CN110643707A

CN110643707A - ESCC-related lncRNA LLNLR-299G3.1 and application thereof

Info

Publication number: CN110643707A
Application number: CN201911024431.0A
Authority: CN
Inventors: 翟日洪; 杨林; 范秀军; 田莉; 宋译; 张宝珍
Original assignee: Shenzhen University
Current assignee: Shenzhen University
Priority date: 2019-10-25
Filing date: 2019-10-25
Publication date: 2020-01-03
Anticipated expiration: 2039-10-25
Also published as: CN110643707B

Abstract

The invention belongs to the technical field of molecular diagnosis, and particularly relates to lncRNA LLNLR-299G3.1 related to Esophageal Squamous Cell Carcinoma (ESCC) and application thereof. The invention proves an lncRNA (LLNLR-299G3.1) with obvious cancer promotion activity through experimental analysis, thereby providing a new target point for a new scheme of individualized and accurate treatment of ESCC. The expression of the lncRNA in ESCC patient plasma exosomes and ESCC cells is respectively obviously higher than that of healthy human plasma exosomes and normal esophageal epithelial cells. Research results show that the lncRNA has obvious capacity of promoting ESCC cell tumor activity; the inhibition of the expression of the lncRNA in a cell and nude mouse transplantation tumor model can obviously inhibit the proliferation and migration capacity of ESCC cells.

Description

ESCC-related lncRNA LLNLR-299G3.1 and application thereof

Technical Field

The invention belongs to the technical field of molecular diagnosis, and particularly relates to an IncRNA LLNLR-299G3.1 related to ESCC and application thereof.

Background

Esophageal cancer is a malignant tumor that occurs in esophageal epithelial tissue, with mortality rates ranked fourth and sixth of all tumor mortality rates in china and worldwide, respectively. China is the world in which the incidence and mortality of esophageal cancer are highest, and more than 50% of cases occur in China among over 50 million people of esophageal cancer found every year worldwide, 90% of which are Esophageal Squamous Cell Carcinoma (ESCC). At present, the total 5-year survival rate of esophageal cancer is only about 10%, but the 5-year survival rate of esophageal cancer in stage I after surgical resection can reach 90%, and the five-year survival rate of patients in stage IV who cannot be operated is almost 0. Therefore, early diagnosis and early treatment are the key to improve the survival rate of esophageal cancer. Due to the lack of typical symptoms in early stage of esophageal cancer and the effective early diagnosis method, more than 85% of patients are diagnosed in middle and advanced stages, and the opportunity of early diagnosis and early treatment is lost. Therefore, finding more effective early diagnostic methods to treat patients early is of great practical significance in reducing ESCC mortality.

At present, esophageal cancer diagnosis mainly depends on means such as imaging (X-ray radiography, CT and the like), mesh pulling cytology, endoscopy (fiberesophagoscope, esophageal ultrasonic endoscope) and pathological biopsy. Although these examination methods have important clinical reference values for the diagnosis of esophageal cancer, they are not ideal for the sensitivity and specificity of diagnosis of early stage ESCC lesions. In order to screen patients for early ESCC at the molecular level and provide scientific basis for early treatment, in recent years, a number of researchers have conducted extensive studies on molecular markers of ESCC at the RNA, DNA, protein, and epigenetic levels, and have discovered molecular markers of some reference value for diagnosis and clinical staging, including molecules such as p53, p21, cyclin D, VEGF, Ki-57, HER-2, Cox-2, c-myc, Bcl-2, CEA, and the like. However, in general, the sensitivity and specificity of ESCC are low in the early stage of diagnosis of the molecular markers, and the detection of most of the markers can be carried out only by pathological biopsy, so that the specimen is difficult to obtain and traumatic. Therefore, the search for markers which have high sensitivity, strong specificity, no wound or minimal invasion and can be found early becomes an important direction for the research of the international tumor molecular markers. However, no molecular marker can be clinically used for early diagnosis of ESCC. Therefore, exploring active molecules playing a key role in the pathological process of ESCC and clarifying the mechanism of ESCC provides a basis for early diagnosis of ESCC, and is an important subject which needs to be solved urgently in clinical and scientific research at present.

Exosomes (exosomes) are small vesicles of about 40-120 nm in diameter that are secreted from the cell into the cell. Exosomes are known as "garbage bags" of cells, responsible for discharging some waste that is not useful to the cells. However, recent studies have found that the exosomes contain biologically active molecules such as RNA. The active molecules not only can provide abundant biological markers for disease diagnosis, but also can mediate substance and information transmission among cells and participate in various important pathophysiological reactions. More importantly, the compositions and the contents of active molecules carried by exosomes from different cell sources are different, and the detection of the active molecules in the exosomes provides a new opportunity for finding disease-specific molecular markers. In vitro studies have shown that cancer cells such as breast, prostate, lung, and bladder cancer can release large amounts of exosomes into the culture medium. In tumor patients, the serum (plasma) exosome concentration of patients with nasopharyngeal carcinoma, melanoma, lung cancer, intestinal cancer, ESCC and the like is found to be obviously higher than that of control people, and the circulating exosome concentration is suggested to have an important relation with the occurrence and development of tumors. However, the relationship between the active molecules carried by circulating exosomes and ESCC and the clinical pathological changes of tumors remains to be explored further.

Of all human RNA sequences, only about 2% of sequences having a function of encoding proteins are sequences that do not encode proteins, and the remaining sequences are referred to as non-coding RNAs (ncrnas). ncrnas can be divided into two broad categories depending on whether they are over 200 nucleotides in length: small-molecule RNA and long non-coding RNA (lncRNA) typified by miRNA. Small ncRNAs account for a small proportion of non-coding sequences, for example, only 2500 human miRNAs. Most non-coding sequences are lncRNAs, and up to 3.7 ten thousand human lncRNAs have been identified. Initially lncRNAs were considered "noise" of genome transcription and did not have any biological function. However, recent studies have shown that lncRNA can regulate gene expression at the transcriptional, posttranscriptional and epigenetic levels by binding to mRNA target molecules, promoters and transcription factors, respectively, and further influence the differentiation, proliferation, metabolism and apoptosis of tumor cells. Of particular interest is the tissue specificity of the expression profile of lncRNA, a feature that makes lncRNA uniquely advantageous as a molecular marker for disease diagnosis.

However, few relevant incrna molecules currently available for diagnosis and/or treatment and/or prognostic assessment of ESCC suggest that existing incrna detection techniques remain to be improved.

Disclosure of Invention

The invention aims to provide an IncRNA LLNLR-299G3.1 related to ESCC and application thereof, and aims to solve the technical problem that the clinical application of the existing ESCC-related IncRNA molecules is limited.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides an IncRNA LLNLR-299G3.1 related to ESCC, and the gene sequence of the IncRNA LLNLR-299G3.1 is shown as SEQ ID No. 1.

The invention also provides application of a reagent for detecting lncRNA LLNLR-299G3.1 in preparing a kit for diagnosing and/or prognostically evaluating ESCC; wherein the gene sequence of the lncRNA LLNLR-299G3.1 is shown as SEQ ID No. 1.

Accordingly, a kit for the diagnostic and/or prognostic evaluation of ESCC comprising reagents for the detection of IncRNA LLNLR-299G 3.1.

Accordingly, an inhibitor for inhibiting the expression of lncRNA LLNLR-299G3.1, said inhibitor comprising at least one of the antisense oligonucleotides shown as SEQ ID No.4, SEQ ID No.5 and SEQ ID No. 6.

Accordingly, use of an inhibitor of lncRNA LLNLR-299G3.1 for the preparation of a medicament for the prevention and/or treatment of ESCC; wherein the gene sequence of the lncRNA LLNLR-299G3.1 is shown as SEQ ID No. 1.

Finally, the invention also provides a medicament for preventing and/or treating ESCC, which comprises an inhibitor capable of inhibiting the expression of IncRNA LLNLR-299G3.1 and a pharmaceutically acceptable carrier; wherein the gene sequence of the lncRNA LLNLR-299G3.1 is shown as SEQ ID No. 1.

The invention proves an lncRNA (LLNLR-299G3.1) with obvious cancer promotion activity through experimental analysis, thereby providing a new target point for a new scheme of individualized and accurate treatment of ESCC. The expression of the lncRNA in ESCC patient plasma exosomes and ESCC cells is respectively obviously higher than that of healthy human plasma exosomes and normal esophageal epithelial cells. Research results show that the lncRNA has obvious capacity of promoting ESCC cell tumor activity; the inhibition of the expression of the lncRNA can obviously inhibit the proliferation and migration capability of ESCC cells. Experiments prove that the antisense oligonucleotide (ASO) for inhibiting the lncRNA LLNLR-299G3.1 has obvious tumor inhibition effect on ESCC nude mouse transplanted tumors. Therefore, the reagent for detecting the lncRNA can be used for preparing a kit for diagnosing and/or prognostically evaluating ESCC, and the inhibitor of the lncRNA can be used for preparing a medicament for preventing and/or treating ESCC.

Drawings

FIG. 1 is a diagram of plasma exosomes extracted in an embodiment of the present invention: wherein A is an exosome morphology graph, B is an exosome particle size distribution graph, and C is a graph of the measurement results of exosome surface positive marker molecules (CD63 and ALIX) and negative marker molecules (calnexin-1);

FIG. 2 is a graph showing the results of the difference in the expression levels of LLNLR-299G3.1 in ESCC patients and normal control plasma exosomes in the example of the present invention (results of RNA sequencing method);

FIG. 3 is a graph showing the results of the difference in the expression levels of LLNLR-299G3.1 in ESCC patients and normal control exosomes according to example of the present invention (qRT-PCR method test results);

FIG. 4 is a graph showing the results of the difference in expression of LLNLR-299G3.1 in ESCC cancer cells and normal esophageal epithelial cells in the present example;

FIG. 5 is a map of a lentiviral shuttle plasmid vector in an example of the invention;

FIG. 6 is a graph showing the results of experiments on the proliferation of ESCC cells (KYSE30) promoted by over-expression of LLNLR-299G3.1 in the present example;

FIG. 7 is a graph showing the results of experiments on the promotion of ESCC cell (KYSE30) migration by over-expression of LLNLR-299G3.1 in the present example;

FIG. 8 is a graph showing the results of experiments on the inhibition of ESCC cell proliferation (KYSE30 and TE1) by LLNLR-299G3.1 under-expression in the present example;

FIG. 9 is a graph showing the results of experiments on the inhibition of ESCC cell migration (KYSE30 and TE1) by LLNLR-299G3.1 under-expression in the present example;

FIG. 10 is a graph showing the results of animal experiments in which the ASO knockdown of LLNLR-299G3.1 expression was used to inhibit the growth of ESCC transplanted tumors in the examples of the present invention.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly apparent, the present invention is further described in detail below with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The lncRNA related to ESCC provided by the embodiment of the invention is LLNLR-299G3.1, and the ID in a genome database (ensemble) is ENSG 00000272448.1; the sequence is shown as follows:

SEQ ID No.1：

TGCTTGACCTCCTTGCTTGGCAACGTGGGCTTCTCCACGGACACCACGCACACCCTGCTGGCCAGCTCCATGTGGAGCTGCTTCTCAAAGAGGCACTGCAGGGTCTTCAAGGGCAGGTGCAGCTTCGGGTAGGACACACGCCCATCCTGCAGGGATGGGGGTAGTGAGGTTGGGGGCTTGCCAGAGGGCGACCTGCCCTCCCAGGACCCCGAGACAGCATGGGTGCACGCGTTTCTGCGTCTCCTGCAAGTTGCTGGTGGCTATCGCTGACGCGGGGAAAGGCGGGCTGCGGGTAAAGTCAGTGCCAGCAGTGCAAACCAAAGGCCTTGACCCTCCTGGCCTCGACCCCTCTAGAAGGGACACTGGGCACCGTGCAGGGGGTGGCAGGGGCGGTGATGCTGGGAGCTGGCAGAGCCTGGGGAGACCGTTCACTGCACCCCCAGATGTTGGCTGTTTTCTCCTCAAACTCAGAACTGTATGAATGTGACCCATCCAGAAATAGATGAATTAAAAATAACAACTAAAAAAAAAAAA。

aiming at the gene sequence of the lncRNA, a primer for amplifying the lncRNA by qRT-PCR is provided, and the sequence is as follows:

SEQ ID No.2：F：CTTCGGGTAGGACACACGCC；

SEQ ID No.3：R：CAGCGATAGCCACCAGCAACT。

the primer can detect the expression level of lncRNA LLNLR-299G3.1 in a biological sample, and provides a basis for further researching the function of lncRNA molecules in ESCC and the research and development of targeted drugs.

In another aspect, the embodiment of the invention also provides an application of a reagent for detecting lncRNA LLNLR-299G3.1 in preparing a kit for diagnosing and/or prognostically evaluating ESCC. The lncRNA provided by the embodiment of the invention has obvious cancer promotion activity, so that a new target is provided for a new scheme of ESCC individualized and accurate treatment, and the detection reagent of the lncRNA can be used for preparing a kit for diagnosing and/or prognostically evaluating NSCLC. Specifically, the reagent for detecting the lncRNA LLNLR-299G3.1 comprises a primer for amplifying the lncRNA LLNLR-299G3.1, wherein the primer is shown as SEQ ID No.2 and SEQ ID No. 3.

Accordingly, a kit for the diagnostic and/or prognostic evaluation of ESCC comprising reagents for the detection of IncRNA LLNLR-299G 3.1. The reagent for detecting the lncRNA LLNLR-299G3.1 comprises a primer for amplifying the lncRNALLNLR-299G3.1, and the primer is shown as SEQ ID No.2 and SEQ ID No. 3; the kit comprises PCR amplification enzyme and PCR amplification buffer solution. The detection reagent also comprises a negative control.

The embodiment of the invention also provides an inhibitor for inhibiting the expression of lncRNA LLNLR-299G3.1, wherein the inhibitor comprises at least one of antisense oligonucleotides shown as SEQ ID No.4, SEQ ID No.5 and SEQ ID No. 6.

In particular, the amount of the solvent to be used,

the SEQ ID No.4 sequence of ASO1 is: GAUAGCCACCAGCAACUUGC, respectively;

the SEQ ID No.5 sequence of ASO2 is: CUUUGGUUUGCACUGCUGGC, respectively;

the SEQ ID No.6 sequence of ASO3 is: UUUCUGGAUGGGUCACAUUC are provided.

Accordingly, the use of an inhibitor of lncRNA LLNLR-299G3.1 for the preparation of a medicament for the prevention and/or treatment of ESCC. The inhibitor comprises at least one of antisense oligonucleotides shown as SEQ ID No.4, SEQ ID No.5 and SEQ ID No. 6.

Finally, the embodiment of the invention also provides a medicine for preventing and/or treating ESCC, which comprises an inhibitor capable of inhibiting the expression of lncRNA LLNLR-299G3.1 and a pharmaceutically acceptable carrier; wherein the gene sequence of the lncRNALLNLR-299G3.1 is shown in SEQ ID No. 1.

The medicine provided by the embodiment of the invention is a composition, and comprises an inhibitor of lncRNA LLNLR-299G3.1, and/or other medicines compatible with the inhibitor, and a pharmaceutically acceptable carrier and/or an auxiliary material. Specifically, the inhibitor comprises at least one of the antisense oligonucleotide shown as SEQ ID NO.4, the antisense oligonucleotide shown as SEQ ID NO.5 and the antisense oligonucleotide shown as SEQ ID NO. 6.

Specifically, the carrier can be liposome nanoparticles, the liposome nanoparticles are coated on the surface of the ASO to form a micro-capsule structure for conveying the ASO, and the liposome nanoparticles and the ASO can be connected through polypeptide to make the ASO more stable.

The inhibitor is an "effective amount" which is an amount that is functional or active in humans and/or animals and acceptable to humans and/or animals. "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents. The term refers to such pharmaceutical carriers: they are not essential active ingredients per se and are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in the composition may comprise liquids such as water, saline, buffers. In addition, auxiliary substances, such as fillers, lubricants, glidants, wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers. The vector may also contain a cell transfection reagent. In the present embodiment, the inhibitor or its transcription gene, or its pharmaceutical composition can be administered to the mammal by various methods well known in the art. Including but not limited to: subcutaneous injection, intramuscular injection, transdermal administration, topical administration, implantation, sustained release administration, and the like; preferably, the mode of administration is parenteral.

The invention is described in further detail with reference to a part of the test results, which are described in detail below with reference to specific examples.

Example 1 discovery of LLNLR-299G3.1

1. Study object selection

Pathologically confirmed ESCC cases meeting the requirements of research conditions, esophagitis patients age-matched with the cases and diagnosed by digestive endoscopy and healthy controls are collected from thoracic surgery of people hospitals in Shenzhen city. Subjects signed an informed consent. The study protocol was approved by the medical ethics committee (approval No.: 2016001).

2. Comparison of plasma exosome lncRNA expression profiles of study subjects (ESCC patients, esophagitis patients, healthy controls)

Peripheral blood of a study object is extracted, the study object is kept stand for 1 hour at the temperature of 4 ℃, 1000g of the study object is centrifuged for 10min to separate plasma, and the separated plasma is stored at the temperature of 80 ℃ below zero to be tested.

Plasma exosomes were isolated using an Exosome Isolation Kit (Ribo-Exosome-Isolation-Kit C10110) comprising the major steps of: cells and cell debris were removed by centrifugation at2,000 Xg for 20 minutes at room temperature. The supernatant was transferred to a new tube, 1/3 volumes of exosome-separating agent was added, mixed well and left to stand at 4 ℃ for 30 minutes, then centrifuged at 15,000 Xg for 2 minutes, the supernatant was removed and PBS buffer was added for subsequent experiments. The isolated exosomes were observed for exosome morphology and size with electron microscope (TEM) (results are shown in a in fig. 1), exosome particle size distribution was analyzed with ZETASIZER Nano-series-Nano-ZS Nano analyzer (Malvern Instruments Ltd, UK) (results are shown in B in fig. 1), exosome surface protein markers (CD63, CD81) were detected with anti-CD63 antibody, anti-CD81 antibody, and flow cytometer, respectively, and the results are shown in C in fig. 1.

Exosome RNA was extracted with TRIzol reagent (Invitrogen life technologies). The extracted RNA was purified using the RNasey Mini Kit (Qiagen p/n 74104). By using

ND-1000 determination of RNA concentration and purity. The extracted RNA was digested with DNase I and digested with Ribozero Magnetic GoldKit is treated by rRNA removal, RNA fragmentation is carried out, first strand cDNA is generated by random primers, and dUTP is added to synthesize second strand cDNA. And (3) carrying out end repair on the double-stranded cDNA by using polymerase, adding A, connecting the ligase with an Illumina specific joint, and carrying out qPCR amplification and purification to obtain an RNA sequencing library.

The constructed RNA sequencing library was subjected to quality inspection by an Agilent 2100Bioanalyzer and quantitative analysis of the library by qPCR. After library qualification, clusters (clusters) were generated on cBot using HiSeq Rapid PE Cluster Kit V2(Illumina) reagent, followed by a paired-end sequencing program (2 × 150bp) run on the HiSeq 3000platform sequencing platform. After obtaining sequencing data (Raw reads), removing short sequences containing linkers according to quality control standards; removing short sequences with the proportion of N being more than 10 percent and removing low-quality short sequences (the number of bases with the quality value QA being less than or equal to 20 accounts for more than 20 percent of the whole short sequences). Reads aligned with the human rRNA sequence database (GeneBank and GENECODE v26) to remove rRNA and obtain high quality data (Cleanreads). Clean read was aligned to the reference human genome (GRCh38) using HISAT2 aligner to generate BAM files. The aligned sequences were assembled with the cufflinks software and their relative abundance was estimated, and the expression level of the gene was calculated using the edgeR-robustsoftware software (FPKM, fragments per foundation of transcript dispersed reads). Expression difference fold change was compared.

Sequencing data of exosome whole genome lncRNAs and mRNAs are subjected to t-test analysis by using a multiple comparison method, and results are corrected by Bonferroni to show that: the plasma exosomes of ESCC patients have 89 IncRNA expression levels which are remarkably different from those of esophagitis groups and healthy control groups (Fold change is ≧ 2.0; Bonferroni-adjusted P < 0.05). Wherein, the P values of the difference between the LLNLR-299G3.1 expression level in the exosomes of the ESCC patients and the P values of the difference between the esophagitis and the normal control are 0.007 and 0.001 respectively, and the difference results of the LLNLR-299G3.1 expression level in the exosomes of the ESCC patients and the normal control are shown in figure 2.

qRT-PCR method for measuring plasma exosome LLNLR-299G3.1 expression quantity

(1) According to the RNA sequencing result, the expression level of LLNLR-299G3.1 in the exosomes of ESCC patients is obviously up-regulated, so that the difference of the expression level of LLNLR-299G3.1 in the plasma exosomes of ESCC patients and healthy controls is further verified by a qRT-PCR method.

(2) Total RNA extraction: shaking 1.5ml centrifuge tube for 2min, and incubating at room temperature for 5 min; adding chloroform according to the ratio of 0.2ml chloroform/1 ml Trizol and fully shaking for 20 s; 12000g, centrifuging for 10min at 4 ℃; sucking the supernatant (0.5ml supernatant/1 ml Trizol), transferring to a new centrifuge tube, and adding isopropanol according to the ratio of 0.5ml isopropanol/1 ml Trizol; mixing, and freezing at-20 deg.C for 30 min; 12000g, centrifuging for 10min at 4 ℃, discarding the supernatant and keeping RNA precipitate; adding 75% ethanol into 1ml of 75% ethanol/1 ml of Trizol to wash RNA precipitate; mixing, and freezing at-20 deg.C for 30 min; 7500g, centrifuging at 4 deg.C for 5min, discarding supernatant, and keeping RNA precipitate; opening the EP tube cover for 10min to allow the residual ethanol to naturally volatilize; add 30. mu.l RNase-free ddH₂Dissolving RNA precipitate for 10min at room temperature; electrophoresis was used to determine the quality of RNA samples, and the OD of each RNA sample was determined using a spectrophotometer₂₆₀/OD₂₈₀And calculating the total RNA concentration and the impurity pollution degree.

(3) Preparation of cDNA: a mixed cDNA reaction solution was prepared using TaKaRa PrimeScript II 1st Strand cDNA Synthesis Kit (D6210A). 700ng of RNA; 1 mul of gene specific primer mix (10 uM); dNTPs Mix (2.5mM) 1.6. mu.l; addition of RNase-free H₂O to a total volume of 14.5. mu.l. The mixture was subjected to a water bath at 65 ℃ for 5 minutes and then placed on ice for 2 minutes. After centrifugation, RT reaction solution was added to the centrifuge tube in order: 5 XFirst-Strand Buffer 4. mu.l; 0.1M DTT 1. mu.l; 0.3 mu l of RNase Ihibitor; SuperScript III RT 0.2. mu.l. After mixing, the mixture was kept at 37 ℃ for 1 minute. Incubation at 50 ℃ for 60 minutes; enzyme inactivation was carried out by incubation at 70 ℃ for 15 minutes; the cDNA was placed in an ice bath for future use or stored at-20 ℃.

(4) qRT-PCR reaction: all cDNA samples were prepared into Real time PCR reaction system. The system is configured as follows: 2 × Master Mix 5 μ l; 10uM PCR specific primer F0.5. mu.l; 10uM PCR specific primer R0.5. mu.l; water was added to the reaction mixture to make a total volume of 8. mu.l. Centrifuge briefly at 5000 rpm. Sample adding: a. add 8. mu.l of the mixture to each well of the 384-PCR plate; b. then adding corresponding 2 mul cDNA; c. carefully sticking a Sealing Film on the Sealing Film, and centrifuging and mixing for a short time; d. placing the prepared PCR plate on ice before setting up the PCR program; the 384-PCR plate was placed on a Real time PCR instrument for PCR reaction.The PCR reaction procedure was as follows: at 95 ℃ for 10 min; 40 PCR cycles (95 ℃, 10 sec; 60 ℃, 60 sec (fluorescence collection)). Respectively carrying out Real time PCR reaction on the target lncRNA or mRNA of each sample and the internal reference beta-actin; data mining 2^-△△The analysis was performed by CT method.

(5) The qRT-PCR determination result is shown in figure 3, and the result shows that the expression of LLNLR-299G3.1 in ESCC patient/healthy control (control) plasma exosome is significantly different between two groups, specifically, the expression level of LLNLR-299G3.1 in ESCC patient plasma exosome is significantly higher than that of LLNLR-299G3.1 in healthy control plasma exosome.

Method for measuring ESCC cancer cell and normal esophagus epithelial cell LLNLR-299G3.1 expression quantity by qRT-PCR method

In the embodiment of the invention, the expression difference of LLNLR-299G3.1 in ESCC cancer cells (EC109, KYSE180, KYSE30, KYSE450, KYSE70 and TE1) and normal esophageal epithelial cells (NE3) is detected by using a qRT-PCR method, and the result is shown in FIG. 4. The results show that the expression level of LLNLR-299G3.1 in various ESCC cancer cells is obviously higher than that of normal esophageal epithelial cells.

Example 2 study of relationship between LLNLR-299G3.1 expression and ESCC cell proliferation and migration

1. Construction of LLNLR-299G3.1 overexpression Stable transformant

Primers (SEQ ID No.2 and SEQ ID No.3) are designed according to the sequence (SEQ ID No.1) of LLNLR-299G3.1 in GeneBank, the full-length human LLNLR-299G3.1 is amplified, and the sequence is directly synthesized on a lentiviral shuttle plasmid vector, namely pHBLV-CMV-MCS-3flag-EF1-ZsGreen-T2A-PURO vector (the map is shown in figure 5). Wherein, LLNLR-299G3.1 insertion interval is arranged between EcoRI and BamHI.

Firstly, amplifying a target gene by using PCR, then digesting the lentiviral shuttle plasmid vector by using EcoRI and BamHI endonucleases, and mixing the target gene with the vector quantity by 3: 1-8: 1 (vector 1. mu.l; target gene 1. mu.l; T4ligase 0.5. mu.l; 10 Xligase Buffer 1. mu.l; ddH)₂O To 10 mu l), completing connection at 14-16 ℃ for 16hrs, transforming escherichia coli competent cell DH5 alpha by a connection product heat shock method, identifying positive colonies by PCR, and performing first-generation sequencing by using sequencing primersAnd (6) verifying. The purified Plasmid was extracted using the Plasmid Maxprep Kit.

The lentiviral shuttle plasmid containing LLNLR-299G3.1 gene and its vector plasmid (pSPAX2, pMD2G) were subjected to high purity endotoxin-free extraction, respectively, and then co-transfected into 293T cells. After 6h after transfection, the medium was replaced with complete medium, after culturing for 48h and 72h, cell supernatants rich in lentiviral particles were collected, centrifuged at 4 ℃ at 2000 × g for 10min to remove cell debris, and then virus supernatants were collected using ultracentrifugation: centrifuging at 82700 Xg for 120min at 4 deg.C, and superionizing to obtain high titer lentivirus superionic liquid.

2. Construction of antisense oligonucleotides (ASO)

Based on the sequence of LLNLR-299G3.1, an oligonucleotide chain (ASO-h-ENST-00000607288-invito;) capable of complementarily binding with LLNLR-299G3.1 was synthesized to inhibit the expression of LLNLR-299G3.1 gene.

3. Cell transfection: the cells were divided into 4 groups

(1) No-load stable transformant control group (Empty Vector, NC),

(2) over-expressing LLNLR-299G3.1 stable transformant group (OE);

(3) ASO group;

(4) ASO control group (ASO-NC).

Cells in the logarithmic growth phase (TE 1 cells and KYSE30 cells as an example) were trypsinized, counted, seeded in a 6-well plate, and diluted with Opti-MEM for transfection. After the cells were overgrown after growing for about 24h, they were transfected with Lipofectamine 2000, respectively. Standing for 10-20 min, standing at 37 deg.C and 5% CO₂And incubating in a cell incubator, and collecting cells for downstream experiment detection after 24-48 hrs.

4. Cell proliferation assay:

logarithmic phase cells were collected, cell suspension concentration was adjusted, 100uL was added to each well, and 2000 cells/well were plated in 96-well plates at 37 ℃ with 5% CO₂Incubate overnight. Balancing EndoFectin-Lenti, plasmid and serum-free DMEM to + 15-25 ℃; diluting a proper amount of plasmid by using a serum-free DMEM medium, and diluting an EndoFectin-Lenti reagent by using the same medium; gently swirlAnd (3) adding the diluted EndoFectin-Lenti reagent dropwise into the plasmid solution, fully mixing uniformly, and standing at room temperature for 10-25 min to form a DNA-EndoFectin-Lenti compound. Dropwise adding the DNA-EndoFectin-Lenti compound into a culture dish while shaking; 37 ℃ and 5% CO₂Culturing for 24 h; add 10. mu.L of CCK8 solution to each well and continue incubation for 1-4h to terminate the culture. Absorbance at 450nm was measured with a microplate reader.

The cell proliferation experiment results are shown in fig. 6 and fig. 7, and compared with an unloaded vector (empty vector), LLNLR-299G3.1 overexpression can remarkably promote KYSE30 cell proliferation activity. After the expression of LLNLR-299G3.1 is inhibited by ASO (ASO1 is SEQ ID No.4 sequence; ASO2 is SEQ ID No.5 sequence; and ASO3 is SEQ ID No.6 sequence), the proliferation activities of TE1 cells and KYSE30 cells are obviously inhibited, and the results are shown in FIG. 8 (wherein, ASO1 LONG and ASO2 LONG are other two ASO sequences designed aiming at different positions of a target gene, and the results show that the effects are not ideal, so the two sequences are removed by screening).

5. Cell invasion assay: grouping when the cells grow to 70-80% and no serum is dissolved for 12-24 h; before the experiment, the transwell chamber paved with extracellular matrix is hydrated by serum-free medium for one night; the transfected cells were digested with pancreatin, suspended in cell suspension, counted on a counting plate, and the number of cells per group was the same and found to be 1.2X 10⁴Per ml; after 700. mu.l of a medium containing 15% FBS serum was added to the lower chamber of the extracellular matrix transwell chamber, the chamber was placed on top of it; adding 200 mul of prepared cell suspension into the upper chamber, and putting the cell suspension into an incubator for 24 h; taking out the upper chamber, placing into another clean 24-well hole, washing with PBS for 3 times, adding 200 μ l of pure methanol, fixing cells for 15min, sucking out, adding 200 μ l of crystal violet, and dyeing for 15 min; sucking out crystal violet, adding PBS and washing for 2 times; the number of cells was calculated by randomly selecting 5 fields using phase contrast microscopy.

Compared with an unloaded vector, LLNLR-299G3.1 overexpression can remarkably promote the migration capacity of TE1 cells and KYSE30 cells. After the expression of LLNLR-299G3.1 is inhibited by ASO (one of ASO1, ASO2 or ASO 3), the migration activity of TE1 cells and KYSE30 cells is obviously inhibited, and the result is shown in FIG. 9.

Example 3 antisense oligonucleotide (ASO) of LLNLR-299G3.1 inhibits growth of nude mouse ESCC transplantable tumors

This example uses liposomal nanoparticles to deliver antisense oligonucleotides (ASO, one of ASO1, ASO2, or ASO 3) against LLNLR-299G3.1 to inhibit the growth of nude mouse ESCC transplanted tumors. Grouping animals: collecting ESCC cell TE1 in logarithmic growth phase, injecting into nude mice subcutaneously at back of 1 × 10⁷And (4) constructing an ESCC animal model by each cell/mouse. When the tumor volume is about 30mm³Thereafter, animals were randomly divided into 4 groups of 5 animals each, and the tail vein was injected with ASO-empty-liposome nanoparticle-linker polypeptide (labeled 2: pCSA-NNPs in FIG. 10), ASO-liposome nanoparticles (labeled 3: ANPs in FIG. 10), respectively; ASO-Liposomal nanoparticle-linker polypeptides (i.e., labeled 4: pCSA-ANPs in FIG. 10), and physiological saline (blank control, labeled 1: control in FIG. 10), were injected every 7 days for 3 times. Measuring the major diameter (a) and the minor diameter (b) of the tumor every 3 days after treatment, calculating the tumor volume (V), and drawing a tumor volume growth curve:

the experimental results are shown in fig. 10, and the results show that the tumor mass of each treatment group is smaller than that of the control group and the no-load control group, and the tumor inhibition rate of each treatment group is statistically different from that of the control group (P < 0.05). The ASO-liposome nanoparticle-linked polypeptide group and the ASO-liposome nanoparticle group have statistical significance (P is less than 0.05), and the fact that the linked polypeptide links the ASO and the liposome nanoparticles is more obvious in tumor inhibition effect compared with the ASO-liposome nanoparticles.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Sequence listing

<110> Shenzhen university

<120> ESCC-related lncRNA LLNLR-299G3.1 and application thereof

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 534

<212> DNA

<213> Homo sapiens

<400> 1

tgcttgacct ccttgcttgg caacgtgggc ttctccacgg acaccacgca caccctgctg 60

gccagctcca tgtggagctg cttctcaaag aggcactgca gggtcttcaa gggcaggtgc 120

agcttcgggt aggacacacg cccatcctgc agggatgggg gtagtgaggt tgggggcttg 180

ccagagggcg acctgccctc ccaggacccc gagacagcat gggtgcacgc gtttctgcgt 240

ctcctgcaag ttgctggtgg ctatcgctga cgcggggaaa ggcgggctgc gggtaaagtc 300

agtgccagca gtgcaaacca aaggccttga ccctcctggc ctcgacccct ctagaaggga 360

cactgggcac cgtgcagggg gtggcagggg cggtgatgct gggagctggc agagcctggg 420

gagaccgttc actgcacccc cagatgttgg ctgttttctc ctcaaactca gaactgtatg 480

aatgtgaccc atccagaaat agatgaatta aaaataacaa ctaaaaaaaa aaaa 534

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cttcgggtag gacacacgcc 20

<210> 3

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cagcgatagc caccagcaac t 21

<210> 4

<211> 20

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gauagccacc agcaacuugc 20

<210> 5

<211> 20

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cuuugguuug cacugcuggc 20

<210> 6

<211> 20

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

uuucuggaug ggucacauuc 20

Claims

1. The IncRNA LLNLR-299G3.1 related to ESCC is characterized in that the gene sequence of the IncRNA LLNLR-299G3.1 is shown as SEQ ID No. 1.

2. The application of the reagent for detecting lncRNA LLNLR-299G3.1 in the preparation of the kit for diagnosing and/or prognostically evaluating ESCC; wherein the gene sequence of the lncRNA LLNLR-299G3.1 is shown as SEQ ID No. 1.

3. The use of claim 2, wherein the reagents for detecting lncRNA LLNLR-299G3.1 comprise primers for amplifying said lncRNA LLNLR-299G3.1, said primers being represented by SEQ ID No.2 and SEQ ID No. 3.

4. A kit for the diagnostic and/or prognostic assessment of ESCC, comprising reagents for the detection of lncRNA LLNLR-299G3.1 according to claim 1.

5. The kit of claim 4, wherein the reagents for detecting lncRNA LLNLR-299G3.1 comprise primers for amplifying the lncRNA LLNLR-299G3.1, the primers being shown in SEQ ID No.2 and SEQ ID No. 3; and/or the presence of a gas in the gas,

the kit comprises PCR amplification enzyme and PCR amplification buffer solution.

6. An inhibitor of the lncRNA LLNLR-299G3.1 expression of claim 1, wherein the inhibitor comprises at least one of the antisense oligonucleotides set forth in SEQ ID No.4, SEQ ID No.5, and SEQ ID No. 6.

Use of an inhibitor of lncRNA LLNLR-299G3.1 for the preparation of a medicament for the prevention and/or treatment of ESCC; wherein the gene sequence of the lncRNA LLNLR-299G3.1 is shown as SEQ ID No. 1.

8. The use of claim 7, wherein the inhibitor comprises at least one of the antisense oligonucleotides set forth as SEQ ID nos. 4, 5, and 6.

9. A medicament for the prevention and/or treatment of ESCC, comprising an inhibitor capable of inhibiting the expression of lncRNA LLNLR-299G3.1 and a pharmaceutically acceptable carrier; wherein the gene sequence of the lncRNA LLNLR-299G3.1 is shown as SEQ ID No. 1.

10. The medicament of claim 9, wherein the inhibitor comprises at least one of the antisense oligonucleotides set forth in SEQ ID nos. 4, 5, and 6.