CN110938691A

CN110938691A - Application of human DUS4L gene and related product

Info

Publication number: CN110938691A
Application number: CN201911221067.7A
Authority: CN
Inventors: 李斌
Original assignee: Lanzhou University; Lanzhou University Second Hospital
Current assignee: Lanzhou University; Lanzhou University Second Hospital
Priority date: 2019-12-03
Filing date: 2019-12-03
Publication date: 2020-03-31
Anticipated expiration: 2039-12-03
Also published as: CN110938691B

Abstract

The invention belongs to the field of biomedical research, and particularly relates to application of a human DUS4L gene as a target in preparation of a lung cancer treatment drug. Extensive and intensive research shows that the RNAi method is adopted to down-regulate the expression of the human DUS4L gene, so that the proliferation of lung cancer cells can be effectively inhibited, the apoptosis of the cells can be promoted, and the growth process of the lung 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 capacity of lung cancer cells, inhibit the lung cancer cell cloning, influence the lung cancer cell cycle, promote the lung cancer cell apoptosis and inhibit the lung cancer cell growth, thereby treating the lung cancer and opening up a new direction for the lung cancer treatment.

Description

Application of human DUS4L gene and related product

Technical Field

The invention belongs to the field of biomedical research, and particularly relates to application of a human DUS4L gene and a related product.

Background

DUS4L is a protein-encoding gene. Gene Ontology (GO) annotation associated with this gene includes oxidoreductase activity and FMN binding. Fusion genes of DUS4L-BCAP29 appeared in multiple tissue samples (Current fusion RNA DUS4L-BCAP29 in non-cancer human tissues and cells. Tang Y, Qin F, Liu A, LiH. oncotarget.2017May 9; 8(19):31415-31423.doi: 10.18632/oncotarget.16329.). And it was reported that DUS4L-BCAP29 fusion gene was significantly highly expressed when inducing human umbilical cord mesenchymal stem cells to undergo neural differentiation (Identification of polymeric RNAs in human amino polypeptides and peptides in neural differentiation. Tang Y, et al. int J Biochem cell biol.2019 Jun; 111:19-26.doi:10.1016/J. biocel. 2019.03.012.epub.2019 Apr 5).

Lung cancer is the most common malignant tumor in recent years, and the rate of incidence of lung cancer is high at the top of various tumors. Despite the emerging therapeutic approaches to lung cancer, 5-year survival rates are only 14.1%, with 60% of patients dying within 1 year after diagnosis. Chemotherapy is the primary treatment for lung cancer, and over 90% of lung cancers require chemotherapy. Chemotherapy can kill tumor cells and also damage normal cells of the human body. Chemotherapy inhibits the bone marrow hematopoietic system, primarily the decline of leukocytes and platelets, and can be treated with granulocyte colony stimulating factor and platelet stimulating factor.

At present, there is no report about the application of DUS4L gene in lung 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 DUS4L gene and related products.

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

in a first aspect of the invention, there is provided the use of the human DUS4L gene as a target in the preparation of a medicament for the treatment of lung cancer.

The human DUS4L gene as a target in preparing the lung cancer treatment drug specifically comprises the following steps: the DUS4L gene is used as an action object, and the drug or preparation is screened to find the drug which can inhibit the expression of the human DUS4L gene and is used as a candidate drug for treating the lung cancer. The DUS4L gene small interfering RNA (siRNA) is obtained by screening human DUS4L gene serving as an action object and can be used as a medicine for inhibiting the proliferation of lung cancer cells. In addition, DUS4L gene can be used as a target of action, for example, for antibody drugs, small molecule drugs, etc.

The lung cancer treatment drug is a molecule capable of specifically inhibiting transcription or translation of a DUS4L gene, or specifically inhibiting expression or activity of a DUS4L protein, so that the expression level of the DUS4L gene in a lung cancer cell is reduced, and the purpose of inhibiting proliferation, growth, differentiation and/or survival of the lung cancer cell is achieved.

The lung cancer therapeutic drug prepared by DUS4L 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 lung cancer therapeutic agent administered is a dose sufficient to reduce transcription or translation of the human DUS4L gene, or to reduce expression or activity of human DUS4L protein. Such that the expression of the human DUS4L gene is reduced by at least 50%, 80%, 90%, 95%, or 99%.

The method for treating lung cancer by adopting the lung cancer treatment medicine mainly achieves the purpose of treating lung cancer by reducing the expression level of human DUS4L gene and inhibiting the proliferation of lung cancer cells. Specifically, in treatment, a substance effective in reducing the expression level of human DUS4L gene is administered to the patient.

In one embodiment, the target sequence of the DUS4L gene is set forth in SEQ ID NO:1 is shown. The method specifically comprises the following steps: 5'-ACATCAGCAATCATAGATT-3' are provided.

In a second aspect of the invention, there is provided the use of an inhibitor of DUS4L in the manufacture of a product having at least one of the following effects:

treating lung cancer;

inhibiting the proliferative capacity of lung cancer cells;

inhibiting the cloning of lung cancer cells;

affecting lung cancer cell cycle;

promoting apoptosis of lung cancer cells;

inhibiting the growth of lung cancer.

The product necessarily comprises the DUS4L inhibitor and the DUS4L inhibitor as an active ingredient of the aforementioned effects.

In the product, the effective component for the above functions can be only the DUS4L inhibitor, and can also comprise other molecules for the above functions.

That is, the DUS4L 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 DUS4L inhibitor can be a nucleic acid molecule, an antibody, a small molecule compound.

As exemplified in the examples herein, the inhibitor of DUS4L can be a nucleic acid molecule that reduces expression of the DUS4L gene in a lung cancer cell. Specifically, it may be a double-stranded RNA or shRNA.

In a third aspect of the invention, a method of treating lung cancer is provided by administering to a subject a DUS4L inhibitor.

The subject may be a mammal or a mammalian lung 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 lung cancer cell may be an ex vivo lung cancer cell.

The subject may be a patient suffering from lung cancer or an individual in whom treatment for lung cancer is desired. Or the subject is an isolated lung cancer cell from a lung cancer patient or an individual expected to treat lung cancer.

The DUS4L inhibitor can be administered to a subject before, during, or after receiving treatment for lung cancer.

The fourth aspect of the invention discloses a nucleic acid molecule for reducing the expression of DUS4L gene in lung 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 the DUS4L gene;

the shRNA contains a nucleotide sequence capable of hybridizing with the DUS4L 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 DUS4L gene.

The target sequence in the DUS4L gene is a segment in the DUS4L gene corresponding to an mRNA segment recognized and silenced by the nucleic acid molecule when the nucleic acid molecule is used for specifically silencing the expression of the DUS4L gene.

Further, the target sequence of the double-stranded RNA is shown as SEQ ID NO:1 is shown. The method specifically comprises the following steps: 5'-ACATCAGCAATCATAGATT-3' are provided. Further, the sequence of the first strand of the double-stranded RNA is shown as SEQ ID NO:2, respectively. Specifically 5'-ACAUCAGCAAUCAUAGAUU-3'.

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

SEQ ID NO:2 is designed by taking the sequence shown in SEQ ID NO. 1 as an RNA interference target sequence and aiming at one strand of small interfering RNA of the human DUS4L gene, and the sequence of the other strand, namely the second strand, is complementary with the sequence of the first strand, and the siRNA can play a role in specifically silencing the expression of endogenous DUS4L gene in lung 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 DUS4L gene.

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

The shRNA can become small interfering RNA (siRNA) after enzyme digestion and processing, and further plays a role in specifically silencing the expression of endogenous DUS4L gene in lung 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: 3, respectively. Specifically 5'-GCACAUCAGCAAUCAUAGAUU CUCGAG AAUCUAUGAUUGCUGAUGUGC-3'.

Further, the DUS4L gene is derived from human.

In the fifth aspect of the invention, the invention discloses a DUS4L gene interfering nucleic acid construct, which comprises a gene segment for coding shRNA in the nucleic acid molecule and can express the shRNA.

The DUS4L gene interfering nucleic acid construct can be obtained by cloning a gene segment coding the human DUS4L gene shRNA into a known vector.

Further, the DUS4L gene interfering nucleic acid construct is a DUS4L gene interfering lentiviral vector.

The DUS4L gene interference lentiviral vector disclosed by the invention is obtained by cloning a DNA fragment for coding the DUS4L gene shRNA into a known vector, wherein the known vector is mostly a lentiviral vector, the DUS4L gene interference lentiviral vector is packaged into infectious viral particles by virus, and then infects lung cancer cells to transcribe the shRNA, and the siRNA is finally obtained by the steps of enzyme digestion processing and the like and is used for specifically silencing the expression of the DUS4L gene.

Further, the DUS4L gene-interfering lentiviral vector further contains a promoter sequence and/or a nucleotide sequence encoding a marker detectable in lung 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 lists a human DUS4L gene interference lentiviral vector constructed by taking pGCSIL-GFP as a vector, and is named as pGCSIL-GFP-DUS 4L-siRNA.

The DUS4L gene siRNA can be used for inhibiting the proliferation of lung cancer cells, and further can be used as a medicine or a preparation for treating lung cancer. The DUS4L gene interference lentiviral vector can be used for preparing the DUS4L gene siRNA. When used as a medicament or formulation for treating lung 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 DUS4L gene interference lentivirus is formed by virus packaging of the DUS4L gene interference nucleic acid construct under the assistance of lentivirus packaging plasmids and cell lines. The lentivirus can infect lung cancer cells and generate small interfering RNA aiming at DUS4L gene, thereby inhibiting the proliferation of the lung cancer cells. The DUS4L gene interference lentivirus can be used for preparing medicaments for preventing or treating lung cancer.

In a seventh aspect of the present invention, there is provided a use of the aforementioned nucleic acid molecule, or the aforementioned DUS4L gene-interfering nucleic acid construct, or the aforementioned DUS4L gene-interfering lentivirus, wherein: is used for preparing a medicament for preventing or treating lung cancer or a kit for reducing the expression of DUS4L gene in lung cancer cells.

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

Further, when the medicament is used for preventing or treating lung cancer in a subject, an effective dose of the medicament needs to be administered to the subject. Using this method, the growth, proliferation, recurrence and/or metastasis of lung cancer is inhibited. Further, at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% fraction of the growth, proliferation, recurrence and/or metastasis of the lung 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 lung cancer, comprising, as active ingredients:

the aforementioned nucleic acid molecules; and/or, the aforementioned DUS4L gene interfering nucleic acid construct; and/or, the aforementioned DUS4L 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 lung cancer in a subject, an effective dose of the composition is administered to the subject. Using this method, the growth, proliferation, recurrence and/or metastasis of lung cancer is inhibited. Further, at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% fraction of the growth, proliferation, recurrence and/or metastasis of the lung cancer is 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 an RNAi target sequence aiming at the human DUS4L gene and constructs a corresponding DUS4LRNAi vector, wherein the RNAi vector pGCSIL-GFP-DUS4L-siRNA can obviously down-regulate the expression of the DUS4L gene at the mRNA level and the protein level. Lentivirus (Lv) is used as a gene operation tool to carry an RNAi vector pGCSIL-GFP-DUS4L-siRNA, so that the RNAi sequence aiming at the DUS4L gene can be efficiently introduced into lung cancer A549 cells in a targeted manner, the expression level of the DUS4L gene is reduced, and the proliferation capacity of the tumor cells is remarkably inhibited. Lentivirus-mediated silencing of DUS4L gene is therefore a potential clinical non-surgical treatment modality for malignancies.

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

extensive and intensive research shows that the RNAi method is adopted to down-regulate the expression of the human DUS4L gene, so that the proliferation of lung cancer cells can be effectively inhibited, the apoptosis of the cells can be promoted, and the growth process of the lung 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 capacity of lung cancer cells, inhibit the lung cancer cell cloning, influence the lung cancer cell cycle, promote the lung cancer cell apoptosis and inhibit the lung cancer cell growth, thereby treating the lung cancer and opening up a new direction for the lung cancer treatment.

Drawings

FIG. 1: RT-PCR detects the target gene reduction efficiency of A549 cell mRNA level.

FIG. 2: western Blot for detecting that the protein level expression of the DUS4L gene is reduced by the A549 cell target.

FIG. 3: the Celigo cell counting method verifies the effect of the DUS4L gene on the proliferation of A549 cells. (upper panel shows recorded cell pictures of Celigo for 5 consecutive days, and lower panel shows the time-dependent cell number curves of shDUS4L group and shCtrl control group)

FIG. 4: the influence of DUS4L gene on the proliferation capacity of A549 cells is detected by a cell clone formation method, the A549 cells are infected by shRNA lentivirus, the clone number is observed after the A549 cells are cultured for 14 days, the left side is a digital camera recording chart, and the right side bar result is displayed by the average value +/-standard deviation of the cell clone number.

FIG. 5-1: after the PI-FACS method detects the expression of the reduced gene, the periodic distribution change of the tumor cells is regulated and controlled.

FIG. 5-2: and (3) a histogram for regulating the change of the period distribution of the tumor cells after detecting the reduction of the expression of the genes by using a PI-FACS method.

FIG. 6-1: a flow apoptosis diagram for detecting the influence of sh DUS4L on A549 cell apoptosis by Annexin V flow apoptosis,

FIG. 6-2: annexin V flow apoptosis bar results of the effect of sh DUS4L on a549 apoptosis are shown as the percent cell mean ± standard deviation.

FIG. 7: EDU cell proliferation assay results are shown as percent cell mean. + -. standard deviation.

In the drawings, there is shown in the drawings,

bar graphs represent the mean of three experiments and error bars represent Standard Deviation (SD).

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

And compared with the target gene shRNA lentivirus treatment group, the shCtrl is not less than 0.01 and P is less than 0.05.

Detailed Description

The inventor of the invention finds that DUS4L gene is obviously highly expressed in lung cancer tumor tissues through extensive and intensive research; the inventor finds that after the expression of the human DUS4L gene is down-regulated by an RNAi method, the proliferation of tumor cells can be effectively inhibited, the apoptosis is promoted, the invasion and the transfer capacity of the tumor cells are reduced, and the growth process of tumors can be effectively controlled, and the research result shows that the DUS4L gene is a protooncogene and can be used as a target point for tumor treatment. The inventor further synthesizes and tests a plurality of siRNAs aiming at DUS4L gene, screens out the siRNA which can effectively inhibit the expression of DUS4L and further inhibit the proliferation and growth of human lung cancer A549 cell, and completes the invention on the basis.

The invention proves the function of the DUS4L gene in the occurrence of lung cancer from the viewpoint of cell function. Transfecting lung cancer cells after constructing a target gene shRNA lentivirus, and comparing with a transfection control lentivirus to detect the expression conditions of mRNA and protein level target genes in two groups of lung cancer cell lines; and then cell proliferation, apoptosis and other detection are carried out through cytofunctional experiments, and the results show that the lung cancer cell proliferation inhibition degree of the shRNA group is obviously higher than that of the control group and the increase degree of the cell apoptosis rate of the shRNA group is higher than that of the control group compared with the control group.

DUS4L inhibitors

Refers to a molecule having an inhibitory effect on DUS 4L. Having inhibitory effects on DUS4L include, but are not limited to: inhibiting the expression or activity of DUS 4L.

Inhibiting DUS4L activity refers to a decrease in the activity of DUS 4L. Preferably, DUS4L 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.

Specifically, inhibiting the expression of DUS4L may be inhibiting the transcription or translation of the DUS4L gene, and specifically, may be: by not transcribing the gene of DUS4L, by reducing the transcriptional activity of the gene of DUS4L, by not translating the gene of DUS4L, or by reducing the level of translation of the gene of DUS 4L.

The regulation of gene expression of DUS4L can be accomplished by one skilled in the art using conventional methods, such as gene knock-outs, homologous recombination, interfering RNA, and the like.

The inhibition of DUS4L gene expression was confirmed by PCR and Western Blot detection of expression level.

Preferably, DUS4L gene expression is reduced by at least 10%, preferably by at least 30%, still more preferably by at least 50%, still more preferably by at least 70%, still more preferably by at least 90%, most preferably by none of the DUS4L gene expression compared to 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 lung cancer

Nucleic acid molecules that reduce the expression of the DUS4L gene in lung cancer cells can be utilized; and/or, a DUS4L gene interfering nucleic acid construct; and/or, DUS4L gene interferes lentivirus, and is used as an effective component for preparing a medicament for preventing or treating lung 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 the human DUS4L Gene

1. Screening of effective siRNA targets against human DUS4L Gene

Calling DUS4L (NM-181581) gene information from Genbank; effective siRNA targets were designed against the DUS4L gene. Table 1-1 lists the effective siRNA target sequences selected against the DUS4L gene.

TABLE 1-1 siRNA target sequences targeting the human DUS4L Gene

SEQ ID NO	TargetSeq(5’-3’)
		1	ACATCAGCAATCATAGATT

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 as an example); 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 DNA Oligo 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; designing universal PCR primers at the upstream and downstream of RNAi sequence in the lentiviral vector, wherein the upstream primer sequence: 5'-CCTATTTCCCATGATTCCTTCATA-3' (SEQ ID NO: 6); the sequence of the downstream primer is as follows: 5'-GTAATACGGTTATCCACGCG-3' (SEQ ID NO: 7), 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, named pGCSIL-GFP-DUS 4L-siRNA.

pGCSIL-GFP-Scr-siRNA negative control plasmid was constructed with negative control siRNA target sequence 5'-TTCTCCGAACGTGTCACGT-3' (SEQ ID NO: 8). When pGCSIL-GFP-Scr-siRNA negative control plasmids are constructed, double-stranded DNAoligo sequences (tables 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 those of pGCSIL-GFP-DUS 4L-siRNA.

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

TABLE 1-4pGCSIL-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 1-6-1PCR reaction System

TABLE 1-7PCR reaction System Programming

3. Packaging DUS4L-siRNA lentivirus

The DNA of RNAi plasmid pGCSIL-GFP-DUS4L-siRNA was extracted using a plasmid extraction kit from Qiagen, Inc., and 100 ng/. mu.l of stock solution was prepared.

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 and washed with PBS solutionWashing, adding 2ml of complete culture medium, and continuing to culture for 48 h. The cell supernatant was collected, and the lentivirus was purified and concentrated by a Centricon Plus-20 centrifugal ultrafiltration device (Millipore) according to the following steps: (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 concentrated solution is shown in SEQ ID NO. 2. The control lentivirus was packaged in the same manner as DUS4L-siRNA lentivirus except that pGCSIL-GFP-Scr-siRNA vector was used instead of pGCSIL-GFP-DUS4L-siRNA vector.

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

Human lung cancer A549 cells in logarithmic growth phase are subjected to pancreatin digestion to prepare cell suspension (the cell number is about 5 multiplied by 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, A549: 10), an appropriate amount of the lentivirus prepared in example 1 is added, the culture medium is replaced 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 DUS4L gene were as follows: an upstream primer 5'-GCCCATTGATTGTTCAGTTTGC-3' (SEQ ID NO: 11) and a downstream primer 5'-AACTCCTGTTGCTTCAGCCTTT-3' (SEQ ID NO: 12). 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: 13) and a downstream primer 5'-CACCCTGTTGCTGTAGCCAAA-3' (SEQ ID NO: 14). 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	0.4
M-MLV-RTase	1.0
		RNase-Free	2.6
Total	10.0

TABLE 2-2Real-time PCR reaction System

Reagent	Volume (μ l)
		SYBR premix ex taq:	6.0
Primer MIX (5 μ M):	0.3
		cDNA	0.6
ddH₂O	5.1
		Total	12.0

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 abundance of expression of the mRNA that infects DUS 4L. Cells infected with the control virus served as controls. The experimental results are shown in fig. 1, and indicate that the expression level of DUS4L mRNA in human lung cancer A549 cells is down-regulated by 64.5%.

Example 3 detection of Gene silencing efficiency by Western Blotting method

1. Extraction of Total cellular proteins

(1) The control virus and the RNAi virus against the DUS4L interfering target were each infected with the cell of interest (a549 cell). Cell samples were received and washed twice with PBS. An appropriate amount of RIPA lysate was taken and PMSF was added to a final concentration of 1mM within a few minutes before use. (using RIPA lysate, instruction chain:http://www.beyotime.com/ripa- lysis-bufferm.htm)

(2) adding appropriate amount of RIPA lysate, and lysing on ice for 10-15 min. Cells were scraped off and transferred to a new EP tube, and then cells were sonicated (20 times at 40W, 1s each, 2s apart).

(3) Centrifugation was carried out at 12000g for 15min at 4 ℃ and the supernatant BCA method was used to determine the Protein concentration (BCA Protein Assay Kit instruction: http:// www.beyotime.com/p0010s. htm)

(4) The protein concentration of each sample was adjusted to be consistent by adding fresh lysate, typically 2. mu.g/. mu.L. Then 6X padding buffer with the volume of 1/5 is added and mixed evenly, the mixture is boiled for 10min in a metal bath with the temperature of 100 ℃, and the mixture is stored for standby at the temperature of 80 ℃ after being centrifuged for a short time.

2.SDS-PAGE

(1) Preparing glue: according to the molecular weight of the target protein, glue with different concentrations is prepared, and the specific system is shown in tables 3-1, 3-2 and 3-3:

TABLE 3-1SDS-PAGE gels (8mL system)

Separating glue (8mL system)	8％	9％	10％	12％	13％	15％
							H₂O	3.7	3.4	3.1	2.6	2.3	1.8
30％PAGE	2.1	2.4	2.7	3.2	3.5	4
							1.5mol/L Tris(pH 8.8)	2	2	2	2	2	2
10％SDS	0.08	0.08	0.08	0.08	0.08	0.08
							10％APS	0.08	0.08	0.08	0.08	0.08	0.08
TEMED	0.005	0.004	0.004	0.004	0.004	0.004

TABLE 3-2SDS-PAGE gels (10mL system)

TABLE 3SDS-PAGE gels

Concentrated gum (5%)	3mL	4mL	5mL
				H₂O	2.1	2.7	3.4
30％PAGE	0.5	0.67	0.83
				1.0mol/L Tris(pH6.8)	0.38	0.5	0.63
10％SDS	0.03	0.04	0.05
				10％APS	0.03	0.04	0.05
TEMED	0.003	0.004	0.005

(2) Loading: after the gel is solidified, the comb is pulled out, the electrophoresis buffer solution is used for cleaning the sample loading hole, and the prepared sample is loaded.

(3) Electrophoresis: concentrating the gel at 80mA for 20 min; the separation gel was 120mA, 1 h.

3. Immunoblotting (Wet transfer)

After the electrophoresis is finished, the protein is transferred to the PVDF membrane by using a transfer electrophoresis device and electrotransfer for 150min under the constant current condition of 300mA at 4 ℃.

4. Antibody hybridization:

(1) and (3) sealing: PVDF membrane was blocked with blocking solution (TBST solution containing 5% skim milk) at room temperature for 1h or overnight at 4 ℃.

(2) Primary antibody incubation: the antibody was diluted with blocking solution and incubated with the blocked PVDF membrane at room temperature for 2h or overnight at 4 ℃ and the membrane was washed 4 times with TBST for 8min each.

(3) And (3) secondary antibody incubation: the corresponding secondary antibody was diluted with blocking solution, the PVDF membrane was incubated at room temperature for 1.5h, and the membrane was washed 4 times with TBST, 8min each.

X-ray development: (use of 20X from CST Co., Ltd.)

Reagent and 20X Peroxide #7003 kit, instruction linked:

https://www.cst-c.com.cn/products/wb-ip-reagents/20x-lumiglo-reagent-and-20x-peroxide/7003？site-search-type＝Products)

(1) the solution A and the solution B in the kit are mixed according to the proportion of 1:1, inverted and mixed evenly, and can be used after being placed for a plurality of minutes.

(2) Taking out the film, wiping the absorbent paper dry, spreading into a cassette, dripping a proper amount of uniformly mixed ECL luminous liquid, spreading a preservative film (avoiding generating bubbles), putting an X-ray film (avoiding the movement of the X-ray film), closing the cassette, and exposing for 1 s-several min (the exposure time needs to be tried for several times, and the exposure time is properly adjusted according to whether the naked eye can see fluorescence and the strength of the fluorescence.

(3) Taking out the X-ray film, placing in developing solution, taking out after banding occurs, rinsing in clear water for several seconds, and placing in fixing solution for at least 2 min.

(4) Taking out the X-ray film, drying and analyzing.

As shown in FIG. 2, Western Blot experiments show that the target has a knockdown effect on the endogenous expression of DUS4L gene, so that the target is an effective target.

Example 4 examination of the proliferation Capacity of tumor cells infected with DUS4L-siRNA lentivirus (Celigo experiment)

Human lung cancer A549 cells in logarithmic growth phase are subjected to pancreatin digestion to prepare cell suspension (the cell number is about 5 multiplied by 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, A549: 10), 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 1500 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. Starting from the day after the plating,the plate readings were performed once a day with a Celigo instrument (Nexcelom) and were performed for 5 consecutive days. Accurately calculating the number of cells with green fluorescence in each scanning pore plate by adjusting input parameters of analyzers; the data were statistically plotted and cell proliferation curves were plotted for 5 days.

The results are shown in FIG. 3. The results show that after each tumor of the lentivirus infection group is cultured in vitro for 5 days, the proliferation speed is obviously slowed down and is far lower than that of tumor cells of a control group, the number of viable cells is reduced by 44.1 percent, and the result shows that the proliferation capacity of human lung cancer A549 cells is inhibited due to DUS4L gene silencing.

Example 5 examination of the clonogenic Capacity of tumor cells infected with DUS4L-siRNA lentivirus (clonogenic assay)

Human lung cancer A549 cells are inoculated in a 12-well plate after being digested by pancreatin, and the cell density is 10-15%. The next day, the medium was changed to fresh medium containing 5ug/ml polybrene. DUS4L-siRNA lentivirus was administered at a multiplicity of infection MOI, a 549: 10 were added to the plates and the medium was changed fresh 12-24h after infection. After infection for 72h, fluorescence is observed under a fluorescence microscope, and the infection efficiency reaches 80%.

After the cells infected with the virus in the logarithmic growth phase are digested by pancreatin, the complete culture medium is re-suspended into cell suspension; after counting the cells, inoculating the cells into a 6-well plate (800 cells/well), continuously culturing the inoculated cells in an incubator for 12 days, changing the liquid every 3day in the middle, and observing the cell state; photographing the cell clone under a fluorescent microscope before the experiment is terminated; at the end of the experiment, cells were fixed with paraformaldehyde, washed with PBS, Giemsa stained, and photographed.

As shown in fig. 4, the number of the clone spots formed by the lung cancer a549 cells was significantly reduced and the volume of the clone spots was significantly reduced after the expression of the gene was reduced by RNA interference (KD group) compared to the control interference (NC group); indicating that gene silencing results in a reduction in the ability of tumor cells to form clones. The plate cloning test detects that after the expression of the gene is reduced, the cloning capacity of the tumor cells is reduced.

Example 6 tumor cell cycle assay (FACS cell cycle assay) of DUS4L-siRNA lentivirus infection

Human lung cancer A549 cells in logarithmic growth phase are subjected to pancreatin digestion to prepare cell suspension (the cell number is about 5 multiplied by 10)⁴/ml) were inoculated in 6-well plates and cultured until the degree of cell confluence reached about 30%. According to the multiplicity of infection MOI, a 549: and 10, adding a proper amount of virus, culturing for 24h, then replacing the culture medium, and collecting the cells of each experimental group in the logarithmic growth phase after the infection time reaches 5 days. If the cells are adherent cells, when the 6cm dish cells of each experimental group grow to the coverage rate of about 80% (the cells do not enter the growth plateau), pancreatin digestion is carried out, the complete culture medium is resuspended into cell suspension, the cells are collected in a 5mL centrifuge tube, and each group is provided with three multiple holes (for ensuring that the number of the cells on the machine is enough, the number of the cells is more than or equal to 106/treatment). And directly collecting the suspension cells. 1300rmp was centrifuged for 5min, the supernatant was discarded, and the cell pellet was washed 1 time with 4 ℃ pre-cooled D-Hanks (pH 7.2-7.4). 1300rmp, 5min centrifugation, 4 ℃ pre-cooled 75% ethanol fixed cells for at least 1 h. 1300rmp centrifugation for 5min to remove fixative, D-Hanks washing cell precipitation, step 2. Preparing a cell staining solution: 40 XPI stock (2 mg/mL): 100 XRNase stock (10 mg/mL): 1 × D-Hanks ═ 25: 10: 1000 cell staining: according to the Cell amount, adding a certain volume of Cell staining solution (0.6-1mL) for resuspension, so that the Cell passing rate during loading is 300-800 cells/s. And (6) performing detection on the machine. And (6) analyzing the data.

As a result, as shown in FIGS. 5-1 and 5-2, the periodic distribution of tumor cells was controlled by detecting the decrease in gene expression by PI-FACS. RNA interference reduced gene expression (KD group) compared to control interference (NC group), with a decrease in cells at G1 phase (P <0.05), no significant change in cells at S phase, and an increase in cells at G2/M phase (P <0.05), suggesting that the DUS4L gene is associated with the cycle distribution of a549 cells.

Example 7 detection of apoptosis levels in tumor cells infected with DUS4L-siRNA lentivirus (FACS apoptosis detection)

Human lung cancer A549 cells are inoculated in a 12-well plate after being digested by pancreatin, and the cell density is 10-15%. The next day, the medium was changed to fresh medium containing 5ug/ml polybrene. Lentiviruses were infected at a multiplicity of MOI, a 549: 10 were added to the plates and the medium was changed fresh 12-24h after infection. After 120h of infection, fluorescence is observed under a fluorescence microscope, and the infection efficiency reaches 90%.

After trypsinizing the cells in logarithmic growth phase, resuspending the complete medium into a cell suspension; collecting the supernatant in the same 5mL centrifuge tube, each group having three multiple holes (the number of cells is not less than 5 × 10 to ensure enough cells on the machine⁵Treatment). 1300rmp for 5min, discard the supernatant and wash the cell pellet with 4 ℃ pre-cooled PBS. The cell pellet was washed once with 1 Xbinding buffer, centrifuged at 1300rmp for 3min, and the cells were collected. 200 μ L of 1 XBinding buffer resuspended cell pellet. Add 10. mu.L Annexin V-APC staining, and keep away from light for 10-15min at room temperature. According to the cell amount, 400-800. mu.L of 1 XBindingbuffer is added, and detection is carried out by an up-flow cytometer. The results were analyzed.

As shown in FIGS. 6-1 and 6-2, the change in the apoptosis ratio of human lung cancer A549 cells was detected by Annexin V single stain assay after the expression of the genes was reduced. It was found that the apoptosis rate of tumor cells increases after down-regulating gene expression. After RNA interference reduced gene expression (KD group) with control interference (NC group), the number of apoptotic tumor cells increased significantly; indicating that gene silencing leads to apoptosis of tumor cells.

Example 8 EDU cell proliferation assay for DUS4L-siRNA Lentiviral-infected tumor cells

Inoculating 3000 cells per well of human lung cancer A549 cells infected with DUS4L-siRNA and its control lentivirus 10 into a 96-well plate, and culturing to normal growth stage, wherein the optimal growth stage is generally three days; the cells were cultured in a cell culture medium at 1000: 1 (reagent A) to prepare an appropriate amount of 50. mu.M EdU medium; adding 100 mu L of 50 mu M EdU culture medium into each hole, incubating for 2 hours, and removing the culture medium; washing the cells with PBS for 1-2 times, 5 minutes each time; the cleaning purpose is to elute the EdU which does not permeate into DNA, the cleaning mode is determined according to different cell types, and the cleaning strength is reduced for cells which are not firmly adhered to the wall; adding 50 μ L of cell fixing solution (PBS containing 4% paraformaldehyde) into each well, incubating at room temperature for 30 min, and discarding the fixing solution; adding 50 mu L of 2mg/mL glycine into each hole, and after incubating for 5 minutes by a decoloring shaker, removing the glycine solution; aims to neutralize paraformaldehyde, ensure a dyeing reaction system, and save the situation when other modes are adopted for cell fixationThis step is omitted; adding 100 mu L PBS into each hole, washing for 5 minutes by a decoloring shaker, and discarding the PBS; add 100. mu.L of 1X per well

Incubating the staining reaction solution (table 3) for 30 minutes in a dark place at room temperature by using a decoloring shaker, and then discarding the staining reaction solution; adding 100 mu L of penetrating agent (PBS of 0.5 percent TritonX-100) to decolor and wash by a shaking table for 2-3 times, each time for 10 minutes, and discarding the penetrating agent; (reinforcing) adding 100 mu L of methanol into each hole for cleaning for 1-2 times, and each time for 5 minutes; PBS washing for 5min for 1 time; deionized water is added according to the proportion of 100: 1, preparing a proper amount of 1X Hoechst33342 reaction solution, and storing in a dark place; adding 100 mu L of 1X Hoechst33342 reaction solution into each hole, keeping out of the sun, incubating at room temperature for 30 minutes; discarding the dyeing reaction solution; adding 100 mu L PBS to clean for 1-3 times; observing immediately after dyeing; if the conditions limit the storage at 4 ℃ in a humid way, the storage time should not exceed 3 days.

The results are shown in fig. 7, where the percentage of EDU-positive cells was decreased in shDUS4L group compared to shCtrl group, indicating a slower proliferation of a549 cells after DUS4L knockdown (p < 0.05).

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

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Lanzhou university second Hospital

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Claims

1. Application of human DUS4L gene as a target in preparing lung cancer treatment medicines.

Use of an inhibitor of DUS4L in the manufacture of a product having at least one of the following effects:

treating lung cancer;

inhibiting the proliferative capacity of lung cancer cells;

inhibiting the cloning of lung cancer cells;

affecting lung cancer cell cycle;

promoting apoptosis of lung cancer cells;

inhibiting the growth of lung cancer.

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

1) the DUS4L inhibitor is a molecule having an inhibitory effect on DUS 4L;

2) the DUS4L inhibitor is the only effective component or one of the effective components of the product;

3) the DUS4L 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 that reduces expression of DUS4L gene in a lung cancer cell, the nucleic acid molecule comprising:

a. a double-stranded RNA comprising a nucleotide sequence capable of hybridizing to the DUS4L gene; or

shRNA containing a nucleotide sequence capable of hybridizing with the DUS4L 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 DUS4L 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 DUS4L gene.

6. The nucleic acid molecule of 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. A DUS4L gene interfering nucleic acid construct comprising a gene fragment encoding an shRNA in the nucleic acid molecule of any of claims 5 to 6, capable of expressing said shRNA.

8. A DUS4L gene interfering lentivirus, which is formed by virus packaging of the interfering nucleic acid construct of claim 7 with the help of a lentivirus packaging plasmid and a cell line.

9. Use of the nucleic acid molecule of any one of claims 5-6, or the DUS4L gene-interfering nucleic acid construct of claim 7, or the DUS4L gene-interfering lentivirus of claim 8, to: is used for preparing a medicament for preventing or treating lung cancer or a kit for reducing the expression of DUS4L gene in lung cancer cells.

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

the nucleic acid molecule of any one of claims 5-6; and/or, the DUS4L gene-interfering nucleic acid construct of claim 7; and/or, the DUS4L gene-interfering lentivirus of claim 8, and a pharmaceutically acceptable carrier, diluent or excipient.