CN107519193B

CN107519193B - Molecular diagnostic marker for early stage esophageal squamous carcinoma and application thereof

Info

Publication number: CN107519193B
Application number: CN201710774432.1A
Authority: CN
Inventors: 任静; 宋宏涛; 石小峰
Original assignee: Qingdao Yangshen Biomedical Co Ltd
Current assignee: Qingdao Yangshen Biomedical Co Ltd
Priority date: 2017-08-31
Filing date: 2017-08-31
Publication date: 2020-04-14
Anticipated expiration: 2037-08-31
Also published as: CN107519193A

Abstract

The invention relates to an early molecular diagnosis marker of esophageal squamous carcinoma and application thereof. The diagnosis caused by the existing esophageal squamous cell carcinoma diagnosis technical level is not clear, so that part of early esophageal squamous cell carcinoma patients possibly lose the optimal treatment opportunity, more accurate indexes are needed for supplement, and effective molecular markers are researched and identified by using a molecular biology method, so that the method is one of key means for assisting the existing clinical diagnosis, guiding clinical intervention and pre-cancer early warning. The early esophageal squamous carcinoma molecular marker mir-1277 and the mature miRNA thereof provided by the invention provide a potential basis for accurate diagnosis of clinical esophageal squamous carcinoma, and have important practical application values.

Description

Molecular diagnostic marker for early stage esophageal squamous carcinoma and application thereof

Technical Field

The invention relates to the field of gene detection, in particular to an early molecular diagnosis marker of esophageal squamous cell carcinoma and application thereof in clinical diagnosis and treatment, and more particularly relates to application of mir-1277 and mature miRNA thereof in preparation of an esophageal squamous cell carcinoma diagnosis preparation.

Background

Esophageal cancer is one of ten common malignant tumors of human, China is one of the countries with high incidence of esophageal cancer, and particularly Esophageal Squamous Cell Carcinoma (ESCC) is the main pathological type of esophageal cancer in China. Esophageal squamous carcinoma develops through a process from normal esophageal mucosa, inflammation, esophageal mucosal epithelial dysplasia, carcinoma in situ, intra-mucosal carcinoma, early invasive carcinoma, and late stage carcinoma. Esophageal cancer is latent, many patients have no symptoms in the early stage, more than 50 percent of patients cannot be resected when diagnosed or have imaging visible metastasis, and the overall survival rate of middle and late stage patients is about 10 percent after 5 years of operation. If the accurate diagnosis method can be found, the patient can intervene in time, and the over-treatment of the patient in the low-risk group is prevented, so that the service life of the surgical patient is prolonged. Studies have shown that by long-term follow-up of patients with early esophageal squamous cell carcinoma who undergo surgical resection, an overall 5-year survival rate of up to eighty percent is found. Therefore, early diagnosis and treatment are more critical to reducing the mortality rate of esophageal squamous carcinoma. The current imaging methods applicable to early diagnosis of esophageal squamous carcinoma include esophageal barium meal radiography, computed tomography and nuclear magnetic resonance imaging, the endoscope technology includes high-magnification high-resolution endoscope, pigment endoscope, narrow-spectrum imaging endoscope, autofluorescence imaging endoscope, laser confocal endoscope, optical continuous X-ray tomography endoscope and endoscopic ultrasound combining the imaging and the endoscope, and the pathological cytology method includes esophageal pull-off cytology examination. These methods each have advantages, but each also show some disadvantages. The unclear diagnosis caused by the technical level is likely to cause the partial early esophageal squamous carcinoma patients to lose the optimal treatment opportunity. The current situation fully shows that the current diagnosis method of esophageal squamous cell carcinoma needs to be supplemented by more accurate indexes, especially for prognosis risk stratification, early cancer accurate diagnosis and precancerous lesion canceration risk early warning of postoperative patients. The research and identification of effective molecular markers by using a molecular biological method is one of the key means for assisting the existing clinical diagnosis, guiding clinical intervention and pre-cancer early warning.

The invention carries out transcriptome sequencing on 3 samples of esophageal squamous carcinoma tissues with metastasis, 3 samples of esophageal squamous carcinoma tissues without metastasis and 6 samples of tissues beside carcinoma, and analyzes by using a bioinformatics method to find some molecular markers related to esophageal squamous carcinoma, wherein the expression level of miR-1277-5p in the tissues of carcinoma is higher than that of the tissues beside carcinoma, and further, the fluorescent quantitative PCR experimental result is consistent with the high-throughput sequencing result. The invention provides a potential molecular marker for accurate diagnosis of clinical esophageal squamous carcinoma, and has important practical application value.

Disclosure of Invention

The invention aims to provide a pharmaceutical composition for treating esophageal cancer, which is characterized by comprising the following components in part by weight:

(a) a compound or composition which down-regulates transcription of mir-1277 and/or its mature miRNA and/or inhibits the activity of mir-1277 and/or its mature miRNA;

(b) a carrier which can be accepted in pharmacy.

The mir-1277 sequence is shown in SEQ ID NO 1: accucccaaauauauauauauauguacguauguguauauaaauguauacguagauauauauguauuuuuggu ggguuu, the mature miRNAs are miR-1277-5p and miR-1277-3p, the sequences are shown in SEQ ID NO 2: aaauauauauauauauguacguau and SEQ ID NO 3: uacguagauauauauguauuuu are provided.

Further, methods employing antisense oligonucleotides, miRNA inhibitors, antagomiRs, miRNA sponges, miRNAErasers, Target Masking, and/or multi-Target antisense oligonucleotides down-regulate transcription of mir-1277 and/or its mature miRNA and/or inhibit activity of mir-1277 and/or its mature miRNA.

Further, the pharmaceutical composition inhibits the growth of esophageal squamous carcinoma cells.

The invention aims to provide application of the pharmaceutical composition in treating esophageal squamous carcinoma.

Further, the sequence of miRNAinhibitor is shown in SEQ ID NO 6.

The invention aims to provide an esophageal cancer diagnostic reagent, which detects the expression condition of mir-1277 and/or mature miRNA thereof in a sample.

Further, esophageal cancer includes esophageal squamous carcinoma and esophageal adenocarcinoma, and is preferably esophageal squamous carcinoma.

The sample is tumor tissue or peripheral blood.

Further, the esophageal squamous carcinoma diagnostic reagent detects the transcription condition of mir-1277 and/or the mature miRNA thereof in the sample based on a high-throughput sequencing method and/or a gene chip method and/or a quantitative PCR method and/or a probe hybridization method.

The high throughput sequencing method mainly refers to the second generation sequencing technology and helicos helicope proposed by 454life sciences, ABI and illumina^TMAnd single molecule sequencing techniques of pacific biosciences.

Preferably, the transcription of mir-1277 and/or its mature miRNA in the sample is detected by a second-generation sequencing method, a single-molecule sequencing method, a northern hybridization method, a miRNA expression profile chip, a ribozyme protection analysis technology, a RAKE method, in-situ hybridization and a microsphere-based flow cytometry.

Preferably, the method for quantitative PCR comprises a primer for specifically amplifying mir-1277 and/or its mature miRNA; the probe-based hybridization methods include probes that hybridize to the nucleic acid sequences of mir-1277 and/or its mature miRNA.

Further, a primer for amplifying miR-1277-5p by a tailing method is a primer with a sequence SEQ ID NO 4: aaatatatatatatatgtacgtat are provided.

The invention also aims to provide application of the mir-1277 and/or the mature miRNA thereof in preparation of an esophageal squamous cell carcinoma diagnosis tool.

Defining:

mirnas are a class of endogenous, highly conserved, non-coding small RNA molecules of about 22 nucleotides in length. The miRNA can specifically bind to the 3' untranslated region (3' UTR) of the target gene via its 5' end, and exert a regulatory effect at the post-transcriptional level. Binding of miRNA to the 3' UTR of the target gene by perfect base-complementary pairing results in degradation of the target mRNA, while binding by imperfect base-complementary pairing inhibits protein translation of the mRNA. In recent years, studies of circulating mirnas as markers of various diseases have been receiving attention. Circulating mirnas are a generic term for mirnas that are present in serum, plasma, some body fluids such as saliva, urine and cerebrospinal fluid, free from tissue. Circulating mirnas have been a new and promising area of research, and research on their use as diagnostic markers for tumors has been a hotspot in recent years.

The method for detecting the expression level of miRNA at present stage mainly comprises a miRNA detection method based on high-throughput sequencing technology, gene chips, nucleotide hybridization and PCR. The miRNA detection method based on the probe hybridization technology is a direct detection method, does not need to pre-amplify sample RNA, and comprises the technologies of a northern hybridization method, a miRNA expression spectrum chip, a ribozyme protection analysis technology, a RAKE method, in-situ hybridization, microsphere-based flow cytometry and the like.

(1) Northern hybridization

The RNA blotting technique is the most classical experimental method for detecting the RNA size of eukaryote and estimating the abundance of the RNA. The basic principle is as follows: firstly fixing miRNA samples on carriers (such as silicon chips, microspheres or membranes and the like), hybridizing the miRNA samples with labeled probes, washing redundant hybridization probes, and then carrying out signal detection; or fixing a DNA probe complementary with a target miRNA sequence on a carrier, hybridizing the DNA probe with a labeled sample miRNA, and detecting a signal. The signal labeling method comprises isotope labeling, fluorescence labeling, nanogold labeling and the like.

(2) miRNA expression profile chip

The principle is also the use of labeled probes to detect target molecules on a solid support. By designing miRNA genes and internal reference sequences on the chip, the expression level of the corresponding miRNA in the sample can be accurately analyzed. The gene chip has the advantage of high flux, and can detect all the expressions of hundreds of genes in the same sample at one time. Liquid phase chips (Liquid chips) developed by Luminex corporation are also called Multi-functional suspension array (MASA), and are a new generation of biochip technology. The liquid phase chip system is formed by taking a plurality of small spheres as main substrates, wherein each small sphere is fixed with different probe molecules, each spherical substrate for marking the probes is provided with a unique color number in order to distinguish different probes, and the small spheres are suspended in a liquid phase system to form the liquid phase chip system. The system can simultaneously carry out rapid qualitative and quantitative analysis on a plurality of different molecules in the same micro sample, and the detection technology is called as FMAP (Flexible multianalyte profiling) technology. The molecular hybridization is carried out in a suspension solution, and the detection speed is extremely high.

(3) Ribozyme Protection Assay (RPA)

The miRNA detection can also adopt a ribozyme protection analysis technology, the marked probe and an RNA sample to be detected are mixed, hybridization is carried out after thermal denaturation, the unhybridized RNA and redundant probe are digested by single-stranded nuclease, the protected RNA molecule is purified after the nuclease is thermally inactivated, and finally the probe is separated through denaturing PAGE electrophoresis and is developed. The new method based on liquid phase hybridization is simple and rapid, has high sensitivity, but can only be used for analyzing the known miRNA.

(4) RAKE method

RAKE (RNA-mediated array-based Klenow enzyme) is a method of hybridizing miRNA with an immobilized DNA probe using Klenow fragment of DNA polymerase I on the basis of miRNA microarray. The RAKE can sensitively and specifically detect the miRNA, and is suitable for rapidly screening all known miRNAs in a large quantity. The miRNA expression profile can be detected in specific cells and tumors. Moreover, the RAKE method also allows miRNA to be isolated from formalin-fixed paraffin-embedded tissues and analyzed, opening the door to miRNA analysis from archived specimens.

(5) In situ hybridization (in situ hybridization)

The in situ hybridization technology can intuitively understand the miRNA expression mode, is a simpler method for observing the time-space expression of the miRNA, and the common marking mode comprises digoxin, biotin, fluorescent marking and the like. Locked nucleic Acid based in situ hybridization (LNA) based in situ hybridization (LNA-ISH) is currently the more commonly used probe format.

(6) Microsphere-based flow cytometry

The method organically combines flow cytometry detection and chip technology, and has the characteristics of high flux, high detection speed, high sensitivity, good specificity and the like.

(7) Real-time fluorescent quantitative PCR technology (Real-time PCR, RT-PCR)

The fluorescence detection PCR instrument can draw a dynamic change curve for the accumulation rate of the amplified sequence in the whole PCR process. The greater the initial concentration of target sequence in the reaction mixture, the fewer PCR cycles (typically expressed in terms of a particular threshold cycle number Ct) are required to obtain a particular yield of amplified product. Since mirnas are only 22nt in length, conventional qRT-PCR is not suitable for amplifying such short fragments. There are several real-time quantitative PCR methods for miRNA, such as tailing method, neck ring method, etc. The neck ring method is an ideal miRNA detection qRT-PCR method: firstly, designing a special stem-loop structure primer, carrying out reverse transcription by taking miRNA to be detected as a template to synthesize a first cDNA chain, wherein one end of the cDNA is a stem-loop primer, the stem-loop structure is opened to increase the length of the cDNA, and then designing a primer by taking the synthesized cDNA as a template to carry out real-time quantitative PCR amplification. qRT-PCR has the advantages of high specificity, good sensitivity, rapidness, simplicity and the like.

(8) Sequencing method

Most known mirnas are found and identified by cDNA clone sequencing. The method requires that a cDNA library of miRNA is constructed firstly, then PCR amplification is carried out, and an amplification product is cloned to an expression vector for sequencing. Takada developed an improved amplification cloning method (miRNA amplification profiling, mRAP) in which a linker was first ligated to the 3' end of the miRNA, followed by reverse transcription using a reverse transcription primer complementary to the linker. Because a particular reverse transcriptase has terminal deoxynucleotidase activity, some nucleotides (mainly deoxycytidylic acid) will be ligated to the 3' end of the reverse transcribed cDNA strand. After annealing of the 5' end linker to the poly (C) sticky end of the cDNA strand, PCR amplification of the cDNA can be achieved by adding a pair of common primers. Because mRAP is highly sensitive, the expression level of miRNA in a small amount of tissues can be directly detected by using cloning and sequencing technology. The tag sequence cloning method is a mirage (miRNA SAGE) cloning method which is developed on the basis of a gene expression Series Analysis (SAGE) technology and has higher detection efficiency.

High-throughput sequencing (also called next generation sequencing) is a revolutionary change to the conventional sequencing, and sequences of hundreds of thousands to millions of DNA molecules are determined at one time, so that the sequencing efficiency is greatly improved. The large-scale sequencing technology greatly improves the reading speed of genetic information of a plurality of species, and provides guarantee for acquiring sequence information of all miRNA and decrypting miRNA maps. High throughput sequencing at the same time makes it possible to perform a detailed global analysis of the transcriptome and genome of a species and is therefore also referred to as deep sequencing.

An RNA-based microRNA gain of function technique is to increase the level of miRNAs by exogenous supplementation of precursor substances for their synthesis. For example, short hairpin-like RNA (shRNA) consistent with the sequence of an endogenous miRNA can be artificially synthesized, polymerase II or III is used as a promoter, a virus is used as a vector to transfect cells, and the cells are modified by Dicer enzyme and then loaded into RISC to perform the function, which is equivalent to increasing the level of pre-miRNA, so that the effect is stable and durable.

The gene specificity miR Mimics technology avoids nonspecific action of miRNA and genes. The artificially synthesized specific oligonucleotide chain complementarily combined with the 3' UTR of the target gene can play the same post-transcriptional regulation role as miRNA.

The pharmaceutically acceptable carrier included in the pharmaceutical composition of the present invention is a carrier generally used in the preparation, and includes lactose (lactose), dextrose (dextrose), sucrose (sucrose), sorbitol (sorbitol), mannitol (mannitol), starch, gum arabic, calcium phosphate, alginate (alginate), gelatin (gelatin), calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone (polyvinylpyrrolidone), cellulose (cellulose), water, syrup, methyl cellulose (methyl cellulose), methyl hydroxybenzoate (methyl hydroxybenzoate), propyl hydroxybenzoate (propyl hydroxybenzoate), talc, magnesium stearate (magnesium stearate), mineral oil (mineral oil), and the like, but is not limited thereto.

The pharmaceutical compositions of the present invention are formulated according to methods that can be readily practiced by those of ordinary skill in the art using pharmaceutically acceptable carriers and/or excipients, and can be prepared in unit dosage form or in multi-volume containers. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oily or aqueous medium, or may be in the form of a extract, powder, granule, tablet or capsule, and may further include a dispersant or stabilizer.

Drawings

FIG. 1 is a graph of relative expression of miR-1277-5p

FIG. 2 is ROC curve diagram of miR-1277-5p for diagnosing esophageal squamous carcinoma

FIG. 3 is a diagram for detecting the influence of miR-1277-5p on cell growth by a CCK8 method

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. Those of ordinary skill in the art will understand that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.

EXAMPLE 1 Collection of samples

Samples of 3 metastatic esophageal squamous carcinoma tissues, 3 non-metastatic esophageal squamous carcinoma tissues and 6 paracarcinoma tissues are all from samples excised by surgery in hospital 2015 from 6 months to 2017 from 1 month, all samples are placed in a liquid nitrogen tank within 10 minutes of being separated, and then transferred to a refrigerator at-80 ℃ for storage.

Example 2 Total RNA extraction

1 extraction method

1) 80mg of the tissue mass were taken, and 800. mu.l of lysine/Binding buffer was added to homogenize the tissue mass using a homogenizer. During the homogenization process, the sample is kept at a low temperature on ice.

2) An additional 1/10 volumes of Homogenate Additive were added to the homogenized tissue samples and left on ice for 10 min.

3) Add the same volume of water saturated phenol as the lysine/Binding buffer, shake for 45s, centrifuge at 10,000 Xg for 5min at room temperature.

4) Carefully remove the supernatant into a new tube, add 1.25 volumes of absolute ethanol, mix well, transfer to a purification column, 10,000 Xg, centrifuge for 15s, and pour off the liquid in the collection tube. Since the maximum volume of the column was only 700. mu.l, this procedure was repeated until all the supernatant was filtered.

5) Adding 700 μ l miRNA eluent 1 into the centrifugal column, centrifuging at room temperature for 15s at 10,000 Xg, pouring off the collected liquid, and replacing with a new collecting tube.

6) This step was repeated once more with 500. mu.l of the eluent 2/3 being applied to the spin column at 10,000 Xg and centrifuged for 10 s.

7) Centrifuge for 1min at 10,000 Xg and discard excess liquid.

8) The above liquid was transferred to a new centrifuge tube, treated with 100. mu.l of 95 ℃ preheated DEPC for 30s, 10,000 Xg, and centrifuged.

9) The concentration of RN A and the ratio of 260nm/280nm were determined using nanodrop.

10) The RNA obtained was stored in a freezer at-80 ℃.

2 extraction criteria

Determination of RNA concentration and 260nm/280nm ratio: the purity requirement of the total RNA is that the OD260/OD280 value should be between 1.8 and 2.2; detection of RNA integrity: the integrity of the RNA was checked by electrophoresis on a 1% agarose gel;

according to the requirements of sequencing companies, the total amount of small RNA sequencing is more than 3 mug, and the concentration is more than 300 ng/mul.

Example 3 sequencing and data analysis

The sequencing library was set up and sequenced on the computer by sequencing company using the HiSeq2000 sequencer from Illumina.

Statistical analysis was performed on data provided by sequencing companies with fdr <0.001, log2(FC) absolute >1, and the difference between the mean values of the two sets of counts was greater than 100. For artificially selecting miRNA with obvious differential expression in filtering, several molecular markers related to esophageal squamous cell carcinoma, which have never been reported before, enter the research scope, wherein miR-1277-5p is differentially expressed between a metastasis group and a diseased group, but the difference is not obvious, and the expression level in the metastasis and diseased cancer tissues is obviously higher than that in paracancerous tissues.

Example 4Real-time PCR detection of miRNA expression in esophageal squamous carcinoma peripheral blood samples

1, sample collection:

peripheral blood samples of 26 esophageal squamous carcinoma patients and 31 healthy control peripheral blood samples come from hospitals, and the diseased groups are confirmed by pathological detection.

2, extraction of miRNA:

GenEluteTM plasma/serum RNA miniprep kit (product number RNB500) from sigma was used, and the detailed procedures were according to the kit instructions.

3miRNA reverse transcription

TABLE 1RT systems

Keeping the temperature of 37 ℃ for 60min on an ABI 9700 type PCR instrument to ensure that the reverse transcription reaction is complete, and stopping the reaction at 95 ℃ for 5 min. 80. mu.l of nucleic-free H was added₂O diluted to 100. mu.l was stored in a-20 ℃ freezer for subsequent experiments.

4 fluorescent quantitative PCR

TABLE 2RT-PCR System

Expression detection of miRNAs 3 parallel tube reactions were set up each time, and the microRNA-specific primer primers are shown in Table 3 with the universal snRNA U6 as internal reference.

PCR procedure: 10min at 95 ℃; 40 cycles (95 ℃ for 10s, 60 ℃ for 30 s). Detecting product specificity by using a melting curve after circulation is finished: the temperature was slowly raised from 60 ℃ to 97 ℃ and fluorescence signals were collected 5 times per ℃ C. 5 statistical analysis

Analysis was performed using originpro8.1 software. Statistical methods the comparison between the means was performed using the t-test, and P <0.05 (significant difference) and P <0.01 (very significant difference) were determined to be statistically significant. The result shows that compared with a healthy control, the miR-1277-5p of the esophageal squamous carcinoma group is obviously increased by about 2.4 times of that of the control group (see figure 1), and the RT-PCR result is consistent with the result of high-throughput data analysis.

Example 5 evaluation analysis of diagnostic efficacy

The method for evaluating the efficiency of a single miRNA molecule or a diagnosis model is to establish a Receiver Operating Characteristic (ROC) curve and judge the diagnosis capability by calculating the Area under the curve (Area UnderCurve). Data about esophageal squamous carcinoma in a TCGA database are downloaded to obtain a data set (comprising 13 control groups and 95 case groups), and then analysis is carried out, and the result shows that the AUC of mir-1277 for diagnosing esophageal squamous carcinoma is 0.779 (see figure 2), which indicates that mir-1277 has a better reference value in diagnosing esophageal squamous carcinoma.

Example 6 culture and transient transfection of esophageal squamous carcinoma cell lines

Firstly, material preparation:

esophageal squamous carcinoma cell line EC109 was purchased from Shanghai cell Bank of Chinese academy of sciences.

Lipofectamine^TM2000Transfection Reagent(Invitrogen)。

RPMI 1640 and DMEM medium were purchased from GIBCO, newborn bovine serum and fetal bovine and serum from PAA.

The miR-1277-5p sequence is issued to a synthesis company, and the company is requested to chemically synthesize miR-1277-5p mimics (SEQ ID NO 5: aaauauauauauauauguacguau), miR-1277-5p inhibitor (SEQ ID NO 6: auacguacauauauauauauauuu) and nonspecific controls thereof.

Second, Experimental methods

1. Cell culture

The esophageal squamous carcinoma cell line EC109 adopts RPMI 1640 medium containing 10% newborn calf serum and is cultured at 37 ℃ and 5% CO₂Subculturing under the condition of saturated humidity, washing the cells for 2 times by PBS when the cells are cultured to the density of about 80 percent of adherent density, digesting the cells by 0.25 percent of pancreatin and stopping the digestion by a complete culture medium containing serum, subculturing according to the required proportion, and performing subsequent experiments by adopting the cells in the logarithmic phase.

2. Cell cryopreservation and recovery

Digesting the cells by using a proper amount of 0.25% pancreatin and stopping the digestion by using a complete culture medium containing serum, adding a cryopreservation solution containing 10% DMSO to prepare a single cell suspension, adding 1mL of the single cell suspension into a sterile cryopreservation tube, gradually cooling, and storing in liquid nitrogen. The freezing procedure is as follows: 30min at 4 ℃, 1-2h at-20 ℃ and overnight at-80 ℃, and then transferring to a liquid nitrogen tank for storage. When the cells are recovered, the cells are quickly thawed in a water bath at 37 ℃ and then transferred to a culture medium for culture. And replacing the culture solution after 12-16 hours for conventional culture.

3. Transient transfection of miRNA

The operation is according to Lipofectamine^TM2000 reagent instructions. Good-growing EC109 cells were seeded in 6-well plates 24h before transfection, and the cell count was about 4X 10⁵L, experiments were carried out with cell confluency of 70-80% on the day of normal culture up to transfection. Adding 100nM miR-1277-5p mimics/miR1277-5p inhibitor into 250 mul serum-free 1640 culture medium, and gently mixing uniformly; another 5. mu.l Lipofectamine diluted with 250. mu.l serum-free 1640 medium^TM2000 liposome, mixing gently; mix and incubate for 20min at room temperature to form the transfection complex: then adding the mixture into a cell culture medium, gently mixing, culturing for 4-6h, and replacing with a complete culture medium containing 10% calf serum. In this case, nonspecific mix Negative Control (mix NC) and inhibitor Negative Control (inhibitor NC) sequences were used as controls.

And extracting total RNA of the cells after culturing for 24-48h, performing reverse transcription to obtain cDNA, and detecting the change of miR-1277-5p expression after transient transfection by real-time quantitative PCR.

4. Results of the experiment

Transient transfection is carried out by adopting a cationic liposome method, and miR-1277-5p mimics or miR-1277-5pinhibitor and a corresponding Control sequence Negative Control (NC) are respectively transfected into the esophageal squamous carcinoma cell strain EC 109. After 48h of transfection, total cellular RNA was extracted. And (3) detecting the expression of miR-1277-5p by real-time quantitative PCR (polymerase chain reaction) by taking U6 as an internal control. The results show that: compared with a control group, after the EC109 transfects miR-1277-5p mimics, the expression of miR-1277-5p is increased by about 3.3 times; after miR-1277-5p inhibitor is transfected, the expression is reduced by nearly 71%. The results show that the expression of miR-1277-5p can be effectively up-regulated or down-regulated by transient transfection of miR-1277-5pmimics and miR-1277-5p inhibitor, and the results are reliable and can be used for subsequent experiments.

Example 7 Effect of miR-1277-5p transfection on growth of human esophageal squamous carcinoma cells

The CCK-8 method was carried out using the Cell Counting Kit-8 Kit of Dojindo, Japan. Cell transfection procedure reference example 6. Viable cells were counted 24h after transfection, old medium was aspirated, and freshly prepared CCK8 detection reagent was added to each well. The detection reagent (100 microliter 1640 culture medium and 10 microliter CCK8 per well) with the corresponding number of the detected wells is prepared, and 100 microliter of the mixture is added into each well after mixing uniformly. Meanwhile, a group of blank controls is set, namely a group of cells which only have CCK8 detection reagent and are not detected. The cells are put into an incubator for continuous culture, and after 2h, an enzyme-labeling instrument detects OD 450. And sequentially adding CCK8 detection reagents at the

same time points

48h, 72h and 96h after transfection, and detecting OD450 after incubation for 2 h. Cell growth curves were plotted against the mean and standard deviation of each group.

And (3) detecting the influence of miR-1277-5p on the in vitro growth of the esophageal squamous cell carcinoma cells by adopting a CCK-8 method. The result shows that the cell growth can be obviously promoted after miR-1277-5p is over-expressed in EC109 cells (miR-1277-5 p mimics are transfected), and the cell survival capability can be reduced after miR-1277-5p is inhibited (miR-1277-5 p inhibitors are transfected) (see figure 3).

While the invention has been described with reference to various preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the essential scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof.

Therefore, the present invention is not intended to be limited to the particular embodiments disclosed herein for carrying out the present invention; but that the invention will include all embodiments falling within the scope of the appended claims.

Sequence listing

<110> Beijing, the deep biometric information technology GmbH

<120> esophageal squamous carcinoma early-stage molecular diagnosis marker and application thereof

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Claims

1. A pharmaceutical composition for treating esophageal cancer, comprising: (a) a compound or composition which down-regulates transcription of mir-1277 and/or its mature miRNA and/or inhibits the activity of mir-1277 and/or its mature miRNA; (b) a carrier which can be accepted in pharmacy.

2. The pharmaceutical composition of claim 1, wherein the mir-1277 and/or its mature miRNA is down-regulated and/or the mir-1277 and/or its mature miRNA activity is blocked by a method employing antisense oligonucleotides, mirnaainhibitors, antagomiRs, miRNA sponges, miRNA Erasers, Target Masking, and/or multi-Target antisense oligonucleotides.

3. Use of a pharmaceutical composition according to claims 1-2 for the manufacture of a medicament for the treatment of esophageal squamous carcinoma.

4. The pharmaceutical composition of claim 2, wherein the miRNA inhibitor sequence is set forth in SEQ ID NO 6.

5. The application of the diagnostic reagent for detecting the expression condition of mir-1277 and/or mature miRNA thereof in the sample in the preparation of an esophageal squamous cell carcinoma diagnostic tool.

6. The use of claim 5, wherein the sample is tumor tissue or peripheral blood.

7. The use according to claim 5, wherein the diagnostic reagent detects the transcription of mir-1277 and/or its mature miRNA in the sample based on high throughput sequencing methods and/or gene chip methods and/or quantitative PCR methods and/or probe hybridization methods.

8. The use according to claim 7, characterized in that the quantitative PCR-based method comprises primers specifically amplifying mir-1277 and/or its mature miRNA; probe-based hybridization methods include probes that hybridize to the nucleic acid sequence of mir-1277 and/or its mature miRNA.

9. The use according to claim 5, wherein the transcription of mir-1277 and/or its mature miRNA in the sample is detected using a second generation sequencing method, a single molecule sequencing method, a northern hybridization method, a miRNA expression profiling chip, a ribozyme protection analysis technique, a RAKE method, in situ hybridization, microsphere-based flow cytometry.