) and 10. Mu.L of RNase A (50X). After the supernatant of the above-mentioned centrifugal sample was aspirated, 450. Mu.L of propidium iodide staining working solution was transferred with a pipette, and the cell sample was gently resuspended and stored in a 37℃water bath in the absence of light.

Before loading, the cell suspension is treated by a 40-mesh screen, the cell fluid is added into a flow type loading tube, a flow cytometer is arranged, and the percentages of G1 phase, S phase and G2/M phase of pcDNA3.1+, pORF-FLAG and pORFput-FLAG groups of cells are recorded respectively.

1.4 cloning experiments

The stable overexpressing cell lines co-GFP, ORF-GFP and ORFm-GFP were digested and the cells were resuspended in complete medium with 15% fetal bovine serum (v/v), counted by means of a blood cell counting plate and diluted to a cell concentration of 1,000 cells/2 mL by means of stepwise dilution. Cells of the stable overexpressing cell line were seeded into 6-well plates, 1,000 cells per well, homogenized well, and placed in a cell incubator for continued culture using a 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, and the cell plate was rinsed with 1mL PBS. Then 600. Mu.L of a 4% fixed formaldehyde solution was added thereto, and the mixture was fixed at room temperature for 30 minutes in a dark place. After absorbing and removing the fixed formaldehyde solution and adding 600. Mu.L of crystal violet solution and incubating for 20min at room temperature, the crystal violet solution is recovered and the cell plate is rinsed with PBS solution. After the plate is naturally dried, the microscope camera is used for shooting and recording data.

1.5 xenograft tumor model experiments

1.5.1 first tumor grafting

Three stable overexpressing cells, co-GFP, ORF-GFP and ORFm-GFP, of HCT116 cells were amplified, digested and the cells were collected. The cell pellet was washed once more with PBS, and after centrifugation, the cells were resuspended with a small amount of ice PBS. After pipetting about 20. Mu.L of the cell suspension and appropriate dilution, it was counted under a microscope using a hemocytometer and the cell suspension concentration was adjusted to 8X 10 with ice PBS ⁶ Each cell/150. Mu.L. 150. Mu.L of the cell suspension was inoculated with a 1mL medical syringe at the underarm subcutaneous site in 4 week female BALB/cNude nude mice, after about 2 weeks, the nude mice developed a visible tumor mass subcutaneously. After about 1 month again, the tumor mass in the nude mice grew large enough to provide enough tumor mass for secondary tumor grafting.

1.5.2 Secondary tumor grafting

Before the experiment, scissors, tweezers and steel balls are wrapped by tinfoil paper, are placed in a baking oven at the high temperature of 120 ℃ for baking for 4 hours, and are naturally cooled, so that ice physiological saline and a cell dish are prepared. After the nude mice are sacrificed in the humane way, the tumors are peeled off, the tissues at the periphery of the tumor blocks are cut off, and the bean curd residue-like tissues in the tumor blocks are also cut off. A small block was taken out of the middle parenchymal part, and RNA and protein were extracted, and the expression level of LINC01234ORF and MBOP were examined. Cutting the remaining major part of the tissue to about 1mm ³ The tissue pieces were placed in ice physiological saline for use. A small opening is cut on the ventral side of a 4-week female BALB/c Nude mouse by scissors, a tissue block is pushed into the small opening, and the tissue block is pushed to an armpit position from the ventral side by a pair of straight forceps, so that the tissue block is prevented from falling out from the small opening in the moving process of the Nude mouse. Several days after the tissue mass was grafted with tumor, close attention was paid to the status of nude mice and their in vivo tissue mass.

1.5.3 data collection

On day 9 after the secondary tumor inoculation, the body weight of the nude mice, as well as the tumor size of the nude mice, were initially recorded for the first time. Tumor volume of nude mice was measured as v=1/2×a×b ² Where a is the length of the tumor and b is the width of the tumor. Continuously recording to eighth time according to frequency recorded every three daysIn nude mice, the tumor is not more than 2 000mm ³ In the case of volume, the nude mice were sacrificed humanly. The tumor is peeled off, and the tissue of the surface and core part of the tumor is removed. The rest tumor parenchyma part is respectively subjected to RNA extraction, protein extraction, immunohistochemical pretreatment and freezing at-80 ℃.

2. Results

2.1 through transwell experiments, the small peptide MBOP has the function of promoting migration. The principle of the Transwell experiment is to induce migration of cells from the top surface of the Transwell chamber to the bottom surface of the Transwell chamber using the serum concentration differences in the culture medium. The experimental results show that the cell number of the small peptide MBOP over-expression group is higher than that of the pcDNA3.1+ group transfected with the empty vector and the promoter mutation plasmid pORFut-FLAG group, which shows that the small peptide MBOP has the function of promoting cell migration (figure 10).

2.2 through scratch experiments, the small peptide MBOP has the function of promoting migration. The principle of scratch experiments is to use the artificial manufacturing of the space interval between adherent cells to induce the migration of cells from dense to cell-free sites. The experimental results show that the cell number of the small peptide MBOP over-expression group is higher than that of the pcDNA3.1+ group transfected with the empty vector and the promoter mutation plasmid pORFut-FLAG group, which shows that the small peptide MBOP has the function of promoting cell migration (figure 11).

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

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

2.5 experiments on xenograft tumor models prove that the small peptide MBOP has the function of promoting proliferation. After the first tumor inoculation, LINC01234ORF and MBOP in tumor mass were tested, demonstrating that stable overexpressing cell lines were able to maintain significant overexpression in vivo (FIG. 14). After secondary tumor inoculation by using the verified tumor mass, the tumor mass volume proliferation of the small peptide MBOP over-expression group is found to be significantly higher than that of the control group and the promoter mutation group, which indicates that the small peptide MBOP has the proliferation promoting function (figure 15).

Sequence listing

<110> university of Zhejiang

<120> Small peptides encoded by Long non-coding RNA LINC01234 and uses thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 258

<212> DNA

<213> Homo sapiens (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> Homo sapiens (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> Homo sapiens (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> Homo sapiens (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. From long-chain non-coding RNALINC01234The application of the coded protein small peptide serving as a tumor marker in preparing a detection reagent for colorectal cancer and cervical cancer targeting MBOP is disclosed, wherein the amino acid sequence of the protein small peptide is shown as SEQ ID No. 2, and the protein small peptide is named MBOP.