CN113930422A

CN113930422A - Application of human IGFL3 gene and related product

Info

Publication number: CN113930422A
Application number: CN202010670926.7A
Authority: CN
Inventors: 蔡丽丽; 曹跃琼
Original assignee: Shanghai Jikai Gene Medical Technology Co ltd
Current assignee: Shanghai Jikai Gene Medical Technology Co ltd
Priority date: 2020-07-13
Filing date: 2020-07-13
Publication date: 2022-01-14

Abstract

The invention belongs to the field of biomedical research, and particularly relates to application of a human IGFL3 gene as a target in preparation of a gastric cancer treatment drug or a gastric cancer diagnosis drug. Extensive and intensive research shows that the expression of human IGFL3 gene is down-regulated by RNAi method to effectively inhibit the proliferation of gastric cancer cells and effectively control the growth process of gastric cancer. The siRNA or the nucleic acid construct containing the siRNA sequence and the lentivirus provided by the invention can specifically inhibit the proliferation rate of gastric cancer cells and inhibit the growth of gastric cancer, thereby treating gastric cancer and opening up a new direction for treating gastric cancer.

Description

Application of human IGFL3 gene and related product

Technical Field

The invention belongs to the field of biomedical research, and particularly relates to application of a human IGFL3 gene and a related product.

Background

IGFL3(Insulin growth factor-like family member 3, IGF-like family member 3) is a protein-encoding gene. Diseases associated with insulin-like growth factor 3 include unilateral or bilateral testicular disease and cryptorchidism. IGFL2 is an important accessory gene for this gene.

There is no report on the use of IGFL3 gene for gastric cancer treatment.

Disclosure of Invention

In order to overcome the problems in the prior art, the invention aims to provide the application of the human IGFL3 gene and a related product.

In order to achieve the above objects and other related objects, the present invention adopts the following technical solutions:

in the first aspect of the invention, the application of the human IGFL3 gene as a target in preparing a gastric cancer treatment drug or a gastric cancer diagnosis drug is provided.

The human IGFL3 gene as a target for preparing gastric cancer treatment medicines specifically comprises the following steps: the IGFL3 gene is used as an action object, and a drug or a preparation is screened to find a drug capable of inhibiting the expression of the human IGFL3 gene as a candidate drug for treating gastric cancer. The IGFL3 gene small interfering RNA (siRNA) is obtained by screening human IGFL3 gene as an action object and can be used as a medicine for inhibiting gastric cancer cell proliferation. In addition, the IGFL3 gene can be used as a target of action, for example, in antibody drugs, small molecule drugs, etc.

The application of the human IGFL3 gene as a target in preparing gastric cancer diagnosis medicines specifically comprises the following steps: the IGFL3 gene expression product is used as a gastric cancer diagnosis index to be applied to the preparation of gastric cancer diagnosis medicaments.

The gastric cancer treatment drug is a molecule which can specifically inhibit the transcription or translation of an IGFL3 gene, or can specifically inhibit the expression or activity of an IGFL3 protein, so that the expression level of the IGFL3 gene in gastric cancer cells is reduced, and the purpose of inhibiting the proliferation, growth, differentiation and/or survival of the gastric cancer cells is achieved.

The gastric cancer therapeutic drug or gastric cancer diagnostic drug prepared from the IGFL3 gene includes but is not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs, antibody drugs, polypeptides, proteins, or interfering lentiviruses.

Such nucleic acids include, but are not limited to: antisense oligonucleotides, double-stranded RNA (dsRNA), ribozymes, small interfering RNA produced by endoribonuclease III or short hairpin RNA (shRNA).

The amount of the gastric cancer treatment drug administered is a dose sufficient to reduce transcription or translation of the human IGFL3 gene, or to reduce expression or activity of the human IGFL3 protein. Such that the expression of the human IGFL3 gene is reduced by at least 50%, 80%, 90%, 95%, or 99%.

The method for treating gastric cancer by adopting the gastric cancer treatment drug achieves the treatment purpose by mainly reducing the expression level of human IGFL3 gene to inhibit the proliferation of gastric cancer cells. Specifically, in treatment, a substance effective in reducing the expression level of human IGFL3 gene is administered to the patient.

In one embodiment, the target sequence of the IGFL3 gene is as set forth in SEQ ID NO:1 and SEQ ID NO:2, respectively. The method specifically comprises the following steps:

5’-TGAGGGTTCTGGGTATGAA-3’(SEQ ID NO：1)；

5’-GTGGGAACAAGATCTACAA-3’(SEQ ID NO：2)。

in a second aspect of the invention, there is provided the use of an inhibitor of IGFL3 in the manufacture of a product having at least one of the following effects:

treating gastric cancer;

inhibiting the proliferation rate of gastric cancer cells;

inhibiting the growth of gastric cancer.

The product necessarily comprises an IGFL3 inhibitor, and an IGFL3 inhibitor as an active ingredient for the aforementioned effects.

In the product, the effective component for the above functions can be only an IGFL3 inhibitor, and can also comprise other molecules for the above functions.

That is, the IGFL3 inhibitor is the only active ingredient or one of the active ingredients of the product.

The product may be a single component material or a multi-component material.

The form of the product is not particularly limited, and can be various substance forms such as solid, liquid, gel, semifluid, aerosol and the like.

The product is primarily directed to mammals. The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, or the like. The primate is preferably a monkey, ape or human.

Such products include, but are not limited to, pharmaceuticals, nutraceuticals, foods, and the like.

The IGFL3 inhibitor may be a nucleic acid molecule, an antibody, a small molecule compound.

As exemplified in the examples herein, the IGFL3 inhibitor can be a nucleic acid molecule that reduces the expression of the IGFL3 gene in gastric cancer cells. Specifically, it may be a double-stranded RNA or shRNA.

In a third aspect of the invention, a method of treating gastric cancer is provided by administering to a subject an IGFL3 inhibitor.

The subject may be a mammal or a mammalian gastric cancer cell. The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, or the like. The primate is preferably a monkey, ape or human. The gastric cancer cell may be an isolated gastric cancer cell.

The subject may be a patient suffering from gastric cancer or an individual in whom treatment is desired. Or isolated gastric cancer cells of a subject who is a gastric cancer patient or an individual expected to treat gastric cancer.

The IGFL3 inhibitor may be administered to a subject before, during, or after receiving treatment for gastric cancer.

The fourth aspect of the invention discloses a nucleic acid molecule for reducing the expression of IGFL3 gene in gastric cancer cells, wherein the nucleic acid molecule comprises double-stranded RNA or shRNA.

Wherein the double-stranded RNA contains a nucleotide sequence capable of hybridizing with an IGFL3 gene;

the shRNA contains a nucleotide sequence capable of hybridizing with the IGFL3 gene.

Further, the double-stranded RNA comprises a first strand and a second strand, the first strand and the second strand are complementary to form an RNA dimer, and the sequence of the first strand is substantially identical to a target sequence in the IGFL3 gene.

The target sequence in the IGFL3 gene is a fragment in the IGFL3 gene corresponding to an mRNA fragment recognized and silenced by the nucleic acid molecule when the nucleic acid molecule is used for specifically silencing the expression of the IGFL3 gene.

Further, the target sequence of the double-stranded RNA is shown as SEQ ID NO:1 and SEQ ID NO:2, respectively. The method specifically comprises the following steps: 5'-TGAGGGTTCTGGGTATGAA-3' (SEQ ID NO: 1); 5'-GTGGGAACAAGATCTACAA-3' (SEQ ID NO: 2).

Further, the sequence of the first strand of the double-stranded RNA is shown as SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The method specifically comprises the following steps:

5’-UGAGGGUUCUGGGUAUGAA-3’(SEQ ID NO：3)；

5’-GUGGGAACAAGAUCUACAA-3’(SEQ ID NO：4)。

further, the double-stranded RNA is small interfering RNA (siRNA).

SEQ ID NO: 3 is one strand of small interfering RNA designed by using the sequence shown in SEQ ID NO. 1 as RNA interference target sequence and aiming at human IGFL3 gene, and the sequence of the other strand, namely the second strand, is complementary with the sequence of the first strand. SEQ ID NO: 4 is one strand of small interfering RNA designed by using the sequence shown in SEQ ID NO. 2 as RNA interference target sequence and aiming at human IGFL3 gene, and the sequence of the other strand, namely the second strand, is complementary with the sequence of the first strand. The siRNA can play a role in specifically silencing the expression of endogenous IGFL3 gene in gastric cancer cells.

The shRNA includes a sense strand segment and an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, the sequences of the sense strand segment and the antisense strand segment are complementary, and the sequence of the sense strand segment is substantially identical to a target sequence in the IGFL3 gene.

Further, the target sequence of the sh RNA is shown as SEQ ID NO:1 and SEQ ID NO:2, respectively.

The shRNA can become small interfering RNA (siRNA) after enzyme digestion and processing, and further plays a role in specifically silencing endogenous IGFL3 gene expression in gastric cancer cells.

Further, the sequence of the stem-loop structure of the shRNA can be selected from any one of the following sequences: UUCAAGAGA, AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, and CCACACC.

Further, the sequence of the shRNA is shown as SEQ ID NO: 5 and SEQ ID NO: and 6. Specifically 5'-UGAGGGUUCUGGGUAUGAACUCGAGUUCAUACCCAGAACCCUCA-3' (SEQ ID NO: 5); 5'-GUGGGAACAAGAUCUACAACUCGAGUUGUAGAUCUUGUUCCCAC-3' (SEQ ID NO: 6).

Further, the IGFL3 gene is derived from human.

In the fifth aspect of the invention, the invention discloses an IGFL3 gene interference nucleic acid construct, which comprises a gene segment for coding shRNA in the nucleic acid molecule and can express the shRNA.

The IGFL3 gene interference nucleic acid construct can be obtained by cloning a gene segment for coding the human IGFL3 gene shRNA into a known vector.

Further, the IGFL3 gene interference nucleic acid construct is an IGFL3 gene interference lentiviral vector.

The IGFL3 gene interference lentiviral vector disclosed by the invention is obtained by cloning a DNA fragment for coding the IGFL3 gene shRNA into a known vector, wherein the known vector is mostly a lentiviral vector, the IGFL3 gene interference lentiviral vector is packaged into infectious viral particles by virus, gastric cancer cells are infected, the shRNA is transcribed, and the siRNA is finally obtained through the steps of enzyme digestion processing and the like and is used for specifically silencing the expression of the IGFL3 gene.

Further, the IGFL3 gene interference lentiviral vector also contains a promoter sequence and/or a nucleotide sequence encoding a marker which can be detected in gastric cancer cells; preferably, the detectable label is Green Fluorescent Protein (GFP).

Further, the lentiviral vector may be selected from the group consisting of: pLKO.1-puro, pLKO.1-CMV-tGFP, pLKO.1-puro-CMV-tGFP, pLKO.1-CMV-Neo, pLKO.1-Neo-CMV-tGFP, pLKO.1-puro-CMV-TagCFP, pLKO.1-puro-CMV-TagYFP, pLKO.1-puro-CMV-TagFP635, pLKO.1-puro-UbC-TurboGFP, pLKO.1-puro-UbC-TagFP635, pLKO-puro-IPTG-1xLacO, pLKO-puro-IPTG-3xLacO, pLP1, pLP2, pLP/VSV-G, pENTR/U6, pLenti6/BLOCK-iT-DEST, pLenti 6-GW/U6-laminsham, pcDNA1.2/V5-GW/lacZ, pLenti6.2/N-Lumio/V5-DEST, pGCSIL-GFP or pLenti 6.2/N-Lumio/V5-GW/lacZ.

The embodiment of the invention specifically enumerates human IGFL3 gene interference lentiviral vectors constructed by taking pGCSIL-GFP as a vector, and the vectors are named as pGCSIL-GFP-IGFL3-siRNA-1 and pGCSIL-GFP-IGFL3-siRNA-2, and the corresponding lentiviruses are named as IGFL3-siRNA lentivirus 1 and IGFL3-siRNA lentivirus 2 respectively.

The IGFL3 gene siRNA can be used for inhibiting the proliferation of gastric cancer cells, and further can be used as a medicine or a preparation for treating gastric cancer. The IGFL3 gene interference lentiviral vector can be used for preparing the IGFL3 gene siRNA. When used as a medicament or formulation for treating gastric cancer, a safe and effective amount of the nucleic acid molecule is administered to a mammal. The particular dosage will also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

In the sixth aspect of the invention, the invention discloses an IGFL3 gene interference lentivirus, which is formed by virus packaging of the IGFL3 gene interference nucleic acid construct under the assistance of a lentivirus packaging plasmid and a cell line. The lentivirus can infect gastric cancer cells and generate small interfering RNA aiming at the IGFL3 gene, thereby inhibiting the proliferation of the gastric cancer cells. The IGFL3 gene interference lentivirus can be used for preparing medicines for preventing or treating gastric cancer.

In a seventh aspect of the present invention, there is provided a use of the aforementioned nucleic acid molecule, or the aforementioned IGFL3 gene-interfering nucleic acid construct, or the aforementioned IGFL3 gene-interfering lentivirus, wherein: is used for preparing a medicine for preventing or treating gastric cancer or a kit for reducing the expression of the IGFL3 gene in gastric cancer cells.

The application of the medicament for preventing or treating the gastric cancer provides a method for treating the gastric cancer, in particular to a method for preventing or treating the gastric cancer in a subject, which comprises the step of administering an effective dose of the medicament to the subject.

Further, when the drug is used for preventing or treating gastric cancer in a subject, an effective dose of the drug needs to be administered to the subject. With this method, the growth, proliferation, recurrence and/or metastasis of the gastric cancer is inhibited. Further, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the growth, proliferation, recurrence and/or metastasis of the gastric cancer is inhibited.

The subject of the method may be a human.

In an eighth aspect of the present invention, there is provided a composition for preventing or treating gastric cancer, comprising, as active ingredients:

the aforementioned nucleic acid molecules; and/or, the aforementioned IGFL3 gene interfering nucleic acid construct; and/or, the aforementioned IGFL3 gene interfering lentivirus, and a pharmaceutically acceptable carrier, diluent or excipient.

The composition may be a pharmaceutical composition.

When the composition is used for preventing or treating gastric cancer in a subject, an effective dose of the composition needs to be administered to the subject. With this method, the growth, proliferation, and recurrence of gastric cancer are inhibited. Further, the growth, proliferation, recurrence of gastric cancer is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% partially inhibited.

The form of the composition is not particularly limited, and may be in the form of various substances such as solid, liquid, gel, semifluid, aerosol, etc.

The subject to which the composition is primarily directed is a mammal. The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, or the like. The primate is preferably a monkey, ape or human.

In conclusion, the invention designs RNAi target sequences aiming at human IGFL3 genes and constructs corresponding IGFL3 RNAi vectors, wherein the RNAi vectors pGCSIL-GFP-IGFL3-siRNA-1 and pGCSIL-GFP-IGFL3-siRNA-2 can obviously down-regulate the expression of the IGFL3 genes at the mRNA level and the protein level. The use of lentivirus (Lv) as a gene manipulation tool to carry RNAi vectors pGCSIL-GFP-IGFL3-siRNA-1 and pGCSIL-GFP-IGFL3-siRNA-2 enables efficient targeted introduction of RNAi sequences against the IGFL3 gene into gastric cancer AGS cells, reduces the expression level of the IGFL3 gene, and significantly inhibits the proliferation ability of the tumor cells. Lentivirus-mediated IGFL3 gene silencing is therefore a potential clinical non-surgical treatment modality for malignancies.

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

the invention discovers through extensive and intensive research that the expression of the human IGFL3 gene can be effectively inhibited and the proliferation of gastric cancer cells and the apoptosis can be promoted after the expression of the human IGFL3 gene is down regulated by adopting an RNAi method, and the growth process of the gastric cancer can be effectively controlled. The siRNA or the nucleic acid construct containing the siRNA sequence and the lentivirus provided by the invention can specifically inhibit the proliferation rate of gastric cancer cells and inhibit the growth of gastric cancer, thereby treating gastric cancer and opening up a new direction for treating gastric cancer.

Drawings

FIG. 1: and RT-PCR is used for detecting the target gene reduction efficiency of AGS cell mRNA level.

FIG. 2: the results of automatic analysis of Celigo cells revealed that the depletion of the IGFL3 gene inhibited the proliferation of gastric cancer cells. (cell lines are AGS cells and cell numbers were counted 1, 2, 3, 4 and 5 days after viral infection)

In the drawings, there is shown in the drawings,

mean values from three experiments were used and error bars indicate Standard Deviation (SD).

P <0.01 for shCtrl compared to target gene shRNA lentivirus treatment group.

Detailed Description

The invention proves the function of the IGFL3 gene in the generation of gastric cancer from the viewpoint of cell function. Through constructing target gene shRNA lentivirus and then transfecting gastric cancer cells, comparing the target gene shRNA lentivirus with transfection control lentivirus, detecting the expression conditions of mRNA and protein level target genes in two groups of gastric cancer cell lines; and then cell proliferation, apoptosis and other detection are carried out through cytofunctional experiments, and the results show that the gastric cancer cell proliferation inhibition degree of the shRNA group is obviously higher than that of the control group compared with the shRNA group and the control group.

According to the research results, a new method for diagnosing and treating the gene is further explored and developed, so that more choices can be provided for the diagnosis and treatment of the gastric cancer patient.

IGFL3 inhibitors

Refers to a molecule having an inhibitory effect on IGFL 3. Having inhibitory effects on IGFL3 include, but are not limited to: inhibiting the expression or activity of IGFL 3.

Inhibition of IGFL3 activity refers to a decrease in IGFL3 activity. Preferably, IGFL3 activity is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, even more preferably by at least 70%, and most preferably by at least 90% as compared to its activity prior to inhibition.

The inhibition of the expression of IGFL3 specifically can be the inhibition of the transcription or translation of IGFL3 gene, and specifically can be the inhibition of: the gene of IGFL3 is not transcribed, or the transcription activity of the gene of IGFL3 is reduced, or the gene of IGFL3 is not translated, or the translation level of the gene of IGFL3 is reduced.

The modulation of gene expression of IGFL3 can be accomplished by one skilled in the art using conventional methods, such as gene knock-out, homologous recombination, interfering RNA, and the like.

The inhibition of gene expression of IGFL3 was verified by PCR and Western Blot detection of the expression level.

Preferably, the IGFL3 gene expression is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, even more preferably by at least 70%, still more preferably by at least 90%, most preferably the IGFL3 gene is not expressed at all, compared to the wild type.

Small molecule compounds

The invention refers to a compound which is composed of several or dozens of atoms and has the molecular mass of less than 1000.

Preparation of medicine for preventing or treating gastric cancer

Nucleic acid molecules that reduce the expression of the IGFL3 gene in gastric cancer cells can be used; and/or, an IGFL3 gene interfering nucleic acid construct; and/or, the IGFL3 gene interferes with lentivirus to be used as an effective component for preparing a medicament for preventing or treating gastric cancer. Generally, the medicament can comprise one or more pharmaceutically acceptable carriers or auxiliary materials besides the effective components according to the requirements of different dosage forms.

By "pharmaceutically acceptable" is meant that the molecular entities and compositions do not produce adverse, allergic, or other untoward reactions when properly administered to an animal or human.

The "pharmaceutically acceptable carrier or adjuvant" should be compatible with the active ingredient, i.e., capable of being blended therewith without substantially diminishing the effectiveness of the drug under ordinary circumstances. Specific examples of some substances that can serve as pharmaceutically acceptable carriers or adjuvants are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium methylcellulose, ethylcellulose and methylcellulose; powdered gum tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyhydric alcohols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting agents, stabilizers; an antioxidant; a preservative; pyrogen-free water; isotonic saline solution; and phosphate buffer, and the like. These materials are used as needed to aid in the stability of the formulation or to aid in the enhancement of the activity or its bioavailability or to produce an acceptable mouthfeel or odor upon oral administration.

In the present invention, unless otherwise specified, the pharmaceutical dosage form is not particularly limited, and may be prepared into injection, oral liquid, tablet, capsule, dripping pill, spray, etc., and may be prepared by a conventional method. The choice of the pharmaceutical dosage form should be matched to the mode of administration.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the respective manufacturers.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts.

Example 1 preparation of RNAi lentivirus against human IGFL3 Gene

1. Screening of effective siRNA target against human IGFL3 Gene

Calling IGFL3 (NM-207393) gene information from Genbank; design effective siRNA target against IGFL3 gene. Table 1-1 lists the effective siRNA target sequences selected against the IGFL3 gene.

TABLE 1-1 siRNA target sequences targeting the human IGFL3 Gene

SEQ ID NO	TargetSeq(5’-3’)
		1	TGAGGGTTCTGGGTATGAA
2	GTGGGAACAAGATCTACAA

2. Preparation of Lentiviral vectors

Synthesizing double-stranded DNA Oligo sequences (Table 1-2) containing Age I and EcoR I enzyme cutting sites at two ends aiming at siRNA targets (taking SEQ ID NO:1 and 2 as examples); the restriction enzymes Age I and EcoR I act on pGCSIL-GFP vector (provided by Shanghai Jikai Gene chemistry Co., Ltd.), linearize it, and identify the enzyme-cleaved fragments by agarose gel electrophoresis.

TABLE 1-2 double-stranded DNAoligo with Age I and EcoR I cleavage sites at both ends

The vector DNA linearized by double digestion (digestion system shown in tables 1-4, 37 ℃ C., reaction 1h) and the purified double-stranded DNA Oligo were ligated by T4 DNA ligase at 16 ℃ C. overnight in an appropriate buffer system (ligation system shown in tables 1-5), and the ligation product was recovered. The ligation product was transformed into calcium chloride prepared fresh E.coli competent cells (transformation protocol reference: molecular cloning protocols second edition, pages 55-56). Dipping the surface of the clone of the strain growing out of the connected transformation product, dissolving the surface in 10 mul LB culture medium, uniformly mixing and taking 1 mul as a template; in a lentiviral vector for SEQ ID NO:1, designing universal PCR primers at the upstream and downstream of the RNAi sequence, wherein the upstream primer sequence is as follows: 5'-GAACAAGATCTACAACCCTTCAG-3' (SEQ ID NO: 11); the sequence of the downstream primer is as follows: 5'-GGGAGATGGGAGATAAGTGAC-3' (SEQ ID NO: 12), in a lentiviral vector against SEQ ID NO:2, designing universal PCR primers at the upstream and downstream of the RNAi sequence, wherein the upstream primer sequence: 5'-GAACAAGATCTACAACCCTTCAG-3' (SEQ ID NO: 13); the sequence of the downstream primer is as follows: 5'-GGGAGATGGGAGATAAGTGAC-3' (SEQ ID NO: 14), and PCR identification experiments were performed (PCR reaction system shown in tables 1-6, reaction conditions shown in tables 1-7). Sequencing and comparing the clones which are identified to be positive by the PCR, wherein the correctly compared clones are the clones which are successfully constructed and are directed at the nucleotide sequence shown in SEQ ID NO:1 and SEQ ID NO:2, and the RNAi expression vectors are named pGCSIL-GFP-IGFL3-siRNA-1 and pGCSIL-GFP-IGFL3-siRNA-2, respectively.

pGCSIL-GFP-Scr-siRNA negative control plasmid was constructed with negative control siRNA target sequence 5'-TTCTCCGAACGTGTCACGT-3' (SEQ ID NO: 15). When pGCSIL-GFP-Scr-siRNA negative control plasmids are constructed, double-stranded DNA Oligo sequences (shown in a table 1-3) containing adhesive ends of Age I and EcoR I enzyme cutting sites at two ends are synthesized aiming at a Scr siRNA target spot, and the rest construction methods, identification methods and conditions are the same as pGCSIL-GFP-IGFL 3-siRNA-1.

TABLE 1-3 double-stranded DNA Oligo with Age I and EcoR I cleavage sites at both ends

TABLE 1-4 pGCSIL-GFP plasmid digestion reaction System

Reagent	Volume (μ l)
		pGCSIL-GFP plasmid (1. mu.g/. mu.l)	2.0
10×buffer	5.0
		100×BSA	0.5
Age I(10U/μl)	1.0
		EcoR I(10U/μl)	1.0
dd H₂O	40.5
		Total	50.0

TABLE 1-5 ligation reaction System of vector DNA and double-stranded DNA Oligo

Reagent	Positive control (μ l)	Self-contained control (μ l)	Connecting group (mu l)
				Linearized vector DNA (100 ng/. mu.l)	1.0	1.0	1.0
Annealed double stranded DNA Oligo (100 ng/. mu.l)	1.0	-	1.0
				10 XT 4 phage DNA ligase buffer	1.0	1.0	1.0
T4 phage DNA ligase	1.0	1.0	1.0
				dd H₂O	16.0	17.0	16.0
Total	20.0	20.0	20.0

TABLE 1-6 PCR reaction System

Reagent	Volume (μ l)
		10×buffer	2.0
dNTPs(2.5mM)	0.8
		Upstream primer	0.4
Downstream primer	0.4
		Taq polymerase	0.2
Form panel	1.0
		ddH₂O	15.2
Total	20.0

TABLE 1-7 PCR reaction System Programming

3. Packaging of IGFL3-shRNA lentivirus

The DNA of RNAi plasmids pGCSIL-GFP-IGFL3-siRNA-1 and pGCSIL-GFP-IGFL3-siRNA-2 was extracted with a plasmid extraction kit from Qiagen corporation to prepare 100 ng/. mu.l of stock solution.

24h before transfection, human embryonic kidney cell 293T cells in logarithmic growth phase were trypsinized and cell density was adjusted to 1.5X 10 in DMEM complete medium containing 10% fetal bovine serum⁵Cells/ml, seeded in 6-well plates at 37 ℃ with 5% CO₂Culturing in an incubator. The cell density can reach 70-80% to be used for transfection. 2h before transfection, the original medium was aspirated and 1.5ml of fresh complete medium was added. Mu.l of Packing Mix (PVM), 12. mu.l of PEI, and 400. mu.l of serum-free DMEM medium were added to a sterilized centrifuge tube according to the instructions of the MISSION Lentiviral Packing Mix kit from Sigma-aldrich, and 20. mu.l of the above-mentioned extracted plasmid DNA was added to the above-mentioned PVM/PEI/DMEM mixture.

The transfection mixture was incubated at room temperature for 15min, transferred to medium of human embryonic kidney 293T cells at 37 ℃ with 5% CO₂Culturing for 16h in an incubator. The medium containing the transfection mixture was discarded, washed with PBS solution, 2ml of complete medium was added and incubation continued for 48 h. Collecting cell supernatantLiquid, Centricon Plus-20 centrifugal ultrafiltration device (Millipore) purified and concentrated lentiviruses, the procedure was as follows: (1) centrifuging at 4 deg.C and 4000g for 10min to remove cell debris; (2) filtering the supernatant with a 0.45 μm filter in a 40ml ultracentrifuge tube; (3) centrifuging at 4000g for 10-15min to obtain the required virus concentration volume; (4) after the centrifugation is finished, separating the filter cup from the lower filtrate collecting cup, reversely buckling the filter cup on the sample collecting cup, and centrifuging for 2min until the centrifugal force is not more than 1000 g; (5) the centrifuge cup is removed from the sample collection cup, and the virus concentrate is obtained. Subpackaging the virus concentrated solution and storing at-80 ℃. The sequence of the first strand of siRNA contained in the virus concentrate is shown in SEQ ID NO. 2. The packaging procedure for the control lentivirus was identical to that for the IGFL3-shRNA lentivirus, except that the pGCSIL-GFP-Scr-siRNA vector was used in place of the pGCSIL-GFP-IGFL3-siRNA-1 vector.

Example 2 detection of Gene silencing efficiency by real-time fluorescent quantitative RT-PCR

Human gastric cancer AGS cells in logarithmic growth phase are trypsinized to prepare cell suspension (the number of cells is about 5X 10)⁴/ml) were inoculated in 6-well plates and cultured until the degree of cell confluence reached about 30%. According to the complex infection value (MOI, AGS: 20), an appropriate amount of the lentivirus prepared in example 1 is added, the medium is changed after 24h of culture, and cells are collected after the infection time reaches 5 days. Total RNA was extracted according to the Trizol protocol of Invitrogen corporation. The RNA was reverse-transcribed to obtain cDNA according to the M-MLV protocol of Promega (reverse transcription reaction system shown in Table 2-1, reaction at 42 ℃ for 1 hour, and then reverse transcriptase was inactivated by water bath for 10min at 70 ℃ in a water bath).

Real-time quantitative detection was carried out using a TP800 Real time PCR instrument (TAKARA). Primers for the IGFL3 gene of lentivirus IGFL3-shRNA lentivirus 1 were as follows: an upstream primer 5'-CAGTCTTCCTCCTCCAGTGTT-3' (SEQ ID NO: 18) and a downstream primer 5'-CGGGAGATGGGAGATAAGTG-3' (SEQ ID NO: 19). Primers for the IGFL3 gene of lentivirus IGFL3-shRNA lentivirus 2 were as follows: an upstream primer 5'-CAGTCTTCCTCCTCCAGTGTT-3' (SEQ ID NO: 20) and a downstream primer 5'-CGGGAGATGGGAGATAAGTG-3' (SEQ ID NO: 21). The housekeeping gene GAPDH is used as an internal reference, and the primer sequences are as follows: an upstream primer 5'-TGACTTCAACAGCGACACCCA-3' (SEQ ID NO: 22) and a downstream primer 5'-CACCCTGTTGCTGTAGCCAAA-3' (SEQ ID NO: 23). The reaction system was prepared in the proportions shown in Table 2-2.

TABLE 2-1 reverse transcription reaction System

Reagent	Volume (μ l)
		5×RT buffer	4.0
10mM dNTPs	2.0
		RNasin	0.5
M-MLV-RTase	1.0
		DEPC H₂O	3.5
Total	11.0

TABLE 2-2 Real-time PCR reaction System

Reagent	Volume (μ l)
		SYBR premix ex taq	10.0
Upstream primer (2.5 μ M):	0.5
		downstream primer (2.5 μ M):	0.5
cDNA	1.0
		ddH₂O	8.0
Total	20.0

the program was a two-step Real-time PCR: pre-denaturation at 95 ℃ for 30 s; then, denaturation is carried out at 95 ℃ for 5s in each step; annealing and extending for 30s at 60 ℃; a total of 40 cycles were performed. Each time reading the absorbance value during the extension phase. After completion of PCR, the DNA double strand was sufficiently bound by denaturation at 95 ℃ for 15 seconds and then cooling to 60 ℃. Melting curves were prepared by increasing the temperature from 60 ℃ to 95 ℃ by 0.5 ℃ for 4 seconds and reading the absorbance. By adopting 2-^ΔΔCtThe assay calculates the expression abundance of lentiviral-infected cell IGFL3 mRNA. Cells infected with the control virus served as controls. As shown in FIG. 1, the results indicated that the expression level of IGFL3 mRNA in human gastric cancer AGS cells infected with IGFL3-shRNA lentivirus 1 (i.e., shIGFL3-1 group) was down-regulated by 56.0%, and the expression level of IGFL3 mRNA in human gastric cancer AGS cells infected with IGFL3-shRNA lentivirus 2 (i.e., shIGFL3-2 group) was down-regulated by 52.3%

Example 3 examination of the proliferative Capacity of tumor cells infected with IGFL3-shRNA lentivirus

Human gastric cancer AGS cells in logarithmic growth phase are trypsinized to prepare cell suspension (the number of cells is about 5X 10)⁴/ml) were inoculated in 6-well plates and cultured until the degree of cell confluence reached about 30%. According to the infection complex number (MOI, AGS: 20), adding a proper amount of virus, culturing for 24h, then replacing the culture medium, and collecting cells of each experimental group in the logarithmic growth phase after the infection time reaches 5 days. Complete medium resuspension into cell suspension (2X 10)⁴Per ml) at a cell density of about 2000 per well, 96-well plates were seeded. Each set of 5 duplicate wells, 100. mu.l per well. After the plate is laid, the plate is placed at 37 ℃ and 5% CO₂Culturing in an incubator. The plate reading was performed once a day with Celigo instrument (Nexcelom) starting the next day after plating, and the plate reading was performed continuously for 5 days. The number of green fluorescent cells in the well plate of each scan was accurately calculated by adjusting the input parameters of analysis settings, and the data was statistically plotted to generate a cell proliferation curve (the results are shown in fig. 2). The results show that after each tumor in the lentivirus infection group is cultured in vitro for 5 days, the proliferation speed is remarkably slowed down and is far lower than that of the tumor cells in the control group, the number of active cells infected with IGFL3-shRNA lentivirus 1 (namely shIGFL3-siRNA-1 group) and IGFL3-shRNA lentivirus 2 (namely shIGFL3-siRNA-2 group) is respectively reduced by 64 percent and 56.4 percent, and the AGS cell proliferation capacity of human gastric cancer caused by IGFL3 gene silencing is inhibited.

While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.

Sequence listing

<110> Shanghai Jikai Gene medicine science and technology Co., Ltd

Application of <120> human IGFL3 gene and related product

<160> 23

<170> SIPOSequenceListing 1.0

<210> 1

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tgagggttct gggtatgaa 19

<210> 2

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gtgggaacaa gatctacaa 19

<210> 3

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ugaggguucu ggguaugaa 19

<210> 4

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gugggaacaa gaucuacaa 19

<210> 5

<211> 44

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ugaggguucu ggguaugaac ucgaguucau acccagaacc cuca 44

<210> 6

<211> 44

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gugggaacaa gaucuacaac ucgaguugua gaucuuguuc ccac 44

<210> 7

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ccgggttgag ggttctgggt atgaactcga gttcataccc agaaccctca actttttg 58

<210> 9

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

aattcaaaaa gttgagggtt ctgggtatga actcgagttc atacccagaa ccctcaac 58

<210> 9

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ccgggtgtgg gaacaagatc tacaactcga gttgtagatc ttgttcccac actttttg 58

<210> 10

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

aattcaaaaa gtgtgggaac aagatctaca actcgagttg tagatcttgt tcccacac 58

<210> 11

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gaacaagatc tacaaccctt cag 23

<210> 12

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gggagatggg agataagtga c 21

<210> 13

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gaacaagatc tacaaccctt cag 23

<210> 14

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gggagatggg agataagtga c 21

<210> 15

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ttctccgaac gtgtcacgt 19

<210> 16

<211> 54

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ccggttctcc gaacgtgtca cgtctcgaga cgtgacacgt tcggagaatt tttg 54

<210> 17

<211> 54

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

aattcaaaaa ttctccgaac gtgtcacgtc tcgagacgtg acacgttcgg agaa 54

<210> 18

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

cagtcttcct cctccagtgt t 21

<210> 19

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

cgggagatgg gagataagtg 20

<210> 20

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

cagtcttcct cctccagtgt t 21

<210> 21

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

cgggagatgg gagataagtg 20

<210> 22

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

tgacttcaac agcgacaccc a 21

<210> 23

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

caccctgttg ctgtagccaa a 21

Claims

1. The human IGFL3 gene is used as a target in the preparation of gastric cancer treatment medicines or gastric cancer diagnosis medicines.

Use of an inhibitor of IGFL3 in the manufacture of a product having at least one of the following effects:

treating gastric cancer;

inhibiting the proliferation rate of gastric cancer cells;

inhibiting the growth of gastric cancer.

3. Use according to claim 2, further comprising one or more of the following features:

1) the IGFL3 inhibitor is a molecule having an inhibitory effect on IGFL 3;

2) the IGFL3 inhibitor is the only effective component or one of the effective components of the product;

3) the IGFL3 inhibitor is selected from double-stranded RNA, shRNA, an antibody or a small molecule compound.

4. Use according to claim 3, further comprising one or more of the following features:

1) the shRNA or double-stranded RNA target sequence is shown as SEQ ID NO:1 is shown in the specification;

2) the double-stranded RNA comprises a first strand and a second strand, wherein the first strand and the second strand are complementary to form an RNA dimer, and the sequence of the first strand is shown as SEQ ID NO:2 is shown in the specification;

3) the nucleotide sequence of the shRNA is shown as SEQ ID NO: 3, respectively.

5. A nucleic acid molecule for reducing expression of IGFL3 gene in gastric cancer cells, the nucleic acid molecule comprising:

a. a double-stranded RNA comprising a nucleotide sequence capable of hybridizing to the IGFL3 gene; or

shRNA containing a nucleotide sequence capable of hybridizing with the IGFL3 gene;

wherein the double-stranded RNA comprises a first strand and a second strand, the first strand and the second strand are complementary to form an RNA dimer, and the sequence of the first strand is substantially identical to a target sequence in the IGFL3 gene; the shRNA includes a sense strand segment and an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, the sequences of the sense strand segment and the antisense strand segment are complementary, and the sequence of the sense strand segment is substantially identical to a target sequence in the IGFL3 gene.

6. The nucleic acid molecule for reducing IGFL3 gene expression in gastric cancer cells according to claim 5, further comprising one or more of the following characteristics:

2) the double-stranded RNA is siRNA, and the sequence of the first strand of the siRNA is shown as SEQ ID NO:2 is shown in the specification;

3) the nucleotide sequence of the shRNA is shown as SEQ ID NO: 3, respectively.

7. An IGFL3 gene interfering nucleic acid construct comprising a gene segment encoding an shRNA in the nucleic acid molecule of any one of claims 5 to 6, capable of expressing the shRNA.

8. An IGFL3 gene-interfering lentivirus, wherein the interfering nucleic acid construct of claim 7 is packaged into a virus with the aid of a lentivirus packaging plasmid or cell line.

9. The nucleic acid molecule of any one of claims 5-6, or the IGFL3 gene-interfering nucleic acid construct of claim 7, or the IGFL3 gene-interfering lentivirus of claim 8, for use in: is used for preparing a medicine for preventing or treating gastric cancer or a kit for reducing the expression of the IGFL3 gene in gastric cancer cells.

10. A composition for preventing or treating gastric cancer, which comprises the following effective substances:

the nucleic acid molecule of any one of claims 5-6; and/or, the IGFL3 gene interfering nucleic acid construct of claim 7; and/or, the IGFL3 gene interfering lentivirus of claim 8, and a pharmaceutically acceptable carrier, diluent or excipient.