CN117925846B

CN117925846B - Biomarker for prognosis evaluation of gastric cancer and application thereof

Info

Publication number: CN117925846B
Application number: CN202410338546.1A
Authority: CN
Inventors: 王晓敏; 武云; 马芷灵; 赵亚茹; 明成
Original assignee: BAOTOU CENTRAL HOSPITAL
Current assignee: BAOTOU CENTRAL HOSPITAL
Priority date: 2024-03-25
Filing date: 2024-03-25
Publication date: 2024-06-11
Anticipated expiration: 2044-03-25
Also published as: CN117925846A

Abstract

The invention provides a biomarker for prognosis evaluation of gastric cancer and application thereof, and the biomarker is used for carrying out intensive research on the expression of DCLK2 and DNAJB in gastric cancer and the influence on malignant biological behaviors such as proliferation, invasion, migration and the like of gastric cancer cells, wherein the expression level of DCLK2 and DNAJB5 is a factor highly related to gastric cancer. DNAJB5 is obviously low-expressed in gastric cancer tissues, and over-expression of DNAJB and DCLK2 can inhibit proliferation, invasion and migration of gastric cancer cells, but silencing DCLK2 can promote proliferation, invasion and migration capability of gastric cancer cells. According to the invention, through revealing the relevance between DNAJB and DCLK2 and gastric cancer diseases, cognition on gastric cancer pathogenesis is improved to a new height, a new drug treatment target is provided for human beings to overcome gastric cancer, and important scientific significance is provided for searching a novel tumor marker or a new treatment strategy related to gastric cancer diagnosis, subsequent drug research and development, clinical treatment and the like.

Description

Biomarker for prognosis evaluation of gastric cancer and application thereof

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a biomarker for prognosis evaluation of gastric cancer and application thereof.

Background

Currently, gastric cancer (GASTRIC CANCER, GC) refers to malignant tumors derived from gastric mucosa, connective tissue, neuroendocrine tissue or lymphoid tissue, and is one of the most common cancers. The etiology of the traditional Chinese medicine composition comprises: (1) dietary lifestyle: the incidence rate of far-end stomach cancer in people eating the smoked and roasted and salted food for a long time is high, and the incidence rate is related to the high content of cancerogens or pre-cancerogens such as nitrite, mycotoxin, polycyclic aromatic compounds and the like in the food; (2) helicobacter pylori (Hp) infection: helicobacter pylori can promote nitrate to be converted into nitrite and nitrosamine so as to be carcinogenic, chronic inflammation of gastric mucosa is caused by Hp infection, and environmental pathogenic factors accelerate excessive proliferation of mucosal epithelial cells, so that distortion and carcinogenesis are caused, toxic products CagA and VacA of helicobacter pylori possibly have a cancer promotion effect, and the detection rate of anti-CagA antibodies in gastric cancer patients is obviously higher than that of general people; (3) precancerous lesions: gastric diseases including gastric polyps, chronic atrophic gastritis and residual stomach after partial gastric resection, which are possibly accompanied by chronic inflammatory processes of different degrees, metaplasias or atypical hyperplasia of gastric mucosa and intestinal epithelium, possibly converted into cancers, the precancerous lesions refer to pathological changes of gastric mucosa which are easy to generate cancerations, are juncture pathological changes in the process of converting benign epithelial tissues into cancers, the abnormal hyperplasia of gastric mucosa epithelium belongs to precancerous lesions, can be divided into light, medium and heavy three degrees according to the abnormal degree of cells, and the severe abnormal hyperplasia is sometimes difficult to distinguish from early gastric cancer with better differentiation; (4) genetic and genetic: genetic and molecular biological researches show that the incidence rate of gastric cancer of relative with blood relationship of gastric cancer patients is 4 times higher than that of a control group, the canceration of gastric cancer is a multi-factor, multi-step and multi-stage development process, the change of oncogenes, cancer suppressor genes, apoptosis related genes, transfer related genes and the like is involved, and the form of gene change is also various. Early symptoms of gastric cancer are atypical, often manifested by upper abdominal discomfort, nonspecific upper gastrointestinal symptoms such as satiety and nausea after eating, and symptoms similar to duodenal ulcer can appear in patients with gastric sinus cancer. Symptoms of cachexia such as pain in upper abdomen, anorexia, debilitation, emaciation, weight loss, etc. can also be hematemesis, black stool, etc. Because early symptoms of gastric cancer are atypical, distant metastasis such as liver, lung, pancreas, bone, and peritoneum is often accompanied by diagnosis.

The examination of gastric cancer can be performed by: (1) X-ray barium meal examination: the application of the digital X-ray gastrointestinal radiography technology is still a common method for diagnosing gastric cancer at present, and diagnosis is usually made by adopting gas-barium double radiography and observing a mucous membrane phase and a filling phase, the main change of early gastric cancer is that the mucous membrane phase is abnormal, and the form of the gastric cancer in the progressive stage is basically consistent with the general typing of the gastric cancer; (2) fibrogastroscopy: the method is the most effective method for diagnosing gastric cancer, adopts a fiber gastroscope with an ultrasonic probe to carry out ultrasonic detection imaging on a lesion area, and is helpful for knowing the tumor infiltration depth and the invasion and metastasis of surrounding organs and lymph nodes; (3) abdominal ultrasound: in gastric cancer diagnosis, abdominal ultrasound is mainly used for observing the conditions of infiltration of adjacent organs of the stomach (particularly liver and pancreas) and lymph node metastasis; (4) spiral CT and positron emission imaging examination: the multi-row spiral CT scanning is combined with the three-dimensional reconstruction and the simulated endoscope technology, is a novel noninvasive examination means, is beneficial to diagnosis of gastric cancer and preoperative clinical staging, can judge the conditions of lymph nodes and distant metastasis by utilizing affinity of gastric cancer tissues to fluorine and deoxidized-D-glucose (FDG) and adopting positron emission imaging (PET), and has higher accuracy; (5) tumor markers: the tumor-associated antigens such as serum CEA, CA50, CA72-4, CA19-9 and the like can be increased, but the sensitivity and the specificity are not high, which is helpful for judging the prognosis of tumor and the curative effect of chemotherapy. In order to better understand the occurrence and mechanism of gastric cancer and to improve the therapeutic effect of gastric cancer, in recent years, intensive studies have been made on molecular markers and signal pathways associated with gastric cancer.

DCLK2 is one of the members of the DCLK protein family, and this gene is located in the chromosomal 4q31.23-q31.3 region, encoding members of the protein kinase superfamily and the biscortical hormone family. The DCLK gene has a highly conserved N-terminal double cortical domain and a protein comprising a C-terminal kinase domain. The N-terminal region is highly homologous to the family of Dicotylectins (DCX), involved in microtubule stabilization and nerve cell migration. The C-terminal kinase domain is highly homologous to the family of multifunctional calmodulin-dependent kinases (calmodulin-DEPENDENT PROTEIN, caMK). Three parallel genes of DCLK have been reported so far, among which the amino acid sequences of DCLK1 and DCLK2 are highly homologous and exhibit similar tissue distribution. At present, more and more researches show that DCLK1 is used as a tumor stem cell marker and is involved in the occurrence and development processes of various cancers. However, little research has been done on DCLK2, which remains essentially on the nervous system: DCLK2 can promote the survival, regeneration and migration of neurons, and DCLK2 gene mutation is related to mental diseases such as epilepsy, hyperkinetic symptom, autism, corpus callosum hypoplasia and the like.

DNAJB5 is a heat shock protein (Heat shock protein, HSP) 40 family member B5, acting as a chaperone. DNAJ proteins are differentially expressed in human tissue, and the human genome contains 49 genes encoding the dnaj proteins, which are subdivided into three subclasses: class I (DNAJA, 4 members), class II (DNAJB, 13 members) and class II (DNAJC, 32 members). There is little research currently done on DNAJB.

Gastric cancer is a highly invasive malignant disease, which is a malignant tumor derived from gastric mucosal epithelium, and is seriously threatened to life safety of patients due to low early screening rate, high clinical morbidity and mortality, and most of gastric cancer is found in middle and late stages due to lack of early screening about gastric cancer. Gastric cancer is a malignant tumor mainly subjected to operation, and the disease control rate and survival rate can be improved by combining postoperative radiotherapy and chemotherapy. It can be found early that whether radical surgery can be performed is critical to patient prognosis. Therefore, the gastric cancer can be detected and screened in early stage, and has very important significance for the discovery and treatment of gastric cancer.

Disclosure of Invention

The invention provides a biomarker for prognosis evaluation of gastric cancer and application thereof, and solves the problems.

The technical scheme of the invention is realized as follows:

A biomarker for prognosis evaluation of gastric cancer is DCLK2 or DNAJB, and the overexpression of DCLK2 and the overexpression of DNAJB can inhibit proliferation, invasion and migration of gastric cancer cells.

Use of a reagent for detecting DCLK2 expression level in the preparation of a product for gastric cancer auxiliary diagnosis.

Use of a reagent for detecting DNAJB expression levels in the preparation of a product for use in the assisted diagnosis of gastric cancer.

Optionally, the reagent for detecting DCLK2 expression level includes a primer pair for detecting DCLK2 gene expression level, and the upstream sequence of the primer pair is SEQ ID NO:1, the downstream sequence is SEQ ID NO:2.

Optionally, the reagent for detecting DNAJB expression level includes a primer pair for detecting DNAJB gene expression level, and the upstream sequence of the primer pair is SEQ ID NO:3, the downstream sequence is SEQ ID NO:4.

Use of an agent that upregulates the transcription of DCLK2 and/or enhances the activity of DCLK2 in the manufacture of a medicament for the treatment of gastric cancer.

Use of an agent that upregulates the transcription of DNAJB and/or enhances the activity of DNAJB in the manufacture of a medicament for the treatment of gastric cancer.

After the technical scheme is adopted, the invention has the beneficial effects that:

1. DCLK2 and DNAJB are obviously low expressed in stomach cancer tissues, and over-expression of DCLK2 and DNAJB can inhibit proliferation, invasion and migration of stomach cancer cells, and silencing of DCLK2 can promote proliferation, invasion and migration capability of stomach cancer cells. Therefore, DCLK2 and DNAJB can become potential targets for treating gastric cancer, and provide a new idea and approach for treating gastric cancer. The expression levels of DCLK2 and DNAJB are a factor highly associated with gastric cancer, and therefore, the expression level index is obtained, so that the probability of suffering from gastric cancer of a subject can be effectively and reasonably predicted, namely, the expression levels of DCLK2 and DNAJB5 can be used as biomarkers for clinically assisting in diagnosing gastric cancer diseases, and when the expression levels of DCLK2 and DNAJB5 are obviously reduced, the subject can be clearly defined as a gastric cancer patient or a high risk group suffering from gastric cancer.

2. By detecting the expression levels of DCLK2 and DNAJB in a subject, the high-risk population of gastric cancer can be effectively screened, the irreversible health damage to the patient caused by rapid development and deterioration of the disease can be effectively prevented, the prognosis of the patient can be reasonably evaluated, a reasonable and effective guiding effect is provided for treatment and rehabilitation, and the growth and development of gastric cancer cells can be assisted and inhibited by promoting the expression of DCLK2 and DNAJB for gastric cancer patients, so that the gastric cancer can be a drug treatment target of gastric cancer.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.

FIG. 1A is a schematic diagram showing the relative expression of DCLK2 in gastric cancer tissue and normal gastric mucosa tissue;

FIG. 1B is a schematic diagram showing the relative expression of DNAJB in gastric cancer tissue and normal gastric mucosal tissue;

FIG. 2 shows the expression of DCLK2 in gastric cancer tissue and normal gastric mucosa tissue;

FIG. 3 is a graph showing the effect of DCLK2 on survival of gastric cancer patients;

FIG. 4 shows DNAJB expression in gastric cancer tissue and normal gastric mucosal tissue;

FIG. 5 is a graph showing the effect of DNAJB on survival of gastric cancer patients;

FIG. 6A shows the expression level of mRNA of DCLK2 in gastric cancer tissue and normal gastric mucosa tissue;

FIG. 6B is a graph showing the mRNA expression levels of DNAJB in gastric cancer tissue and normal gastric mucosal tissue;

FIG. 7A shows the expression of DCLK2 in different cell lines;

FIG. 7B is DNAJB showing expression in different cell lines;

FIG. 8A is a comparison of the expression levels of DCLK2 and DNAJB in normal stomach tissue and stomach cancer cells;

FIG. 8B is a quantitative analysis and comparison of the expression level gray scale in DCLK2 normal stomach tissue and gastric cancer cells after correction with GAPDHA;

FIG. 8C is a quantitative analysis comparison of the expression levels in DNAJB5 normal stomach tissue and gastric cancer cells after correction with GAPDHA;

FIG. 9 shows the results of RT-PCR screening for optimal knockdown target sequences;

FIG. 10A is a quantitative analysis comparison of transcript levels after GAPDHA correction for DCLK2 silencing and overexpression in gastric cancer cells;

FIG. 10B is a quantitative analysis comparison of transcript levels after GAPDHA correction for silencing and overexpression of DNAJB in gastric cancer cells;

FIG. 11A is a comparison of expression levels of DCLK2 and DNAJB after silencing and overexpression in gastric cancer cells;

FIG. 11B is a graph showing the comparison of gray scale quantitative analysis of expression levels of DCLK2 after silencing and overexpression in gastric cancer cells after correction with GAPDHA;

FIG. 11C is a quantitative analysis comparison of the gray scale of expression levels of DNAJB5 after silencing and overexpression in gastric cancer cells after correction with GAPDHA;

FIG. 12A is an image of the results of CCK8 experiments;

FIG. 12B is a bar graph of CCK8 experimental results;

FIG. 13A is an image of the results of a Transwell cell invasion assay;

FIG. 13B is a bar graph of the results of a Transwell cell invasion assay;

FIG. 14A is an image of the results of a Transwell cell migration experiment;

FIG. 14B is a bar graph of the results of a Transwell cell migration experiment.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Without particular explanation, the cell lines listed in the context of the present invention, including GES-1, HGC-27, MKN-45, NCI-N87, and KATOIII, were cultured after purchase from the national academy of sciences while being cryopreserved in liquid nitrogen and used for subsequent experiments. All of the reagents used in the present invention are commercially available. The experimental methods used in the present invention, such as DNA extraction, primer design, PCR, tumor cell culture, cell proliferation, cell migration experiments, cell invasion experiments, cell survival experiments, western Blot (Western Blot), etc., are all conventional methods and techniques in the art.

The present invention uses SPSS 26.0 software to process and analyze data. n represents the number of samples, all experiments in the invention are performed three times, experimental data are represented by' X+/-S, and rank sum test and analysis of variance are adopted for comparison among groups. P <0.05 judged the difference to be significant statistically. Representative results of the selection in the biological assay replicates are presented in the figures of the context.

Example 1, cell resuscitation and expansion

Resuscitates the cells, quickly thaws at 37 ℃, then moves 1mL of cell suspension into a centrifuge tube, and centrifugally processes for 5min under the condition of 1000rmp at room temperature, and discards the supernatant; the cells were suspended in a complete medium containing 10% fetal bovine serum, inoculated into a petri dish, and were stirred and mixed well, and cultured at 37℃under 5% CO ₂ saturated humidity. Passaging is performed when the cell density reaches 80%; the medium was discarded and washed once with PBS; then adding 1-2mL of 0.25% trypsin, discarding pancreatin after cell digestion is completed, adding complete culture medium, blowing and mixing uniformly to prepare single cell suspension according to the following ratio of 1:2 and expanded at 37℃with 5% CO ₂ saturated humidity.

EXAMPLE 2 investigation of expression levels of DCLK2 and DNAJB in gastric cancer tissues

Based on UALCAN database, the invention discovers that compared with other types of tumors, the expression of DCLK2 and DNAJB5 genes in STAD (gastric cancer) tissues and the expression of normal gastric mucosa tissues have larger difference (figures 1A-1B), and the DCLK2 and DNAJB genes possibly have research value in gastric cancer. In fig. 1, in each set of data, the left bar represents normal tissue, the right bar represents tumor tissue, the TCGA sample is from the TCGA database, the chinese language of TCGA is collectively referred to as cancer genomic map, which is a cancer genomic project with a milestone meaning, resulting in molecular data of approximately 20,000 primary tumors and matching normal tissue from 11328 patients of 33 cancer types. Further analysis found that: in fig. 3, the upper line represents the medium-low expression samples (n=294), the lower line represents the high expression samples (n=98) of DCLK2, the expression of DCLK2 in STAD (n=415) is significantly reduced (p=0.0099, fig. 2) compared to normal tissue (n=34), and the prognosis survival rate of DCLK2 high expression (n=98) STAD patients is lower than that of medium-low expression (n=294) patients (p=0.021, fig. 3), the difference being statistically significant. Similarly, in fig. 5, the upper line represents the medium-low expressing samples (n=293), the lower line represents the high DCLK2 expressing samples (n=99), the DNAJB expression in STAD is significantly reduced (p=0.0019, fig. 4), and the survival rate of the pre-treatment of DNAJB5 high expressing (n=99) STAD patients is lower than that of medium-low expressing (n=293) patients (p=0.0062, fig. 5), the difference being of significant statistical significance.

MRNA expression of DCLK2 and DNAJB5 was further verified in GEPIA database. Of these, the STAD sample 408 cases, the normal gastric mucosa sample 211 cases, the gastric cancer group on the left in FIG. 6A, the normal group on the right, the gastric cancer group on the left in FIG. 6B, and the normal group on the right. The analysis results show that: the expression levels of mRNA of DCLK2 and DNAJB in STAD tissues were lower than those of normal gastric mucosal tissues (p <0.05, fig. 6A-6B).

Example 3 RT-PCR detection

Gastric cancer cells (HGC-27, MKN-45, NCI-N87 and KATOIII) and human gastric mucosal epithelial cells (GES-1) were cultured in six well plates, respectively, and 1X 10 ⁶ gastric cancer cells (HGC-27, MKN-45, NCI-N87 and KATOIII) were collected, respectively, when the cells were grown to the logarithmic phase.

1. Extraction of total RNA from cells

1X 10 ⁶ cell samples were collected using a 1.5mL centrifuge tube. 1mL of Trizol solution was added thereto, and the mixture was homogenized well, and left to stand at room temperature for 5 minutes. 0.2mL of chloroform was added and vigorously shaken for 15s, followed by standing for 3min. Centrifugation was performed at 12000r/min at 4℃for 10min and the supernatant was taken. Adding 0.5mL of isopropanol, fully and uniformly mixing, and standing on ice for 20-30min. Centrifuging at 12000r/min at 4deg.C for 10min, and discarding supernatant. 1mL of 75% ethanol solution was added, and the mixture was centrifuged at 7500g at 4℃for 5min, and the supernatant was discarded. Air-drying at room temperature or blow-drying in a super clean bench for 5min, and adding a proper amount of Rnase-free H ₂ O solution for dissolution.

2. RNA concentration determination

TABLE 1RNA concentration determination

Sample numbering	A260/A280	Concentration (ng/mL)
			GES-1	2.054	520.800
HGC-27	2.016	812.800
			MKN-45	1.954	303.200
NCI-N87	2.041	872.000
			KATOIII	1.940	384.800

3. CDNA Synthesis

The following reagents were added to a 0.2mL PCR tube, and after thorough mixing, each was incubated at room temperature (37 ℃) for 5min, at 42℃for 60min, at 70℃for 10min, followed by termination and storage of the above solutions at-20 ℃. The reverse transcription reaction system is shown in Table 2 below:

TABLE 2 reverse transcription reaction System

Reagent(s)	Volume (mL)
		Total RNA	5.0
Random Primer	1.0
		Rnase-free ddH ₂ O70℃temperature bath 5 min, quick ice bath 10 sec	5.0
5×Reaction Buffer	4.0
		dNTP Mix (10 mmol/L)	2.0
Rnase inhibitor (20 U/mL)	1.0
		Reverse Transcriptase (10 U/mL)	2.0
Total volume of	20.0

4. Polymerase chain reaction PCR

(1) Primer synthesis: the primers used for Real-time PCR were designed and synthesized by the Shanghai Ind PRIMER PREMIER 5.0.0 software using the housekeeping gene Gapdh as an internal reference, as shown in Table 3 below.

TABLE 3 Real-timePCR primer sequences

Primer(s)	Sequence (5 '-3')	PCR product size (bp)
			DNAJB5(human)-F	SEQ ID NO：3	197
DNAJB5(human)-R	SEQ ID NO：4	197
			DCLK2(human)-F	SEQ ID NO：1	301
DCLK2(human)-R	SEQ ID NO：2	301
			Gapdh（human）-RT-F	SEQ ID NO：5	197
Gapdh（human）-RT-R	SEQ ID NO：6	197

Reagents for detecting the expression level of DCLK2 include a primer pair for detecting the expression level of DCLK2 gene, the upstream sequence of the primer pair is SEQ ID NO:1, the downstream sequence is SEQ ID NO:2.

Reagents for detecting DNAJB expression levels include a primer pair for detecting DNAJB gene expression levels, the upstream sequence of the primer pair being SEQ ID NO:3, the downstream sequence is SEQ ID NO:4.

(2) Real time-PCR reaction system

And preparing a reaction solution according to the RT-PCR reaction system. ddH ₂ O, SYBRGreen QPCR MASTER Mix, forward primer, REVERSE PRIMER, and cDNA template were added to the PCR tube, respectively, and thoroughly mixed. The reaction system was prepared as in table 4 below.

TABLE 4 RT-PCR reaction System

Component (A)	Volume (mL)
		ddH₂O	7.0
SYBRGreen qPCR Master Mix（2×）	10.0
		Forward primer 10 mM	1.0
Reverse primer 10 mM	1.0
		cDNA	1.0
Total volume	20.0

(3) PCR amplification conditions

TABLE 5 Real time-PCR reaction conditions

Project	Temperature (temperature)	Time of	Cycle number
				Pre-denaturation	95℃	10min	1
Denaturation (denaturation)	95℃	20sec	40
				Annealing extension	55℃	20sec	40
	72℃	20sec	40
				Melting curve acquisition	95℃	15sec	1
	60℃	60sec	1
					95℃	15sec	1

RT-PCR experimental results show (FIG. 7A-FIG. 7B): the expression level of the target gene DNAJB in HGC-27, MKN-45, NCI-N87 and KATOIII is obviously lower than that in the GES-1 group, the expression level of the target gene DCLK2 in HGC-27, MKN-45 and KATOIII is obviously lower than that in the GES-1 group, and the expression level of the target gene DCLK2 in NCI-N87 is obviously higher than that in the GES-1 group.

Example 4 Western immunoblot (Western Blot) detection

1. Protein extraction and quantification

(1) The cell samples collected from GES-1, HGC-27, MKN-45, NCI-N87 and KATOIII are added with precooled RIPA lysate containing PMSF, fully mixed, centrifuged for 10min after being fully cracked, and the supernatant is taken for protein quantification and stored in a refrigerator at-80 ℃.

(2) Protein quantification was performed.

(3) Taking an ELISA plate, adding reagents according to the following table 6, and drawing a standard curve;

TABLE 6 dosage of added reagents

Hole number	0	1	2	3	4	5	6	7
									Protein standard solution (mu L)	0	1	2	3	4	5	6	7
Deionized water (mu L)	20	19	18	17	16	15	14	13
									Content of corresponding protein (μg)	0	5	10	15	20	25	30	35

(4) Preparing a proper amount of BCA working solution by the BCA reagent A and the reagent B according to the volume ratio of 50:1, fully and uniformly mixing, and then adding 200 mu L of BCA working solution into each hole respectively;

(5) Oscillating the ELISA plate for 30s, standing at 37 ℃ for 30min, measuring absorbance at 562nm and drawing a standard curve;

(6) 2.5. Mu.L of the protein to be measured and 17.5. Mu LPBS (diluted 8 times) were added to the ELISA plate, then 200. Mu.L of BCA working solution was added thereto, and the plate was shaken for 30 seconds, allowed to stand at 37℃for 30 minutes, absorbance at 562nm was measured, and the corresponding protein concentration (mg/mL) was found from the absorbance measured, thereby determining the loading amount.

2. Western blot (Western blot) detection

(1) Protein samples were prepared: adding 5 XSDS loading buffer solution into the extracted protein, fully and uniformly mixing, denaturing by boiling water bath for 5min, centrifuging for 5min at 12000r/min, and directly loading the obtained protein sample for detection or storing at-80 ℃.

(2) Preparation of polyacrylamide gel: determining the concentration of the separating glue, cleaning and airing the glass plate, inserting the glass plate into a clamp in an aligned manner, clamping, slowly injecting the separating glue along the glass plate, then injecting a proper amount of water, pouring out the water after the glue is solidified, sucking the residual water by using filter paper, filling concentrated glue into the residual space, inserting a comb, cooling in a refrigerator at 4 ℃, and pulling out the comb after the gel is solidified.

TABLE 7 preparation of separation gel and concentrated gel

Reagent(s)	Concentrated gum 5%	Separation gel 12%	10% Of separating gel
				ddH₂O	1.4mL	1.6mL	1.9mL
30% Acrylamide mixed solution	0.33mL	2.0mL	1.7mL
				1.5M Tris-HCl（pH8.8）	—	1.3mL	1.3mL
1.0M Tris-HCl（pH6.8）	0.25mL	—	—
				10% SDS	0.02mL	0.05mL	0.05mL
10% Ammonium persulfate	0.02mL	0.05mL	0.05mL
				TEMED	0.002mL	0.002mL	0.002mL
Total volume of	2mL	5mL	5mL

(3) Loading: the electrophoresis apparatus was assembled, and buffer was poured in, and marker and 30. Mu.g of protein were injected into the sample wells, respectively.

(4) Electrophoresis: and stopping electrophoresis when bromophenol blue approaches the bottom of the gel, and performing transfer.

(5) Cutting glue: taking out the gel, and cutting the gel by taking a marker as a control.

(6) Transferring: the PVDF membrane and the filter paper are cut into the same size as the gel, the PVDF membrane is soaked in methanol solution for 15s, the solution is put into ultrapure water after being semitransparent, the solution is kept stand for 2min, and then the PVDF membrane is put into a transfer buffer solution for balancing for 15min. The foam-rubber cushion, the filter paper, the gel, the PVDF film, the filter paper and the foam-rubber cushion are stacked together in this order, and bubbles between the layers are removed by using a glass rod, and then the foam-rubber cushion is placed in a film transfer tank, and the transfer buffer is poured.

(7) Closing: the membranes were rinsed 5min with TBST solution, 3 times, and then slowly shaken with 5% skim milk powder at 37℃for 2h.

(8) Incubating primary antibodies: the primary dilution ratio was determined according to the instructions and incubated overnight at 4 ℃.

(9) Secondary antibody incubation: after the end of the primary antibody incubation, the incubation was rinsed 3 times for 1min each with TBST solution. PVDF membrane was placed in the secondary antibody solution and incubated for lh with slow shaking at 37 ℃. The solution was rinsed with TBST for 5min and 3 times.

(10) Color development: an appropriate amount of ECL luminescent liquid was added to the film and a photograph was taken.

The detection result of Western Blot (Western Blot) is consistent with RT-PCR experiment, the content of DCLK2 in HGC-27, MKN-45 and KATOIII is obviously lower than that in GES-1 group, and the content of DCLK2 in NCI-N87 is obviously higher than that in GES-1 group. The content of the target protein DNAJB in HGC-27, MKN-45, NCI-N87, KATOIII was significantly lower than that in the GES-1 group (FIG. 8A, FIG. 8B and FIG. 8C).

Example 5 screening of optimal target sequences by RT-PCR

1. The experiments were divided into five groups, respectively ：A：NCI-N87;B：NCI-N87+siRNA-NC;C：NCI-N87+siRNA-DCLK2-1;D：NCI-N87+siRNA-DCLK2-2;E：NCI-N87+siRNA-DCLK2-3.

(1) NCI-N87 cells were expanded. After the cells are grown, they are inoculated into six-well plates, and the cell concentration is adjusted to 5×10 ⁵ cells/well, so that the cell density reaches about 80% the next day. Each well of the six well plate with cells cultured was replaced with 2mL of fresh medium containing serum and antibiotics.

(2) Taking a clean centrifuge tube, sequentially adding 125 mu L of 1640 culture solution without antibiotics and serum and 100pmol of siRNA into cells in each hole of six holes to be transfected, and lightly blowing by a gun to fully and uniformly mix the cells; then, 4. Mu.L Lipo8000 ™ transfection reagent was added thereto, and the mixture was thoroughly mixed by gentle blowing with a gun, and stored at room temperature for 6 hours to stabilize the mixture.

(3) According to the dosage of 125 mu L Lipo8000 ™ transfection reagent-siRNA mixture in each well, uniformly dripping the mixture into the whole well, fully mixing the mixture, continuously culturing the mixture for 48 hours, and collecting each group of cell samples for subsequent detection.

2. RT-PCR screening of optimal knockdown target sequences

RNA extraction, reverse transcription into cDNA, primer design, and Q-PCR amplification, and the specific method is the same as RT-PCR detection.

TABLE 8 knockdown primer sequences

Primer	Sequence（5'-3'）
		DCLK2-RT-F:	SEQ ID NO：1
DCLK2-RT-R:	SEQ ID NO：2
		Gapdh(human)-F	SEQ ID NO：5
Gapdh(human)-R	SEQ ID NO：6

PCR amplification conditions: 94℃for 10min, (94℃for 20 seconds, 55℃for 20 seconds, 72℃for 20 seconds) 40 cycles.

DCLK2 interference experiments were performed by selecting NCI-N87 gastric cancer cell lines, and DCLK2 expression was significantly reduced after transfection of siRNA-DCLK2 compared to NCI-N87 group, wherein the siRNA-DCLK2-2 knockdown effect was the best (FIG. 9).

Example 6, cell group transfection

Experimental grouping ：A：NCI-N87;B：NCI-N87+NC;C：NCI-N87+siRNA-DCLK2;D：NCI-N87+OE-NC;E：NCI-N87+OE-DCLK2;F：NCI-N87;G：NCI-N87+OE-NC;H：NCI-N87+OE-DNAJB5.

1. RT-PCR verification effect

TABLE 9 grouping transfection primer sequences

2. Western Blot (Western Blot) for detecting transfection effect

Protein sample extraction, electrophoresis, membrane transfer, sealing, primary antibody incubation, secondary antibody incubation and development, and the specific method is similar to Western immunoblotting (Western Blot) detection.

Cells were transfected in groups to construct overexpression vectors for DCLK2 and DNAJB. RT-PCR results showed that: compared to NCI-N87, DNAJB of NCI-N87+ OE-DNAJB and DCLK2 of NCI-N87+ OE-DCLK2 showed significantly increased expression and DCLK2 of NCI-N87+ siRNA-DCLK2 showed significantly decreased expression (fig. 10A-10B). The transfection effect was further verified using Western Blot (Western Blot) assay, and the results remained consistent with RT-PCR assays (fig. 11A, 11B and 11C).

Example 7, CCK8 detection of cell proliferation

After the time required for the cell culture of each group, 10. Mu.L of CCK8 was added to each well and incubated at 37℃for 3 hours, and 1 hour for most cases. The time is determined according to the experimental conditions such as the cell type, the cell density and the like; the absorbance of each well was measured by an enzyme-labeled instrument, OD 450nm.

The cell proliferation capability is one of important indexes of tumor development, and can reflect the growth speed and proliferation activity of tumor cells. The influence of DCLK2 and DNAJB over expression on the proliferation capacity of gastric cancer cells is verified by CCK8 experiments, and the result shows that: the NCI-N87+siRNA-DCLK2 group significantly promoted cell proliferation compared to the NCI-N87 group, and the NCI-N87+OE-DCLK2 group and the NCI-N87+OE-DANJB group significantly inhibited cell proliferation (FIGS. 12A-12B).

Example 8 Transwell detection of cell invasion and migration

1. Cell invasion assay

(1) 3ML PBS was added to each treated NCI-N87 cell, 0.25% pancreatin was digested and collected, centrifuged at 1000rpm for 5min and the supernatant was taken, rinsed twice with PBS, and residual serum was washed off.

(2) The serum-free 1640 medium resuspended cells and counted, and the cell concentration therein was diluted to 3X 10 ⁵/mL for use.

(3) Thawing Matrigel one day in advance at 4deg.C, pre-cooling 24-well culture plate, gun head and Transwell chamber overnight at-20deg.C;

(4) Matrigel was diluted to 1mg/mL with serum-free medium on ice;

(5) Pre-cooling 800 mu L of 10% FBS 1640 culture medium (containing double antibody) at 4 ℃ into a 24-well plate, placing the 24-well plate into a Transwell chamber, vertically adding 100 mu L of Matrigel with a final concentration of 1mg/mL, incubating for 4-5h at 37 ℃ to enable the Matrigel to be dried into gel, respectively inoculating 200 mu L of each group of cell suspension into the Transwell upper chamber, and culturing for 48h in a 5% CO ₂ incubator at 37 ℃;

(6) Taking out the Transwell, washing the cell once, and fixing the cells for 1h by using 70% ice-ethanol;

(7) Staining was performed using 0.5% crystal violet dye, left at room temperature for 20min, washed with pbs, and then the non-migrated cells on the upper chamber side were wiped clean with cotton balls, observed under a microscope and photographed.

2. Cell migration experiments

(2) Cells were resuspended in serum-free 1640 medium and counted, and the concentration of cells therein was diluted to 3X 10 ⁵/mL to leave for use.

(3) Mu.L of 1640 culture medium (containing double antibody) of 10% FBS is added into a 24-well plate, placed into a transwell chamber, 200uL of each group of cell suspension is respectively inoculated into the transwell upper chamber after 1h, and then cultured in a 5% CO ₂ incubator at 37 ℃ for 48h;

(4) Taking out transwell, cleaning the cell once, and fixing the cells for 1h by using 70% ice-ethanol;

(5) Dyeing with 0.5% crystal violet dye solution, standing for 20min at room temperature, washing with PBS, cleaning the non-migrated cells at one side of the upper chamber with cotton ball, observing under microscope, and photographing;

The ability of cells to invade and migrate is one of the important indicators of malignant biological behavior of gastric cancer cells, and has a critical effect on the development and metastasis of gastric cancer. According to the invention, through a Transwell experiment, the influence of DCLK2 and DNAJB over-expression on the invasion and migration capability of gastric cancer cells is verified.

The results of the Transwell cell invasion experiments showed that the NCI-n87+sirna-DCLK2 group significantly promoted cell invasion, the NCI-n87+oe-DCLK2 group and the NCI-n87+oe-DANJB group significantly inhibited cell invasion compared to the NCI-N87 group (fig. 13A-13B).

The results of the Transwell cell migration experiments showed that the NCI-n87+sirna-DCLK2 group significantly promoted cell migration compared to the NCI-N87 group, and the NCI-n87+oe-DCLK2 group and NCI-n87+oe-DANJB group significantly inhibited cell migration (fig. 14A-14B).

From the above results, it is clear that the expression of DCLK2 and DNAJB in normal tissues is significantly higher than that of gastric cancer tissues, and it is further clear that DCLK2 and DNAJB5 can be used as biomarkers for detecting gastric cancer, and the prognosis can be reasonably predicted by detecting the expression levels of DCLK2 and DNAJB5 in a patient, and gastric cancer can be assisted by enhancing the expression of DCLK2 and DNAJB. If the expression level of DCLK2 and DNAJB in stomach tissue obtained by gastroscopy is obviously reduced, which indicates that the stomach tissue possibly has cancer or precancerous lesions, further medical treatment should be carried out, and meanwhile, the identification of the expression level of DCLK2 and DNAJB5 is carried out on the tissue excised by surgical large pathology, so that the diagnosis of gastric cancer patients is facilitated. During the treatment of gastric cancer patients, the activity of cancer cells can be inhibited by enhancing the gene expression of DCLK2 and DNAJB, thereby achieving the effect of treating gastric cancer.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims

Application of DNAJB5 over-expression vector in preparing medicine for treating gastric cancer.
2. The use according to claim 1, wherein DNAJB is a biomarker for prognosis evaluation of gastric cancer, and overexpression of DNAJB5 is capable of inhibiting proliferation, invasion and migration of gastric cancer cells.