CN108721316B - Application of marker miR-652-5p in medicines and kits for metastasis, prognosis and treatment of esophageal squamous carcinoma - Google Patents

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to application of a marker miR-652-5p in medicines and kits for metastasis, prognosis and treatment of esophageal squamous cell carcinoma. The invention discloses an application of microRNA-652-5p in preparing a medicine for inhibiting tumor, an application of microRNA-652-5p in preparing a medicine for inhibiting esophageal squamous cell carcinoma metastasis and an application of a molecular marker microRNA-652-5p in preparing a diagnostic kit for predicting esophageal squamous cell carcinoma metastasis. The invention can effectively detect the molecular level to judge the mark of the metastasis and the targeted therapy of the esophageal squamous cell carcinoma patient by utilizing the molecular biology technology, thereby providing convenience for individual therapy of the diseases. Meanwhile, the method has important guiding significance for the development of follow-up clinical research.

Description

Application of marker miR-652-5p in medicines and kits for metastasis, prognosis and treatment of esophageal squamous carcinoma

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to application of a marker miR-652-5p in medicines and kits for metastasis, prognosis and treatment of esophageal squamous cell carcinoma.

Background

Esophageal cancer is one of the most serious malignant tumors harming the health of people in China, and according to the latest statistical result of the world health organization in 2013: the incidence rate of esophageal cancer is 5 th in malignant tumor and the death rate is 4 th in China. The number of people dying from esophageal cancer in China is 19,7472, and is more than half of the total death number of esophageal cancer all over the world. Meanwhile, because of the difference of east and west people and the difference of pathogenesis and other aspects of Esophageal cancer, the characteristics of Esophageal cancer in China are obviously different from those of low-incidence areas (lower chest segment and multiple adenocarcinoma) in Europe and America and the like, the number of breast mid-segment and squamous cell carcinoma is more, and the proportion of Esophageal Squamous Cell Carcinoma (ESCC) is up to more than 90%. However, the molecular mechanism of the occurrence and development of esophageal cancer in China is not clear, so that the esophageal cancer cannot be treated by aiming at the cause of the disease. Therefore, the molecular mechanism of the occurrence and development of esophageal cancer is actively explored, and the molecular mechanism plays an important role in preventing and treating esophageal squamous cell carcinoma.

By utilizing the molecular biology technology, the molecular level detection can be effectively carried out to judge the signs of metastasis, prognosis and targeted therapy of malignant tumor patients. Thereby providing convenience for individual treatment of the diseases.

MicroRNA is a small piece of single-stranded non-coding RNA of about 19-22 nucleotides in length. It controls gene expression by binding the complementary sequence of the 3' untranslated region of its target gene through defective base pairing, thereby down-regulating expression of the target gene at the transcriptional or translational level. Micrornas have recently become important and revolutionary conserved regulators of many pathophysiological processes, from growth to cancer. The effect of the same microRNA may differ in different malignancies. One microRNA can have a plurality of target genes belonging to different paths, and one gene can be regulated and controlled by several microRNAs. Scientists have developed techniques to identify specific gene expression markers that are relevant to tumor staging and patient prognosis to improve prognosis and treatment. Comprehensive transcriptional studies have been used to identify prognostic markers based on gene expression, but none have been clinically applied to date. microRNA tumor profiles appear to enable a more accurate determination of the classification of tumor subtypes compared to classical mRNA profiles. Increasing evidence supports specific microRNA markers for solid tumors.

Meanwhile, more and more target genes of microRNA play an important role in the occurrence of esophageal squamous cell carcinoma, such as miR-17, miR-20a, miR-92b and the like. Currently, scientists have limited recognition of microRNA target genes. In the molecular framework, the mature microRNA becomes a microRNA-induced silencing complex (miRISC) after being charged. This complex contains the Argonaute family (a large family of proteins) that react with complementary sites usually located in the untranslated region at the 3' end of the target gene. The current model suggests that the reaction of microRNA with its target gene originates from a short 6 to 8 nucleotide fragment called "seed sequence" located at the 5' end of the microRNA. The MicroRNA-induced silencing complex is capable of reconfiguring target genes to a specific interval in which translation arrest and mRNA decay are addressed. Numerous studies have demonstrated microRNA-induced mRNA destabilization. Combined computational prediction, measurement of mRNA expression profiles represents an effective method for identifying functional microRNA-target relationships. In esophageal squamous carcinoma, many different micrornas dysregulate, may have oncogenic or oncosuppressive effects, and predict prognosis. Identification of microRNA capable of predicting esophageal squamous carcinoma patient prognosis is an important finding, which indicates that microRNA may play an important role in tumor progression. The metastasis development and prognosis may be significantly different for patients with esophageal squamous cell carcinoma of similar clinical pathological characteristics or of the same stage. Molecular markers can help physicians identify high-risk patients for administering individualized treatment to patients, thereby improving survival.

The inventor researches esophageal squamous carcinoma in China by using microRNA genes and a qRT-PCR technology to find that the expression level of microRNA-652-5p in an esophageal squamous carcinoma tissue is obviously lower than that of a paracancer normal tissue, and the expression level of microRNA-652-5p in peripheral blood of an esophageal squamous carcinoma patient is obviously lower than that of a normal healthy person; the expression level of the microRNA is obviously and negatively related to clinical staging and lymph node metastasis. Research shows that the microRNA-652-5p can regulate and control the growth and cell invasion and transfer capacity of esophageal squamous carcinoma cell strains. The overexpression of the microRNA-652-5p can inhibit the proliferation of esophageal squamous carcinoma cells and the invasion and metastasis capacity of the cells. Conversely, the down-regulation of microRNA-652-5p leads to the increase of the capacity. The evidence also provides evidence for the role of the microRNA-652-5p in the invasion and metastasis and treatment of esophageal squamous cell carcinoma. Therefore, the microRNA-652-5p has a very definite cancer inhibition effect, and a cancer inhibition preparation aiming at the microRNA-652-5p can be developed clinically as clinical treatment.

Tumor molecular markers (Tumor markers) are chemical species that reflect the presence of tumors. They are not existed in normal adult tissue but only in embryonic tissue, or their content in tumor tissue is greatly greater than that in normal tissue, and their existence or quantity can indicate the nature of tumor, so that it can know the tissue generation, cell differentiation and cell function of tumor, and can help diagnosis, classification, prognosis and treatment guidance of tumor.

Disclosure of Invention

In order to solve the technical problem that the risk of esophageal cancer metastasis cannot be judged by effectively performing molecular level detection at present, the invention provides a molecular marker microRNA-652-5p for predicting esophageal squamous carcinoma metastasis and application thereof in medicines and kits.

In order to solve the technical problem, the invention is solved by the following technical scheme:

the invention provides the application of the following substances in preparing the medicine for inhibiting tumor:

a）microRNA-652-5p；

b) a recombinant plasmid containing a non-coding gene of microRNA-652-5 p.

Wherein the sequence of the microRNA-652-5p is as follows: 5'-CAACCCUAGGAGAGGGUGCCAUU-3' are provided.

In one possible embodiment in combination with the first aspect, the tumor is esophageal cancer.

In a second aspect, the invention provides a use of a substance comprising:

a）microRNA-652-5p；

b) a recombinant plasmid containing a non-coding gene of microRNA-652-5 p.

In one possible embodiment, in combination with the second aspect, the esophageal cancer is esophageal squamous carcinoma.

In a third aspect, the invention provides molecular markers for esophageal cancer metastasis, prognosis and therapy prediction

The molecular marker is microRNA-652-5 p.

With reference to the third aspect, in one possible embodiment, the esophageal cancer is esophageal squamous carcinoma.

The fourth aspect of the invention provides a molecular marker microRNA-652-5p for preparing an esophageal cancer metastasis

The application of the diagnosis kit for prognosis and treatment prediction.

With reference to the fourth aspect, in one possible embodiment, the esophageal cancer is esophageal squamous carcinoma.

The molecular marker is microRNA-652-5p, which is called miR-652-5p for short.

Compared with the prior art, the invention has the following beneficial technical effects:

1) the invention provides a molecular marker miR-652-5p, which can effectively perform molecular level detection to judge the esophageal squamous cell carcinoma metastasis condition and target treatment by using a molecular biology technology, and further provides convenience for individually treating the diseases.

2) The invention provides application of a molecular marker miR-652-5p in preparation of a diagnostic kit for predicting esophageal squamous cell carcinoma metastasis, and the kit not only has high accuracy, but also has important guiding significance for development of subsequent clinical research.

Drawings

FIG. 1 is a graph comparing the expression level of miR-652-5p in esophageal squamous carcinoma tissues and paracarcinoma normal esophageal tissues.

FIG. 2 is a graph comparing the expression level of miR-652-5p in the tissue with or without lymph node metastasis esophageal squamous carcinoma.

FIG. 3 is a graph comparing the expression level of miR-652-5p in esophageal squamous carcinoma tissues in stages III + IV and I + II.

FIG. 4 is a graph comparing the expression level of miR-652-5p in peripheral blood of patients with and without esophageal squamous cell carcinoma.

FIG. 5 is a graph of the expression ratio of miR-652-5p in peripheral blood of patients with esophageal squamous cell carcinoma who have lymph node metastasis.

FIG. 6 is a graph comparing the expression level of miR-652-5p in peripheral blood of patients with esophageal squamous carcinoma at stages III + IV and I + II.

FIG. 7 is a comparison graph of survival rate of patients with miR-652-5p low expression group whose survival time is significantly lower than that of patients with miR-652-5p high expression group.

FIG. 8 is a graph of miR-652-5p high-expression of the invention capable of inhibiting cell proliferation of esophageal squamous carcinoma cell strains.

FIG. 9 is a diagram of miR-652-5p high-expression energy for inhibiting cell plate clone formation of esophageal squamous carcinoma cell lines.

FIG. 10 is a graph of miR-652-5p high-expression of the invention capable of inhibiting cell invasion and metastasis of esophageal squamous carcinoma cell lines.

Detailed Description

The invention is further described with reference to the following drawings and examples, which are not intended to limit the invention in any way.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The experimental methods under specific conditions not specified in the examples are generally conventional in the art.

The detection method related to the embodiment comprises the following steps of microRNA probe design, extraction, in-situ hybridization, qRT-PCR verification, cell proliferation, clone colony formation and invasion transfer experiment. In the future clinical application, only two technologies of microRNA extraction and qRT-PCR are used. Both methods are routine for those skilled in the art, therefore, the model is easily generalized in the clinic.

The method for searching markers of esophageal squamous carcinoma metastasis, prognosis and targeted therapy in the embodiment is as follows:

patient and specimen

Tissue specimens of esophageal squamous carcinoma patients operated in our hospital from 1 month and 1 month in 2009 to 12 months and 30 months in 2013 and paracarcinoma normal esophageal tissue paraffin specimens more than 5 cm away from tumor tissues are collected for 100 cases, and finally the follow-up time is 2017 and 12 months. 110 cases of peripheral blood of esophageal squamous carcinoma patients and 57 cases of peripheral blood of normal healthy patients operated in our hospital from 1 month 1 day of 2011 to 12 months and 30 days of 2013 are collected and stored at-80 ℃. Clinical staging is carried out according to the TNM staging standard formulated by the International anticancer Union (UICC) in 2009, all patients do not receive any radiotherapy before operation, and all patients receive postoperative adjuvant chemotherapy after operation.

Second, RNA extraction, quality detection, chip hybridization and data processing

Paraffin embedded tissue processed by lemon oil fine dewaxing

Pretreatment of paraffin-embedded tissue samples: if the paraffin-embedded tissue is sliced, about ten slices with the thickness of not more than 50 μm are selected, surrounding paraffin is removed as much as possible, and the slices are placed in a 1.5mL centrifuge tube.

If the paraffin-embedded tissue is a single piece, the tissue can be gently scraped off with a sharp blade and placed in a 1.5mL centrifuge tube to avoid scraping off paraffin as much as possible. The amount of the scraped tissue is controlled within 100 mg.

Dewaxing lemon olein:

adding 1ml of limonene (Limonen) into the centrifuge tube, and shaking and mixing uniformly for 5min at 55 ℃. Centrifuge at 13,200 rpm for 2min at room temperature and discard the supernatant. This step was performed 3 times in total.

Adding 1ml of absolute ethyl alcohol into a centrifuge tube, shaking and uniformly mixing for 2min at 55 ℃, centrifuging for 2min at room temperature of 13,200 rpm, and discarding the supernatant. This step was repeated once.

The supernatant was discarded, centrifuged briefly at room temperature, and the remaining absolute ethanol was aspirated by a pipette.

The tube was placed in a vacuum desiccator and pumped to dry the tissue to a dry powder.

(II) extraction of RNA and microRNA of esophageal tissue specimen

1. Extraction of tissue and peripheral blood RNA

The whole procedure was carried out exactly as described in the Trizol kit, in a RNase-free environment, using test tips and solutions that were routinely treated with 1% DEPC water (Tris excluded) at room temperature overnight. Wherein DEPC (diethyl pyrocarbonate) is named in Chinese.

(1) Respectively taking 50-lOOmg of each of the frozen esophageal cancer tissue and paracancer normal tissue, putting the frozen esophageal cancer tissue and paracancer normal tissue into a 1.5ml Eppendorf tube in which 0.5ml of Trizol lysate is added in advance, continuously grinding the frozen esophageal cancer tissue and paracancer normal tissue into a slurry state by using a homogenizer, and adding the Trizol lysate to 1 ml.

(2) Incubating for 5 minutes at 15-30 ℃ to completely dissolve the nucleoprotein complex, adding 0.2 ml of chloroform into 1ml of Trizol reaction solution, strongly shaking for 15 seconds, and incubating for 2-3 minutes at 15-30 ℃.

(3) After centrifugation at 12,000g for 15 minutes at 4 ℃ the supernatant was transferred to another Eppendorf tube at a volume of about 60% of the volume of the Trizol reaction solution added.

(4) And (3) RNA precipitation: 0.5ml of isopropanol is added to every 1ml of Trizol, and the mixture is incubated for lO minutes at 15-30 ℃.

(5) Centrifuging at 2-8 ℃ and less than or equal to 12 at 000g for 10 minutes.

(6) The supernatant was discarded and the lml rinse was carried out 2 times with 70% ethanol (made with DEPC treated triple distilled water).

(7) 7 at 2-8 ℃, and centrifuging for 5 minutes at 500 g.

(8) The RNA was vacuum dried and dissolved in 50. mu.l DEPC water. Storing at-70 deg.C for use.

(9) RNA quantification and electrophoretic identification

A. Mu.l of RNA solution is taken and added with 100 mu.l of water, after being mixed evenly, the ratio of A260 to A280 to A260/A280 is measured by an ultraviolet-visible spectrophotometer, and the RNA concentration is read. Should be 1.8 to 2.0.

B.1.2% formaldehyde denaturing agarose gel electrophoresis for identifying RNA quality:

0.3% for electrophoresis tankH ₂0₂Soaking for 30 min, washing with DEPC water and air drying.

② preparing gel (20ml) containing 0.24g of agarose, 17.4ml of RNAase-free water, 2ml of 10 XMOPS, 0.6ml of 37% formaldehyde and EB lml, adding water into the agarose, heating and melting the agarose in a microwave oven, adding 10 XMOPS, and adding the formaldehyde and the EB after the gel is cooled to 60 ℃. Then pouring gel into the gel groove, inserting a comb, horizontally placing for use after solidification.

The non-RNAase water is DEPC treated water, which is obtained by inhibiting or removing RNAase possibly existing in water by using DEPC, and then sterilizing the water at high temperature to decompose DEPC in the water, thereby removing harm to human.

Wherein MOPS is 3- (N-malineline) propanesulfonic acid 10.3g, adding 50mM NaAc 400ml, adjusting pH to 7.0 with 2M NaOH, adding 0.5M EDTA 10ml, adding DEPC H₂O to 500 ml. Sterile suction filtering, and storing at room temperature in dark place.

Wherein EB is the English abbreviation for ethidium bromide.

Thirdly, the gel is pre-electrophoresed for 5min, and the voltage is reduced to 5V/cm.

Fourthly, taking a proper amount of RNA, adding 2 mul of electrophoresis buffer solution (10X), 3.5 mul of formaldehyde and 0 mul of formamide into the RNA, mixing the RNA evenly, preserving the temperature for 10min at 60 ℃, and quickly cooling the RNA on ice. Adding 3 mul of loading buffer solution, mixing evenly, and loading a proper amount of sample into the gel sample application hole. And simultaneously preparing the RNA standard product.

Taking a picture under an ultraviolet lamp after electrophoresis is finished.

Two bands, 28S and 18S, were seen after electrophoresis, and 28S: an 18S ratio greater than 2 indicates that the RNA is not degraded.

2. Extraction of cellular small RNA

Extraction of cell line small molecular RNA (less than or equal to 200nt) is extracted by a mirvana (TM) miRNA extraction kit according to an instruction. The method is briefly described as follows:

cells (a)<10⁷) After collection, lysis was performed with 600. mu.l lysis/binding buffer (appropriate amount of tissue was taken: (<250mg) was ground to a powder in liquid nitrogen, transferred to 600. mu.l lysis/binding buffer for lysis), 60. mu.l of the RNA homogenate was added and lOmin was placed on ice. Adding 600 μ l of phenol/chloroform, mixing vigorously for 30-60s, and centrifuging at 10000rpm for 5min at room temperature (complete stratification). The supernatant was carefully pipetted into a new RNase-free 1.5ml centrifuge tube.

Adding anhydrous ethanol with volume of l/3, completely mixing, adding into a centrifugal column (700 μ l each time) in a collecting tube, centrifuging lmin at room temperature at 10000rpm, and collecting eluate.

Adding 2/3 volume of anhydrous ethanol into the collected effluent, completely mixing, adding into another new centrifugal column in a collecting tube, centrifuging lmin at l0000rpm at room temperature, and discarding the effluent.

Adding 700 mu l miRNA washing solution 1 into the centrifugal column, and centrifuging for 5-10s for rinsing.

The plate was rinsed twice with 500. mu.l of 2/3 as described above. The lmin is centrifuged again to completely remove the ethanol.

Mu.l of the eluent at room temperature was added thereto, and the lmin was centrifuged at l0000rpm to recover RNA.

After the RNA is diluted by 25 times, the values of OD260 and OD280 of the RNA are measured by an ultraviolet spectrophotometer, and the OD260/0D280 is calculated, and when the ratio is more than 1.8, the RNA purity is better. The concentration of RNA was also calculated from OD 260.

Transient transfection of trimic and mimic

Preparing micic or inhibitor: 250 mu l of 1 × universal buffer solution is added into 5nmol of double-stranded siRNA to obtain a micic or inhibitor mother solution with the concentration of 20 mu M, and the mixture is stored at-20 ℃.

Secondly, cells with good growth state are taken, the cells are inoculated in a culture dish with the thickness of 60mm (without adding antibiotics) one day before transfection, and the cell density reaches about 30% during transfection.

Preparing the following composite: solution A: diluting the mimic or the inhibitor with proper concentration in 500 mu l of serum-free culture medium, and gently mixing the mixture evenly; and B, liquid B: mu.l Lipofectamine 2000 (gently mixed before use) was diluted in 500. mu.l serum-free medium and mixed well. Incubate at room temperature for 5 min.

And mixing the diluted liposome with the diluted micic or inhibitor, gently mixing the mixture, and incubating the mixture at room temperature for 20 minutes (the complex can keep stable within 6 hours at room temperature).

Fifthly, adding 1000 mul of the mixed compound into a cell culture dish, adding a serum-free culture medium to 5ml, and gently mixing. After 6 hours, the original medium was discarded and replaced with a medium containing 10% serum.

Sixthly, collecting cells after 48 hours and carrying out corresponding experiments such as Western blot, RT-PCR, MTS and the like.

Fourth, MTS method detects cell proliferation curve

When the cells grow to the logarithmic growth phase, cell transfection or corresponding treatment is carried out, the cells are collected and diluted into cell suspension with proper concentration, the cell suspension is added into a 96-well cell culture plate, each well contains 5000 cells/100 mu l, and the culture is continued in an incubator for proper time. For each experimental group, 6 parallel wells were made and the wells containing medium alone were used as blanks. Four time points are set: 0h (measured after the cells are plated and adhered to the wall), 24h, 48h, 72h and 96 h. The cell activity is measured by adopting an MTS colorimetric method, 15 mul of MTS reagent (500 mu g/ml) is added into each hole, the light absorption value (OD) is measured at the position of 570 nm of wavelength by using an enzyme-labeled spectrophotometer after the culture is continued for 2 hours, the zero adjustment is carried out by using a blank hole, and the higher the OD value is, the more the cell number is.

Fifth, clone colony formation experiment

Carrying out cell transfection or cell treatment according to experimental requirements.

② prepare a large dish, add 10ml culture medium per well, and 800 cells.

③ the cells are cultured for 10-14 days under standard conditions, and the clone formation is observed.

And fourthly, terminating the culture when the cells form macroscopic clones, removing the culture medium, carefully cleaning the cells for 2 times by using PBS, adding 4% formaldehyde for fixation, fixing for 15min at a hole of 1ml, removing the fixing solution, slowly washing by using running water, and adding 1ml of crystal violet staining solution for dyeing for 3 min. The staining solution was slowly washed off with running water, dried in a fume hood, and photographed.

Sixthly, chemotaxis chamber experiment (Transwell)

(ii) counting cells and adding 5X 10 to the chamber⁵For each cell, 500ul of serum-free medium was added to the upper chamber, and 800ul of 10% serum-containing medium was added to the lower chamber.

And moving for 12-18h, discarding culture solution in the upper chamber and the lower chamber of the filter membrane, washing with preheated PBS, slightly blowing and beating the PBS to achieve the effect of washing the lower surface of the filter membrane, and repeating for 1 time.

③ moving the lower chamber into 600 microlitres of 4 percent paraformaldehyde, and immersing the lower surface of the filter membrane in the fixed cells for 15 min.

Fourthly, removing the fixing liquid, inverting the transwell chamber to enable the lower surface of the filter membrane to be upward, and naturally drying.

Fifthly, directly dripping a plurality of Giemsa dye drops on the lower surface of the filter membrane of the inverted transwell chamber after air drying for 10 min.

Sixthly, washing by distilled water, wiping the non-migrated cells on the surface of the small chamber by a cotton ball, and observing and counting under an inverted microscope.

Seventhly, small-scale extraction of plasmid

1-5ml (high copy number plasmid) or 10ml (low copy number plasmid) of the bacterial culture was centrifuged for 5 minutes at 10,000g using a desk centrifuge. The supernatant was discarded and the tube was inverted on a paper towel to aspirate the remaining culture medium.

② 250 mul cell suspension is added and whirling or blowing is carried out to fully suspend the cells. It is critical to suspend the cells sufficiently. If not in the centrifuge tube, transfer the suspended cells to a 1.5ml sterile centrifuge tube. Add 250. mu.l of cell lysate and invert the tube 4 times for thorough mixing (without vortex). Incubation until the cell suspension is clear takes approximately 1-5 minutes. Note that: it is important to observe partial clarification of the lysate before adding the alkaline protease solution (step 3); however, the incubation time is not more than 5 minutes.

③ add 10. mu.l of alkaline protease solution and invert the tube 4 times for thorough mixing. Incubate at room temperature for 5 minutes.

Alkaline proteases are capable of inactivating nucleases and other proteins released during bacterial lysis that can affect the quality of the isolated plasmid. Add 350. mu.l of Wizard Plus SV neutralizing solution and quickly invert the tubes 4 times to mix thoroughly (without vortex shaking). The cell lysate was centrifuged at maximum speed (about 14,000 g) for 10 minutes at room temperature. Clear lysates (approximately 850 μ l) were transferred to a prepared spin column. Without agitation or any white precipitate was transferred with the supernatant.

And fourthly, centrifuging the supernatant for 1 minute at the maximum speed by a centrifuge at room temperature. Remove the centrifuge tube from the collection tube and discard the liquid in the collection tube. The column was reinserted into the collection tube. 750 μ l of column wash previously diluted with 95% alcohol was added. Centrifuge at maximum speed for 1 minute at room temperature. Remove the centrifuge tube from the collection tube and discard the liquid in the collection tube. The column was reinserted into the collection tube.

And fifthly, repeating the cleaning step by using 250 mul of column cleaning solution. Centrifuge at maximum speed for 2 minutes at room temperature.

The column was transferred to a fresh 1.5ml sterile centrifuge tube, taking care not to transfer any of the column wash with the column. If the column cleaning liquid is stuck on the centrifugal column, the centrifugal column is centrifuged again for 1 minute at the maximum speed. The column was transferred to a new 1.5ml sterile centrifuge tube. Mu.l of nuclease-free water was added to the spin column to elute plasmid DNA. Centrifuge at maximum speed for 1 minute at room temperature.

Sixthly, after the plasmid DNA is eluted, taking the centrifugal column out of a 1.5ml disinfection centrifugal tube and discarding the centrifugal column. The aqueous DNA solution without buffer was stable at-20 ℃ and below. The DNA in TE buffer was stable at 4 degrees. If the DNA is to be stored in TE buffer, 11. mu.l of 10 XTE buffer is added to 100. mu.l of the eluted DNA. The centrifuge tube was covered and the purified plasmid DNA was stored at-20 ℃ or below.

Eighth, plasmid extraction and identification

The plasmid concentration was determined using an Amersham Bioscience Gene Quant concentration determinator. DNA has a maximum absorption peak at 260nm, protein has a maximum absorption peak at 280nm, and salts and small molecules are concentrated at 230 nm. Thus, the DNA concentration can be determined spectrophotometrically at a wavelength of 260nm, with an OD of 1.0 corresponding to about 50. mu.g/ml of double-stranded DNA. If the 1cm light path is used, the DNA sample is diluted by 100 times by deionized water and water is used as a blank control, and the concentration of the sample before dilution can be calculated according to the OD260 value read at the moment:

DNA (mg/ml) =50 × OD260 reading × dilution multiple/1000;

the OD 260/OD 280 of the pure DNA was 1.8, and the purity of the DNA was estimated from the OD 260/OD 280 values. If the ratio is higher, RNA is contained, and if the ratio is lower, residual protein is present. The ratio OD 230/OD 260 should be between 0.4 and 0.5, with higher ratios indicating residual salt.

Nine, statistical analysis

All data of the experiment are obtained by repeating the experiment for more than 3 times. The data processing software is Prism 5 and Word Excel. The data were statistically analyzed by SPSS16.0, and P <0.05 was considered statistically significant.

The embodiment also provides an application of the following substances in preparing a medicine for inhibiting tumor:

a）microRNA-652-5p；

b) a recombinant plasmid containing a non-coding gene of microRNA-652-5 p.

Wherein the sequence of the microRNA-652-5p is as follows: 5'-CAACCCUAGGAGAGGGUGCCAUU-3' are provided.

Preferably, the tumor is esophageal cancer.

The embodiment also provides application of the following substances in preparing a medicament for inhibiting esophageal cancer metastasis:

a）microRNA-652-5p；

b) a recombinant plasmid containing a non-coding gene of microRNA-652-5 p.

Preferably, the esophageal cancer is esophageal squamous carcinoma.

In the embodiment, the method for searching the marker microRNA-652-5p for esophageal squamous cell carcinoma metastasis and targeted therapy has the following experimental effects: the expression condition of the gene miR-652-5p is used as a marker for the metastasis, prognosis and targeted therapy of esophageal squamous cell carcinoma.

(1) The expression condition is used as a marker for judging the metastasis of esophageal squamous cell carcinoma.

1) The expression level of miR-652-5p in the esophageal squamous carcinoma tissue is obviously lower than that of a paracancer normal tissue, and the expression level of miR-652-5p has no obvious correlation with the age, sex, tumor differentiation degree and tumor size of a patient, but has obvious negative correlation with clinical stage and lymph node metastasis.

2) The expression level of miR-652-5p in peripheral blood of an esophageal squamous carcinoma patient is obviously lower than that of a normal healthy person, the expression level of miR-652-5p has no obvious correlation with the age, sex, tumor differentiation degree and tumor size of the patient, but has obvious negative correlation with clinical staging and lymph node metastasis. Survival analysis shows that the survival time of the patients in the miR-652-5p low-expression group is remarkably lower than that in the high-expression group.

Cox risk ratio model analysis shows that miR-652-5p expression can be used as a marker for poor prognosis of esophageal squamous carcinoma patients. By the prognosis model, the esophageal squamous carcinoma metastasis can be divided into two types of low-risk and high-risk, different types adopt different treatment schemes, low-risk lesions can adopt local means such as operation, local radiotherapy and the like, and for high-risk lesions, treatment is relatively aggressive, so that the prognosis model has epoch-making significance for changing the treatment mode of the esophageal squamous carcinoma.

(2) Influence of miR-652-5p on proliferation, migration and invasion capacity of esophageal squamous carcinoma cells.

Research shows that miR-652-5p can regulate growth of esophageal squamous carcinoma cell lines and cell invasion and transfer capacity. The miR-652-5p overexpression can inhibit the cell proliferation and cell invasion and metastasis capacities of esophageal squamous carcinoma. Conversely, miR-652-5p down-regulation leads to an increase in this capacity. The method also provides evidence for the role of miR-652-5p in invasion and metastasis and treatment of esophageal squamous carcinoma. Therefore, the miR-652-5p has a very definite cancer inhibition effect, and a cancer inhibition preparation aiming at the miR-652-5p can be developed clinically as clinical treatment.

Specific clinical effects see fig. 1-10, as follows:

referring to FIG. 1, the expression level of miR-652-5p in esophageal squamous carcinoma tissues is obviously lower than that in paracarcinoma normal esophageal tissues.

Referring to FIG. 2, the expression level of miR-652-5p is obviously in negative correlation with lymph node metastasis, and the expression level of miR-652-5p in the esophageal squamous carcinoma tissue with lymph node metastasis is lower than that in the esophageal squamous carcinoma tissue without lymph node metastasis.

Referring to FIG. 3, the expression level of miR-652-5p is obviously negatively correlated with clinical stages, and the expression level of miR-652-5p in the esophageal squamous carcinoma tissue of stage III + IV is lower than that in the esophageal squamous carcinoma tissue of stage I + II.

Referring to FIG. 4, the expression level of miR-652-5p in the peripheral blood of patients with esophageal squamous carcinoma is obviously lower than that of normal healthy people.

Referring to FIG. 5, the expression level of miR-652-5p in the peripheral blood of patients with esophageal squamous carcinoma is obviously negatively correlated with lymph node metastasis. The expression level of miR-652-5p is obviously and negatively correlated with lymph node metastasis, and the expression level of miR-652-5p in peripheral blood of a lymph node metastasis person esophageal squamous carcinoma patient is obviously lower than that of a lymph node metastasis person esophageal squamous carcinoma patient peripheral blood.

Referring to FIG. 6, the expression level of miR-652-5p in peripheral blood of esophageal squamous carcinoma is obviously inversely related to clinical stage. The miR-652-5p expression level is obviously and negatively related to clinical stages, and the miR-652-5p expression level in the peripheral blood of the esophageal squamous carcinoma patient in the III + IV stage is obviously lower than that in the peripheral blood of the esophageal squamous carcinoma patient in the I + II stage.

Referring to FIG. 7, survival analysis showed that the survival of miR-652-5p low expression group patients was significantly lower than that of the high expression group.

Referring to FIG. 8, the high expression of miR-652-5p can inhibit the cell proliferation of esophageal squamous carcinoma cell lines.

Referring to FIG. 9, the high expression of miR-652-5p can inhibit the cell plate clone forming ability of esophageal squamous carcinoma cell lines.

Referring to FIG. 10, high expression of miR-652-5p can inhibit invasion and metastasis of esophageal squamous carcinoma cell lines.

The embodiment provides a molecular marker for esophageal carcinogenesis metastasis, prognosis and treatment prediction, wherein the molecular marker is microRNA-652-5 p.

Preferably, the esophageal cancer is esophageal squamous carcinoma.

In the embodiment, the molecular marker microRNA-652-5p is used for preparing the metastasis and prognosis of esophageal cancer

Use in a diagnostic kit for therapy prediction. Preferably, the esophageal cancer is esophageal squamous carcinoma.

The diagnostic kit in this embodiment is prepared according to a conventional method.

The prognosis in this example is detection, efficacy assessment or monitoring of metastatic relapse.

In the embodiment, the kit of the molecular marker microRNA-652-5p monitors the metastasis and recurrence of esophageal squamous cell carcinoma.

10 patients with confirmed esophageal squamous carcinoma by pathological examination were selected and followed up. The patient samples are from esophageal squamous carcinoma patients in people hospitals in Tangshan City, and all collected patients are effectively treated, so that clinical data and follow-up resources are complete. Firstly collecting blood of 10 patients six weeks after esophageal squamous carcinoma treatment, detecting the expression level of the corresponding gene in the blood, checking once every three months, tracking nine months, and detecting four times.

The relative expression quantity of the corresponding gene in the blood of 10 patients with esophageal squamous cell carcinoma is detected by using the kit. Judging whether the esophageal squamous carcinoma patient has metastasis and relapse according to the changed level of the relative expression quantity of the corresponding gene in the blood sample at 6 weeks, 3 months, 6 months and 9 months after the esophageal squamous carcinoma patient is treated compared with that before the treatment. The judgment standard is that when the relative expression quantity of the gene after treatment is reduced by more than or equal to 35 percent compared with that before treatment, the metastasis relapse is judged; when the relative expression level of the gene after the treatment was decreased by less than 35% compared to that before the treatment, survival without progression was judged.

The results of the detection judgment of the kit are shown in Table 1.

Table 1: detection judgment result of kit

As is clear from Table 1, the clinical diagnosis results showed that 7 of 10 patients with esophageal squamous carcinoma developed metastatic recurrence after treatment, and 3 patients had no progression. The kit for monitoring esophageal squamous carcinoma can be used for detecting clinical symptoms and physical signs earlier, and further provides reference convenience for doctors to treat the diseases individually.

The details not described in the present specification are within the common general knowledge of those skilled in the art.

Although the present invention has been described in detail with reference to the preferred embodiments, it should be understood that various modifications and adaptations of the present invention may occur to those skilled in the art without departing from the spirit and scope of the present invention.

Claims (3)

1. The application of the following substances in preparing the medicine for treating esophageal cancer metastasis:

a）microRNA-652-5p；

b) recombinant plasmids containing non-coding genes of microRNA-652-5 p; the sequence of the microRNA-652-5p is as follows: 5'-CAACCCUAGGAGAGGGUGCCAUU-3', respectively; the esophageal cancer is esophageal squamous carcinoma.

2. The application of the molecular marker microRNA-652-5p for esophageal cancer metastasis, prognosis and treatment prediction in preparing a diagnostic kit for esophageal cancer metastasis, prognosis and treatment prediction.

3. Use according to claim 2, wherein the esophageal cancer is esophageal squamous carcinoma.

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