CN106701900B

CN106701900B - Long-chain non-coding RNA HERC2P3 gene and application thereof in gastric cancer

Info

Publication number: CN106701900B
Application number: CN201510787049.0A
Authority: CN
Inventors: 高勇; 李砚东; 解永松
Original assignee: Shanghai East Hospital
Current assignee: Shanghai East Hospital
Priority date: 2015-11-16
Filing date: 2015-11-16
Publication date: 2020-03-20
Anticipated expiration: 2035-11-16
Also published as: CN106701900A

Abstract

The invention discloses application of a long-chain non-coding RNA HERC2P3 gene in diagnosis and treatment of gastric cancer. Specifically, the invention proves that the expression of the LncRNA-HERC2P3 gene in the stomach cancer tissue is obviously higher than that of the tissue beside the cancer, and the growth of the stomach cancer cells can be obviously inhibited by silencing the LncRNA-HERC2P3 gene, so that the LncRNA-HERC2P3 gene and the expression product thereof can be used as a marker for diagnosing the stomach cancer and a drug target for treating the stomach cancer, and the diagnosis of the stomach cancer is more accurate and rapid. Therefore, the LncRNA-HERC2P3 gene and the application thereof provide a new therapeutic target and an effective new medicine for preventing and treating gastric cancer.

Description

Long-chain non-coding RNA HERC2P3 gene and application thereof in gastric cancer

Technical Field

The invention belongs to the technical field of gene therapy and diagnosis, and particularly relates to a long-chain non-coding RNA gene and application of an expression product thereof in diagnosis and treatment of gastric cancer.

Background

China is a country with high incidence of gastric cancer, and the morbidity and mortality of China are respectively high in the 3 rd and 2 nd of all malignant tumors. In 2008, 46.3 thousands of new-onset gastric cancer in China accounts for 46.8 percent of the world; about 73.7 thousands of patients with stomach cancer die in the same year all over the world, wherein 35.2 thousands of patients die in China, which accounts for 47.8% of the world. Gastric tumors are rarely found or diagnosed in the preclinical stage, and particularly in China, over 80% of gastric cancers are in the advanced stage when diagnosed, so that the prognosis is generally poor. Gastroscopy is the main screening means of early gastric cancer, but the cost of gastroscopy is expensive, and simultaneously brings much pain to patients, and currently, only Japan is used for screening asymptomatic people all over the world. To overcome this difficulty, many biomarkers are widely used for the diagnosis of gastric tumors, including oncogenes, tumor suppressor genes, DNA repair-related genes, and regulatory genes and their expression products related to cell cycle and cell adhesion. However, these biomarkers are not highly sensitive or specific for early diagnosis of gastric cancer. Therefore, the discovery of new tumor markers with higher sensitivity and specificity is the key to improving the early diagnosis level of gastric cancer.

In recent years, with the completion of the human ENCODE program, it has been found that non-coding RNAs play an important role in cell life activities. Non-coding RNAs are a class of functional RNA molecules that do not encode proteins and can be divided into two broad categories according to their length: short-segment non-coding RNA (including siRNA, miRNA, piRNA, etc.) and long-segment non-coding RNA (LncRNA). miRNAs are a more studied group of short-fragment non-coding RNAs, and they are non-coding single-stranded small molecule RNAs with a size of about 22 bases, which, by complementary binding with the 3' non-coding region of the target gene mRNA, cause degradation or transcriptional repression of the target gene mRNA, and regulate the expression of the gene at the post-transcriptional level. LncRNA refers to RNA greater than 200nt in length, located in the nucleus or cytoplasm, most with conserved secondary structure, splicing patterns, and subcellular localization. LncRNA exists widely and regulates intracellular signaling systems, and can regulate the expression level of genes at multiple levels, and currently known mechanisms of action include chromatin modification, transcriptional regulation, post-transcriptional regulation (e.g., precursor mRNA processing), and the like.

LncRNA is also closely related to the development of cancer, and like mirnas, LncRNA represents another important molecular source for disease diagnosis and treatment. The current research shows that the difference of the expression level of lncRNA or the expression of specific lncRNA of certain cancer types can be used as a new molecular marker for tumor diagnosis and treatment.

Therefore, there is an urgent need in the art to find more associations between LncRNA and gastric cancer, and thus to develop a new method for diagnosing and/or treating gastric cancer.

Disclosure of Invention

The present inventors provide a use of LncRNA-HERC2P3 in diagnosis and treatment of gastric cancer.

In a first aspect of the invention, the use of the isolated lncRNA-HERC2P3 or a detection reagent thereof for preparing a chip, a reagent or a kit for diagnosing gastric cancer is provided; the lncRNA-HERC2P3 is selected from:

(a) a sequence as shown in SEQ ID No. 1;

(b) lncRNA-HERC2P3 complementary to the sequence shown in SEQ ID NO. 1.

In another preferred embodiment, the lncRNA-HERC2P3 is isolated from blood and/or tissue of a human or non-human mammal.

In another preferred embodiment, the blood is plasma and/or serum.

In another preferred embodiment, the blood does not include blood cells.

In another preferred embodiment, the non-human mammal is a mouse, rat, rabbit, pig, cow, sheep, etc.

In another preferred embodiment, the tissue comprises tumor tissue, especially gastric cancer tissue.

In another preferred embodiment, the lncRNA-HERC2P3 is isolated from human.

In another preferred example, the chip includes:

a solid support; and oligonucleotide probes orderly fixed on the solid phase carrier, wherein the oligonucleotide probes specifically correspond to the sequence shown in SEQ ID NO. 1.

In a second aspect of the invention, a kit is provided, said kit comprising a detection reagent for lncRNA-HERC2P3, and instructions for use,

wherein, the lncRNA-HERC2P3 is selected from:

(a) a sequence as shown in SEQ ID No. 1;

(b) lncRNA-HERC2P3 complementary to the sequence shown in SEQ ID NO. 1.

In another preferred embodiment, the kit also contains lncRNA-HERC2P3 as a positive control.

In another preferred example, the detection reagent for lncRNA-HERC2P3 comprises a primer pair, a probe and a chip for specifically amplifying the sequence shown in SEQ ID NO. 1.

In another preferred example, the primer pair for specifically amplifying the sequence shown in SEQ ID NO. 1 comprises the sequences shown in SEQ ID NO. 2-3.

In a third aspect of the invention, there is provided an inhibitor of lncRNA-HERC2P3, which specifically inhibits the expression and/or activity of lncRNA-HERC2P3 in vitro and/or in vivo;

wherein, the lncRNA-HERC2P3 is selected from:

(a) a sequence as shown in SEQ ID No. 1;

(b) lncRNA-HERC2P3 complementary to the sequence shown in SEQ ID NO. 1.

In another preferred embodiment, the inhibitor comprises antisense nucleic acid, siRNA, miRNA and shRNA of the sequence shown in SEQ ID NO. 1.

In another preferred embodiment, the siRNA is shown in SEQ ID NO. 4-5 or SEQ ID NO. 6-7.

In another preferred embodiment, the shRNA is shown in SEQ ID NO. 8-9.

In a fourth aspect of the invention, there is provided a use of the lncRNA-HERC2P3 inhibitor of the third aspect of the invention for preparing a pharmaceutical composition for treating gastric cancer and/or gastric cancer metastasis.

In another preferred embodiment, the pharmaceutical composition comprises an inhibitor of lncRNA-HERC2P3 and a pharmaceutically acceptable carrier.

In the fifth aspect of the invention, a pharmaceutical composition for treating gastric cancer and/or gastric cancer metastasis is provided, wherein the pharmaceutical composition comprises the inhibitor of lncRNA-HERC2P3 of the third aspect of the invention and a pharmaceutically acceptable carrier.

In a sixth aspect of the present invention, there is provided a method for screening a candidate compound for a drug for treating gastric cancer and/or gastric cancer metastasis, the method comprising:

in the test group, the candidate compound is added into a gastric cancer cell culture system, and the expression and/or activity E1 of the sequence shown in SEQ ID No. 1 is determined; in a control group, the candidate compound is not added into a gastric cancer cell culture system, and the expression and/or activity E0 of the sequence shown in SEQ ID No. 1 is determined;

wherein, when E1 is significantly higher than E0, the candidate compound is indicated to be a drug capable of treating gastric cancer and/or gastric cancer metastasis.

In another preferred embodiment, said significantly higher is E1/E0 ≧ 1.5, preferably E1/E0 ≧ 2.

In another preferred embodiment, the method further comprises further testing the candidate compound obtained in step (a) for its effect on the growth and/or migration of gastric cancer cells.

In a seventh aspect of the invention, there is provided a method of non-therapeutically inhibiting gastric cancer cell growth and/or migration in vitro by culturing gastric cancer cells in the presence of an inhibitor according to the third aspect of the invention and/or a pharmaceutical composition according to the fifth aspect of the invention, thereby inhibiting gastric cancer cell growth and/or migration.

In the eighth aspect of the present invention, a method for diagnosing gastric cancer and/or gastric cancer metastasis is provided, wherein the expression and/or activity of lncRNA-HERC2P3 in a sample from a desired subject is detected, and when the expression and/or activity of lncRNA-HERC2P3 in the sample is significantly higher than that of a control, the subject is indicated to be likely to have gastric cancer and/or gastric cancer metastasis;

wherein, the lncRNA-HERC2P3 is selected from:

(a) a sequence as shown in SEQ ID No. 1;

(b) lncRNA-HERC2P3 complementary to the sequence shown in SEQ ID NO. 1. .

In a ninth aspect of the present invention, there is provided a method of treating gastric cancer and/or gastric cancer metastasis, comprising the steps of: administering to a subject in need thereof a safe and effective amount of an inhibitor according to the third aspect of the invention and/or a pharmaceutical composition according to the fifth aspect of the invention.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1A shows that the qPCR result in example 1 shows that the expression level of LncRNA-HERC2P3 in gastric cancer cells is significantly reduced after the LncRNA-HERC2P3 gene is silenced.

FIG. 1B shows that in example 1, the growth and proliferation ability of gastric cancer cells was significantly reduced in the siRNA group targeting LncRNA-HERC2P3, compared to the negative control group.

FIG. 2A shows that in example 2, cell invasion assay qualitatively indicates that the invasion ability of stomach cancer cells silencing LncRNA-HERC2P3 gene is obviously reduced.

FIG. 2B shows that the number of cells in the experimental group with the LncRNA-HERC2P3 gene silenced in example 2 is significantly reduced compared to the control group.

FIG. 3A shows the transfection efficiency of shHERC2P3 group and shNC group under white light and fluorescence microscopy (X100) in example 3, and a lentiviral vector with a knockout of LncRNA-HERC2P3 gene was successfully transfected in SGC-7901 gastric cancer cells.

FIG. 3B shows that in the shHERC2P3 group versus the shNC group in example 3, the expression of LncRNA-HERC2P3 gene is significantly reduced.

FIG. 4A shows the ability of silenced LncRNA-HERC2P3 gene to inhibit tumor formation of gastric cancer cell SGC-7901 subcutaneously in nude mice. The LncRNA-HERC2P3 silencing group was smaller and lighter than the control group, both groups having statistical significance, both in terms of volume and tumor weight.

Fig. 4B shows that the number of visible tumors in the lung of shHERC2P3 group mice was significantly reduced relative to the control group.

FIG. 5 is a schematic diagram showing the real-time quantitative PCR detection of the expression of LncRNA-HERC2P3 gene in the cancer tissues and the paracarcinoma tissues of 30 patients with gastric cancer in example 5, wherein "N" refers to the paracarcinoma tissues and "C" refers to the gastric cancer tissues. The results showed that the expression level of LncRNA-HERC2P3 gene was higher in the cancer tissues of 13 patients (43.3%) than in the corresponding paracarcinoma tissues.

Detailed Description

The present inventors have conducted extensive and intensive studies and, for the first time, have unexpectedly found that a long-chain non-coding RNA, HERC2P3, which is closely related to the onset and metastasis of gastric cancer, is highly expressed in gastric cancer tissues and highly expressed in paracancerous and normal tissues, and thus can be used as a detection marker for assisting gastric cancer or metastasis thereof. In addition, the inventor further shows through experiments that the lncRNA can be very effectively inhibited from gastric cancer or the metastasis thereof, so that the lncRNA can be used as a drug target for gastric cancer treatment.

Non-coding RNA (Non-coding RNAs)

As used herein, the term "non-coding RNA" refers to an RNA that does not encode a protein. In the human genome, most are non-coding RNAs. Only 2% of the transcripts produced in the human genome are codable RNAs, the remaining 98% are non-coding RNAs, and they are functional RNA molecules that cannot be translated into protein. These non-coding RNAs are widely involved in physiological and pathological activities of human body and closely related to a plurality of tumors.

Among non-coding RNAs, molecules having a regulatory effect are mainly classified into two types according to their sizes: short non-coding RNAs (including siRNA, miRNA, piRNA) and Long non-coding RNAs (Long non-coding RNAs, LncRNA). These ncRNAs are widely involved in almost all physiological and pathological activities of human body, including participating in, regulating or mediating the process of generation and development of a plurality of tumors.

Long non-coding RNA (LncRNA)

As used herein, the terms "LncRNA", "Long non-coding RNA", "Long non-coding RNA" and "Long non-coding RNA" are synonymous and used interchangeably and refer to a fragment of RNA transcribed by RNA polymerase II that does not encode a protein and is generally greater than 200bp in length.

In the human genome sequence, 4% -9% of the transcripts produced are long non-coding rnas (lncrnas), many of which are only present at specific developmental stages and are tissue or cell specific, and which can regulate their associated protein-coding genes in various ways at different levels and are involved in inducing disease development.

The present invention provides a long non-coding RNA isolated from blood and/or tissue.

As used herein, the term "blood" may be plasma, serum, but does not include blood cells.

As used herein, the term "isolated" refers to a substance that is separated from its original environment (which, if it is a natural substance, is the natural environment). If the polynucleotide or polypeptide in its native state in a living cell is not isolated or purified, the same polynucleotide or polypeptide is isolated or purified if it is separated from other substances coexisting in its native state.

LncRNA can be processed from precursor LncRNA, which can be cleaved to generate mature LncRNA, which may be substantially complementary to at least a portion of the mRNA encoding the gene.

As used herein, "substantially complementary" means that the sequences of nucleotides are sufficiently complementary to interact in a predictable manner, such as to form secondary structures (e.g., stem-loop structures). Typically, two "substantially complementary" nucleotide sequences are complementary to each other for at least 70% of the nucleotides; preferably, at least 80% of the nucleotides are complementary; more preferably, at least 90% of the nucleotides are complementary; further preferably, at least 95% of the nucleotides are complementary; such as 98%, 99% or 100%. Generally, two sufficiently complementary molecules may have up to 40 mismatched nucleotides between them; preferably, there are up to 30 mismatched nucleotides; more preferably, there are up to 20 mismatched nucleotides; further preferred, there are up to 10 mismatched nucleotides, such as 1, 2, 3, 4, 5, 8, 11 mismatched nucleotides.

As used herein, a "stem-loop" structure, also referred to as a "hairpin" structure, refers to a nucleotide molecule that can form a secondary structure comprising a double-stranded region (stem) formed by two regions (on the same molecule) of the nucleotide molecule flanking a double-stranded portion; it also includes at least one "loop" structure comprising non-complementary nucleotide molecules, i.e., a single-stranded region. The double-stranded portion of the nucleotide remains double-stranded even if the two regions of the nucleotide molecule are not completely complementary. For example, an insertion, deletion, substitution, etc., can result in the non-complementarity of a small region or the small region itself forming a stem-loop structure or other form of secondary structure, however, the two regions can still be substantially complementary and interact in a predictable manner to form a double-stranded region of the stem-loop structure. The stem-loop structure is well known to those skilled in the art, and usually, after obtaining a nucleic acid having a nucleotide sequence of a primary structure, those skilled in the art can determine whether the nucleic acid can form a stem-loop structure.

The lncRNA has a sequence shown in SEQ ID No. 1.

Long-chain non-coding RNA HERC2P3 gene

As used herein, the terms "lncRNA-HERC 2P 3", "long non-coding RNA HERC2P3 gene", "long RNA of the invention", or "lncRNA of the invention" are used interchangeably to refer to the HERC2 pseudogene 3 represented by the sequence shown in SEQ ID No. 1 or its complement, which does not encode a protein. HERC2 is a large protein with HECT and RLD domains with E3 ubiquitin ligase activity. Its mutation has been found to be associated with autism and to date no association with tumors has been reported. The gene has a plurality of pseudogene sequences, and HERC2P3 is one of the pseudogene sequences. The function of HERC2P3 has not been reported to date.

Antisense oligonucleotides

As used herein, the terms "antisense oligonucleotide", "antisense-oligonucleotides", "AS-Ons", or "ASO" are synonymous. According to the LncRNA sequences provided by the present invention, antisense oligonucleotides thereof can be designed, which can down-regulate the amount of the corresponding LncRNA or the expression of LncRNA in vivo.

In the present invention, the "antisense oligonucleotide" also includes modified antisense nucleotides obtained by means such as nucleic acid lock or nucleic acid chain skeleton modification technology, the modification does not substantially change the activity of the antisense oligonucleotide, and preferably, the modification can improve the stability, activity or therapeutic effect of the antisense oligonucleotide.

After the antisense oligonucleotides are transferred into human or non-human mammals, the antisense oligonucleotides can obviously down regulate the expression of related LncRNA.

Polynucleotide constructs

According to the human LncRNA sequence provided by the present invention, a polynucleotide construct that can be processed into LncRNA that can affect the expression of the corresponding mRNA after being introduced, i.e., the polynucleotide construct can up-regulate the amount of the corresponding LncRNA in vivo, can be designed. Accordingly, the present invention provides an isolated polynucleotide (construct) that can be transcribed into LncRNA by human cells.

In a preferred embodiment of the invention, the polynucleotide construct comprises a structure of formula I:

Seq_{forward direction}-X-Seq_{Reverse direction}The compound of the formula I is shown in the specification,

in the formula I, the compound is shown in the specification,

Seq_{forward direction}Is a nucleotide sequence capable of expressing the LncRNA in cells, Seq_{Reverse direction}Is and Seq_{Forward direction}A substantially complementary nucleotide sequence; alternatively, Seq_{Reverse direction}Is a nucleotide sequence capable of expressing the LncRNA in cells, Seq_{Forward direction}Is and Seq_{Forward direction}A substantially complementary nucleotide sequence;

x is at Seq_{Forward direction}And Seq_{Reverse direction}A spacer sequence therebetween, and the spacer sequence and Seq_{Forward direction}And Seq_{Reverse direction}Are not complementary;

the structure of formula I, when transferred into a cell, forms a secondary structure of formula II:

in the formula II, the compound is shown in the specification,

in formula II, Seq_{Forward direction}、Seq_{Reverse direction}And X is as defined above;

i is expressed in Seq_{Forward direction}And Seq_{Reverse direction}The base complementary pairing relationship is formed between the two.

Typically, the polynucleotide construct is located on an expression vector. Accordingly, the present invention also includes a vector comprising said LncRNA, or said polynucleotide construct. The expression vector usually further contains a promoter, an origin of replication, and/or a marker gene.

Methods well known to those skilled in the art can be used to construct the expression vectors required by the present invention. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as kanamycin, gentamicin, hygromycin, ampicillin resistance.

Chip and method for manufacturing the same

The LncRNA expression profiling chip usually contains up to several hundred probes, covers various RNAs, and utilizes the principle of DNA double-strand homologous complementation to detect the content of various RNAs in a sample at the whole genome level. Therefore, the transcription level of the RNA in the whole genome range in the sample to be tested can be detected at the same time.

By utilizing the LncRNA sequence, a corresponding LncRNA chip can be prepared, and the expression profile and the regulation mode of the LncRNA can be further researched.

In another aspect, the present invention also provides a chip for analyzing an LncRNA expression profile. The LncRNA chip of the present invention comprises: a solid support; and oligonucleotide probes orderly fixed on the solid phase carrier, wherein the oligonucleotide probes specifically correspond to the sequence shown in SEQ ID NO. 1, and in addition, the lncRNA chip can also contain probes aiming at other known gastric cancer related markers.

Specifically, suitable probes can be designed based on the LncRNA of the present invention, and immobilized on a solid support to form an "oligonucleotide array". By "oligonucleotide array" is meant an array having addressable locations (i.e., locations characterized by distinct, accessible addresses), each addressable location containing a characteristic oligonucleotide attached thereto. The oligonucleotide array may be divided into a plurality of subarrays as desired.

The solid phase carrier can adopt various common materials in the field of gene chips, such as but not limited to nylon membranes, glass slides or silicon wafers modified by active groups (such as aldehyde groups, amino groups and the like), unmodified glass slides, plastic sheets and the like.

The LncRNA chip can be prepared by a conventional method for manufacturing a biochip known in the art. For example, if a modified glass slide or silicon wafer is used as the solid support, and the 5' end of the probe contains a poly-dT string modified with an amino group, the oligonucleotide probe can be prepared into a solution, and then spotted on the modified glass slide or silicon wafer by using a spotting instrument, arranged into a predetermined sequence or array, and then fixed by standing overnight, so as to obtain the miRNA chip of the invention. If the nucleic acid does not contain amino modifications, the preparation can also be referred to: the "Gene diagnostic technique-non-Radioactive operation Manual" edited by Wangshen five; l.l.erisi, v.r.i.er, p.o.brown.expansion of the metabolic and genetic control of genetic compression a genetic scale, science, 1997; 278:680 and maliren, jiang china main edition biochip, beijing: chemical industry Press, 2000, 1-130.

Detection kit

The invention also provides a kit, and the kit contains the chip. The kit can be used for detecting the expression of the LncRNA in blood.

Preferably, the kit further comprises a marker for marking the RNA sample and a substrate corresponding to the marker.

In addition, the kit may further include various reagents required for RNA extraction, PCR, hybridization, color development, and the like, including but not limited to: an extraction solution, an amplification solution, a hybridization solution, an enzyme, a control solution, a color development solution, a washing solution, an antibody, and the like.

In addition, the kit can also comprise an instruction book and/or chip image analysis software.

LncRNA inhibitor

As used herein, the terms "active ingredient of the invention", "inhibitor of the invention", "LncRNA inhibitor" are used interchangeably and refer to a substance capable of inhibiting or reducing LncRNA expression or activity.

Representative incrna inhibitors include antisense nucleic acids, small molecule compounds, mirnas, shrnas, or sirnas, and the like.

Having obtained the lncRNA target of the present invention, lncRNA inhibitors can be prepared and/or screened by conventional techniques of the present invention. The candidate range for screening lncRNA inhibitors of the present invention may include any of the commonly used databases known in the art, such as compound databases and the like.

One preferred lncRNA inhibitor of the invention is siRNA or shRNA with a sequence shown in SEQ ID No. 1, preferably, the siRNA is shown in SEQ ID No. 4-5 or SEQ ID No. 6-7; the shRNA is shown in SEQ ID No. 8-9.

Pharmaceutical composition

The invention also provides a pharmaceutical composition which contains a safe and effective amount of LNCRNA inhibitor and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions, such as tablets and capsules, can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, LNCRNA inhibitors of the invention can also be used with other therapeutic agents.

In using the pharmaceutical composition, a safe and effective amount of the LNCRNA inhibitor of the present invention is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms/kg body weight, and in most cases no more than about 8 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 1 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

The invention has the beneficial effects

The experiment of the invention proves that the expression of LncRNA-HERC2P3 gene in gastric cancer tissue is obviously higher than that of paracancer tissue, and the growth of gastric cancer cells can be obviously inhibited by silencing LncRNA-HERC2P3 gene, so that LncRNA-HERC2P3 gene and its expression product can be used as a marker for diagnosing gastric cancer and a drug target for treating gastric cancer, and the diagnosis of gastric cancer is more accurate and rapid. In a word, the LncRNA-HERC2P3 gene and the application thereof provide a new therapeutic target and an effective new medicine for preventing and treating gastric cancer.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

The general method comprises the following steps:

(1) clinical tissue sample acquisition

Gastric cancer and tissues adjacent to the cancer were obtained from surgically treated gastric cancer patients and informed consent was signed with the patients prior to obtaining the samples. Once the stomach cancer tissue excised by operation is separated, the tissue beside the cancer except the primary tumor focus and the surrounding 5cm is quickly cut, put into liquid nitrogen for quick freezing, moved to a refrigerator at minus 80 ℃ for storage, and stored in the liquid nitrogen during transportation. Both cancer and paracancerous tissue are ultimately diagnosed by a pathologist. Samples were classified as class I-III according to Edmondson classification criteria.

(2) Tissue and cell RNA extraction

The RNA was extracted using TRIzol Reagent (Invitrogen) by the following procedure:

1) cleaning the containers such as mortar, pestle and homogenizer, and respectively using ddH₂O and DEPC H₂O rinsing, then baking in an oven at 180 ℃ for about 4 hours to remove RNase;

2) adding a proper amount of liquid nitrogen into a mortar for precooling, quickly taking out the tissue from the liquid nitrogen, cutting the tissue into 50-100mg, and grinding the tissue into powder in the mortar;

3) transferring the ground tissue powder to an RNase-free EP tube as completely as possible by using a curette, adding a proper volume (1ml) of TRIzol reagent in advance to the EP tube, and fully homogenizing;

4) standing at room temperature for 5 minutes, proportionally adding chloroform (200. mu.l/1 ml TRIzol) into a centrifuge tube, rapidly and violently shaking for 15 seconds, standing at room temperature for 2-3 minutes, and centrifuging at 4 ℃ under 12000 Xg for 15 minutes;

5) transferring the upper aqueous phase into a new RNA enzyme-free EP tube as far as possible, adding isopropanol with the same volume, reversing and uniformly mixing for 5 times, standing for 10 minutes at room temperature, centrifuging for 10 minutes at 4 ℃ under the condition of 12000 Xg, and then, detecting RNA precipitation;

6) pouring off the supernatant, adding 75% ethanol (1ml/1ml TRIzol), mixing, washing RNA, centrifuging at 4 deg.C for 5min at 7500 Xg;

7) discarding the supernatant, removing residual ethanol as much as possible, and naturally drying the precipitate for 5-10min (taking care not to completely dry); adding 30-50 μ l DEPC H₂O, blowing and sucking for several times, and dissolving RNA precipitate;

8) measuring the concentration and purity OD 260/280(1.8-2.0) of RNA by an enzyme-labeling instrument; gel electrophoresis was performed to observe whether degradation occurred or not, and the samples were stored at-80 ℃.

Extracting cell line RNA, collecting cells in logarithmic growth phase, sucking culture solution, adding TRIzol reagent (1ml TRIzol/10 cm) in corresponding amount according to the area of culture dish²) The cells were lysed and blown several times, and the lysed cells were collected in an RNase-free EP tube, and the remaining RNA was isolated and purified by the chloroform-isopropanol method according to the above steps 4) to 8).

(3) Reverse transcription of RNA

Reverse transcription was performed with M-MLV Reverse Transcriptase (Promega) as follows:

1) the following components were added to the nuclease-free EP tube:

the mixture was placed in a PCR apparatus at 70 ℃ for 5 minutes and then immediately cooled on ice for 5 min.

2) The following components are added into the system:

after mixing gently, the mixture was placed in a PCR instrument at 37 ℃ for 60 min.

The cDNA obtained by the reversion was stored at 4 ℃.

(4) Real-time quantitative PCR

Real-time quantitative PCR reaction use

Premix Ex Taq^TM(Perfect Real Time) kit (TaKaRa Biotechnology Co., Ltd. Dalian, China) using Thermal cycleDicce^TMReal Time System (TP800 Real-Time fluorescent quantitative PCR instrument, TaKaRa) was performed. The length of the amplification product of the quantitative PCR is preferably 80bp to 150bp (can be extended to 300 bp).

The reaction system is as follows:

dissolution curve analysis step:

95℃ 15sec

60℃ 30sec

95℃ 15sec

the dissociation time was 4 sec.

The fluorescence background signal and the threshold value adopt default values set by an instrument and are automatically generated after each PCR reaction is finished, the Ct value represents the cycle number of the fluorescence signal in each reaction tube when the fluorescence signal reaches the set threshold value (10 times of the baseline fluorescence intensity), each template of the target gene HERC2P3 is subjected to 3 multitubules, the obtained Ct value is averaged, the Ct average value of the HERC2P3 gene subtracts the Ct average value of the internal reference gene (β -actin) of the corresponding template to obtain the delta Ct of a delta Ct. gastric cancer group minus the delta Ct of the corresponding cancer collateral tissue to obtain the delta Ct value, and the multiple relation of the HERC2P3 genes in the gastric cancer group and the cancer collateral group uses 2P3 genes^-ΔΔCtAnd (4) showing.

(5) Determination of cell growth curves

1) Different kinds of HCC cells are grown according to 3-5 × 10³The total amount of cells was calculated per 100. mu.l/well, and after digesting the cells sufficiently, the cells were diluted to the desired concentration and seeded in a 96-well plate. Inoculating cells in each group with three wells every day for 5-7 days;

2) to be thinCell status and number were observed after cells were substantially adherent. Color reaction was carried out with CCK-8 developer (Cell Counting Kit-8, DOJINDO, Japan) by adding 10. mu.l of CCK-8 to 100. mu.l of the culture medium at 37 ℃ with 5% CO₂And (3) placing the incubator for incubation for 1h, measuring the absorbance at 450nm by using an enzyme-labeling instrument, recording, and determining the actual initial density of the cells as a relative zero point of growth.

3) Changing the liquid half a day or every other day, which is determined by the experiment requirement;

4) observing the cell morphology under a microscope, measuring at fixed time intervals, and recording the cell growth condition;

5) typically 5 to 7 days. After the measurement is finished, data are collected and processed, and a graph is drawn by Excel.

(6) Cell clone formation assay

1) Transfection: using Lipofectamine^TM2000(Invitrogen), overexpressing or silencing the expression of HERC2P3 gene in the cells;

2) after culturing the transfected cells in a 6-well plate or a 35mm culture dish for 24h by using a normal culture solution, digesting and counting the cells, inoculating the cells to a 100mm culture dish according to a certain number (different cell strains are different in number), continuously culturing for 24h, and then adding G418 (600-;

3) culturing for 2-3 weeks, changing fresh culture solution every 3-5 days, and adding G418 for screening until macroscopic cell clone is formed;

4) sucking out the culture solution in the culture dish, washing twice with 1 × PBS, dyeing with Coomassie brilliant blue R-250 for 2h, washing with water, and decolorizing with Coomassie brilliant blue dye-decolorizing solution for 30-60 min;

5) colony formation staining results were photographed and cell clones on each dish were counted according to the same criteria (cell clone size).

(7) Nude mouse tumorigenesis experiment

1) The mice are 5-6 week male BLAB/c nu nude mice, provided by Shanghai Si Laike laboratory animals Co., Ltd, and are bred in southern model animal culture center;

2) the treated cells are taken and inoculated under the skin of the mouse in the same quantity (different cell types are inoculated in different numbers), and in order to avoid errors caused by individual differences, the same cells can be inoculated in the same mouse in a left-right symmetrical mode through different treatments;

3) after a visible tumor appears, the size of the tumor is monitored every 3 days, the long diameter and the short diameter of the tumor are read by a vernier caliper, and the tumor volume is calculated according to the following formula: volume is 0.5 x major diameter x minor diameter²；

4) After continuously monitoring for about 7-8 times, the data are collated and counted.

Example 1: silencing the expression of LncRNA-HERC2P3 inhibits the growth of gastric cancer cells.

To further verify the growth inhibitory function of silencing LncRNA-HERC2P3 on gastric cancer cells, the inventors examined changes in cell growth by silencing its expression by RNA interference. The siRNA for interfering the expression of LncRNA-HERC2P3 is synthesized by Shanghai Ji code pharmaceutical Co., Ltd, and has the sequence si-1: sense strand 5-CACCAAGACAUUAUGCGGAdTdT-3 (SEQ ID No.: 4); antisense strand 5-UCCGCAUAAAGUCUUGGUGdTdT-3(SEQ ID No.: 5). Si-2: the strand is 5-CUCAUCAACAAGUACAUCAdTdT-3(SEQ ID NO: 6) and the antisense strand is 5-UGAUGUACUUGUGAUGAGdTdT-3 (SEQ ID NO: 7). The NC nonspecific nucleotide sequence used as an interference control was: sense strand 5-UUCCCGAACGUCACGUdT-3 (SEQ ID No.: 10); the antisense strand 5-ACGUGACACGUCGGAGAAdTdT-3 (SEQ ID No.: 11). Real-time quantitative PCR detection shows that the artificially synthesized interfering siRNA can effectively reduce the expression level of LncRNA-HERC2P3 (figure 1A), and a growth curve experiment also proves that the LncRNA-HERC2P3 can inhibit the growth of gastric cancer cells SGC-7901 (figure 1B), thereby strongly supporting the conclusion that the target LncRNA-HERC2P3 can inhibit the growth of tumor cells.

Example 2: silencing expression of LncRNA-HERC2P3 inhibits gastric cancer cell invasion.

To further understand the relationship between LncRNA-HERC2P3 and gastric cancer cell invasion, the present inventors transfected siRNA targeting LncRNA-HERC2P3 into AGS and SGC-7901 cells and verified the invasion ability of the transfected cells through transwell experiments. Each 8-chamber is filled with 3X 10⁴Individual cells, stained after 48h and counted by observation. The inventor finds that the invasion of stomach cancer cells for silencing LncRNA-HERC2P3 geneThe ability to attack was significantly diminished (fig. 2A). Cell counts quantitatively indicated that the number of cells in the experimental group silencing LncRNA-HERC2P3 gene was significantly reduced compared to the control group (fig. 2B).

Example 3: successfully transfect a lentiviral vector silencing LncRNA-HERC2P3 into SGC-7901 cells.

Transfection efficiency experiments under white light and fluorescence microscopy after transfection of the silencing LncRNA-HERC2P3 lentiviral vector into SGC-7901 cells indicated that both positive and negative control groups of cells were well transfected (fig. 3A). The qPCR results further showed that the expression amount of LncRNA-HERC2P3 was significantly less in the shHERC2P3 group than in the shNC group.

Example 4: LncRNA-HERC2P3 influences the blood migration ability of gastric cancer cells in nude mice.

In vitro experiments have clearly shown that Srrp35 inhibits the growth and invasion of gastric cancer cells, and then the inventor utilizes a nude mouse model to research the influence of silencing LncRNA-HERC2P3 on the metastatic capacity of gastric cancer cells in nude mice. Mixing 1.5X 10⁶SGC-7901 cells silencing LncRNA-HERC2P3 and control cells were injected symmetrically into the tail vein of nude mice. After 6 weeks, nude mice were sacrificed and lung tissue was taken out and weighed. The results show that silencing LncRNA-HERC2P3 effectively inhibits the metastatic tumorigenicity ability of SGC-7901 cells in nude mice, both in the volume and weight of a single tumor (FIG. 4A) and in the tumor number of lung tissues (FIG. 4B).

Example 5: the expression level of LncRNA-HERC2P3 in the gastric cancer tissue sample has a clear tendency of being up-regulated.

The inventor previously found through a large number of experimental studies that LncRNA-HERC2P3 is obviously up-regulated in gastric cancer tissues, in 30 pairs of clinical samples, 13 pairs of samples of LncRNA-HERC2P3 expression in gastric cancer tissues is obviously higher than that of corresponding paracancer tissues through real-time quantitative PCR detection, the up-regulation rate reaches 43.3%, and statistical analysis shows that P is less than 0.01 (FIG. 5), thereby illustrating the reliability of the result.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. Use of an isolated lncRNA-HERC2P3 detection reagent for preparing a chip, a reagent or a kit for diagnosing gastric cancer; the lncRNA-HERC2P3 is selected from:

(a) 1 as shown in SEQ ID NO;

(b) lncRNA-HERC2P3 complementary to the sequence shown in SEQ ID NO. 1;

the detection reagent of lncRNA-HERC2P3 comprises a primer pair for specifically amplifying a sequence shown by SEQ ID NO. 1, wherein the primer pair for specifically amplifying the sequence shown by SEQ ID NO. 1 is a sequence shown by SEQ ID NO. 2-3.

2. The use according to claim 1, wherein the lncRNA-HERC2P3 is isolated from blood and/or tissue of a human or non-human mammal.

3. Use according to claim 2, wherein the blood is plasma and/or serum.

4. The use of claim 2, wherein the blood does not comprise blood cells.

5. The use of claim 2, wherein said tissue comprises tumor tissue.

6. The use of claim 2, wherein the lncRNA-HERC2P3 is isolated from a human.

7. The use according to claim 2, wherein the tissue is gastric cancer tissue.

8. A kit comprising a detection reagent for IncRNA-HERC 2P3, and instructions for use,

wherein, the lncRNA-HERC2P3 is selected from:

(a) 1 as shown in SEQ ID NO;

(b) lncRNA-HERC2P3 complementary to the sequence shown in SEQ ID NO. 1;

9. The kit of claim 8, further comprising lncRNA-HERC2P3 as a positive control.

10. An isolated inhibitor of lncRNA-HERC2P3, wherein said inhibitor specifically inhibits the expression and/or activity of lncRNA-HERC2P3 in vitro and/or in vivo;

wherein, the lncRNA-HERC2P3 is selected from:

(a) 1 as shown in SEQ ID NO;

(b) lncRNA-HERC2P3 complementary to the sequence shown in SEQ ID NO. 1;

the inhibitor is siRNA shown as SEQ ID NO. 4-5 or SEQ ID NO. 6-7.

11. Use of the incrna-HERC 2P3 inhibitor according to claim 10 for the preparation of a pharmaceutical composition for the treatment of gastric cancer and/or gastric cancer metastasis.

12. A pharmaceutical composition for treating gastric cancer and/or gastric cancer metastasis, comprising the inhibitor of lncRNA-HERC2P3 of claim 10 and a pharmaceutically acceptable carrier.

13. A method of non-therapeutically inhibiting gastric cancer cell growth and/or migration in vitro by culturing gastric cancer cells in the presence of the inhibitor of claim 10, and/or the pharmaceutical composition of claim 12, thereby inhibiting gastric cancer cell growth and/or migration.