CN115261482B

CN115261482B - Application of miR-4256 in treatment, diagnosis and prognosis evaluation of gastric cancer

Info

Publication number: CN115261482B
Application number: CN202211219194.5A
Authority: CN
Inventors: 王敏; 汤绍辉; 陈埏芳; 李玉婷; 冯淑芬
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2022-10-08
Filing date: 2022-10-08
Publication date: 2022-12-09
Anticipated expiration: 2042-10-08
Also published as: CN115261482A

Abstract

The invention relates to application of miR-4256 in treatment, diagnosis and prognosis evaluation of gastric cancer. The invention deeply researches the pathogenesis and development mechanism of gastric cancer, and finds that the expression level of miR-4256 is a factor highly associated with gastric cancer; the invention also discovers that miR-4256 is complementarily combined with the HDAC5 gene promoter, thereby promoting the expression of HDAC 5. According to the invention, by disclosing the relevance of miR-4256 and gastric cancer diseases, the cognition of gastric cancer pathogenesis is improved to a new height, a new drug treatment target point is provided for human beings to overcome gastric cancer, a novel tumor marker related to gastric cancer diagnosis or a new treatment strategy is searched, the research and development of subsequent drugs, clinical treatment and the like are facilitated, and the method has important scientific significance.

Description

Application of miR-4256 in treatment, diagnosis and prognosis evaluation of gastric cancer

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to application of miR-4256 in treatment, diagnosis and prognosis evaluation of gastric cancer.

Background

Gastric cancer is one of the common malignant tumors that seriously endanger human health. Although surgery, intervention and molecular targeted therapies have made significant progress over the last few decades, the survival rate of gastric cancer patients remains unsatisfactory, mainly due to the susceptibility to metastasis and recurrence. Therefore, further exploration of the exact mechanism of gastric cancer progression is urgently needed in order to find new GC diagnostic biomarkers and to develop effective therapeutic interventions.

mirnas are a type of non-coding RNA encoded by the genome that is about 19-24 nucleotides in length. The miRNA functions as a negative regulator of gene expression, and complementarily binds to 3' utr of mRNA of a target gene to suppress gene expression at a post-transcriptional level. miRNA is complementary and combined with target gene promoter, and activates or inhibits gene expression at transcription level, thereby participating in generation and development of human malignant tumor, including gastric cancer. mirnas can be stably present in the blood circulation and specifically enriched in exosomes. With the development of molecular biology and sequencing technologies, circulating exosome miRNA has become a focus and focus of current attention in the field of tumor research. Research shows that compared with normal tissues, the tumor tissues secrete more exosomes, even can be 10 times higher than the normal tissues, and participate in regulation and control of tumor microenvironment and tumor immune escape. In addition, researches also find that the proportion of miRNA in the exosome is higher than that of the exosome-derived cell, which indicates that miRNA is specifically enriched in the exosome. More importantly, exosomes derived from tumor cells and miRNA contained in the exosomes exist in all body fluids, and due to the special structure of a lipid bilayer cell membrane, the miRNA, mRNA and other substances contained in the exosomes are not easy to degrade and damage; in addition, researches show that extracellular miRNA and Argonaute (Ago) protein can form a compound so as to prevent the miRNA from being damaged by degrading enzyme, and the miRNA is easy to extract and obtain, so that circulating exosome miRNA is considered to be an ideal liquid biopsy tool.

The mechanism of miRNA regulation of gene expression is very complex, mainly including two major regulatory mechanisms at post-transcriptional level and at transcriptional level. miRNA-mediated post-transcriptional level regulation mechanisms occur in the cytoplasm, such as miRNA targeting gene mRNA 3' utr inhibits gene expression (classical mode). On the other hand, miRNA-mediated transcriptional level regulation mechanisms are performed in the nucleus, including miRNA-targeted gene promoters up-regulating or down-regulating gene expression. miRNA-mediated gene expression regulation is largely dependent on the function of the Ago protein. The miRNA is complementarily combined with a target gene promoter and guides Ago protein to be enriched in the target gene promoter, so that the activation histone modification and RNA polymerase II are enriched in the promoter, the target gene expression is promoted, or the inhibition histone modification is enriched in the promoter, and the target gene expression is inhibited. In the prior art, the pathogenesis factors of the gastric cancer are not clear, the related action and influence of miRNA in the prevention, treatment and prognosis processes of the gastric cancer are few researches, and the clinical treatment lacks an effective treatment means, so that the search of key miRNA related to the gastric cancer treatment is of vital significance.

Disclosure of Invention

The invention aims to solve the technical problems that the gastric cancer pathogenesis is unclear and the advanced prevention is difficult in the prior art, and therefore, the miRNA, namely miR-4256, highly related to the occurrence and development of the gastric cancer is provided. By taking miR-4256 as a marker, the occurrence and development processes of gastric cancer diseases can be effectively predicted, so that high-incidence people of gastric cancer are screened out for reasonable early prevention, the occurrence probability and severity of the diseases are remarkably reduced, and the damage to human health is reduced.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the invention provides application of a reagent for detecting the expression level of miR-4256 in preparation of a product for auxiliary diagnosis and/or prognosis evaluation of gastric cancer.

Preferably, the reagent for detecting the expression level of the miR-4256 comprises a primer pair for detecting the expression level of the miR-4256 gene.

Preferably, the upstream sequence of the primer pair is ACACTCCAGCTGGGATCTGACCTGATG, and the downstream sequence is CTCAACTGGTGTCGTGGA.

It is understood that, without being particularly specified, in the context of the present invention, said primers and/or primer pairs refer to PCR primers used for the synthesis of the cDNA strand of the miR-4256 gene in a PCR, and thus for the detection of the expression level of the miR-4256 gene. In addition to the primers and/or primers listed in the present invention, the skilled person is fully capable of designing corresponding primers and/or primer pairs according to the gene sequence of miR-4256 by means of conventional methods in the art including, but not limited to, molecular biology, and screening the designed primers and/or primer pairs by means of conventional experiments, as long as specific detection of miR-4256 expression level can be achieved.

The invention provides an application of an active reagent for down-regulating the transcription of miR-4256 and/or a mature miRNA thereof and/or inhibiting miR-4256 and/or a mature miRNA thereof in preparing a medicine for preventing and/or treating gastric cancer.

It is to be understood that, without being particularly specified, in the context of the present invention, the agent that down-regulates the transcription of miR-4256 and/or its mature miRNA and/or inhibits miR-4256 and/or its mature miRNA refers to a substance capable of specifically reducing the expression level of miR-4256, for example, a method using antisense oligonucleotides, antagomiRs, miRNA sponges, miRNA Erasers, target Masking and/or multi-Target, the antisense oligonucleotides down-regulates the transcription of miR-4256 and/or its mature miRNA and/or inhibits the activity of miR-4256 and/or its mature miRNA, as long as knock-down and/or knock-out of miR-4256 can be achieved.

Preferably, the active reagent sequence for down-regulating the transcription of miR-4256 and/or its mature miRNA and/or inhibiting miR-4256 and/or its mature miRNA is: ACCUCUCUUCACAGGUCAGAU.

The third aspect of the invention provides a kit for auxiliary diagnosis and/or prognosis evaluation of gastric cancer, which comprises a primer pair for detecting the expression level of miR-4256 gene.

Preferably, the kit further comprises one or more of PCR enzymes, PCR buffers, dNTPs, fluorogenic substrates.

Preferably, the fluorogenic substrate is selected from Syber Green or a fluorescently labeled probe.

The fourth aspect of the present invention provides a chip for auxiliary diagnosis and/or prognosis evaluation of gastric cancer, comprising a solid support, and an oligonucleotide probe immobilized on the solid support; the oligonucleotide probe comprises a primer pair for specifically detecting the expression level of miR-4256.

The fifth aspect of the invention provides an application of the miR-4256 mimic in preparation of a product for promoting HDAC5 protein expression.

Preferably, the miR-4256 mimetic is selected from one or more of the pri-miRNA, pre-miRNA, mature miRNA, dsmiRNA of miR-4256.

Preferably, the sense strand sequence of the miR-4256 mimetic is: 5 'AUCUGACCUGAUGAAGGU-3', and the sequence of the antisense chain is as follows: 3'-CUUCAUCAGGUCAGAUUU-5'.

The invention provides a pharmaceutical composition for preventing and/or treating gastric cancer, which comprises an active agent for down-regulating the transcription of miR-4256 and/or its mature miRNA and/or inhibiting miR-4256 and/or its mature miRNA, and a pharmaceutically acceptable carrier.

Preferably, the active agent sequence for down-regulating the transcription of miR-4256 and/or its mature miRNA and/or inhibiting miR-4256 and/or its mature miRNA is: ACCUCUCUCAGGUCAGAU.

Research finds that the exosome miRNA is closely related to the generation and development of gastric cancer. By comparing the expression profiles of miRNA in early GC patients and healthy human serum exosomes, the expression of miR-92b-3p, miR-146b-5p, miR-9-5p and let-7g-5p in early GC patients is obviously up-regulated, and the possibility that the miRNA of exosomes participates in gastric cancer is suggested. A similar research method is adopted to identify the miRNA expression profile of exosomes in postoperative abdominal dropsy of a patient with gastric cancer resection, and the miR-21 and miR-1225-5p expression levels are found to be closely related to tumor serosa infiltration and can be used as biomarkers for abdominal membrane metastasis and recurrence after curative gastric cancer resection. And the GC cell exosome miR-155-5p can promote the proliferation and migration of GC cells by inhibiting the expression of TP53INP1 gene. The research shows that the exosome miRNA is possible to be a marker for noninvasive diagnosis, targeted therapy and prognosis judgment of gastric cancer.

However, at present, no mature gastric cancer detection kit and targeted drugs based on miRNA are on the market, and one of the main reasons is that the regulation mechanism of miRNA in gastric cancer is not very clear. One of the important advances in recent miRNA research is to mediate gene expression regulation at the transcriptional level in the nucleus, and the major modes of regulation include the following: targeting a gene promoter or non-coding promoter RNA, thereby up-regulating or down-regulating gene expression; targeting gene mRNA 5' UTR; gene enhancers are targeted, but their detailed molecular mechanisms are not clear.

According to the invention, a large number of researches show that the expression level of miR-4256 in a gastric cancer patient is obviously different from that of a normal person, so that the expression level of miR-4256 is presumed to have high relevance to occurrence, development, treatment, prognosis and the like of gastric cancer. According to the invention, experiments further show that the expression of miR-4256 in gastric cancer cells is remarkably increased, the HDAC5 is a target gene of miR-4256 is determined, and the expression of HDAC5 in gastric cancer cells is remarkably increased. The method is based on a gene chip, 3 stomach cancer cells and 1 normal stomach mucosa epithelial cells are detected, expression data of miRNA and mRNA of the stomach cancer cells and the expression data are obtained, bioinformatics analysis and literature research are carried out simultaneously, backup miR-4256 is selected, and target gene HDAC5 is respectively predicted to carry out molecular biology verification and target verification, and the result shows that miR-4256 provided by the invention is closely related to stomach cancer, can be used for prevention and detection and clinical diagnosis and treatment, and has good clinical application value.

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

(1) The invention deeply researches the pathogenesis and development mechanism of gastric cancer, and finds that the expression level of miR-4256 is a factor highly associated with gastric cancer; furthermore, the expression level index of the miR-4256 in the body of the subject is obtained by detecting the miR-4256 in the subject, so that the probability of the stomach cancer suffering of the subject can be effectively and reasonably predicted, namely the expression level of the miR-4256 can be used as a biomarker for clinically assisting in diagnosis of the stomach cancer disease, and when the expression level of the miR-4256 is obviously increased, the subject can be determined to be a stomach cancer patient or a high-risk group suffering from the stomach cancer.

(2) By detecting the expression level of miR-4256 in a subject, the high-risk people with gastric cancer can be effectively screened out, the rapid development and deterioration of diseases can be effectively prevented from causing impossible health damage to patients, the prognosis of the patients can be reasonably evaluated, and a reasonable and effective guiding function is provided for treatment and rehabilitation.

(3) The invention also discovers that miR-4256 and HDAC5 gene promoter are complementarily combined, thereby promoting HDAC5 expression, further influencing the progress of gastric cancer, and defining that miR-4256/HDAC5 signal axis has important function in the generation and the progress of GC. The relevance of miR-4256 and gastric cancer diseases is disclosed, the cognition of gastric cancer pathogenesis is improved to a new height, a new drug treatment target point is provided for human beings to overcome gastric cancer, a new direction is provided for searching a novel tumor marker related to gastric cancer diagnosis or a new treatment strategy to control gastric cancer, the method is beneficial to subsequent drug research and development, clinical treatment and the like, and the method has important scientific significance.

Drawings

FIG. 1 is a schematic representation of miRNA expression profiles of gastric cancer patients and healthy control serum exosomes.

Fig. 2 is a schematic diagram showing analysis results of 218 differentially expressed mirnas in serum exosomes of gastric cancer patients and healthy controls.

FIG. 3 is a schematic diagram of the results of analysis of prediction of target genes and GO from the startBase database.

FIG. 4 shows the miR-4256 sequence and the complementary sequence of the seed sequence and HDAC5 gene promoter.

FIG. 5 is a schematic diagram showing the expression level of miR-4256 in gastric cancer cells and gastric mucosal epithelial cells.

FIG. 6 is a graph showing the expression levels of miR-4256 and HDAC5 in gastric cancer tissues and paracarcinoma tissues.

FIG. 7 is a graph showing the correlation analysis result between miR-4256 expression level and HDAC 5.

FIG. 8 is a diagram showing the effect of miR-4256 overexpression or inhibition on the expression level of HDAC5 mRNA in gastric cancer cells.

FIG. 9 is a schematic diagram showing the effect of miR-4256 overexpression or inhibition on the expression level of HDAC5 protein in gastric cancer cells.

FIG. 10 is a graph showing the effect of miR-4256 overexpression or inhibition on the luciferase activity of the HDAC5 gene promoter-driven reporter plasmid.

FIG. 11 is a schematic diagram showing the effect of miR-4256 overexpression or inhibition on gastric cancer cell proliferation.

FIG. 12 is a graph showing the effect of miR-4256 overexpression or inhibition on SGC-7901 cell migration and invasion.

FIG. 13 is a schematic diagram of the results of the effect of miR-4256 overexpression or inhibition on HGC-27 cell migration and invasion.

FIG. 14 is a graph showing the effect of miR-4256/HDAC5 signal axis on gastric cancer cell proliferation.

FIG. 15 is a graph showing the effect of miR-4256/HDAC5 signaling axis on SGC-7901 cell migration and invasion.

FIG. 16 is a schematic diagram showing the effect of miR-4256/HDAC5 signal axis on HGC-27 cell migration and invasion.

FIG. 17 is a schematic diagram showing the effect of miR-4256 overexpression or inhibition on tumor growth in mice.

Detailed Description

In order to make the objects, technical solutions and effects of the present invention clearer and clearer, the present invention is described in further detail below with reference to examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Cell lines listed in the context of the present invention, including SGC-7901, HGC-27, AGS and GES-1, are purchased from the agency of American Type Culture Collection (ATCC, manassas, VA, USA) and cultured according to the ATCC guidelines, unless otherwise specified. All cell lines were identified by short tandem repeat analysis of the chinese typical culture collection (wuhan) and verified for the presence of mycoplasma contamination using a PCR assay kit (shanghai Biothrive Sci) while being cryopreserved in liquid nitrogen and used for subsequent experiments. The reagents used in the invention are all obtained by commercial sale, and the adopted miR-4256 inhibitor is a single-stranded RNA molecule designed and synthesized based on miR-4256 sequence. For the use of clinical specimens, informed consent is signed with patients, and related procedures and methods meet medical ethical requirements and quality management specifications of clinical trials of medicines. The experimental methods used in the present invention, such as DNB model construction, bioinformatics analysis, DNA extraction, genome sequencing, primer design, PCR, western blot, tumor cell culture, cell migration experiments, cell invasion experiments, cell survival experiments, animal experiments, immunohistochemistry, and the like, are all conventional methods and techniques in the art.

Representative results from selection of biological experimental replicates are presented in the context figure, and data are presented as mean ± SD and mean ± SEM as specified in the figure. All experiments were repeated at least three times. Data were analyzed using GraphPad Prism 5.0 or SPSS 22.0 software. And comparing the difference of the average values of two or more groups by adopting conventional medical statistical methods such as t test, chi-square test, variance analysis and the like. p < 0.05 was considered a significant difference.

Example 1 screening of biomarkers

Serum exosome miRNA of 7 gastric cancer patients and 9 healthy volunteers are detected in high flux by adopting an Illumina HiSeq 2000 type gene sequencing system, a miRNAs differential expression profile is obtained, and data processing analysis is carried out. After the miRNA original data background is corrected by transcriptome data analysis software, t-test is carried out to obtainpValues, then merged using Fisher's testpAnd screening the differential expression miRNA, wherein the detection result is shown in figure 1-2. The results show that there are a total of 218 differentially expressed mirnas in serum exosomes of gastric cancer patients compared to healthy subjects (p< 0.05) (see FIG. 2), wherein 139 miRNAs with up-regulated expression (fold Change ≧ 1), 79 miRNAs with down-regulated expression (fold Change less than or equal to 0.5), miR-4256 is the miRNA with the most significant up-regulated expression (log 2 (fold Change) is 3.696959,pvalue is 0.000189).

Then, the analysis of prediction of target genes by using a starBase database and GO shows that the target genes of miRNA with differential expression are significantly enriched in biological processes such as histone modification and covalent modification of chromatin, and the miR-4256 is suggested to be possibly involved in regulation and control of histone modification (see figure 3). The results together suggest that the expression level of miR-4256 has high correlation with gastric cancer.

Further, a 2kb DNA fragment upstream of the transcription start site of the histone modification related enzyme gene is extracted from the NCBI database, the target genes of differentially expressed miRNA are predicted by algorithms such as mirtarbas, miRDB, targetScan and the like, and sequence alignment finds that miR-4256 is possibly complementarily combined with the HDAC5 gene promoter, which indicates that miR-4256 is possibly capable of regulating HDAC5 expression, namely HDAC5 is a potential target gene of miR-4256 (see FIG. 4).

Example 2 study of miR-4256 expression level in human gastric cancer cells and tissues

(1) Respectively culturing gastric cancer cells (SGC-7901, HGC-27 and AGS) and human gastric mucosal epithelial cells (GES-1) in 6-well plate, and respectively collecting 1 × 10 cells when the cells grow to logarithmic phase ⁶ Gastric cancer cells (SGC-7901, HGC-27 and AGS) and human gastric mucosal epithelial cells (GES-1).

(2) Adding 1mL Trizol solution into the cell sap, blowing, uniformly mixing to fully crack the cells, and standing for 5min; adding 200 μ L chloroform, shaking vigorously for 30s to remove water phase and organic phase, and standing at room temperature for 2min.

(3) Centrifuging at 12000g for 10min at 4 deg.C to obtain three layers, wherein RNA in the upper water phase is transferred to a new RNase-free EP tube; adding equal volume of isopropanol, gently mixing well, and standing at-20 deg.C for 10min.

(4) Centrifuging at 12000g at 4 deg.C for 10min, collecting RNA precipitate, collecting supernatant, washing with 75% ethanol twice, air drying on ultra-clean bench, and adding 20-60 μ L DEPC water to dissolve precipitate; purity control OD of Total RNA ₂₆₀ /OD ₂₈₀ The value is 1.8-2.2, and the integrity of RNA is checked by electrophoresis on 1% agarose gel, and the RNA is stored at-80 ℃.

(5) Mu.g of total RNA was taken as template RNA, and EasyScript First-Strand cDNA Synthesis SuperMix (TransGen, cat. AE 301-02) was used for reverse transcription, the reaction system of reverse transcription is shown in the following Table 1, and the reaction conditions were: keeping the temperature at 25 ℃ for 10min; keeping the temperature at 50 ℃ for 30min; keeping the temperature at 85 ℃ for 5min.

TABLE 1 reverse transcription reaction

Reaction component	Concentration of	Volume (uL)
			Oligo（dT）	-	1.0
Random primer	-	1.0
			EasyScript First-Strand cDNA Synthesis SuperMix	2x	10.0
Total RNA	-	1.0
			H2O	-	Supply to 20

(6) The mRNA expression levels of miR-4256 and HDAC5 genes in gastric cancer cells (SGC-7901, HGC-27 and AGS) and human gastric mucosal epithelial cells (GES-1) are detected by adopting fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction), wherein the reaction conditions are as follows: an upstream primer miR-4256-F: ACACTCCAGCTGGGATCTGACCTGATG, downstream primer miR-4256-R: CTCAACTGGTGTCGTGGA; the upstream primer HDAC5-F: TGAACCCAAACTTGAAGTGCG, downstream primer HDAC5-R: CGCTGTTACACACGGACGA; 3 parallel tube reactions are set for each time of miRNAs expression detection, and universal snRNA U6 is used as an internal reference; procedure for RT-PCR amplification of mirnas: circulating for 40 times at 95-10min, 95-10s, 60-31s and 70-10 s; 3 parallel tube reactions are set for each time of mRNAs expression detection, and GAPDH is used as an internal reference; procedure for RT-PCR amplification of mRNA: 95-30s, 95-15s, 60-34 s, circulating for 40 times; the qRT-PCR reaction process was performed according to the Power SYBR Green PCR Master Mix instructions, the qPCR reaction conditions: 95-10min, 95-10s, 60-31s and 70-10 s, circulating for 40 times, and selecting U6 as internal reference. The band of interest was determined by melting curve analysis and electrophoresis, and relative quantification was performed by Δ Δ CT. The fluorescent quantitative PCR (RT-PCR) reaction system is shown in Table 2 below.

TABLE 2 fluorescent quantitative PCR (RT-PCR) reaction System

Reaction component	Concentration of	Volume (uL)
			cDNA（1:20）	-	5.0
Upstream primer	-	0.5
			Downstream primer	-	0.5
Power SYBR Green PCR Master Mix	2x					10
			dH2O	-	4.0

The detection results are shown in the figure. The results show that the expression level of miR-4256 in gastric cancer cells is significantly higher than that of normal gastric mucosal epithelial cells (p < 0.05) (see FIG. 5), which is consistent with the miRNA chip results.

Then, the expression conditions of miR-4256 and HDAC5 in the tumor and the tissues beside the tumor of the gastric cancer patient are detected 36 by fluorescent quantitative RT-PCR, and the specific steps are as follows:

(1) Taking 50mg of tissue to 1.5mL of EP tube, adding 1mL of Trizol, fully homogenizing, and standing for 5min at room temperature;

(2) Adding 0.2ml chloroform, shaking for 15-30s, standing for 2-3min;

(3) Centrifuging at 4 deg.C for 12000g × 15min, and sucking up the upper aqueous phase, about 400 μ L into an enzyme-free EP tube;

(4) Adding isopropanol with the same volume, mixing the liquid in the tube, and standing at room temperature for 10min;

(5) Centrifuging at 4 ℃ for 12000g multiplied by 15min, and obtaining white feather-shaped sediment at the bottom of the tube, namely RNA;

(6) Discarding the supernatant, adding 1mL of ice 75% ethanol, flicking the tube wall slightly to float the RNA in the tube wall, turning the RNA upside down, and fully washing;

(7) Centrifuging at 4 deg.C, 7500g × 6min, carefully sucking to remove supernatant, and repeating the steps for 2-3 times;

(8) Adding appropriate amount of (20-40 μ L) enzyme-free water, dissolving RNA, taking 2 μ L to determine concentration, and storing the rest at-80 deg.C;

(9) Mu.g of RNA was extracted in accordance with the concentration to be measured, and then reverse-transcribed into cDNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche, cat. No. 489703001) in accordance with the instructions, followed by the above-mentioned method to carry out fluorescent quantitative RT-PCT detection.

The results are shown in FIG. 6. The result shows that compared with the tissues beside the cancer, the expression level of miR-4256 in the gastric cancer tissues is obviously increased, and the expression level of HDAC5 in the gastric cancer tissues is also obviously increased. Correlation analysis of the above results shows that the expression level of miR-4256 is positively correlated with HDAC5 expression (see FIG. 7).

Further, the results of miR-4256 analysis and clinical pathological feature analysis in the body of the gastric cancer patient are shown in the following table 3. The result shows that the high expression of miR-4256 is related to the poor differentiation degree of lymph nodes and tumor tissues of a gastric cancer patient and the later TNM stage, and the miR-4256 can be used as a marker for detecting gastric cancer.

TABLE 3 correlation analysis of relative expression level of miR-4256 of 36 gastric cancer patients and clinical pathological characteristics

Index (es)	Number of cases	Relative expression of miR-4256
			Lymph node metastasis
Negative of	14	0.57 (0.23-1.93) *
			Positive for	22	1.90 (1.12-3.03)
TNM staging
			I-Stage II	17	1.21 (0.19-1.79) *
III- Stage IV	19	2.05 (0.83-3.32)
			Peritoneal metastasis
Negative of	25	1.43 (0.41-2.04)
			Positive for	11	2.32 (0.61-3.59)
Venous invasion
			Negative of	23	1.64 (0.36-2.83)
Positive for	13	1.25 (0.56-2.68)
			Degree of tumor differentiation
Moderate/high differentiation	12	0.42 (0.08-1.52) *
			Low differentiation	24	1.93 (0.93-3.04)

Example 3 study of interaction of miR-4256 with HDAC5

(1) miR-4256 mimic (sequence of sense strand: 5 'AUCUGACCUGAUGAGGU-3' and sequence of antisense strand: 3 '-CUUCAUCAGGUCAGAUUU-5'), miR-4256 inhibitor (sequence: 5 'ACCUCAUCAGGUCAGAU-3') and empty vector (scambled control, sc) were transfected into SGC-7901 and HGC-27 cells, respectively.

(2) After transfection is carried out for 24h, 1mL Trizol solution is added into cell sap, the cell is blown and uniformly mixed to ensure that the cells are fully cracked, and the mixture is kept stand for 5min; adding 200 μ L chloroform, shaking vigorously for 30s to remove water phase and organic phase, and standing at room temperature for 2min.

(3) Centrifuging at 12000g at 4 deg.C for 10min to obtain three layers, wherein RNA in the upper water phase is transferred to a new RNase-free EP tube; adding equal volume of isopropanol, gently mixing well, and standing at-20 deg.C for 10min.

(4) Centrifuging at 12000g at 4 deg.C for 10min, collecting RNA precipitate, collecting supernatant, washing with 75% ethanol twice, and ultrafilteringAir drying on a clean bench, and adding 20-60 mu L of DEPC water to dissolve the precipitate; purity control OD of Total RNA ₂₆₀ /OD ₂₈₀ The value is 1.8-2.2, and the integrity of RNA is checked by electrophoresis on 1% agarose gel, and the RNA is stored at-80 ℃.

(5) Mu.g of total RNA was used as template RNA, and reverse transcription and fluorescent quantitative RT-PCT assay were performed under the conditions described in example 2, and the results are shown in FIG. 8. The results show that in two gastric cancer cells, after miR-4256 is overexpressed by miR-4256 mimic, the expression level of intracellular HDAC5 mRNA is obviously increased (p is less than 0.01, vs blank control (bc)); after the miR-4256 inhibitor is used for inhibiting the expression of the miR-4256 in the cells, the expression level of the intracellular HDAC5 mRNA is obviously reduced (p < 0.01, vs blank control (bc)).

Subsequently, researching the relation between the expression levels of the miR-4256 and HDAC5 proteins in the cells, and specifically comprising the following steps:

(2) After 24h of transfection, the original medium was discarded and washed 2 times with PBS.

(3) Cell lysis solution (containing PMSF) was added and lysed on ice for 30min.

(4) Scraping cells with cells, transferring into a sterilized centrifuge tube, centrifuging at 12000rpm at 4 deg.C for 10min, collecting supernatant to obtain cell lysate, and extracting total cell proteins according to kit instruction (Shanghai Bin Yun Tian Biotechnology Co., ltd.). Each histone concentration (Thermo) was measured using BCA protein quantitation. Each histone was diluted to an equal concentration using a cell lysate, mixed with 5 × loading buffer at a volume ratio of 4.

(5) Extracting total protein 20 μ g, performing SDS-PAGE gel electrophoresis (100V, 90min) with 10% concentration, transferring to PVDF membrane with constant current of 300mA for 100 min; 5% bsa incubation at rt for 2h on a shaker, incubation of HDAC5 primary antibody (1) 1000) overnight (Abcam) at 4 ℃, TBST washing 3 times for 5min each, incubation of secondary antibody (1 20000) at room temperature for 1h after dilution with blocking solution; TBST was washed 3 times, each at 7min, and the ECL luminescence kit (Santa Cruz) was exposed in an exposure apparatus, and the net optical density of the bands was analyzed using a gel Image processing system (Image-Pro Plus 6.0) with internal reference GAPDH as a control.

The detection results are shown in fig. 9. The results show that in two gastric cancer cells, after miR-4256 is overexpressed by miR-4256 mimic, the expression level of the intracellular HDAC5 protein is obviously increased; after the miR-4256 inhibitor is used for inhibiting the expression of miR-4256 in the cell, the expression level of the HDAC5 protein in the cell is obviously reduced, and the result is consistent with the result of the HDAC5 mRNA in the cell, which indicates that the miR-4256 can promote the expression of the HDAC5 mRNA and protein.

Further research shows that miR-4256 transiently overexpresses and obviously up-regulates the luciferase activity of the reporter plasmid driven by the HDAC5 gene promoter, and miR-4256 knockdown obviously down-regulates the luciferase activity of the reporter plasmid driven by the HDAC5 gene promoter; on the other hand, miR-4256 transient overexpression or knockdown did not affect mutant HDAC5 gene promoter-driven reporter plasmid luciferase activity, suggesting that miR-4256 directly targets HDAC5 gene promoter to up-regulate reporter gene expression (see fig. 10).

Example 4 Effect of miR-4256 on gastric cancer cell function

(1) SGC-7901 and HGC-27 cells transfected with miR-4256 mix (5 '-CUUCAUCCUGAUGAGGU-3' for sense strand sequence and 3'-CUUCAUCAGGUCAGAUUU-5' for antisense strand sequence), miR-4256 inhibitor (5 '-CUUCAUCAGGUCAGAU-3' for sequence) or empty vector (sc) and SGC-7901 and HGC-27 cells without any treatment were taken as blank controls (bc group), trypsinized and counted, and appropriate cell densities were selected according to doubling times of the respective cells and seeded in 96-well plates (3 repeats).

(2) Culturing in 37 deg.C incubator, collecting cells at 24h, 48h, 72h and 96h, adding 10 μ L CCK-8 per well, incubating the culture plate in incubator for 1-4h, measuring absorbance at 450nm, and evaluating proliferation status of cells.

The results of the experiment are shown in FIG. 11. The results show that the miR-4256 can significantly promote the proliferation of gastric cancer cells after the miR-4256 is transiently overexpressed by miR-4256 mimic compared with a blank control group (bc) or an empty vector group (sc) ((pLess than 0.5), and can effectively reduce the proliferation of gastric cancer cells after the miR-4256 inhibitor is used for inhibiting miR-4256 (p＜0.01）。

Subsequently, the cell migration and invasion experiments were carried out using the above SGC-7901 and HGC-27 cells transfected with miR-4256 mimic (5 'AUCUGACCUGAUGAAGGU-3' for the sense strand sequence and 3'-CUUCAUCAGGUCAGAUUU-5' for the antisense strand sequence), miR-4256 inhibitor (5 'ACCUCAUCAGGUCAGAU-3') or empty vector (scambled control, sc), and SGC-7901 and HGC-27 cells which were not subjected to any treatment as a blank control, as follows:

transwell cell invasion assay

(1) One day before the experiment, a tube of Matrigel that had been dispensed was placed in a refrigerator at 4 ℃ overnight from-20 ℃ in advance, and allowed to melt from a solid state to a liquid state.

(2) A10% Matrigel was prepared on ice, 50. Mu.L of the upper face of the bottom membrane of the transwell cell was coated and placed at 37 ℃ for 30min to allow the Matrigel to solidify.

(3) After trypsinizing the cells in logarithmic growth phase, the basal medium was resuspended into a cell suspension, counted and the cell density was adjusted to 5X 10 ⁵ one/mL.

(4) The basal medium in the chamber and in the 24-well plate was aspirated and discarded, 200. Mu.L of cell suspension was added to the upper chamber of the transwell chamber, and 600. Mu.L of complete medium (basal medium +10% fetal bovine serum) was added to the lower chamber of the 24-well plate.

(5) The plates were incubated at 37 ℃ CO ₂ The incubation was continued for 16 hours in the incubator.

(6) The cell was taken out, rinsed 2 times with PBS, fixed in a 24-well plate with 4% paraformaldehyde for 20min, and stained with crystal violet solution for 15min.

(7) The cells and matrigel in the upper layer of the microporous membrane of the chamber were carefully wiped off with a cotton swab and photographed under an inverted microscope.

Transwell cell migration assay

The cells with a pore size of 8 μm and a 24-well plate from corning corporation transwell migration were used.

(1) The chamber was placed in a 24-well plate and 1mL basal medium was added per well for wetting.

(2) After trypsinizing the cells in logarithmic growth phase, the basal medium was resuspended into a cell suspension, counted and the cell density was adjusted to 5X 10 ⁵ One per mL.

(3) The basal medium in the chamber and in the 24-well plate was aspirated and discarded, 200. Mu.L of cell suspension was added to the upper chamber of the transwell chamber, and 600. Mu.L of complete medium (basal medium +10% fetal bovine serum) was added to the lower chamber of the 24-well plate.

(4) The plates were incubated at 37 ℃ CO ₂ The cultivation was continued for 14 hours in the incubator.

(5) The cell was taken out, rinsed 2 times with PBS, fixed in a 24-well plate with 4% paraformaldehyde for 20min, and stained with crystal violet solution for 15min.

(6) The cells in the upper microporous membrane layer of the chamber were carefully wiped off with a cotton swab and photographed under an inverted microscope.

The results are shown in FIGS. 12-13, respectively. The results show that compared with a blank control group (bc) or an empty vector group (sc), the miR-4256 can obviously promote the migration and invasion of gastric cancer cells after being transiently overexpressed by miR-4256 mimicpLess than 0.01), and after miR-4256 inhibitor is used for inhibiting miR-4256, the migration and invasion of gastric cancer cells can be effectively reduced (p＜0.01）。

Further, HDAC5 gene overexpression plasmids (HDAC 5-OE), negative control plasmids (HDAC 5-CON), miR-4256 inhibitor plasmids and empty vectors (scrambled control, sc) were constructed to transfect SGC-7901 and HGC-27 cells of gastric cancer cells, respectively, and the cell proliferation, migration and invasion experiments were repeated, with the results shown in FIGS. 14-16. The results showed that compared to the empty vector group and the negative control group, the effect on HDAC5 was observedAfter overexpression, the cell proliferation, migration and invasion capacity of gastric cancer cells can be remarkably promoted (p< 0.01); on the basis of HDAC5 overexpression, inhibition of miR-4256 expression by miR-4256 inhibitor can effectively reverse the promotion effect of HDAC5 on proliferation, migration and invasion of gastric cancer cells (p＜0.01）。

Example 5 study of Effect of miR-4256 on tumor growth in vivo

(1) One day before the experiment, the matrix gel which is subpackaged is put into a refrigerator at 4 ℃ overnight from-20 ℃ in advance, so that the matrix gel is melted from a solid state into a liquid state;

(2) After digesting SGC-7901 cells transfected with an empty vector (sc), SGC-7901 cells transfected with miR-4256 inhibitor (sequence: 5- ⁷ Per mL;

(3) Mixing the cell suspension, sucking 100. Mu.L, inoculating to Balb/c-nu right dorsal subcutaneous tissue for 4 weeks, i.e. inoculating 2X 10 subcutaneous tissue to each nude mouse ⁶ A cell; among them, group 1 mice were injected subcutaneously with SGC-7901 cells transfected with an empty vector (sc), group 2 mice were injected subcutaneously with SGC-7901 cells transfected with miR-4256 inhibitor plasmid (agomir), group 3 mice were injected subcutaneously with SGC-7901 cells transfected with miR-4256 imic plasmid (agomir), and 6 mice each group.

(4) Observing the growth and mental conditions of the mice every day, measuring the tumor size of each group of mice every 4 days after the mice form tumors, and calculating the tumor volume, wherein the tumor volume is calculated by adopting the following formula: volume (mm) ³ ）=Length（mm）×Width ² （mm ² ）/2。

(5) Mice were sacrificed 23 days after cell injection, groups of mice were stripped of tumors and photographed.

The results of the experiment are shown in FIG. 17. The results show that compared with the empty vector group (scrambled control), the tumor growth rate in nude mice over-expressed by miR-4256 in vivo (agomir group) is obviously faster, while in the group inhibiting miR-4256 (agomir group), the growth rate of the tumor and the size of the tumor are obviously and effectively controlled; the miR-4256 is shown to promote the growth of gastric cancer cell xenograft tumor of nude mice, and when the expression of miR-4256 is inhibited, the growth of tumor can be obviously inhibited.

The results show that the miR-4256 is highly expressed in normal stomach tissues or cells and is lowly expressed in stomach cancer tissues, and the miR-4256 level in the serum exosomes of the stomach cancer patients is obviously higher than that of healthy subjects, so that the miR-4256 can be used for early diagnosis of stomach cancer diseases. Meanwhile, by analyzing a clinical sample, the miR-4256 high expression is related to the poor differentiation degree of lymph nodes and tumor tissues of a gastric cancer patient and the later TNM stage, and the miR-4256 can be further determined to be used as a marker for detecting gastric cancer, so that the prognosis of the gastric cancer can be reasonably predicted by detecting the expression level of the miR-4256 in the body of the patient, and meanwhile, a personalized treatment scheme is selected to improve the clinical treatment effect. In addition, in the stomach cancer cell with high miR-4256 expression, the cell proliferation, migration or invasion performance is obviously improved compared with that of the conventional stomach cancer cell, and when the expression of miR-4256 is inhibited, the functional activities of the stomach cancer cell can be effectively reduced, so that the remarkable inhibiting effect on the stomach cancer cell can be generated by inhibiting the expression of miR-4256, the remarkable inhibiting activity on the generation, development, transfer and the like of the stomach cancer can be further realized, a new medicine treatment target point is provided for human beings to overcome the diseases such as the stomach cancer, a new direction is provided for the subsequent medicine research and development, clinical treatment and the like, and the high-efficiency miR-4256 gastric cancer cell has extremely high social value and market application prospect.

The above detailed description section specifically describes the analysis method according to the present invention. It should be noted that the above description is only for the purpose of helping those skilled in the art better understand the method and idea of the present invention, and not for the limitation of the related contents. The present invention can be appropriately adjusted or modified by those skilled in the art without departing from the principle of the present invention, and the adjustment and modification should also fall within the protection scope of the present invention.

Claims

1. Application of a reagent for detecting miR-4256 expression level in preparation of a product for gastric cancer auxiliary diagnosis.

2. The use of claim 1, wherein the reagent for detecting the expression level of miR-4256 comprises a primer pair for detecting the expression level of miR-4256 gene, the upstream sequence of the primer pair is ACACTCCAGCTGGGATCTGACCTGATG, and the downstream sequence of the primer pair is CTCAACTGGTGTCGTGGA.

3. The application of an agent for reducing the transcription of miR-4256 and/or inhibiting the activity of miR-4256 in the preparation of a medicine for treating gastric cancer; the reagent is oligonucleotide, and the sequence of the oligonucleotide is ACCUUCAUCAGGUCAGAU.