CN111068056A

CN111068056A - Application of human DNAJC24 gene and related product

Info

Publication number: CN111068056A
Application number: CN201911412051.4A
Authority: CN
Inventors: 郭华; 陈鹏; 刘志勇; 林丽; 陈璐; 陈丽维; 罗艺; 刘东明; 张翠翠
Original assignee: Tianjin Medical University Cancer Institute and Hospital
Current assignee: Tianjin Medical University Cancer Institute and Hospital
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2020-04-28

Abstract

The invention belongs to the field of biological medicine research, and particularly relates to application of a human DNAJC24 gene as a target in preparation of a lung cancer treatment drug or a lung cancer diagnosis drug. The invention discovers that the proliferation of the lung cancer cell can be effectively inhibited and the apoptosis can be promoted after the expression of the human DNAJC24 gene is down-regulated by adopting an RNAi method, 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 rate of lung cancer cells, promote the apoptosis of the lung cancer cells, inhibit the cloning of the lung cancer cells, inhibit the invasion of the lung cancer cells, inhibit the metastasis of the lung cancer cells and inhibit the growth of the lung cancer cells, thereby treating the lung cancer and opening up a new direction for treating the lung cancer.

Description

Application of human DNAJC24 gene and related product

Technical Field

The invention belongs to the field of biomedical research, and particularly relates to application of a human DNAJC24 gene and a related product.

Background

Diphtheria amide is a unique post-translational histidine modification, found only in translation elongation factor-2 (EEF2), which is well conserved from archaea to humans, and is the target for the glycosylation and inactivation of EEF2 ADP-ribosylation by Diphtheria Toxin (DT) and exotoxin A, whereas DNAJC24 is one of the enzymes involved in diphtheria acylation in EEF 2. The disease associated with DNAJC24 is Aniridia 1, the pathways associated with which include gamma-carboxylation of proteins, hydroxyproline formation and arylsulfatase activation and metabolism. Many acute lymphoblastic leukemia patients have been found to be resistant to the Immunotoxin HA22, and it was found that the expression level of the DANJC24 gene promoter region is significantly reduced due to hypermethylation, and diphtheria acylation of EEF2 protein is not possible, so that the effect of HA22 is inhibited in an ALL cell line resistant to HA22 (Immunotoxin resistance via conversion analysis of the DPH4 promoter is a unique individual property.Wei H, ethyl. Proc Natl Acad Sci U S.2012May 1; 109(18): 6898-.

Lung cancer is one of the most rapidly growing malignancies and the most life-threatening to the health of the human population. Lung cancer includes Small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC accounts for 85% of cancer cases in lung cancer, and can be further classified into three categories including Adenocarcinoma (ADC), Squamous Cell Carcinoma (SCC), and large cell carcinoma by histopathological heterogeneity studies.

No related report of DNAJC24 gene for treating lung cancer exists at present.

Disclosure of Invention

In order to overcome the problems in the prior art, the invention aims to provide application of a human DNAJC24 gene and a related product.

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

in the first aspect of the invention, the application of the human DNAJC24 gene as a target in preparing a lung cancer treatment drug or a lung cancer diagnosis drug is provided.

The application of the human DNAJC24 gene as a target in preparing a lung cancer treatment drug specifically comprises the following steps: the DNAJC24 gene is used as an action object, and the drug or preparation is screened to find out the drug which can inhibit the expression of the human DNAJC24 gene and is used as a candidate drug for treating the lung cancer. The DNAJC24 gene small interfering RNA (siRNA) is obtained by screening human DNAJC24 gene as an action object and can be used as a medicine with the function of inhibiting the proliferation of lung cancer cells. In addition, DNAJC24 gene can be used as an object of action, such as antibody drug, small molecule drug, and the like.

The application of the human DNAJC24 gene as a target in preparing the lung cancer diagnosis medicine specifically comprises the following steps: the DNAJC24 gene expression product is used as a lung cancer diagnosis index and applied to the preparation of lung cancer diagnosis medicaments.

The lung cancer treatment drug is a molecule which can specifically inhibit the transcription or translation of a DNAJC24 gene or specifically inhibit the expression or activity of a DNAJC24 protein, so that the expression level of the DNAJC24 gene in a lung cancer cell is reduced, and the purposes of inhibiting the proliferation, growth, differentiation and/or survival of the lung cancer cell are achieved.

The lung cancer therapeutic drug or lung cancer diagnostic drug prepared from the DNAJC24 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 drug administered is an amount sufficient to reduce transcription or translation of the human DNAJC24 gene, or to reduce expression or activity of the human DNAJC24 protein. Such that the expression of the human DNAJC24 gene is reduced by at least 50%, 80%, 90%, 95%, or 99%.

The method for treating the lung cancer by adopting the lung cancer treatment medicine mainly achieves the aim of treating by reducing the expression level of the human DNAJC24 gene to inhibit the proliferation of lung cancer cells. Specifically, in the treatment, a substance effective in reducing the expression level of the human DNAJC24 gene is administered to a patient.

In one embodiment, the target sequence of the DNAJC24 gene is as set forth in SEQ ID NO:1 is shown. The method specifically comprises the following steps: 5'-AAGTACAGATGTACCAGCA-3' are provided.

In a second aspect of the invention, there is provided the use of a DNAJC24 inhibitor in the preparation of a product having at least one of the following effects:

treating lung cancer;

inhibiting the rate of proliferation of lung cancer cells;

promoting apoptosis of lung cancer cells;

inhibiting the cloning of lung cancer cells;

inhibiting lung cancer cell invasion;

inhibiting lung cancer cell metastasis;

inhibiting the growth of lung cancer.

The product necessarily comprises the DNAJC24 inhibitor, and the DNAJC24 inhibitor is used as an effective component of the effects.

In the product, the effective component for the above functions can be only a DNAJC24 inhibitor, and can also comprise other molecules for the above functions.

That is, the DNAJC24 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 DNAJC24 inhibitor can be a nucleic acid molecule, an antibody, a small molecule compound.

As exemplified in the examples herein, the DNAJC24 inhibitor may be a nucleic acid molecule that reduces expression of DNAJC24 gene in a lung cancer cell. Specifically, it may be a double-stranded RNA or shRNA.

In a third aspect of the invention, there is provided a method of treating lung cancer by administering to a subject a DNAJC24 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 DNAJC24 inhibitor may 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 DNAJC24 gene in a lung cancer cell, wherein the nucleic acid molecule comprises double-stranded RNA or shRNA.

Wherein the double-stranded RNA contains a nucleotide sequence capable of hybridizing with a DNAJC24 gene;

the shRNA contains a nucleotide sequence capable of hybridizing with a DNAJC24 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 DNAJC24 gene.

The target sequence in the DNAJC24 gene is a segment in the DNAJC24 gene corresponding to an mRNA segment which is recognized and silenced by the nucleic acid molecule when the nucleic acid molecule is used for specifically silencing the expression of the DNAJC24 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'-AAGTACAGATGTACCAGCA-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'-AAGUACAGAUGUACCAGCA-3'.

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

SEQ ID NO:2 is one strand of small interfering RNA designed by using the sequence shown in SEQ ID NO. 1 as an RNA interference target sequence and aiming at the human DNAJC24 gene, the sequence of the other strand, namely the second strand, is complementary to the sequence of the first strand, and the siRNA can play a role in specifically silencing the expression of endogenous DNAJC24 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 DNAJC24 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 endogenous DNAJC24 gene expression 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'-AAGUACAGAUGUACCAGCA CUCGAG UGCUGGUACAUCUGUACUU-3'.

Further, the DNAJC24 gene is derived from human.

In the fifth aspect of the invention, a DNAJC24 gene interfering nucleic acid construct is disclosed, which comprises a gene segment for coding shRNA in the nucleic acid molecule and can express the shRNA.

The DNAJC24 gene interfering nucleic acid construct can be obtained by cloning a gene segment for coding the human DNAJC24 gene shRNA into a known vector.

Further, the DNAJC24 gene interference nucleic acid construct is a DNAJC24 gene interference lentiviral vector.

The DNAJC24 gene interference lentiviral vector disclosed by the invention is obtained by cloning a DNA fragment for coding the DNAJC24 gene shRNA into a known vector, wherein the known vector is mostly a lentiviral vector, the DNAJC24 gene interference lentiviral vector is packaged into infectious viral particles by viruses, then lung cancer cells are infected, the shRNA disclosed by the invention is further transcribed, and the siRNA is finally obtained through the steps of enzyme digestion processing and the like and is used for specifically silencing the expression of the DNAJC24 gene.

Further, the DNAJC24 gene interference lentiviral vector also contains a promoter sequence and/or a nucleotide sequence encoding a marker which can be detected in a lung cancer cell; 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-1 xLacO, pLKO-puro-IPTG-3xLacO, Tet-puro, Tet-pLKO-TpLKO-dXLacPG, pDNAJVdLKO-5, pLKO-dLKO-5, pLKO-5-dXLacGW, pLKO-5/35, pLKO-LKO-5, pLKO-LepLKO-5, pLKO-3-5, pLKO-5, pGCSIL-GFP or pLenti 6.2/N-Lumio/V5-GW/lacZ.

The embodiment of the invention specifically lists a human DNAJC24 gene interference lentiviral vector constructed by taking pGCSIL-GFP as a vector, and is named as pGCSIL-GFP-DNAJC 24-siRNA.

The DNAJC24 gene siRNA disclosed by the invention 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. DNAJC24 gene interference lentiviral vector can be used for preparing the DNAJC24 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.

The sixth aspect of the invention discloses a DNAJC24 gene interference lentivirus, which is prepared by carrying out virus packaging on the DNAJC24 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 DNAJC24 gene, thereby inhibiting the proliferation of the lung cancer cells. The DNAJC24 gene interference lentivirus can be used for preparing medicines for preventing or treating lung cancer.

In a seventh aspect of the invention, there is provided a use of the nucleic acid molecule, or the DNAJC24 gene interfering nucleic acid construct, or the DNAJC24 gene interfering lentivirus, wherein: is used for preparing a medicament for preventing or treating lung cancer or a kit for reducing the expression of DNAJC24 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 DNAJC24 gene interfering nucleic acid constructs; and/or, the aforementioned DNAJC24 gene interferes with 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 a human DNAJC24 gene and constructs a corresponding DNAJC24RNAi vector, wherein the RNAi vector pGCSIL-GFP-DNAJC24-siRNA can obviously down-regulate the expression of the DNAJC24 gene at the mRNA level and the protein level. Lentivirus (lentivirus, abbreviated as Lv) is used as a gene operation tool to carry an RNAi vector pGCSIL-GFP-DNAJC24-siRNA, so that RNAi sequences aiming at DNAJC24 genes can be efficiently introduced into lung cancer A549 and NCI-H1299 cells in a targeted manner, the expression level of the DNAJC24 genes is reduced, and the proliferation capacity of the tumor cells is obviously inhibited. Thus lentivirus-mediated DNAJC24 gene silencing is a potential clinical non-surgical treatment modality for malignancies.

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

the invention discovers that the proliferation of the lung cancer cell can be effectively inhibited and the apoptosis can be promoted after the expression of the human DNAJC24 gene is down-regulated by adopting an RNAi method, 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 rate of lung cancer cells, promote the apoptosis of the lung cancer cells, inhibit the cloning of the lung cancer cells, inhibit the invasion of the lung cancer cells, inhibit the metastasis of the lung cancer cells and inhibit the growth of the lung cancer cells, thereby treating the lung cancer and opening up a new direction for treating the lung cancer.

Drawings

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

b: western Blot detection of the protein level expression condition of the DNAJC24 gene reduced by the A549 cell target.

C: RT-PCR detects the target gene reduction efficiency of NCI-H1299 cell mRNA level.

D: western Blot detection shows that the NCI-H1299 cell target reduces the protein level expression of DNAJC24 gene.

FIG. 2 wherein A: the cell automatic analysis result reveals that the reduction of the DNAJC24 gene inhibits the proliferation capacity of the lung cancer cell. (cell line is A549 cell, cell number is counted 1, 2, 3, 4 and 5 days after virus infection)

B: the cell automatic analysis result reveals that the reduction of the DNAJC24 gene inhibits the proliferation capacity of the lung cancer cell. (cell line is NCI-H1299 cell, cell number was counted 1, 2, 3, 4 and 5 days after virus infection)

Fig. 3 wherein A, B: the MTT method reveals that the reduction of DNAJC24 gene inhibits the proliferation ability of lung cancer cells. (cell line is A549 cell)

C. D: the MTT method reveals that the reduction of DNAJC24 gene inhibits the proliferation ability of lung cancer cells. (cell line is NCI-H1299 cell)

FIG. 4 wherein A: the influence of DNAJC24 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 shRNA lentivirus is cultured for 8 days, the left side is a digital camera record chart, and the right side bar result is displayed by the average value +/-standard deviation of the cell clone number.

B: the influence of the DNAJC24 gene on the proliferation capacity of the NCI-H1299 cell is detected by a cell clone formation method, the NCI-H1299 cell is infected by shRNA lentivirus, the clone number is observed after the cell is cultured for 8 days, the left side is a digital camera record chart, and the right side bar result is shown by the average value of the cell clone number +/-standard deviation.

FIG. 5 wherein A: annexin V-APC flow apoptosis test the influence of sh DNAJC24 on A549 cell apoptosis, the left side is a schematic diagram of flow apoptosis, and the right side bar chart result is shown by the average value of cell percentage +/-standard deviation.

B: annexin V-APC flow apoptosis assay sh DNAJC24 effect on NCI-H1299 apoptosis, schematic flow apoptosis on the left, and histogram results on the right are shown as the mean value of cell percentage. + -. standard deviation.

FIG. 6 wherein A: invasion Invasion experiments show that the Invasion capacity of A549 cells is reduced after DNAJC24 gene expression is down-regulated.

B: invasion Invasion experiments show that the Invasion capacity of NCI-H1299 cells is reduced after the expression of DNAJC24 gene is down-regulated.

FIG. 7 wherein A: migration Migration experiment shows that the Migration capacity of A549 cells is reduced after the expression of DNAJC24 gene is down-regulated.

B: migration Migration experiment shows that the cell Migration capacity of NCI-H1299 is reduced after the expression of DNAJC24 gene is down-regulated.

FIG. 8 wherein A: scratch healing experiments show that the transfer capacity of A549 cells is reduced after the expression of DNAJC24 gene is down-regulated.

B: scratch healing experiments show that the transfer capacity of NCI-H1299 cells is reduced after the expression of DNAJC24 gene is down-regulated.

In the figure, the bar graph represents the mean of three experiments and the error bars represent the Standard Deviation (SD).

P <0.001 for shCtrl compared to target gene shRNA lentivirus treatment group.

And compared with the target gene shRNA lentivirus treatment group, the shCtrl has the P of more than or equal to 0.001 and less than 0.01.

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 invention proves the function of the DNAJC24 gene in the generation of lung cancer from the perspective 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.

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

DNAJC24 inhibitors

Refers to a molecule having an inhibitory effect on DNAJC 24. Having inhibitory effects on DNAJC24 include, but are not limited to: inhibiting the expression or activity of DNAJC 24.

Inhibiting DNAJC24 activity refers to decreasing DNAJC24 viability. Preferably, DNAJC24 viability is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, even more preferably by at least 70%, most preferably by at least 90% compared to prior to inhibition.

Specifically, inhibiting the expression of DNAJC24 may be inhibiting the transcription or translation of DNAJC24 gene, and specifically, may be: making the gene of DNAJC24 untranscribed, or reducing the transcriptional activity of the gene of DNAJC24, or making the gene of DNAJC24 untranslated, or reducing the translational level of the gene of DNAJC 24.

The regulation of gene expression of DNAJC24 can be performed by one skilled in the art using conventional methods, such as gene knock-out, homologous recombination, interfering RNA, and the like.

The inhibition of the gene expression of DNAJC24 can be verified by PCR and Western Blot detection of the expression level.

Preferably, DNAJC24 gene expression is reduced by at least 10%, preferably by at least 30%, even more preferably by at least 50%, even more preferably by at least 70%, even more preferably by at least 90%, most preferably DNAJC24 gene is not expressed at all, 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 DNAJC24 gene in lung cancer cells can be utilized; and/or, DNAJC24 gene interfering nucleic acid constructs; and/or DNAJC24 gene interferes lentivirus to be used as an effective component for preparing the medicine 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 human DNAJC24 Gene

1. Screening effective siRNA target point aiming at human DNAJC24 gene

Calling DNAJC24 (NM-181706) gene information from Genbank; designing effective siRNA target points aiming at DNAJC24 gene. Table 1-1 lists the effective siRNA target sequences screened against DNAJC24 gene.

TABLE 1-1 siRNA target sequences targeting the human DNAJC24 gene

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

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, and is named pGCSIL-GFP-DNAJC 24-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 DNA Oligo sequences (shown in 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-DNAJC 24-siRNA.

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

TABLE 1-4 pGCSIL-GFP plasmid digestion reaction System

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

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

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

TABLE 1-6-1PCR reaction System

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

TABLE 1-7 PCR reaction System Programming

3. Packaging DNAJC24-siRNA lentivirus

The DNA of RNAi plasmid pGCSIL-GFP-DNAJC24-siRNA was extracted with a plasmid extraction kit from Qiagen, 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. According to SAs an instruction of the MISSION Lentiviral Packaging Mix kit from igma-aldrich, 20. mu.l Packaging Mix (PVM), 12. mu.l PEI, and 400. mu.l serum-free DMEM medium were added to a sterilized centrifuge tube, and 20. mu.l of the above-mentioned extracted plasmid DNA was added to the above-mentioned PVM/PEI/DMEM mixture.

The transfection mixture was incubated at room temperature for 15min, transferred to medium of human embryonic kidney 293T cells at 37 ℃ with 5% CO₂Culturing for 16h in an incubator. The medium containing the transfection mixture was discarded, washed with PBS solution, 2ml of complete medium was added and incubation continued for 48 h. 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 packaging process of the control lentivirus is the same as that of the DNAJC24-siRNA lentivirus, and only pGCSIL-GFP-Scr-siRNA vector is used for replacing pGCSIL-GFP-DNAJC24-siRNA vector.

Example 2 examination of silencing efficiency of tumor cells infected with DNAJC24-siRNA lentivirus

Human lung cancer cells A549 and NCI-H1299 in logarithmic growth phase were trypsinized to prepare a cell suspension (about 5X 10 cells in number)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 and NCI-H1299: 10), a proper amount of the lentivirus prepared in example 1 is added, the culture medium is replaced after 24 hours of culture, and the cells are collected after the infection time reaches 5 days.

a) Gene silencing efficiency detection by real-time fluorescent quantitative RT-PCR method

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 DNAJC24 gene were as follows: an upstream primer 5'-CCAGCAGGAACAGTGGAGGA-3' (SEQ ID NO: 11) and a downstream primer 5'-CCACATCTGCAACTCAGATAAA-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
		DEPC H₂O	2.6
Total	10.0

TABLE 2-2 Real-time PCR reaction System

Reagent	Volume (μ l)
		SYBR premix ex taq	6.0
Primer MIX (5. mu.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 15 s; then, denaturation is carried out at 95 ℃ for 5s in each step; annealing and extending for 30s at 60 ℃; a total of 45 cycles were performed. Each time reading the absorbance value during the extension phase. After the PCR was completed, the DNA was denatured at 95 ℃ for 1min, and then cooled to 55 ℃ to allow the DNA double strands to be sufficiently bound. Melting curves were prepared by increasing the temperature from 55 ℃ to 95 ℃ by 0.5 ℃ for 4 seconds and reading the absorbance. By using 2^-ΔΔCtThe assay calculates the expression abundance of the infected DNAJC24 mRNA. Cells infected with the control virus served as controls. The experimental results are shown in FIG. 1-A and FIG. 1-C, which indicate that DNAJC2 exists in human lung cancer A549 cell4 the expression level of mRNA was down-regulated by 51.1%; the expression level of DNAJC24 mRNA in lung cancer NCI-H1299 cells was down-regulated by 50.6%.

b) Western Blotting method for detecting gene silencing efficiency

1. Extraction of Total cellular proteins

(1) 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 as follows:

tables 3-18 mL fractions of separation gel

TABLE 3-210 mL fractions of the separation gel

Tables 3-3 different concentrations of 5% concentrated gum component

(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/20 x-lumiglo-reagents-and-20 x-p-oxyhydroxide/7003? site-search-type product)

(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.

The results are shown in FIGS. 1-B and 1-D, which indicate that the two targets have a knockdown effect on the endogenous expression of the target gene and are effective targets.

Example 3 examination of the proliferation potency of tumor cells infected with DNAJC24-siRNA lentivirus

a) Celigo experiment

Human lung cancer cells A549 and NCI-H1299 in logarithmic growth phase were trypsinized to prepare a cell suspension (about 5X 10 cells in number)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 and NCI-H1299: 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)⁴/ml), 96-well plates were seeded at a cell density of about 1500/well (A549), 1200/well (NCI-H1299). Each set of 5 duplicate wells, 100. mu.l per well. After the plate is laid, the plate is placed at 37 ℃ and 5% CO₂Culturing in an incubator. The plate reading was performed once a day with Celigo instrument (Nexcelom) starting the next day after plating, and the plate reading was performed continuously for 5 days. The number of green fluorescent cells in the well plate of each scan was accurately calculated by adjusting the input parameters of analysis settings, and the data was statistically plotted to generate cell proliferation curves (the results are shown in FIGS. 2-A and 2-B). The result shows that after the slow virus infects the tumor and the cell is cultured in vitro for 5 days, the proliferation speed is obviously slowed down and is far lower than that of the tumor cell of a control group, the number of the viable cells is respectively reduced by 60.8 percent (A549) and 73.8 percent (NCI-H1299), and the DNAJC24 gene silencing leads the proliferation capacity of the human lung cancer A549 and NCI-H1299 cells to be inhibited.

b) MTT assay

Human lung cancer cells A549 and NCI-H1299 in logarithmic growth phase were trypsinized to prepare a cell suspension (about 5X 10 cells in number)⁴/ml) were inoculated in 6-well plates and cultured until the degree of cell confluence reached about 30%. Adding appropriate amount of virus according to the infection complex number (MOI, A549: 10, NCI-H1299: 10), culturing for 24H, changing culture medium, and collecting the strain in logarithmic growthAfter trypsinization of the cells of each experimental group of phase, the complete medium was resuspended into a cell suspension and counted. Determining the density of plated cells (2000 cells/well) according to the growth speed of the cells, repeating each group by 3-5, uniformly plating, observing the cell density of each experimental group under a microscope after the cells are completely precipitated, fixing one group if the density is not uniform, finely adjusting the amount of the cells of other groups, plating again (for example, plating again after the cells of the Con group are found to be more and the cell amount is reduced), and putting the cells into a cell culture box for culture. Starting the day after plating, 20. mu.L of 5mg/mL MTT was added to the wells 4h before termination of the culture without changing the medium. After 4h, the culture was completely aspirated, and the formazan particles were dissolved by adding 100. mu.L of LDMSO, taking care not to aspirate the formazan particles at the bottom of the well plate. Oscillating for 2-5min with oscillator, and detecting OD value with enzyme labeling instrument 490/570 nm. And (6) carrying out data statistical analysis.

The results are shown in FIGS. 3-A/B and 3-C/D, and the results show that after the tumor is infected by the lentivirus, the proliferation speed is remarkably reduced and is far lower than that of the tumor cells of a control group, the number of viable cells is respectively reduced by 54.4 percent (A549) and 66.3 percent (NCI-H1299), and the DNAJC24 gene silencing causes the proliferation capacities of the human lung cancer A549 and NCI-H1299 cells to be inhibited.

Example 4 detection of the clonogenic Capacity of tumor cells infected with DNAJC24-siRNA lentivirus

Human lung cancer A549 and NCI-H1299 cells are trypsinized and inoculated into a 12-well plate, and the cell density is 10-15%. The next day, the medium was changed to fresh medium containing 5ug/ml polybrene. DNAJC24-siRNA lentiviruses were added to the plates according to the multiplicity of infection (MOI, A549: 10, NCI-H1299: 10) and the medium was changed fresh after 12-24H of 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 8 days, changing the liquid every 3 days 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 FIGS. 4-A and 4-B, the number of clones formed by human lung cancer A549 and NCI-H1299 cells is significantly reduced and the volume of the clones is significantly reduced after the RNA interference reduces the expression of the gene (KD group) compared with the control interference (NC group); the DNAJC24 gene silencing is shown to cause the reduction of the cloning capability of human lung cancer A549 and NCI-H1299 cells. The plate cloning test detects that after the expression of the gene is reduced, the cloning capacity of the tumor cells is reduced.

Example 5 detection of apoptosis level of tumor cells infected with DNAJC24-siRNA lentivirus

Human lung cancer A549 and NCI-H1299 cells are inoculated in a 6-well plate after being digested by pancreatin, and the cell density is 10-15%. The next day was changed to fresh medium. DNAJC24-siRNA lentiviruses were added to the plates according to the multiplicity of infection (MOI, A549: 10, NCI-H1299: 10) and the medium was changed fresh after 12-24H of 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. 5-A and 5-B, it was demonstrated that the proportion of apoptosis in tumor cells increased after down-regulation of gene expression. Following reduced gene expression by RNA interference (KD group), the number of apoptotic tumor cells increased significantly compared to control interference (NC group); indicating that gene silencing leads to apoptosis of tumor cells.

Example 6 detection of tumor cell invasion level of infecting DNAJC24-siRNA lentivirus

Placing the invasion chamber in an incubator to reach room temperature; sterilizing tweezers with 70% ethanol, and treating the transwell chamber with the tweezers; adding 300 mul of warm serum-free culture medium into the chamber, and standing at room temperature for 1-2 h to rehydrate the ECM layer (excellular Matrix); preparation of 1.0X 10⁶A cell suspension/ml (in serum-free medium); after rehydration at step 3, carefully remove the medium from the chamber; add 500. mu.l of medium containing 10% FBS to the lower chamber; add 300. mu.l of the cell suspension prepared in step 4 to each chamber; culturing in a tissue culture box for 48 h; gently remove non-invasive cells with cotton swabs; add 500. mu.l of staining solution to the wells of the plate; soaking the small chamber in staining solution for 20min to stain the lower surface of the membrane to invade the cell; the infusion chamber was rinsed several times in a large cup. Air-dry cell microscope photo: for each cell, a field of view was randomly selected and 4 pictures were taken at 100X and 9 pictures taken at 200X. Counting by 200X pictures, performing data analysis, and comparing the difference of the cell invasion capacities of the experimental group and the non-control group: calculating the number of invasion metastatic cells (Migratory cells per field) of each group, obtaining a p value by T-Test analysis, and judging whether the difference is significant (p is different)<0.05, there was a significant difference, otherwise there was no significant difference).

As a result, as shown in FIGS. 6-A and 6-B, the tumor cell invasion ability was decreased after RNA interference decreased the expression of DNAJC24 gene (KD group) compared to the control interference (NC group).

Example 7 detection of tumor cell migration and movement levels of infecting DNAJC24-siRNA lentivirus

a) Migration experiment of Migration

The kit was removed, the desired number of chambers were placed in a new 24-well plate, 100. mu.L serum-free medium was added to the upper chamber, and the plate was placed in an incubator at 37 ℃ for 1 h. Serum-free cell suspensions were prepared and counted, and the cell number was adjusted according to the preliminary experiment, typically 8 x 10⁴Perwell (24 well plate). The medium was carefully removed from the upper chamber and 100. mu.L of cell suspension was added, and 600. mu.L of 30% FBS medium was added to the lower chamber. At the same time, the cell suspension was used to spread an MTS96 well plate, approximately 5000 cells were seeded per well, and OD570 was measured after seeding as a transfer reference. The incubation was carried out in an incubator at 37 ℃ for a period of time (the specific time was adjusted according to the preliminary experiment). And reversely buckling the chamber on absorbent paper to remove the culture medium, lightly removing non-transferred cells in the chamber by using a cotton swab, dripping 2-3 drops of Giemsa staining solution to the lower surface of the membrane to stain the transferred cells for 3-5min, soaking and washing the chamber for several times, and airing in the air. Taking a picture by a microscope: for each transwell cell, fields of view were randomly selected and 4 pictures were taken at 100X and 9 pictures at 200X. Counting by 200X pictures, performing data analysis, and comparing the difference of cell transfer capacity of the experimental group and the control group: calculating the number of transferred cells (Migratorycels per field) of each group, obtaining a p value by T-Test analysis, and judging whether a significant difference (p) exists or not<0.05, there was a significant difference, otherwise there was no significant difference).

The results are shown in FIG. 7-A and FIG. 7-B, which indicate that the tumor cell migration motility was decreased after RNA interference decreased the expression of DNAJC24 gene (KD group) compared to the control interference (NC group).

b) Scratch healing test

According to the experimentally designed groups (KD: infection DNAJC24-siRNA lentivirus group, NC: infection Scr-siRNA lentivirus group), about 5X 10 of each was added to the wells⁴The infected cells were treated to achieve a cell confluence of 90% or more the next day. The low-concentration serum culture medium is changed the next day, the central part of the lower end of the 96-well plate is aligned by using a scratch instrument, and the lower end of the 96-well plate is slightly pushed upwards to form a scratch. Serum-free medium is used to gently rinse 2-3 times, and low-concentration serum medium (e.g., 0.5% FBS) is added to photograph. 37 ℃ and 5% CO₂Culturing in an incubator, and taking pictures at proper time points (generally 0h, 8h, 16h, 24h and the like can be selected) according to pre-experiments. Fluorescence microscopy photographs (with the central shaded area of the 96-well as the reference, with the scratch in the middle of the picture) were taken and the cell mobility of each group was calculated from the post-scratch pictures.

The results are shown in FIG. 8-A and FIG. 8-B, which indicate that the tumor cells have reduced ability to migrate directionally after RNA interference reduces the expression of DNAJC24 gene (KD group) compared to the control interference (NC group).

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.

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Claims

1. The application of the human DNAJC24 gene as a target in preparing a lung cancer treatment drug or a lung cancer diagnosis drug.

Use of a DNAJC24 inhibitor for the preparation of a product having at least one of the following effects:

treating lung cancer;

inhibiting the rate of proliferation of lung cancer cells;

promoting apoptosis of lung cancer cells;

inhibiting the cloning of lung cancer cells;

inhibiting lung cancer cell invasion;

inhibiting lung cancer cell metastasis;

inhibiting the growth of lung cancer.

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

1) the DNAJC24 inhibitor is a molecule having an inhibitory effect on DNAJC 24;

2) the DNAJC24 inhibitor is the only effective component or one of the effective components of the product;

3) the DNAJC24 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 a DNAJC24 gene in a lung cancer cell, the nucleic acid molecule comprising:

a. a double-stranded RNA containing a nucleotide sequence capable of hybridizing with a DNAJC24 gene; or

shRNA which contains a nucleotide sequence capable of hybridizing with a DNAJC24 gene;

wherein the double-stranded RNA comprises a first strand and a second strand, the first strand and the second strand are complementary together to form an RNA dimer, and the sequence of the first strand is substantially identical to a target sequence in the DNAJC24 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 DNAJC24 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 DNAJC24 gene interfering nucleic acid construct containing a gene segment coding shRNA in the nucleic acid molecule of any one of claims 5-6 and capable of expressing said shRNA.

8. A DNAJC24 gene interference lentivirus is prepared from the interference nucleic acid constructor of claim 7 through virus packing with the help of lentivirus packing plasmid and cell line.

9. The nucleic acid molecule of any one of claims 5-6, or the DNAJC24 gene interfering nucleic acid construct of claim 7, or the use of the DNAJC24 gene interfering lentiviruses of claim 8, to: is used for preparing a medicament for preventing or treating lung cancer or a kit for reducing the expression of DNAJC24 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 DNAJC24 gene interfering nucleic acid construct of claim 7; and/or, the DNAJC24 gene interfering lentivirus of claim 8, and a pharmaceutically acceptable carrier, diluent or excipient.