CN111647660B

CN111647660B - Application of Linc01559 in diagnosis and treatment of gastric cancer

Info

Publication number: CN111647660B
Application number: CN202010659227.2A
Authority: CN
Inventors: 仓顺东; 徐登飞; 任英杰; 陈雪姣; 陈昱名; 李梦圆; 刘莉娜; 张均硕; 闫君雅; 冯骁
Original assignee: Henan Provincial Peoples Hospital
Current assignee: Henan Provincial Peoples Hospital
Priority date: 2020-07-09
Filing date: 2020-07-09
Publication date: 2021-03-16
Anticipated expiration: 2040-07-09
Also published as: CN111647660A

Abstract

The invention belongs to the technical field of medical biology, and discloses a molecular marker Linc01559 for auxiliary diagnosis of gastric cancer. Linc01559 is highly expressed in cancer tissues of gastric cancer and is low expressed in paracancer normal tissues; moreover, the expression level of Linc01559 in the gastric cancer cell line is significantly higher than that of the normal gastric cell line; by knocking down the expression level of the Linc01559 gene, the proliferation, migration and invasion of gastric cancer cells can be obviously inhibited. Therefore, Linc01559 can be used as a molecular marker for auxiliary diagnosis of gastric cancer; by detecting the expression level of Linc01559 in the sample, the auxiliary diagnosis of the gastric cancer can be carried out, and a reference basis is provided for a clinician to diagnose the gastric cancer.

Description

Application of Linc01559 in diagnosis and treatment of gastric cancer

Technical Field

The invention belongs to the technical field of medical biology, and particularly relates to application of Linc01559 in diagnosis and treatment of gastric cancer.

Background

Gastric Cancer (GC) is one of the common malignant tumors in our country, the incidence rate of which is the fourth of the worldwide malignant tumors, and the mortality rate of which is the third of the solid tumor tumors. In recent years, GC has become one of the malignant tumors with a fast-rising incidence in China, and 95 ten thousand new cases occur every year. Because there is no obvious subjective symptom in the early stage of gastric cancer, the main radical treatment means of gastric cancer is still the early surgical treatment, and the digestive tract and general symptoms gradually appear along with the further development of lesions are not specific to gastric cancer, so that most gastric cancer patients are diagnosed in the later stage, the overall prognosis of gastric cancer is relatively poor, and the five-year survival rate is less than 10%. This is closely related to the lack of specific diagnostic targets for GC. The search for key molecules for early accurate diagnosis and target therapy of gastric cancer is a problem to be solved urgently.

Long non-coding RNA (lncRNA) is a type of non-coding RNA with the transcript length of more than 200bp, and is firstly discovered by Okazaki et al in the large-scale sequencing process of a mouse full-length complementary DNA (cDNA) library. They are an important class of regulatory molecules in the human genome, which exert their biological functions in a variety of ways. There is a great deal of evidence that lncRNA is involved in the progression of human malignancies in either a pro-tumor or anti-tumor mode of action. It mainly realizes the regulation and control of gene expression from three layers, mainly including posttranscriptional regulation, transcriptional regulation and epigenetic regulation to silence or activate genes. Specifically, transcription occurs in an upstream promoter region of a protein coding gene, the transcription expression of a downstream gene is interfered, the activity of RNA polymerase II is inhibited, and chromatin reconstruction or histone modification is mediated; combining with mRNA of protein coding gene to interfere mRNA cutting mature process or generate endogenous siRNA under Dicer enzyme action, thereby regulating and controlling related gene expression; the lncRNA can also be combined with a specific protein to regulate the activity of the corresponding protein and change the cytoplasmic localization of the protein, and then the lncRNA participates in the processes of regulating apoptosis, invasion and the like and plays an important regulating role in eukaryotic cell genomes.

Since stomach cancer seriously threatens human health, researchers in various countries around the world search for effective methods for treating stomach cancer through various research approaches or means. In recent years, more and more researches show that lncRNA plays a significant role in the occurrence and development of malignant tumors, so that the search for lncRNA closely related to the occurrence and development of gastric cancer has important significance in the mechanism research, clinical diagnosis and treatment of gastric cancer.

Disclosure of Invention

One of the purposes of the invention is to provide a molecular marker for auxiliary diagnosis of gastric cancer, Linc 01559; the other purpose of the invention is to provide the application of the quantitative detection reagent of the Linc01559 gene in the preparation of products for auxiliary diagnosis of gastric cancer; it is a further object of the present invention to provide a product for diagnosis of gastric cancer; the fourth purpose of the invention is to provide the application of the inhibitor of the functional expression of the Linc01559 gene in the preparation of the medicine for treating gastric cancer; the fifth purpose of the invention is to provide a medicine for treating gastric cancer.

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

the invention firstly provides a molecular marker for auxiliary diagnosis of gastric cancer, and the molecular marker is Linc01559(NCBI gene ID: 283422).

The expression conditions of the Linc01559 gene in the cancer tissue of the gastric cancer and the matched paracancer normal tissue are detected through real-time fluorescent quantitative PCR, and the expression level of the Linc01559 gene in the cancer tissue of the gastric cancer is found to be remarkably higher than that of the matched paracancer normal tissue; it is shown that the Linc01559 gene has different degrees of expression up-regulation in gastric cancer tissues.

The expression conditions of the Linc01559 gene in a gastric cancer cell line and a normal gastric cell line are detected by real-time fluorescent quantitative PCR, and the expression level of the Linc01559 gene in the gastric cancer cell line is found to be remarkably higher than that of the normal gastric cell line; the Linc01559 genes are shown to be up-regulated in the gastric cancer cell lines to different degrees.

The invention also provides application of the quantitative detection reagent of the Linc01559 gene in preparation of a product for auxiliary diagnosis of gastric cancer.

According to the application, the product preferably detects the expression level of the Linc01559 gene in a sample through real-time quantitative PCR, in-situ hybridization, northern blotting, a chip or a high-throughput sequencing platform.

According to the application, the product preferably contains a specific primer for amplifying the Linc01559 gene or a probe hybridized with the Linc01559 gene.

According to the above-mentioned application, preferably, the product for detecting the expression level of the Linc01559 gene in the sample by real-time quantitative PCR comprises a pair of specific primers for amplifying the Linc01559 gene.

According to the above-mentioned application, preferably, the product for detecting the expression level of the Linc01559 gene in the sample by in situ hybridization comprises a probe that hybridizes to the nucleotide sequence of the Linc01559 gene.

According to the application, preferably, the product for detecting the expression level of the Linc01559 gene in the sample by Northern blotting comprises a probe hybridized with the nucleotide sequence of the Linc01559 gene.

According to the above-mentioned application, preferably, the detection of the expression level of the Linc01559 gene in the sample by the chip comprises a probe that hybridizes with a nucleotide sequence of the Linc01559 gene.

According to the above-mentioned application, preferably, the nucleotide sequence of the specific primer for amplifying the Linc01559 gene is shown as SEQ ID NO.1 (5'-TCCCTCAGCCAAGTCCTTCCTTAC-3') and SEQ ID NO.2 (5'-GTCCAGTTCATGCTCTGACAGTCC-3').

According to the above-mentioned use, preferably, the nucleotide sequence of the probe that hybridizes to the Linc01559 gene is shown in SEQ ID NO.3 (5'-GTCTACTTATTGTATATTA-3').

According to the above-mentioned application, preferably, the sample is a sample including (but not limited to) tissue, cell, body fluid (blood, lymph). More preferably, the sample is tissue, blood.

According to the above-mentioned use, preferably, the product is a chip, a preparation or a kit.

The invention also provides a product for gastric cancer diagnosis, which comprises a reagent for detecting the expression level of the Linc01559 gene in a sample.

According to the product, preferably, the reagent comprises a reagent for detecting the expression level of Linc01559 by RT-PCR, real-time quantitative PCR, in-situ hybridization, Northern blotting, a chip or a high-throughput sequencing platform.

According to the product, preferably, the reagent for detecting the expression level of the Linc01559 gene through real-time quantitative PCR comprises a pair of specific primers for amplifying the Linc 01559.

According to the product, preferably, the reagent for detecting the expression level of the Linc01559 gene through in situ hybridization comprises a probe hybridized with a Linc01559 nucleotide sequence.

According to the product, preferably, the reagent for detecting the expression level of the Linc01559 gene by Northern blotting comprises a probe hybridized with a Linc01559 nucleotide sequence.

According to the product, preferably, the reagent for detecting the expression level of the Linc01559 gene by the chip comprises a probe hybridized with the Linc01559 nucleotide sequence.

According to the above products, preferably, the nucleotide sequences of the specific primers for amplifying the Linc01559 gene are shown as SEQ ID NO.1 (5'-TCCCTCAGCCAAGTCCTTCCTTAC-3') and SEQ ID NO.2 (5'-GTCCAGTTCATGCTCTGACAGTCC-3').

According to the above product, preferably, the nucleotide sequence of the probe that hybridizes to the Linc01559 gene is shown in SEQ ID NO.3 (5'-GTCTACTTATTGTATATTA-3').

According to the above products, preferably, the sample includes, but is not limited to, tissue, cells, body fluids (blood, lymph). More preferably, the sample is tissue, blood.

The product according to the above, preferably the product comprises (but is not limited to) a chip, a preparation or a kit. Further, the chip includes a gene chip; the kit comprises a gene detection kit. The gene chip comprises a solid phase carrier and oligonucleotide probes fixed on the solid phase carrier, wherein the oligonucleotide probes comprise oligonucleotide probes for detecting the expression level of the Linc01559 gene. The gene detection kit comprises a specific primer for detecting the expression level of the Linc01559 gene.

In the present invention, unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. In the present invention, the term "primer" refers to a nucleic acid sequence having a free 3' hydroxyl group which is capable of binding complementarily to a template and enabling reverse transcriptase or DNA polymerase to initiate template replication. Primers are nucleotides having a sequence complementary to a nucleic acid sequence of a specific gene.

The invention also provides application of the inhibitor for the functional expression of the Linc01559 gene in preparation of a medicine for treating gastric cancer.

According to the above application, preferably, the inhibitor comprises siRNA and/or shRNA specifically targeting Linc01559 gene.

According to the above application, preferably, the siRNA specifically targeting Linc01559 gene comprises siRNA1 and siRNA 2; the nucleotide sequence of the siRNA1 is shown as SEQ ID NO.4 (5'-GAUUAUUUAUUGUCUACUU-3') and SEQ ID NO.5 (5'-AAGUAGACAAUAAAUAAUC-3'); the nucleotide sequence of the siRNA2 is shown as SEQ ID NO.6 (5'-UGAUUAUUUAUUGUCUACU-3') and SEQ ID NO.7 (5'-AGUAGACAAUAAAUAAUCA-3').

The invention also provides a medicine for treating gastric cancer, which contains an inhibitor of the functional expression of the Linc01559 gene.

According to the medicament, preferably, the inhibitor comprises siRNA and/or shRNA specifically targeting Linc 01559.

According to the medicament, preferably, the siRNA specifically targeting the Linc01559 gene comprises siRNA1 and siRNA 2; the nucleotide sequence of the siRNA1 is shown as SEQ ID NO.4 (5'-GAUUAUUUAUUGUCUACUU-3') and SEQ ID NO.5 (5'-AAGUAGACAAUAAAUAAUC-3'); the nucleotide sequence of the siRNA2 is shown as SEQ ID NO.6 (5'-UGAUUAUUUAUUGUCUACU-3') and SEQ ID NO.7 (5'-AGUAGACAAUAAAUAAUCA-3').

According to the above medicament, preferably, the medicament further comprises a pharmaceutically acceptable carrier and/or an auxiliary material.

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

(1) the invention discovers for the first time that the expression quantity of the Linc01559 gene in the cancer tissue of the gastric cancer is obviously up-regulated compared with the normal tissue beside the cancer, and the expression level of the Linc01559 gene in the gastric cancer cell line is obviously higher than that of a normal gastric cell line, so that the Linc01559 gene can be used as a molecular marker for auxiliary diagnosis of the gastric cancer; by detecting the expression level of Linc01559 in the sample, the early diagnosis of the gastric cancer is realized, and a reference basis is provided for a clinician to diagnose the gastric cancer.

(2) The invention also discovers that the Linc01559 gene is related to the proliferation, migration and invasion capacity of the gastric cancer cells, the Linc01559 gene can promote the proliferation, migration and invasion of the gastric cancer cells, and the Linc01559 gene is knocked down to have an obvious inhibiting effect on the proliferation, migration and invasion of the gastric cancer cells; therefore, by inhibiting the expression level of the Linc01559 gene in the patient, the effect of treating gastric cancer can be achieved.

(3) The siRNA sequence of the specific targeting Linc01559 gene provided by the invention can efficiently inhibit or knock down the expression of Linc01559 in target cells, inhibit the proliferation, migration and invasion of gastric cancer, and further inhibit the growth of gastric cancer cells, so that the siRNA sequence can be used for treating gastric cancer and has important significance in the treatment of gastric cancer.

Drawings

FIG. 1 is a graph showing the expression levels of Linc01559 in cancer tissues of gastric cancer and their paired paracancerous normal tissues.

FIG. 2 is a graph showing the expression levels of Linc01559 in a gastric cancer cell line and a normal gastric cell line.

FIG. 3 is a graph of the transfection results of Linc 01559.

FIG. 4 is a graph showing the effect of Linc01559 on the cell proliferation potency of gastric cancer cell lines.

FIG. 5 is a graph showing the results of the effect of Linc01559 on cell migration of a gastric cancer cell line.

FIG. 6 is a graph showing the results of the effect of Linc01559 on cell invasion of gastric cancer cell lines.

Detailed Description

The present invention will be described in further detail with reference to the following examples, which are not intended to limit the scope of the present invention.

The experimental methods in the following examples, which do not indicate specific conditions, all employ conventional techniques in the art, or follow the conditions suggested by the manufacturers; the reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

The first embodiment is as follows: expression condition research of Linc01559 in cancer tissue of gastric cancer and paracancer normal tissue matched with cancer tissue

1. Collecting samples:

60 cancer tissues of patients with primary gastric cancer diagnosed by histopathology and corresponding paracancer normal tissue samples of >3cm are collected, in order to prevent RNA degradation, the samples are cut immediately when cut by surgery, washed twice by PBS, placed into a freezing tube, immediately placed into liquid nitrogen, and finally transferred to a refrigerator at minus 80 ℃ for long-term storage. No radiotherapy or chemotherapy is performed on 60 gastric cancer patients before operation, and the operation is the preferred treatment scheme. Each patient signed an informed consent prior to sampling, according to the rules prescribed by the ethical review board.

2. Experimental methods

The qRT-PCR method is adopted to detect the expression level of Linc01559 in cancer tissues of 60 cases of gastric cancer patients and corresponding paracancer normal tissues, and the specific operation steps are as follows:

(1) total RNA extraction:

1) the collected cancer tissues of the gastric cancer and the corresponding paracancer normal tissue samples are taken out from liquid nitrogen, the samples are placed on ice for thawing, TRIzol (1ml of TRIzol is added to 50-100 mg of tissues) is added into each tube according to the weight, then two small steel balls are added, a cover is tightly covered, the mixture is placed into a frame prepared by a multi-sample tissue grinder, the grinder is placed, screws are screwed, the parameters are adjusted to be 60Hz, the time is 1min, and the tissues are fully ground.

2) The ground sample was taken out and placed on ice for 20min for sufficient lysis.

3) The TRIzol-digested tissue or cell sample was placed symmetrically in a 4 ℃ centrifuge at 12000rpm for 10min at 8 ℃.

4) Spraying RNase spray scavenger onto the tabletop, and wiping with paper towel after 5 min. The sample from the centrifuge was removed, the supernatant transferred to a new labeled 1.5mL EP tube, chloroform added (0.2 mL of chloroform was required for 1mL TRIzol-infiltrated tissue), the lid closed and shaken vigorously for 15s, and allowed to stand for 5 min.

5) After the standing is finished, the sample is put into the 4 ℃ centrifuge again at 12000rpm for 15min at 8 ℃.

6) After centrifugation the sample was removed and the upper aqueous phase carefully aspirated into a new EP tube, complying with the principle of little or no more.

7) Adding equal amount of isopropanol, reversing, mixing for 15s, and placing in a refrigerator at-20 deg.C for more than one hour or overnight;

8) after 1h or the next day, the EP tube is taken out and placed into a 4 ℃ centrifuge at 12000rpm for 10min and 8 ℃ (75% ethanol is prepared by using absolute ethanol and DEPC water in advance and placed on ice for precooling).

9) And (3) after the centrifugation is finished, removing the supernatant, adding 1mL of pre-cooled 75% ethanol into each tube, tightly covering the cover, slightly flicking the tube wall to separate the white precipitate from the tube wall, and centrifuging at 12000rpm for 5min in a 4 ℃ centrifuge.

10) The ethanol in the EP tube is sucked up as far as possible, and the white precipitate in the tube is cooled to be semitransparent.

11) Adding 50-100u1RNase free H2O, dissolving, mixing, and gently blowing to dissolve RNA completely.

12) The concentration and purity of the RNA was determined using a NanoDrop 2000 assay.

(2) Reverse transcription to synthesize cDNA:

1) the extracted RNA was thawed on ice, at a volume not exceeding 500ng, calculated as concentration.

2) The forceps were burned on an alcohol burner for about 10 seconds, and after cooling, 0.2ml PCR tubes of RNAse free were removed from the lunch box.

3) 5 XgDNA Eraser Buffer and 5 XPrimeScript RT Master Mix in the PrimeScript RT Master Mix (Perfect Real Time) kit were removed, centrifuged briefly, and placed on ice.

4) The reaction solution for removing genomic DNA was prepared according to the composition in Table 1 and was carried out on ice:

TABLE 1 reaction solution System for removing genomic DNA

Reagent	Amount of the composition used
		5×gDNA Eraser Buffer	2μl
gDNA Eraser	1μl
		Total RNA	*
RNase Free dH₂O	up to 10μl

5) RT reaction solution was prepared according to the composition in Table 2, and was carried out on ice:

TABLE 2 reverse transcription reaction System

Reagent	Amount of the composition used
		Reaction solution of step 4)	10μl
PrimeScript RT Enzyme Mix	1μl
		RT Primer Mix	1μl
5×PrimeScript Buffer 2(for Real Time)	4μl
		RNase Free dH₂O	4μl
Total	20μl

6) After the prepared mixed solution is mixed softly and uniformly, a PCR instrument is opened, the following procedures are set according to the following cycle, samples are put into the instrument one by one, and a reverse transcription reaction is started, wherein the reverse transcription reaction procedure is as follows: 15min at 37 ℃ (reverse transcription reaction), 5sec at 85 ℃ (reverse transcriptase inactivation reaction). And (3) timely taking out RT reaction liquid, namely cDNA (complementary deoxyribonucleic acid), and diluting the RT reaction liquid in a ratio of 1:10 for qPCR (when the RT reaction liquid is added into a next Real Time PCR reaction system, the addition amount of the RT reaction liquid does not exceed 1/10(V/V) of the Real Time PCR reaction volume).

(3) And (3) fluorescent quantitative detection:

relative expression levels of Linc01559 in cancer tissues of the gastric cancer patients and corresponding paracancer normal tissues are detected by Real Time PCR reaction by taking GAPDH as an internal reference, and the expression difference between the cancer tissues and the corresponding paracancer normal tissues is determined.

The nucleotide sequence of the specific amplification primer of Linc01559 is as follows:

an upstream primer: 5'-TCCCTCAGCCAAGTCCTTCCTTAC-3' (SEQ ID NO. 1);

a downstream primer: 5'-GTCCAGTTCATGCTCTGACAGTCC-3' (SEQ ID NO. 2).

The nucleotide sequence of the specific amplification primers for GAPDH is as follows:

an upstream primer: 5'-CTGGGACGACATGGAGAAAA-3', respectively;

a downstream primer: 5'-AAGGAAGGCTGGAAGAGTGC-3' are provided.

Specific amplification primers of Linc01559 and GAPDH are designed and synthesized by biological engineering (Shanghai) company.

The Real-time PCR reaction system (20uL system) was prepared in the proportions shown in Table 3.

TABLE 3Real-time PCR reaction System

Name (R)	Volume of
		FastStart Universal SYBR Green Master(Rox)	10μl
Upstream primer (10. mu.M)	0.5μl
		Downstream primer (10. mu.M)	0.5μl
cDNA	1μl
		ddH₂O	To 20. mu.l

Real-Time PCR was performed by the two-step method and melting curves were generated and programmed as in Table 4.

TABLE 4 two-step Real-Time PCR reaction System Programming

According to the original detection result of RealTimePCR, according to 2^ s^-δδCtThe method calculates the relative expression level of Linc01559, namely the difference of the transcription level of the target gene Linc01559 in each cancer tissue sample relative to the matched paracancer control sample.

3. Data processing and analysis:

the experiments were performed in 3 replicates, the results were represented as mean ± sd, and were statistically analyzed using SPSS18.0 statistical software, with the difference between the two using the t-test, and considered statistically significant when P < 0.05.

4. Results of the experiment

Real-Time PCR detected the expression level of Linc01559 in cancer tissues of 60 patients with gastric cancer and corresponding paracancer normal tissues, and the detection result is shown in FIG. 1. As can be seen from fig. 1, the expression level of Linc01559 in the gastric cancer tissue is significantly higher than that in the paired paracancer normal tissue, and the difference has significant statistical significance (P < 0.001). Therefore, the expression level of Linc01559 is related to the occurrence of gastric cancer, and can be used as a molecular marker of gastric cancer for clinical auxiliary diagnosis of gastric cancer.

Example two: expression of Linc01559 in gastric cancer cell and normal gastric cell strains

1. Cell selection and culture:

culturing human normal gastric cell strain GSE-1, gastric cancer cell line SNU-16, BGC-823, MGC-803, AGS, MKN-45 and SGC-7901; the cells are routinely cultured in 1640 cell culture medium containing 10% FBS, 1% penicillin and streptomycin, wherein GSE-1 is in high-sugar DMEM medium instead of 1640 medium, at 37 deg.C and 5% CO₂And culturing under saturated humidity. Cells were plated every 2 days with 1: passage 3.

2. The experimental method comprises the following steps:

the qRT-PCR is adopted to detect the expression level of Linc01559 in gastric cancer cells and normal gastric cell strains, and the specific operation steps are as follows:

(1) extracting cell RNA:

total RNA from cells was extracted by TRIzo1 method.

Collecting cells in logarithmic phase, adding 1mL of TRIzo1, and mixing uniformly; the cells were disrupted and the DNA sheared by repeated aspiration with a 1m1 syringe and left to stand at room temperature for 5min, the remaining steps were the same as in example one and will not be described further.

(2) Reverse transcription to synthesize cDNA:

the specific procedures for synthesizing cDNA by reverse transcription are the same as those in the first embodiment, and are not repeated herein.

(3) And (3) fluorescent quantitative detection:

the specific operation steps of the fluorescence quantitative detection are the same as those in the first embodiment, and are not described herein again.

3. Data processing analysis

All data are expressed as mean standard deviation (mean ± SD). Two groups were compared using two-sided Student' st test, and three and above groups were analyzed using one-way anova. All results were plotted using GraphPad Prism 6Software, at a test level of P <0.05, and considered statistically significant when P < 0.05.

4. Results of the experiment

The qRT-PCR detects the expression level of the Linc01559 gene in gastric cancer cells and normal gastric cell strains, and the detection result is shown in figure 2. As can be seen from FIG. 2, compared with GSE-1 cells of normal cells of gastric mucosa, the expression of the Linc01559 gene in gastric cancer cell strains BGC-823, MGC-803, AGS, MKN-45 and SGC-7901 is remarkably increased, which indicates that the expression level of the Linc01559 gene in gastric cancer cells is remarkably increased.

Wherein, the expression level of the Linc01559 gene in the MKN-45 cell is higher, so the MKN-45 cell line is selected as a tool cell for subsequent experimental study.

Example three: silencing of Linc01559 gene

1. Cell culture:

human gastric cancer cell line MKN-45, in an amount of 10%1640 cell culture medium of fetal bovine serum and 1% penicillin and streptomycin at 37 deg.C and 5% CO₂And culturing in an incubator with relative humidity of 90%. Cells were changed 1 time every 2 days and passaged by digestion with digestive enzymes.

siRNA design

siRNA sequence against Linc01559 gene:

negative control siRNA (siRNA-NC) sequence: the siRNA-NC sequence is provided by Ruibo, Guangzhou, product number: siN 0000001-1-5.

siRNA 1：

The sense strand is: 5'-GAUUAUUUAUUGUCUACUU-3' (SEQ ID NO. 4);

the antisense strand is: 5'-AAGUAGACAAUAAAUAAUC-3' (SEQ ID NO. 5).

siRNA 2：

The sense strand is: 5'-UGAUUAUUUAUUGUCUACU-3' (SEQ ID NO. 6);

the antisense strand is: 5'-AGUAGACAAUAAAUAAUCA-3' (SEQ ID NO. 7).

siRNA 3：

The sense strand is: 5'-GCCAUGACCUUGAAUAAGU-3', respectively;

the antisense strand is: 5 '-ACUUAUCAAGGUCZUGGC-3'.

3. Cell transfection:

cells were transfected according to the transfection reagent Lipofectamine 3000reagent (Invitrogen) instructions. The method comprises the following specific steps:

(1) the cells were aligned at 2.0X 10⁵The cells were inoculated into six-well cell culture plates, cultured for 24 hours at 37 ℃ in a 5% CO2 incubator to reach a cell confluency of 50%, and 1.8mL of fresh medium was replaced before transfection.

(2) Transfection: lipofectamine 3000reagent (invitrogen) was removed from the 4 ℃ refrigerator in advance, siRNA1, siRNA2, siRNA3, and siRNA-NC were removed from the-20 ℃ refrigerator, and dissolved on ice, and EP tubes were prepared in advance and labeled, and 150ul Opti-MEM medium was added to each EP tube.

1) And siRNA group:

a. add 5ul siRNA to EP tube containing 150ul Opti-MeM medium (tube (r));

b. add 5ul Lipofectamine 3000reagent (invitrogen) to EP tube containing 100ul Opti-MEM medium (tube @);

c. completely sucking out the liquid in the first tube by using a liquid transfer gun, adding the liquid into the second tube, and incubating for 5min at room temperature;

d. then a liquid transfer gun is used for sucking out all liquid in the No.2 tube, the liquid is uniformly dripped into a 10cm dish, the mixture is uniformly mixed by shaking, and the 10cm dish is placed back into a 37 ℃ incubator for continuous culture.

2) Negative control group (siRNA-NC group):

a. add 5ul siRNA-NC to EP tube containing 150ul Opti-MEM medium (tube # q);

b. 5ul Lipofectamine 3000reagent (invitrogen) was added to an EP tube containing 100ul Opti-MeM medium (tube # d);

c. sucking out all liquid in the tube III by using a liquid transfer gun, adding the liquid into the tube IV, and incubating for 5min at room temperature;

d. and then, sucking out all the liquid in the tube No. IV by using a liquid transfer gun, uniformly dripping the liquid into a 10cm dish, uniformly mixing the liquid and the dish by shaking, and putting the 10cm dish back into the 37 ℃ incubator for continuous culture.

qRT-PCR detection of expression level of Linc 01559:

(1) extracting cell RNA:

the specific operation of extracting cell RNA is the same as the example, and is not repeated here.

(2) Reverse transcription to synthesize cDNA:

(3) And (3) fluorescent quantitative detection:

5. The experimental results are as follows:

the results of the experiment are shown in FIG. 3. As can be seen from FIG. 3, compared with the negative control group, the expression level of Linc01559 in the siRNA-transfected experimental group is significantly reduced, wherein the interference effect of siRNA1 and siRNA2 is better.

Example four: effect of Linc01559 Gene on cell proliferation, migration and invasion of gastric cancer cells

1. CCK8 test for cell proliferation

(1) Cell culture:

the specific operation of cell culture is the same as that of the examples, and is not repeated here.

(2) Cell transfection:

the specific procedures for cell transfection are the same as those in the examples, and are not described herein.

(3) CCK8 detection of cell proliferation

1) Experiments are divided into 3 groups, namely a negative control group (siRNA-NC group), a siRAN 1 group and a siRAN2 group, wherein each group is provided with 4 multiple wells.

2) After transfection, MKN-45 cells were cultured for 72 hours, cells were counted by digestion with 0.25% trypsin, and the cells were seeded in a 96-well plate at a concentration of 6X 10³M1, adding 100u1 per hole;

3) adding 10u of 1 CCK8 reagent into each hole of a 96-hole plate under the condition of keeping out of the sun, shaking the culture plate for 1min, putting the culture plate into an incubator for continuous culture, taking out after 2h, and measuring the 0D value at the 450nm wavelength of an enzyme-labeling instrument.

(4) Results of the experiment

The results of the experiment for detecting cell proliferation by CCK8 are shown in FIG. 4. As can be seen from FIG. 4, cell proliferation was significantly inhibited in the experimental group transfected with siRNA1 and siRAN2 (P <0.05) compared to the negative control group (siRNA-NC group). Therefore, the knock-down Linc01559 has obvious inhibition effect on the proliferation capacity of MKN-45 cells.

2. Transwell cell migration and invasion assay

The influence of the target gene on the migration ability of the cells was verified by examining the migration of the target cells into the serum-containing medium in the Transwell chamber.

(1) The experimental method comprises the following steps:

1) after melting the Matrigel in an ice bath under the aseptic condition, using a serum-free 1640 culture medium according to the ratio of 1: matrix 1 gel was diluted at 7 ratios (no gel was applied for migration experiments, and gel was applied for invasion experiments), added slowly to the bottom of the upper chamber in Transwell 11, and the 24-well plate with the Transwell wells was moved into a 37 ℃ cell incubator for incubation or placed horizontally at room temperature until it solidified into a gel.

2) The experiment was divided into 3 groups, negative control (siRNA-NC group), siRAN 1 group, siRAN2 group, each group was provided with 3 multiple wells, the number of cells added to the upper chamber was 5X 10⁴Cell suspension with individual cells free of serum (migration experiment), 1X 10⁵A cell suspension without serum for each cell (invasion experiment) is added into a lower chamber, 700u1 medium containing 10% fetal calf serum is added into the lower chamber, and the cell suspension is cultured in a constant temperature incubator at 37 ℃ for 48 hours.

3) And taking out the Transwell chamber, washing with PBS for 3 times, fixing with 4% paraformaldehyde for 30min, washing with PBS for 3 times, adding crystal violet, dyeing for 8-12min, observing the coloration intensity under a microscope, discarding the crystal violet solution, washing with purified water, placing under a fluorescence microscope, observing, photographing and counting.

(2) Data processing and analysis:

all data are expressed as mean-squared standard deviation. Two groups were compared using two-sided Student' st test, and three and above groups were analyzed using one-way anova. All results were plotted using GraphPad Prism 6Software, with P <0.05 as the test level, and differences of <0.05 were statistically significant.

(3) The experimental results are as follows:

the results of the Transwell chamber cell migration assay are shown in FIG. 5. As seen in FIG. 5, the cell migration ability of the experimental group transfected with siRNA1 and siRNA2 was significantly reduced (P <0.05) as compared with the negative control group (siRNA-NC group). Therefore, the knock-down Linc01559 has obvious inhibition effect on the migration capability of MKN-45 cells.

The results of the Transwell cell invasion assay are shown in figure 6. As shown in FIG. 6, the cell invasion ability of the experimental group transfected with siRNA1 and siRNA2 was significantly reduced (P <0.05) as compared with the negative control group (siRNA-NC group). Therefore, the knock-down Linc01559 has obvious inhibition effect on the cell invasion capacity of MKN-45.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the present invention, but rather as the following description is intended to cover all modifications, equivalents and improvements falling within the spirit and scope of the present invention.

Sequence listing

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Application of <120> Linc01559 in diagnosis and treatment of gastric cancer

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Claims

Application of a quantitative detection reagent of a Linc01559 gene in preparation of a product for auxiliary diagnosis of gastric cancer.
2. The use of claim 1, wherein the product detects the expression level of the Linc01559 gene in a sample by real-time quantitative PCR, in situ hybridization, Northern blotting, a chip, or a high throughput sequencing platform.
3. The use of claim 1, wherein the product comprises a specific primer for amplifying the Linc01559 gene or a probe for hybridizing with the Linc01559 gene.
4. The use of claim 3, wherein the nucleotide sequence of the specific primer for amplifying the Linc01559 gene is shown as SEQ ID No.1 and SEQ ID No. 2; the nucleotide sequence of the probe hybridized with the Linc01559 gene is shown as SEQ ID NO. 3.
5. The use of claim 2, wherein the sample comprises a tissue, a cell, a body fluid; the product is a chip, a preparation or a kit.
Application of an inhibitor of Linc01559 gene functional expression in preparation of a medicine for treating gastric cancer.
7. The use of claim 6, wherein the inhibitor comprises an siRNA and/or shRNA that specifically targets the Linc01559 gene.
8. The use of claim 7, wherein the siRNA specifically targeting the Linc01559 gene comprises siRNA1 and siRNA 2; the nucleotide sequence of the siRNA1 is shown as SEQ ID NO.4 and SEQ ID NO. 5; the nucleotide sequence of the siRNA2 is shown in SEQ ID NO.6 and SEQ ID NO. 7.
9. The medicine for treating the gastric cancer is characterized by comprising an inhibitor of the functional expression of a Linc01559 gene; the inhibitor is siRNA specifically targeting Linc01559, and the siRNA specifically targeting Linc01559 gene comprises siRNA1 and siRNA 2; the nucleotide sequence of the siRNA1 is shown as SEQ ID NO.4 and SEQ ID NO. 5; the nucleotide sequence of the siRNA2 is shown as SEQ ID NO.6 and SEQ ID NO. 7.
10. The medicament for treating gastric cancer according to claim 9, wherein the medicament further comprises a pharmaceutically acceptable carrier and/or adjuvant.