CN110938691B

CN110938691B - Application of human DUS4L gene and related products

Info

Publication number: CN110938691B
Application number: CN201911221067.7A
Authority: CN
Inventors: 李斌; 李政; 李�杰
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: 2023-07-07
Anticipated expiration: 2039-12-03
Also published as: CN110938691A

Abstract

The invention belongs to the field of biomedical research, and particularly relates to application of a human DUS4L gene serving as a target in preparation of a lung cancer therapeutic drug. The invention is widely and deeply researched, and discovers that the RNAi method is adopted to down regulate the expression of human DUS4L gene, can effectively inhibit the proliferation of lung cancer cells, promote apoptosis and can effectively control the growth process of lung cancer. The siRNA or the nucleic acid construct containing the siRNA sequence and the slow virus provided by the invention can specifically inhibit the proliferation capability of lung cancer cells, inhibit lung cancer cell cloning, influence lung cancer cell cycle, promote lung cancer cell apoptosis and inhibit lung cancer cell growth, thereby treating lung cancer and opening up a new direction for lung cancer treatment.

Description

Application of human DUS4L gene and related products

Technical Field

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

Background

DUS4L is a protein-encoding gene. Gene Ontology (GO) annotations associated with this gene include oxidoreductase activity and FMN binding. Fusion genes of DUS4L-BCAP29 were found in various tissue samples (Recurrent fusion RNA DUS L-BCAP29 in non-cancer human tissues and cells, tang Y, qin F, liu A, li H.Oncostarget.2017May 9;8 (19): 31415-31423.Doi: 10.18632/oncotarget.16329.). And it has been reported that the DUS4L-BCAP29 fusion gene is remarkably highly expressed when inducing neural differentiation of human umbilical cord mesenchymal stem cells (Identification of chimeric RNAs in human infant brains and their implications in neural differentiation. Tang Y, et al, int J Biochem Cell biol.2019jun;111:19-26.Doi:10.1016/J. Biocel.2019.03.012.Epub 2019Apr 5).

Lung cancer is the malignant tumor with the highest incidence in recent years, and the incidence rate of lung cancer is high at the beginning of various tumors. Although the treatment of lung cancer varies day by day, its 5-year survival rate is only 14.1% and 60% of patients die within 1 year after diagnosis. Chemotherapy is the primary treatment for lung cancer, with more than 90% of lung cancers requiring chemotherapy. Chemotherapy can kill tumor cells and also has damage to normal cells of human body. Chemotherapy can inhibit the decrease of bone marrow hematopoietic system, mainly leucocyte and platelet, and can be used for treating granulocyte colony stimulating factor and platelet stimulating factor.

There is currently no report on the use of the DUS4L gene for lung cancer treatment.

Disclosure of Invention

In order to overcome the problems of the prior art, the present invention aims to provide the use of the human DUS4L gene and related products.

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

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

The human DUS4L gene serving as a target is specifically used for preparing lung cancer therapeutic drugs: the DUS4L gene is taken as an acting object, and medicines or preparations are screened to find medicines capable of inhibiting the expression of the human DUS4L gene as medicines for treating lung cancer. The DUS4L gene small interfering RNA (siRNA) is obtained by taking the human DUS4L gene as an acting object to be screened, and can be used as a medicament with the effect of inhibiting lung cancer cell proliferation. In addition, DUS4L gene can be the subject of action, such as antibody drugs, small molecule drugs, and the like.

The lung cancer therapeutic drug is a molecule capable of specifically inhibiting the transcription or translation of the DUS4L gene or specifically inhibiting the expression or activity of the DUS4L protein, thereby reducing the expression level of the DUS4L gene in lung cancer cells and achieving the purpose of inhibiting the proliferation, growth, differentiation and/or survival of the lung cancer cells.

The lung cancer therapeutic drug prepared by DUS4L gene includes but is not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemicals, antibody drugs, polypeptides, proteins or interfering lentiviruses.

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

The lung cancer therapeutic agent is administered in an amount sufficient to reduce transcription or translation of the human DUS4L gene, or to reduce expression or activity of the 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 drug mainly achieves the aim of treatment by reducing the expression level of human DUS4L gene and inhibiting the proliferation of lung cancer cells. Specifically, at the time of treatment, a substance effective to reduce the expression level of human DUS4L gene is administered to the patient.

In one embodiment, the DUS4L gene has a target sequence as set forth in SEQ ID NO: 1. The method comprises the following steps: 5'-ACATCAGCAATCATAGATT-3'.

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

treating lung cancer;

inhibit the proliferation of lung cancer cells;

inhibiting lung cancer cell cloning;

affecting lung cancer cell cycle;

promoting apoptosis of lung cancer cells;

inhibit lung cancer growth.

The product necessarily includes a DUS4L inhibitor and has the DUS4L inhibitor as an active ingredient for the aforementioned efficacy.

In the product, the active ingredient which plays the role can be only DUS4L inhibitor, and other molecules which can play the role can also be contained.

That is, the DUS4L inhibitor is the only active ingredient or one of the active ingredients of the product.

The product can be a single component substance or a multi-component substance.

The form of the product is not particularly limited, and may be solid, liquid, gel, semifluid, aerosol, or the like.

The subject to which the product is primarily directed is a mammal. The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, etc. 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 may be a nucleic acid molecule, an antibody, a small molecule compound.

As exemplified by the examples of the present invention, the DUS4L inhibitor may be a nucleic acid molecule that reduces the expression of the DUS4L gene in lung cancer cells. Specifically, it may be a double-stranded RNA or an shRNA.

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

The subject may be a mammal or a lung cancer cell of a mammal. The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, etc. 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 desiring treatment for lung cancer. Or the subject is an isolated lung cancer cell of a lung cancer patient or an individual desiring treatment for lung cancer.

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

In a fourth aspect, the invention discloses a nucleic acid molecule that reduces expression of a DUS4L gene in a lung cancer cell, the nucleic acid molecule comprising 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 that are complementary together 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 fragment in the DUS4L gene corresponding to the mRNA fragment 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. The method comprises the following steps: 5'-ACATCAGCAATCATAGATT-3'. Further, the sequence of the first strand of the double-stranded RNA is shown in SEQ ID NO: 2. Specifically 5'-ACAUCAGCAAUCAUAGAUU-3'.

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

SEQ ID NO:2 is one strand of small interfering RNA designed by taking the sequence shown in SEQ ID NO. 1 as an RNA interference target sequence and aiming at human DUS4L genes, 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 genes in lung cancer cells.

The shRNA includes a sense strand segment and an antisense strand segment, and a stem-loop structure linking the sense strand segment and the antisense strand segment, the sequences of the sense strand segment and the antisense strand segment being complementary, and the sequence of the sense strand segment being substantially identical to a target sequence in a DUS4L gene.

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

The shRNA can become small interfering RNA (siRNA) after enzyme digestion processing, thereby playing a role in specifically silencing the expression of endogenous DUS4L genes in lung cancer cells.

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

Further, the sequence of the shRNA is shown as SEQ ID NO: 3. Specifically 5'-GCACAUCAGCAAUCAUAGAUU CUCGAG AAUCUAUGAUUGCUGAUGUGC-3'.

Further, the DUS4L gene is of human origin.

In a fifth aspect, the invention discloses a DUS4L gene interfering nucleic acid construct comprising a gene fragment encoding an shRNA in the aforementioned nucleic acid molecule, capable of expressing the shRNA.

The DUS4L gene interfering nucleic acid construct can be obtained by cloning a gene fragment encoding 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 slow virus vector disclosed by the invention is obtained by cloning a DNA fragment for encoding the DUS4L gene shRNA into a known vector, wherein most of the known vectors are slow virus vectors, the DUS4L gene interference slow virus vector is packaged into infectious virus particles by viruses, then lung cancer cells are infected, the shRNA is transcribed, and finally the siRNA is obtained through the steps of enzyme cutting and the like and is used for specifically silencing the expression of the DUS4L gene.

Further, the DUS4L gene interfering lentiviral vector also contains a promoter sequence and/or a nucleotide sequence encoding a marker detectable in lung cancer cells; preferably, the detectable label is a 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-tGFP, pLKO.1-puro-CMV-TagCFP, pLKO.1-puro-CMV-TagYFP, pLKO.1-puro-CMV-TagRFP, pLKO.1-puro-CMV-TagFP635, pLKO.1-puro-UbC-TurboGFP, pLKO.1-puro-UbC-TagFP635 any one of pLKO-puro-IPTG-1xLacO, pLKO-puro-IPTG-3xLacO, pLP1, pLP2, pLP/VSV-G, pENTR/U6, pLenti6/BLOCK-iT-DEST, pLenti 6-GW/U6-lamrishma, pcDNA1.2/V5-GW/lacZ, pLenti6.2/N-Lumio/V5-DEST, pGCSIL-GFP or pLenti6.2/N-Lumio/V5-GW/lacZ.

The embodiment of the invention specifically enumerates a human DUS4L gene interference slow virus vector constructed by taking pGCSIL-GFP as a vector, and is named pGCSIL-GFP-DUS4L-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 preparation for treating lung cancer. DUS4L gene interfering lentiviral vectors can then be used to prepare 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 should 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 a sixth aspect of the invention, a DUS4L gene interfering lentivirus is disclosed, wherein the DUS4L gene interfering nucleic acid construct is packaged by virus with the aid of a lentivirus packaging plasmid and a cell line. The lentivirus can infect lung cancer cells and produce small interfering RNA directed against the DUS4L gene, thereby inhibiting proliferation of lung cancer cells. The DUS4L gene interference lentivirus can be used for preparing medicines for preventing or treating lung cancer.

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

The use of the medicament for preventing or treating lung cancer provides a method for treating lung cancer, in particular for preventing or treating lung cancer in a subject, comprising 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 is required to be administered to the subject. With this method, the growth, proliferation, recurrence and/or metastasis of the lung 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 lung cancer is inhibited.

The object of the method may be a person.

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

the nucleic acid molecules as described above; and/or, the aforementioned DUS4L gene interfering nucleic acid construct; and/or, the aforementioned DUS4L gene interferes with the lentivirus, as well as pharmaceutically acceptable carriers, diluents or excipients.

The composition may be a pharmaceutical composition.

When the composition is used to prevent or treat lung cancer in a subject, an effective amount of the composition is required to be administered to the subject. With this method, the growth, proliferation, recurrence and/or metastasis of the lung 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 lung cancer is inhibited.

The form of the composition is not particularly limited, and may be solid, liquid, gel, semifluid, aerosol, or the like.

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

In summary, the invention designs an RNAi target sequence aiming at the human DUS4L gene, and constructs a corresponding DUS4L RNAi vector, wherein the RNAi vector pGCSIL-GFP-DUS4L-siRNA can obviously reduce the expression of the DUS4L gene at the mRNA level and the protein level. The RNAi sequence aiming at the DUS4L gene can be efficiently introduced into the lung cancer A549 cells in a targeted manner by using lentiviruses (abbreviated as Lv) as a gene manipulation tool to carry an RNAi vector pGCSIL-GFP-DUS4L-siRNA, so that the expression level of the DUS4L gene is reduced, and the proliferation capacity of the tumor cells is obviously inhibited. Lentivirus-mediated DUS4L gene silencing is thus a potential clinical non-surgical treatment modality for malignant tumors.

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

the invention is widely and deeply researched, and discovers that the RNAi method is adopted to down regulate the expression of human DUS4L gene, can effectively inhibit the proliferation of lung cancer cells, promote apoptosis and can effectively control the growth process of lung cancer. The siRNA or the nucleic acid construct containing the siRNA sequence and the slow virus provided by the invention can specifically inhibit the proliferation capability of lung cancer cells, inhibit lung cancer cell cloning, influence lung cancer cell cycle, promote lung cancer cell apoptosis and inhibit lung cancer cell growth, thereby treating lung cancer and opening up a new direction for lung cancer treatment.

Drawings

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

Fig. 2: western Blot detects that A549 cell targets reduce the expression condition of DUS4L gene protein level.

Fig. 3: celigo cytometry validated the effect of the DUS4L gene on A549 cell proliferation. (the upper graph shows Celigo continuous 5 days recorded cell pictures, and the lower graph shows the time-dependent curve of the cell numbers of the shDUS4L group and the shCtrl control group)

Fig. 4: cell clone formation method detects the influence of DUS4L gene on A549 cell proliferation capacity, shRNA lentivirus infects A549 cells, after culturing for 14 days, the clone number is observed, the left side is a digital camera record chart, and the right side column-shaped result is shown by mean value of cell clone number +/-standard deviation.

Fig. 5-1: after the PI-FACS method detects and reduces the expression of genes, the periodic distribution change of tumor cells is regulated.

Fig. 5-2: after PI-FACS method detects the reduced gene expression, the histogram of the periodic distribution change of the tumor cells is regulated.

Fig. 6-1: flow apoptosis schematic diagram of Annexin V flow apoptosis detection sh DUS4L effect on A549 cell apoptosis,

fig. 6-2: columnar results of Annexin V flow apoptosis assay sh DUS4L effect on a549 apoptosis are shown as percent mean ± standard deviation.

Fig. 7: EDU cell proliferation assay results are shown as percent mean ± standard deviation of cells.

In the drawings of which there are shown,

the bar graph represents the average of three experiments and the error bars represent Standard Deviation (SD).

* shCtrl has P <0.01 compared to the target gene shRNA lentivirus treatment group.

* Compared with the target gene shRNA lentivirus treatment group, the shCtrl has the P of more than or equal to 0.01 and less than or equal to 0.05.

Detailed Description

Through extensive and intensive studies, the inventors of the present invention have found that, in lung cancer tumor tissues, the DUS4L gene is significantly highly expressed; the inventor finds that the expression of the human DUS4L gene can be effectively inhibited after the RNAi method is adopted to down regulate the expression of the human DUS4L gene, the proliferation of tumor cells can be effectively inhibited, the apoptosis can be promoted, the invasion and transfer capacity of the tumor cells can be reduced, the growth process of the tumor can be effectively controlled, and the research result shows that the DUS4L gene is a protooncogene and can be used as a target point of tumor treatment. The inventor further synthesizes and tests a plurality of siRNAs aiming at DUS4L genes, screens out siRNAs which can effectively inhibit the expression of DUS4L and further inhibit the proliferation and growth of human lung cancer A549 cells, and completes the invention on the basis.

The invention confirms the role of DUS4L gene in lung cancer occurrence from the aspect of cell functional science. The expression condition of mRNA and protein level target genes in two groups of lung cancer cell lines is detected by constructing target gene shRNA lentivirus and then transfecting lung cancer cells and comparing the target gene shRNA lentivirus with a transfection control lentivirus; and then, cell proliferation, apoptosis and other detection are carried out through a cell functional experiment, and the result shows that the shRNA group is compared with the control group, the lung cancer cell proliferation inhibition degree of the shRNA group is obviously higher than that of the control group, and the apoptosis rate increase degree is higher than that of the control group.

DUS4L inhibitors

Refers to molecules that have an inhibitory effect on DUS 4L. Having inhibitory effects on DUS4L includes, but is not limited to: inhibiting expression or activity of DUS 4L.

Inhibiting DUS4L activity refers to decreasing DUS4L activity. Preferably, the 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 that prior to inhibition.

Inhibition of DUS4L expression may specifically be inhibition of transcription or translation of the DUS4L gene, and may specifically be referred to as: the DUS4L gene is either not transcribed, or the transcriptional activity of the DUS4L gene is reduced, or the DUS4L gene is not translated, or the level of translation of the DUS4L gene is reduced.

Conventional methods for modulating gene expression of DUS4L, such as gene knockout, homologous recombination, interfering RNA, etc., can be used by those skilled in the art.

The inhibition of gene expression of DUS4L can be verified by PCR and Western Blot detection.

Preferably, the DUS4L 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%, yet more preferably by at least 90%, and most preferably the DUS4L gene is not expressed at all, as compared to the wild type.

Small molecule compounds

The present invention refers to a compound having a molecular mass of 1000 or less, which is composed of several or several tens of atoms.

Preparation of medicine for preventing or treating lung cancer

Nucleic acid molecules that reduce expression of the DUS4L gene in lung cancer cells can be utilized; and/or, the DUS4L gene interferes with the nucleic acid construct; and/or DUS4L gene interfering slow virus, as effective component, for preparing medicine for preventing or treating lung cancer. Typically, the medicament will include, in addition to the active ingredient, one or more pharmaceutically acceptable carriers or excipients, as required by the different dosage forms.

By "pharmaceutically acceptable" is meant that the molecular entity and composition 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. it can be blended therewith without substantially reducing the efficacy of the drug in the usual manner. Specific examples of some substances which may be pharmaceutically acceptable carriers or excipients are sugars, such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methyl cellulose, ethyl cellulose and methyl cellulose; tragacanth powder; 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; polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifying agents, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting and stabilizing agent; an antioxidant; a preservative; non-thermal raw water; isotonic saline solution; and phosphate buffer, etc. These substances are used as needed to aid stability of the formulation or to aid in enhancing the activity or its bioavailability or to produce an acceptable mouthfeel or odor in the case of oral administration.

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

Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention. The test methods in the following examples, in which specific conditions are not noted, are generally conducted under conventional conditions or under conditions recommended by the respective manufacturers.

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. 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, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.

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

EXAMPLE 1 preparation of RNAi lentiviruses against human DUS4L Gene

1. Screening for effective siRNA targets against human DUS4L genes

Retrieving DUS4L (nm_ 181581) gene information from Genbank; efficient siRNA targets against DUS4L gene were designed. Table 1-1 lists the effective siRNA target sequences screened 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

Double-stranded DNA Oligo sequences (tables 1-2) containing the Age I and EcoR I cleavage site sticky ends at both ends are synthesized aiming at siRNA targets (taking SEQ ID NO:1 as an example); the restriction enzymes Age I and EcoR I were used to linearize pGCSIL-GFP vector (available from Shanghai Ji Kai Gene chemical technologies Co., ltd.) and the cut fragments were identified by agarose gel electrophoresis.

TABLE 1-2 double-stranded DNA Oligo containing Age I and EcoR I cleavage sites at both ends

The vector DNA, which was digested with double enzymes and digested with T4 DNA ligase (cleavage system shown in tables 1 to 4, 37 ℃ C., 1h of reaction) was ligated to the purified double-stranded DNA Oligo, and the ligation was performed overnight at 16 ℃ in a suitable buffer system (ligation system shown in tables 1 to 5) to recover the ligation product. The ligation products were transformed into fresh E.coli competent cells prepared from calcium chloride (transformation protocol: see second edition of molecular cloning protocol pages 55-56). Dipping a surface of a clone growing with a transformation product, dissolving in 10 μl of LB culture medium, uniformly mixing, and taking 1 μl as a template; upstream and downstream of the RNAi sequence in the lentiviral vector, universal PCR primers were designed, upstream primer sequences: 5'-CCTATTTCCCATGATTCCTTCATA-3' (SEQ ID NO: 6); downstream primer sequence: 5'-GTAATACGGTTATCCACGCG-3' (SEQ ID NO: 7) and performing PCR identification experiments (the PCR reaction systems are shown in tables 1-6, and the reaction conditions are shown in tables 1-7). Sequencing and comparing the clones positive to the PCR identification, and comparing the correct clones to obtain the sequence of the sequence shown in SEQ ID NO:1, named pGCSIL-GFP-DUS4L-siRNA.

pGCSIL-GFP-Scr-siRNA negative control plasmid was constructed, the negative control siRNA target sequence was 5'-TTCTCCGAACGTGTCACGT-3' (SEQ ID NO: 8). When constructing pGCSIL-GFP-Scr-siRNA negative control plasmid, double-stranded DNAOligo sequences (tables 1-3) with Age I and EcoR I enzyme cutting sites at two ends and sticky ends are synthesized aiming at Scr siRNA targets, and other construction methods, identification methods and conditions are the same as those of pGCSIL-GFP-DUS4L-siRNA.

Tables 1-3 double-stranded DNA Oligo containing the sticky ends of the Age I and EcoRI cleavage sites at both ends

Table 1-4pGCSIL-GFP plasmid cleavage reaction System

Reagent(s)	Volume (mul)
		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 H ₂ O	40.5
		Total	50.0

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

Reagent(s)	Positive control (μl)	Self-connecting control (mul)	Connection 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×T4 phage DNA ligase buffer	1.0	1.0	1.0
T4 phage DNA ligase	1.0	1.0	1.0
				dd H ₂ O	16.0	17.0	16.0
Total	20.0	20.0	20.0

TABLE 1-6-1PCR reaction System

TABLE 1-7 Programming of PCR reaction System

3. Packaging DUS4L-siRNA lentiviruses

DNA of RNAi plasmid pGCSIL-GFP-DUS4L-siRNA was extracted with a plasmid extraction kit from Qiagen, and 100 ng/. Mu.l of the stock solution was prepared.

24h before transfection, human embryonic kidney 293T cells in logarithmic growth phase were digested with trypsin and cell density was adjusted to 1.5X10% in DMEM complete medium containing 10% fetal bovine serum ⁵ Cells/ml, seeded in 6-well plates, 37 ℃,5% CO ₂ Culturing in an incubator. And the cell density reaches 70-80% and can be used for transfection. 2h before transfection, the original medium was aspirated and 1.5ml of fresh complete medium was added. 20. Mu.l of Packing Mix (PVM), 12. Mu.l of PEI, 400. Mu.l of serum-free DMEM medium, 20. Mu.l of the extracted plasmid DNA were added to the PVM/PEI/DMEM mixture as described in Sigma-aldrich company MISSION Lentiviral Packaging Mix kit.

Incubating the above transfection mixture at room temperature for 15min, transferring into culture medium of human embryo kidney 293T cells, 37 ℃ and 5% CO ₂ Culturing in an incubator for 16h. The medium containing the transfection mixture was discarded, washed with PBS solution, and 2ml of complete medium was added to continue the culture for 48 hours. Cell supernatants were collected, and lentiviruses purified and concentrated by a Centricon Plus-20 centrifugal ultrafiltration device (Millipore) as follows: (1) centrifuging at 4 ℃ for 10min at 4000g to remove cell debris; (2) The supernatant was filtered through 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 centrifugation, 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 1000g; (5) The centrifuge cup is removed from the sample collection cup and the virus concentrate is present in the sample collection cup. Packaging the virus concentrate, and storing at-80deg.C. The sequence of the first strand of siRNA contained in the virus concentrate is shown as SEQ ID NO. 2. The packaging process of the control lentivirus is the same as that of DUS4L-siRNA lentivirus, and pGCSIL-GFP-Scr-siRNA vector is used for replacing pGCSIL-GFP-DUS4L-siRNA vector.

Example 2real-time fluorescent quantitative RT-PCR method for detecting silencing efficiency of Gene

Human lung cancer A549 cells in logarithmic growth phase were subjected to pancreatin digestion to prepare cell suspension (cell number about 5X 10) ⁴ Per ml) was inoculated into 6-well plates and cultured until the cell fusion reached about 30%. According to the complex value of infection (MOI, A549: 10), a suitable amount of lentivirus prepared in example 1 was added, the medium was changed after culturing for 24 hours, and after the infection time reached 5 days, the cells were collected. Total RNA was extracted according to Trizol protocol from Invitrogen. RNA was reverse transcribed to obtain cDNA according to the M-MLV protocol from Promega (reverse transcription reaction system see Table 2-1, 42℃for 1h, followed by inactivation of reverse transcriptase in a water bath at 70℃for 10 min).

Real-time quantitative detection was performed using a Real time PCR instrument model TP800 (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 taken 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(s)	Volume (mul)
		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(s)	Volume (mul)
		SYBR premix ex taq:	6.0
Primer MIX (5 μm):	0.3
		cDNA	0.6
ddH ₂ O	5.1
		Total	12.0

the procedure was set as two-step Real-time PCR: pre-denaturation at 95 ℃ for 30s; then each step is denatured at 95 ℃ for 5s; annealing and extending at 60 ℃ for 30s; a total of 40 cycles were performed. The absorbance was read each time during the extension phase. After the PCR was completed, the DNA was denatured at 95℃for 15 seconds, and then cooled to 60℃to allow the DNA double strand to bind sufficiently. Starting from 60 ℃ to 95 ℃, increasing the temperature by 0.5 ℃ in each step, keeping for 4s, and simultaneously reading the light absorption value to prepare a melting curve. By 2- ^ΔΔCt Analysis calculated the abundance of expression of the DUS4L mRNA infected. Cells infected with control virus served as controls. The experimental results are shown in FIG. 1, which demonstrate that the expression level of DUS4L mRNA in human lung cancer A549 cells was down-regulated by 64.5%.

EXAMPLE 3Western Blotting method for detecting silencing efficiency of Gene

1. Extraction of total cell proteins

(1) The control virus and RNAi virus against the DUS4L interference target were separately infected with the cells of interest (A549 cells). Cell samples were received and washed twice with PBS. An appropriate amount of RIPA lysate was taken and PMSF was added over the last few minutes before use to give a final concentration of PMSF of 1mM. (RIPA lysate used, instruction chain:http://www.beyotime.com/ripa- lysis-bufferm.htm)

(2) Adding proper amount of RIPA lysate, and performing ice lysis for 10-15min. Cells were scraped off and transferred into new EP tubes, and then sonicated (20 times 40W total, 1s each, 2s intervals).

( 3) Centrifugation was performed at 12000g at 4℃for 15min, and the protein concentration was measured by the supernatant BCA method (BCA Protein Assay Kit instructions are linked: http:// www.beyotime.com/p0010s.htm )

(4) The addition of fresh lysate adjusts the protein concentration for each sample to be consistent, typically 2. Mu.g/. Mu.L. Then adding 1/5 volume of 6X locking buffer, mixing, decocting in 100 deg.C metal bath for 10min, centrifuging briefly, and preserving at-80deg.C.

2.SDS-PAGE

(1) And (3) glue preparation: the glues with different concentrations are prepared according to the molecular weight of the target protein, and the specific systems are shown in tables 3-1, 3-2 and 3-3:

TABLE 3-1SDS-PAGE separating gel (8 mL system)

Separation gel (8 mL system)	8％	9％	10％	12％	13％	15％
							H ₂ O	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 separating gel (10 mL system)

TABLE 3SDS-PAGE gel

Concentrated glue (5%)	3mL	4mL	5mL
				H ₂ O	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 glue 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 80mA for 20min; the gel was separated 120mA for 1h.

3. Immunoblot (Wet turn)

After electrophoresis, the protein was transferred to PVDF membrane by using a transfer electrophoresis apparatus and electroblotting at 4℃for 150min under 300mA constant current.

4. Antibody hybridization:

(1) Closing: the PVDF membrane was blocked with blocking solution (TBST solution containing 5% skimmed milk) at room temperature for 1h or overnight at 4 ℃.

(2) Incubation resistance: the blocking solution diluted antibodies, then with blocked PVDF membrane room temperature incubation 2 hours or 4 degrees overnight, and with TBST membrane washing 4 times, each time 8 minutes.

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

X-ray development: (using CST Co. 20X

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

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

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

(2) Taking out the film, wiping the water absorbing paper, spreading the film into a magazine, dripping a proper amount of well mixed ECL luminous solution, spreading a preservative film (avoiding generating bubbles), putting an X-ray film (avoiding the movement of the X-ray film), closing the magazine, exposing for 1 s-several minutes (the exposure time needs to be more than several times, and properly adjusting the exposure time according to whether the naked eyes can see fluorescence and the intensity of the fluorescence).

(3) Taking out the X-ray film, putting into a developing solution, taking out after the strip appears, rinsing in clear water for a few seconds, and putting into a fixing solution for at least 2min.

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

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

Example 4 detection of proliferation potency of DUS4L-siRNA lentivirus-infected tumor cells (Celigo experiment)

Human lung cancer A549 cells in logarithmic growth phase were subjected to pancreatin digestion to prepare cell suspension (cell number about 5X 10) ⁴ Per ml) was inoculated into 6-well plates and cultured until the cell fusion reached about 30%. According to the complex infection (MOI, A549: 10), adding appropriate amount of virus, culturing for 24 hr, changing culture medium, collecting each virus in logarithmic phase after infection time reaches 5 daysExperimental group cells. Complete medium resuspension of the adult cell suspension (2X 10) ⁴ Per ml), 96-well plates were seeded at a cell density of about 1500 cells per well. Each group had 5 duplicate wells, 100 μl per well. After being paved, the mixture is placed at 37 ℃ and 5 percent of CO ₂ Culturing in an incubator. The plates were read once daily with a Celigo instrument (Nexcelom) starting the next day after plating and were continuously tested for 5 days. Accurately calculating the number of cells with green fluorescence in each scanning hole plate by adjusting the input parameters of analysis settings; statistical plots were made on the data to plot 5 day cell proliferation curves.

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

Example 5 detection of the clonogenic Capacity of tumor cells that infect DUS4L-siRNA lentiviruses (clonogenic experiments)

Human lung cancer A549 cells are inoculated into 12-hole plates after being digested by pancreatin, and the cell density is 10-15%. The next day was changed to fresh medium containing 5ug/ml polybrene. DUS4L-siRNA lentivirus was tested according to the complex MOI of infection, A549:10 are added into the culture plate, and fresh culture medium is replaced after 12 to 24 hours of infection. After 72h of infection, fluorescence is observed under a fluorescence microscope, and the infection efficiency reaches 80%.

After pancreatin digestion of the cells after infection with virus in the logarithmic growth phase, the complete medium is resuspended into a cell suspension; inoculating the cells into a 6-hole plate (800 cells/hole) after counting, continuously culturing the inoculated cells in an incubator for 12 days, and replacing liquid at 3day intervals and observing the cell state; photographing the cell clone under a fluorescence microscope before the experiment is terminated; cells were fixed with paraformaldehyde at the end of the experiment, and after washing the cells with PBS, giemsa was stained and photographed.

As shown in fig. 4, the number of clonotypes formed by lung cancer a549 cells was significantly reduced and the volume of clonotypes significantly reduced after expression of the RNA interference-reduced gene (KD set) as compared to control interference (NC set); indicating that gene silencing results in a decrease in the ability of tumor cells to form clones. After the expression of the reduced gene was detected in the plate clone formation assay, the clonality of tumor cells was decreased.

Example 6 tumor cell cycle detection (FACS cell cycle detection) of DUS4L-siRNA lentivirus infected

Human lung cancer A549 cells in logarithmic growth phase were subjected to pancreatin digestion to prepare cell suspension (cell number about 5X 10) ⁴ Per ml) was inoculated into 6-well plates and cultured until the cell fusion reached about 30%. Based on the complex MOI of infestation, a549:10, adding a proper amount of virus, culturing for 24 hours, replacing a culture medium, and collecting each experimental group cell in a logarithmic growth phase after the infection time reaches 5 days. If the cells are adherent cells, when the coverage rate of the cells of each experimental group of 6cm dish cells is about 80% (the cells do not enter the growth platform stage), the cells are digested by pancreatin, the complete culture medium is resuspended into a cell suspension, the cells are collected in a 5mL centrifuge tube, and three compound holes are arranged in each group (in order to ensure that the number of the cells on the machine is enough, and the number of the cells is more than or equal to 106/treatment). In the case of suspension cells, they are collected directly. 1300rmp was centrifuged for 5min, the supernatant was discarded, and the cell pellet was washed 1 time with 4℃pre-chilled D-Hanks (pH=7.2 to 7.4). The cells were fixed with 1300rmp, 5min centrifugation, 75% ethanol pre-chilled at 4℃for at least 1h.1300rmp centrifugation for 5min to remove fixative, D-Hanks washed the cell pellet once, step 2. Preparing a cell staining solution: 40 XPI mother liquor (2 mg/mL): 100X RNase mother liquor (10 mg/mL): 1 xd-hanks=25: 10: staining 1000 cells: according to the Cell quantity, adding a certain volume of Cell staining solution (0.6-1 mL) to re-suspend so that the Cell passing rate is 300-800 Cell/s when the machine is on. And (5) detecting on the machine. And (5) data analysis.

As shown in FIGS. 5-1 and 5-2, the PI-FACS method was used to control the change in the periodic distribution of tumor cells after detecting the decrease in gene expression. After the RNA interference reduced gene expression (KD set) compared to control interference (NC set), G1 phase cells decreased (P < 0.05), cells in S phase were not significantly altered, cells in G2/M phase increased (P < 0.05), suggesting that DUS4L gene was associated with the periodic distribution of a549 cells.

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

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

After digestion of the cells in the logarithmic growth phase with pancreatin, the complete medium is resuspended into a cell suspension; collecting the supernatant cells in the same 5mL centrifuge tube, and arranging three compound holes (in order to ensure that the number of the cells on the machine is enough, the number of the cells is more than or equal to 5 multiplied by 10) ⁵ Treatment). 1300rmp was centrifuged for 5min, the supernatant was discarded and the cell pellet was washed with PBS pre-chilled at 4 ℃. The cell pellet was washed once with 1 Xbinding buffer, centrifuged at 1300rmp for 3min and the cells were collected. 200 μL of 1 Xbinding buffer resuspended cell pellet. Add 10. Mu.L Annexin V-APC staining, keep out of light for 10-15min at room temperature. According to the cell amount, 400-800 mu L of 1×binding buffer is added, and the detection is performed by an up-flow cytometer. The results were analyzed.

The results are shown in FIG. 6-1 and FIG. 6-2, and the Annexin V single-stain method shows the change of apoptosis ratio of human lung cancer A549 cells after the expression of the reduced gene was detected. The apoptosis proportion of tumor cells was found to increase after down-regulating gene expression. Compared with control interference (NC group), after RNA interference reduces the expression of genes (KD group), the number of apoptotic tumor cells is obviously increased; indicating that gene silencing leads to apoptosis of tumor cells.

Example 8 detection of proliferation of tumor cells EDU cell infecting DUS4L-siRNA lentiviruses

Human lung cancer A549 cells infected with DUS4L-siRNA and a control lentivirus 10 thereof are inoculated into a 96-well plate by 3000 cells per well, and cultured to a normal growth stage, and the method is generally optimal for three days; cell culture medium was used at 1000:1 (reagent A) and preparing a proper amount of 50 mu M EdU culture medium; mu.L of 50. Mu.M EdU medium was added to each well and incubated for 2 hours, and the medium was discarded; PBS washes cells for 1-2 times, each time for 5 minutes; the cleaning purpose is to elute EdU which is not infiltrated with DNA, the cleaning mode is determined according to different cell types, and cells with weak adhesion are required to reduce the cleaning strength; mu.L of cell fixative (i.e., containingPBS of 4% paraformaldehyde) for 30 min at room temperature, and removing the fixative; adding 50 mu L of 2mg/mL glycine into each hole, and after a decoloration shaking table is used for incubating for 5 minutes, discarding glycine solution; the aim is to neutralize paraformaldehyde and ensure a dyeing reaction system, and when other modes are adopted for cell fixation, the step can be omitted as appropriate; adding 100 mu L of PBS into each hole, washing for 5 minutes by a decolorizing shaker, and discarding the PBS; 100. Mu.L of 1X was added to each well

The staining reaction solution (table 3) was discarded after incubation for 30 minutes in a dark, room temperature and decoloration shaker; adding 100 mu L of penetrant (PBS of 0.5% TritonX-100), decolorizing and shaking table and cleaning for 2-3 times, 10 minutes each time, and discarding the penetrant; (boost) 100 μl of methanol was added to each well for 1-2 washes, 5 minutes each time; PBS was washed 1 time, 5 minutes each; deionized water is mixed according to 100:1, preparing a proper amount of 1X hoechst33342 reaction solution, and preserving in a dark place; 100 mu L of 1X Hoechst33342 reaction solution is added into each hole, and the mixture is incubated for 30 minutes at room temperature in the dark; discarding the dyeing reaction liquid; adding 100 mu L PBS for cleaning for 1-3 times; observing immediately after dyeing; if the condition is restricted, the light is protected from 4 ℃ and the storage is moist, but the storage should not be carried out for more than 3 days.

The results are shown in fig. 7, which shows a decrease in the percentage of EDU positive cells in the shDUS4L group compared to the shCtrl group, indicating a slow proliferation of a549 cells after DUS4L knockdown (p < 0.05).

While the invention has been described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and additions may be made without departing from the scope of the invention. Equivalent embodiments of the present invention will be apparent to those skilled in the art having the benefit of the teachings disclosed herein, when considered in the light of the foregoing disclosure, and without departing from the spirit and scope of the invention; meanwhile, any equivalent changes, modifications and evolution of the above embodiments according to the essential technology of the present invention still fall 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

Use of a dus4l inhibitor for the preparation of a product having at least one of the following effects:

treating lung cancer;

inhibit the proliferation of lung cancer cells;

inhibiting lung cancer cell cloning;

promoting apoptosis of lung cancer cells;

the method is characterized in that lung cancer is lung adenocarcinoma, the inhibitor is shRNA, and the target sequence of the shRNA is shown in SEQ ID NO: 1.
2. The use according to claim 1, wherein the sequence set forth in SEQ ID NO:1, synthesizing double-stranded DNA Oligo sequences with two ends containing Age I and EcoR I restriction enzyme cutting sites sticky ends, wherein the double-stranded DNA Oligo sequences are shown in SEQ ID NO:4 and SEQ ID NO: shown at 5.
3. A composition for preventing or treating lung adenocarcinoma, which contains, as an active substance:

the inhibitor for use according to claim 1 or 2, together with a pharmaceutically acceptable carrier, diluent or excipient.
4. Use of a DUS4L inhibitor for use as claimed in claim 1 or 2 in the manufacture of a medicament for the prophylaxis or treatment of lung adenocarcinoma.