CN115786345A - siRNA for inhibiting CCR4 gene expression and application thereof - Google Patents

Abstract

The invention discloses siRNA for inhibiting CCR4 gene expression and application thereof, belonging to the field of biological medicine, wherein the disclosed siRNA sequence is shown in SEQ ID NO:1 and SEQ ID NO:2, the siRNA can specifically inhibit CCR4 target gene expression, thereby blocking CCL17 action and relieving pulmonary fibrosis; the siRNA has obvious curative effect on pulmonary fibrosis diseases, small toxic and side effect and safe use.

Description

siRNA for inhibiting CCR4 gene expression and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to siRNA for specifically inhibiting CCR4 gene expression and application thereof.

Background

Pulmonary fibrosis is the terminal change of a large group of lung diseases characterized by fibroblast proliferation and massive extracellular matrix aggregation with inflammation injury and tissue structure destruction, is one of four diseases of respiratory diseases, and is the most serious pathological state of the lung. The pathological changes are mostly manifested by the initial inflammation of the lower respiratory tract, as well as damage to alveolar epithelial cells and vascular endothelial cells, with proliferation of fibroblasts and type II alveolar cells, cytokine release, deposition of extracellular matrix proteins and collagen, and finally lung changes. Pulmonary alveoli of lungs of patients with pulmonary fibrosis are gradually replaced by fibrous substances, so that lung tissues become hard and thick, the gas exchange capacity of lungs is gradually lost, the patients are difficult to breathe due to different degrees of oxygen deficiency, and finally, the patients die due to exhaustion of breath. The pulmonary fibrosis has complex etiology and unknown pathogenesis, the existing drugs and methods for treating pulmonary fibrosis are very limited, the curative effect is very good, the prognosis is very poor, and the 5-year survival rate is only 50%.

CCR4 is the only known receptor for the chemokine CCL 17. The CCL17-CCR4 axis plays a crucial role in the pathogenesis of immune diseases. The CCL17-CCR4 axis is reported to be involved in a variety of inflammatory diseases, including dermatitis, allergic asthma, atherosclerosis, colitis, and arthritis. In recent years, the CCL17-CCR4 axis has been shown to play an important role in the pathogenesis of fibrotic diseases.

The early results show that the CCL17 level in the lung of patients with pulmonary fibrosis and mice with pulmonary fibrosis induced by bleomycin is increased, and particularly the CCL17 expression in alveolar macrophages is up-regulated. Mechanistic studies have shown that CCL17 interacts with its receptor CCR4 on fibroblasts, thereby activating TGF- β/Smad signaling pathways, promoting fibroblast activation and tissue fibrosis. These results suggest that the CCL17-CCR4 axis is involved in the progression of pulmonary fibrosis, that targeting CCR4 can inhibit fibroblast activation and tissue fibrosis, and that inhibition of CCR4 is a potential target for the treatment of pulmonary fibrosis. Therefore, the research and development of the substance for inhibiting the expression of CCR4 have good prospect of patent drugs for treating pulmonary fibrosis.

Disclosure of Invention

Because the pathogenesis of pulmonary fibrosis is complex, an effective treatment drug is lacked at present, and the invention aims to provide an anti-fibrosis RNA interference drug, which adopts RNAi technology to reduce the expression of CCR4 gene at mRNA level, further inhibits the activation of fibroblast to generate excessive extracellular matrix, and effectively treats pulmonary fibrosis.

The siRNA sequence can be obtained by direct chemical synthesis, in vitro transcription, plasmid amplification, viral replication, etc., and thus the present invention shall include the aforementioned forms of use comprising said sequence.

In order to solve the technical problem of the invention, the invention provides the following technical scheme.

The invention mainly aims to provide an siRNA sequence which can be specifically combined with CCR4mRNA to reduce CCR4 expression in a targeted mode, so that fibroblast activation is inhibited to generate excessive extracellular matrix, wherein the RNA sequence of the siRNA is shown as the following sequence, or a sequence with the same function obtained by chemically modifying, replacing, deleting or adding any position of the RNA sequence.

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

5'-AGAGTCCTAATGATCATGGTT-3’(SEQ ID NO:2)；

And two T at the tail end of the 3' end are thymine deoxyribonucleotide.

Further, the siRNA molecule is an siRNA molecule in which at least one strand of the RNA single strand is complementary after any chemical modification as shown in 1) to 13) below;

1) Phosphorothioate modification of the phosphate backbone;

2) 2' -methoxy modification of ribose or deoxyribose;

3) 2' -fluoro modification of ribose or deoxyribose;

4) Modifying locked nucleic acid;

5) Open loop nucleic acid modification;

6) Modifying indole;

7) 5-methylcytosine modification of bases;

8) 5-ethynyluracil modification of the base;

9) Single-stranded 5' terminal cholesterol modification;

10 Single-stranded 3' terminal galactose modification;

11 Single chain 5' terminal polypeptide modification;

12 Single-stranded 5' end phosphorylation modification;

13 Single-stranded 5' end fluorescent label modification.

The second objective of the invention is to provide a DNA molecule capable of producing the siRNA molecule.

The third purpose of the invention is to provide the application of the siRNA molecule for inhibiting CCR4 gene expression in the preparation of drugs for treating pulmonary fibrosis.

The "pulmonary fibrosis" described in the present invention is pulmonary fibrosis which is conventional in the art. The pulmonary fibrosis is preferably pulmonary fibrosis characterized by a pathological change in idiopathic pulmonary fibrosis, resulting from a variety of factors. Wherein the pulmonary fibrosis preferably refers to pulmonary fibrosis of human or animals, and the symptoms of the pulmonary fibrosis more preferably include: pulmonary inflammation caused by pulmonary fibrosis, and pulmonary function deterioration caused by pulmonary fibrosis. The cause of the pulmonary fibrosis is preferably: pulmonary fibrosis caused by lung injury, pulmonary fibrosis caused by dust, or pulmonary fibrosis caused by a drug, preferably bleomycin.

Wherein the pulmonary fibrosis is preferably primary (specific) pulmonary fibrosis, i.e. pulmonary fibrosis of unknown cause; or secondary pulmonary fibrosis, i.e. pulmonary fibrosis secondary to the original disease, more preferably pulmonary function deterioration, pulmonary inflammation and pulmonary injury in pulmonary fibrosis. The disease of pulmonary fibrosis preferably comprises Chronic Obstructive Pulmonary Disease (COPD), idiopathic pulmonary fibrosis or interstitial pneumonia.

The term "treatment" as used herein means to reduce the degree of pulmonary fibrosis, or to cure pulmonary fibrosis to normalize it, or to slow down the progression of pulmonary fibrosis.

The drug for treating pulmonary fibrosis is preferably a pharmaceutical composition, and the dosage form of the pharmaceutical composition is not particularly limited and is a dosage form conventional in the art. The dosage form of the pharmaceutical composition is preferably solid, semi-solid or liquid. The pharmaceutical composition may also be in the form of an aqueous solution, a non-aqueous solution or a suspension. The dosage form of the pharmaceutical composition is preferably tablet, capsule, granule, injection or infusion. The route of administration of the pharmaceutical composition is conventional in the art, and is preferably injection or oral administration. Wherein the mode of administration by injection preferably comprises: intravenous, intramuscular, intraperitoneal, intradermal or subcutaneous routes.

The fourth purpose of the invention is to provide a drug for treating pulmonary fibrosis, wherein the drug contains the siRNA for inhibiting CCR4 gene expression, or further comprises a pharmaceutically acceptable carrier or excipient.

In the treatment of pulmonary fibrosis, the pharmaceutical composition of the present invention can be used alone or in combination with other drugs.

Further, the pulmonary fibrosis includes primary pulmonary fibrosis or secondary pulmonary fibrosis.

Further, the pulmonary fibrosis is drug-induced pulmonary fibrosis.

Further, the drug-induced pulmonary fibrosis is drug-induced pulmonary fibrosis caused by bleomycin.

Further, the product is used for treating pulmonary fibrosis by treating pulmonary inflammation, pulmonary function deterioration or pulmonary injury.

The fifth purpose of the invention is to provide the application of the siRNA in preparing any preparation for targeted reduction of CCR4 expression, inhibition of CCL17 effect and reduction of excessive extracellular matrix generated by fibroblast activation.

The invention has the beneficial technical effects that: the siRNA can reduce CCR4 expression in a targeted manner, inhibit the action of CCL17 and reduce excessive extracellular matrix generated by fibroblast activation, so that the siRNA can be applied to preparation of anti-pulmonary fibrosis drugs. The medicine prepared by the siRNA has the advantages of obvious curative effect, small toxic and side effect and safe use in the treatment of pulmonary fibrosis diseases.

Drawings

FIG. 1 is a pathological picture of the staining of Masson pine in example 5;

FIG. 2 shows the visualization of the bands in example 5 using ChemiDoxTM XRS + and Image LabTM software (Bio-Rad, hercules, california, USA) and a chemiluminescent detection reagent (Tanon).

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, the present invention is described in detail with reference to the accompanying drawings and the detailed description thereof.

The reagents and starting materials used in the present invention are commercially available.

PBS, as used in the examples, refers to phosphate buffer at a concentration of 0.1M and a pH of 7.2.

The room temperature described in the examples is a room temperature conventional in the art, and preferably 15 to 30 ℃.

The experimental results are expressed by mean +/-standard error, and the significant difference is considered to be generated when p is less than 0.05 and the extremely significant difference is considered to be generated when p is less than 0.01 through parameter or nonparametric variance tests.

Example 1 preparation of an animal model of pulmonary fibrosis

1.1 Main reagents and laboratory animals

Bleomycin used for the experiments was purchased from Japan Chemicals, lot X81040.

The compounds used in the experiments were purchased from Sigma, unless otherwise specified.

SPF grade C57BL/6 mice (male, 6-8 weeks old, 16-18 g) used for the experiments were purchased from Schlekshirta laboratory animals, inc. of Hunan province.

1.2 preparation of animal model with pulmonary fibrosis

Male C57BL/6 (week-old 6-8 week) mice were fasted overnight, anesthetized with sodium pentobarbital (45 mg/kg, i.p.), and given intratracheally with bleomycin (1U/kg) for 6 total doses at 14 day intervals.

The specific scheme is as follows: fixing the prone position of a mouse after anesthesia, irradiating the neck position of the mouse by using a cold light source, pulling the tongue of the mouse outwards by using a right-hand forceps, opening the oral cavity as far as possible by using a left-hand forceps until the glottis is exposed, inserting a 20G trocar into the trachea of the mouse under the mediation of a guide wire, injecting about 50 mu L of bleomycin into the trachea by using a micro-injection needle, rapidly rotating and standing for 5 minutes so as to enable the bleomycin to uniformly enter the left and right lung lobes. The whole operation is carried out at a surgical operating table at about 60 ℃. The same amount of normal saline for injection was injected into the trachea in the sham operation group.

Example 2 screening of siRNA which best inhibits expression of CCR4 by real-time fluorescent quantitative PCR method

The method comprises the following specific steps:

1. spreading human fibroblasts on a six-hole plate;

2. after cell attachment, 100pmol CCR4-siRNA and CCR4-siRNA-2 were transiently transfected using Lipofectamine RNAiMax (Invitrogen) with siNC as control. The CCR4-siRNA sequence is as follows:

5 'CCATGATCATTAGGACTCTTT-3'; shown in SEQ ID NO. 1;

5' AGAGTCCTAATGATCATGGTT-; shown in SEQ ID NO. 2;

the CCR4-siRNA-2 sequence is as follows:

5 'GCAAGATCGTTTCATGGATTT-3'; shown in SEQ ID NO. 3;

5' ATCCATGAAACGATCTTGCTT-; shown in SEQ ID NO. 4;

the two T at the 3' end of the four sequences are thymine deoxyribonucleotide.

3. After transfection for 48h, cells were harvested and RNA was extracted. The method for rapidly extracting the kit (Shanghai Yishuai biology) by adopting RNA comprises the following steps

(1) The medium was blotted dry and washed once with 1mL of PBS.

(2) Add 500. Mu.L of lysine Buffer, aspirate 10 times vigorously, transfer to an EP tube, vortex for 10 seconds to lyse cells well.

(3) The lysed cells were added with equal volume of absolute ethanol and mixed well, and then the liquid was added to the binding column in the kit.

(4) Centrifuge at 4000 Xg for one minute and discard the liquid.

(5) 600 μ L of Wash Buffer,12000 Xg was used for one minute of centrifugation and the liquid was discarded.

4. Then the extracted RNA is reversely transcribed into cDNA, and the steps of adopting a rapid reverse transcription kit (Shanghai Yiniao organisms) are as follows:

(1) Taking 100ng-2 μ g total RNA, adding 2 μ LDNA enzyme, adding water to 16 μ L, and gently beating with pipette for 5-10 times to mix well.

(2) The reaction was carried out at room temperature for 5 minutes, and then placed on ice for use.

(3) Then 4. Mu.L of 5 XTT Mix was added to the system.

(4) Blow and beat for 10 times by a liquid transfer gun, and mix well.

(5) The reaction was carried out at 42 ℃ for 15 minutes. qPCR was performed using 5-10 fold dilution as template.

qPCR primers:

GAPDH

forward 5 'GTCTCCTCTGACTTCAACAGCG-3'; shown in SEQ ID NO. 5;

reverse 5' -ACCACCTGTTGCTGTAGCCAA-3; shown in SEQ ID NO. 6;

CCR4

forward 5 'CTCTCTGGCTTTGTTCACTGCTGC-3'; shown in SEQ ID NO. 7;

reverse 5' -AGCCCAGTATTGGCAGAGACA-3; see SEQ ID NO. 8.

qPCR primers for human GAPDH and CCR4 were designed and qPCR was performed using the cDNA obtained by reverse transcription as template. The qPCR adopted system was: KAPA enzyme 10. Mu.L, primer 200nM, template 50ng, water 8. Mu.L.

The results show that: the siRNA can obviously down-regulate the expression of CCR4 in fibroblasts. The results are shown in Table 1.

TABLE 1CCR 4mRNA levels of CCR4-siRNA treated fibroblasts

Grouping	Expression level of CCR4	P value
			A: siNC group	1.23±0.08
Group B of CCR4-siRNA	0.12±0.04	<0.001(A VS.B)
			Group C CCR4-siRNA-2	0.86±0.02	＞0.05(B VS.C)

Example 3 detection of siRNA inhibition of fibroblast activation Using real-time fluorescent quantitative PCR

The method comprises the following specific steps:

1. spreading human fibroblasts on a six-hole plate;

2. after cell attachment, 100pmol CCR4-siRNA was transiently transfected using Lipofectamine RNAiMax (Invitrogen) with siNC as control.

After 3.48 hours, cells were harvested and RNA extracted after 3 hours of stimulation with PBS or recombinant human CCL17 (50 ng/ml), respectively. The method for rapidly extracting the RNA from the kit (Shanghai Yiniao organisms) comprises the following steps

(1) The medium was blotted dry and washed once with 1ml of PBS.

(2) Add 500. Mu.L of lysine Buffer, aspirate 10 times vigorously, transfer to an EP tube, vortex for 10 seconds to lyse cells well.

(3) The lysed cells were mixed well with an equal volume of absolute ethanol and the liquid was then added to the binding column in the kit.

(4) Centrifuge at 4000 Xg for one minute and discard the liquid.

(5) 600 μ L of Wash Buffer,12000 Xg was used for one minute of centrifugation and the liquid was discarded.

4. Then the extracted RNA is reversely transcribed into cDNA, and the steps of adopting a rapid reverse transcription kit (Shanghai Yiniao organisms) are as follows:

(1) Taking 100ng-2 μ g total RNA, adding 2 μ LDNA enzyme, adding water to 16 μ L, and gently beating with pipette for 5-10 times to mix well.

(2) The reaction was carried out at room temperature for 5 minutes and placed on ice until use.

(3) Then 4. Mu.L of 5 XTT Mix was added to the system.

(4) Blow and beat for 10 times by a liquid transfer gun, and mix well.

(5) The reaction was carried out at 42 ℃ for 15 minutes. qPCR was performed using 5-10 fold dilution as template.

qPCR primers:

GAPDH

Forward:5'-GTCTCCTCTGACTTCAACAGCG-3'；

Reverse:5'-ACCACCCTGTTGCTGTAGCCAA-3

ACTA2：

5' CTATGCCTCTGGACGCAACT-; shown as SEQ ID NO. 9;

reverse 5' -CAGATCCAGACGCATGATGGA-3. Shown in SEQ ID NO. 10;

qPCR primers for human GAPDH and ACTA2 were designed and qPCR was performed using the cDNA obtained by reverse transcription as template. The qPCR adopted system was: KAPA enzyme 10. Mu.L, primer 200nM, template 50ng, water 8. Mu.L.

The results show that: the CCR4-siRNA is used for knocking down CCR4, so that the activation of a fibroblast induced by CCL17 can be inhibited, and the expression of ACTA2 is reduced. The results are shown in Table 2.

TABLE 2 ACTA2 mRNA levels in CCR4-siRNA treated fibroblasts

Grouping	Amount of ACTA2 expression	P value
			A: PBS group	0.90±0.07
CCL17+ sinC group	1.10±0.06	<0.05(A VS.B)
			CCL17+ CCR4-siRNA group	0.16±0.08	<0.001(B VS.C)

Example 4 detection of inhibition of fibroblast migration by siRNA using the Transwell method

1. Spreading human fibroblasts on a six-hole plate; after cell attachment, 100pmol CCR4-siRNA was transiently transfected using Lipofectamine RNAiMax (Invitrogen) with siNC as control.

Transwell migration experiment was performed in a Transwell chamber with a filter membrane pore size of 8 μm (Corning). The 24-well culture plate was inserted into a chamber whose lower surface was precoated with 10. Mu.g/ml fibronectin. Injecting the treated human fibroblast cells into the upper chamber at a rate of 1 × 10 per well for 48 hr ⁴ The cells were simultaneously supplemented with PBS or CCL17 (50 ng/ml) as stimulating factors in the lower chamber medium (MEM medium +0.4% FBS), respectively.

After 2.12h, non-migrating cells were removed from above the screen with a cotton swab. After fixation with 4% paraformaldehyde, stained with crystal violet staining solution and counted in 4 random fields under an optical microscope.

The results show that: the CCR4-siRNA is used for knocking down CCR4, so that the migration promoting effect of CCL17 on fibroblasts can be inhibited. The results are shown in Table 3

TABLE 3 migration number of CCR4-siRNA treated fibroblasts

Grouping	Number of migrating cells	P value
			A: PBS group	35.25±1.73
CCL17+ siNC group	78.5±3.24	<0.001(A VS.B)
			CCL17+ CCR4-siRNA group	41.75±2.217	<0.001(B VS.C)

Example 5 verification of the anti-pulmonary fibrosis effect of siRNA using pulmonary fibrosis animal models

5.1 the animal models prepared in example 1 were administered in groups 10 days after molding, and the groups and administration are shown in Table 4:

TABLE 4 grouping administration after model building of pulmonary fibrosis animal model

5.2 Masson staining Pathology imaging analysis

Masson dyeing is a classic and authoritative method for dyeing collagen fibers, and the dyed muscle fibers are red, while the collagen fibers are blue, so that the muscle fibers and the collagen fibers are mainly distinguished.

The lung tissue of the right lower lobe of the animal was taken and fixed with 4% paraformaldehyde and then embedded in paraffin. The section of the largest cross section of the wax block embedding lung tissue is stained by masson to observe fibrosis. High-definition pathological pictures (100 times) specially stained by masson pine are obtained by applying a high-definition element color pathological graph and text analysis system Spot Advanced 3.0, and the results are shown in figure 1. The results show that the blue collagen fiber area in the lung tissue of mice is significantly increased after bleomycin administration, and significantly decreased after CCR4-siRNA administration.

5.3 determination of hydroxyproline content in mice with pulmonary fibrosis

Hydroxyproline accounts for 13.4% of collagen, accounts for a very small amount of elastin, and is absent in other proteins, so that the content of collagen is detected by hydroxyproline. Detecting the content of the hydroxyproline in the left lung of the animal, and evaluating the condition of the pulmonary fibrosis. The specific method comprises the following steps: taking all the lung lobes on the left side of the model animal prepared in the example 1, recording the wet weight, ultrasonically homogenizing with physiological saline to prepare 10% tissue homogenate, taking about 150 mu L homogenate supernatant, adding 500 mu L alkali hydrolysate, vortex and mixing uniformly, treating for 40min under the condition of 120 ℃ and 0.1Kpa by an alkali hydrolysis method (the method refers to the instruction of a kit of Nanjing Biotechnology Limited company, and is slightly changed), adjusting the pH value, fixing the volume, treating with active carbon, and taking the supernatant. Hydroxyproline determination was performed according to the instructions (chloramine-T method). The results are shown in table 5, and from table 5, it can be seen that CCR4-siRNA treatment can significantly reduce the pulmonary hydroxyproline content of fibrotic mice, indicating that fibrotic pathology is improved.

TABLE 5CCR4-siRNA reduces hydroxyproline content in pulmonary tissue of pulmonary fibrosis mice

5.4Western blot detection of alpha-SMA protein in mice with pulmonary fibrosis

1. Using RIPA buffer (Beyotime, P0013C) containing protease inhibitor (MCE, HY-K0010), grinding beads were added for lysis in a tissue grinder, about 4-10mg of total tissue protein was harvested, and each histone was adjusted to the same concentration. Adding 5 xSDS gel sample buffer solution proportionally, mixing, denaturing at 95 deg.C for 10min, and quickly cooling in ice bath. Centrifuging at 12000rpm for 2min at room temperature to obtain supernatant as precipitated protein sample, and performing SDS-polyacrylamide gel electrophoresis on part or all of the protein sample.

2. The protein is separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membrane. After blocking with skim milk, the PVDF membrane was incubated with anti-GAPDH and α -SMA antibodies overnight at 4 ℃ and then with horseradish peroxidase-labeled secondary antibody. Bands were visualized using ChemiDoxTM XRS + and Image LabTM software (Bio-Rad, hercules, california, USA) and chemiluminescent detection reagents (Tanon). The results are shown in FIG. 2. The optical density values of the bands were analyzed by Western-blot analysis software (gelPro 32), and the results are shown in Table 6. The results in table 6 show that after treatment with CCR4-siRNA, the expression level of α -SMA in fibrotic mouse lung tissue can be significantly reduced, indicating that the fibrotic pathology is improved.

TABLE 6CCR4-siRNA reduces pulmonary fibrosis mouse lung tissue alpha-SMA expression

Name of protein	Model set	Group for administration of CCR4-siRNA	P value
				Pulmonary fibrosis tissue alpha-SMA expression	34819.34±15.73	12929.14±5.32	<0.001

In the experiment, the result of pathological examination, pathological imaging and other means is analyzed, and the result shows that the CCR4-siRNA can obviously inhibit pulmonary fibrosis caused by bleomycin; the contents of hydroxyproline and collagen in pulmonary tissues of the mice with pulmonary fibrosis are obviously reduced. The experimental results prove that the CCR4-siRNA has excellent treatment prospect in the aspect of pulmonary fibrosis.

The results of the above examples show that the siRNA of the present invention has significant effect of resisting pulmonary fibrosis diseases, and can be used as an active ingredient for preparing drugs for resisting pulmonary fibrosis.

The above-described embodiments are only intended to illustrate the preferred embodiments of the present invention, and not to limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims (10)

1. An siRNA molecule for inhibiting CCR4 gene expression, comprising: the sense strand and the antisense strand of the siRNA molecule are respectively the sequences shown as follows:

5'-CCATGATCATTAGGACTCTTT-3’；

5'-AGAGTCCTAATGATCATGGTT-3’；

and two T at the tail end of the 3' end are thymine deoxyribonucleotides.

2. An siRNA molecule according to claim 1, wherein: siRNA molecules, wherein at least one strand of the RNA single strands is subjected to any chemical modification shown in the following 1) to 13) and then is complemented;

1) Phosphorothioate modification of the phosphate backbone;

2) 2' -methoxy modification of ribose or deoxyribose;

3) 2' -fluoro modification of ribose or deoxyribose;

4) Modifying locked nucleic acid;

5) Open loop nucleic acid modification;

6) Modifying indole;

7) 5-methylcytosine modification of a base;

8) 5-ethynyluracil modification of the base;

9) Single-chain 5' end cholesterol modification;

10 Single-stranded 3' terminal galactose modification;

11 Single chain 5' terminal polypeptide modification;

12 Single-stranded 5' end phosphorylation modification;

13 Single-stranded 5' end fluorescent label modification.

3. A DNA molecule capable of producing the siRNA molecule of claim 1 or 2.

4. Use of the siRNA molecule inhibiting CCR4 gene expression according to claim 1 or 2 for the preparation of a medicament for the treatment of pulmonary fibrosis.

5. The use of claim 4, wherein the pulmonary fibrosis comprises primary pulmonary fibrosis or secondary pulmonary fibrosis.

6. Use according to claim 4, wherein the pulmonary fibrosis comprises drug-induced pulmonary fibrosis, in particular drug-induced pulmonary fibrosis induced by bleomycin.

7. A medicament for treating pulmonary fibrosis, which comprises the siRNA for inhibiting CCR4 gene expression of claim 1 or 2, or further comprises a pharmaceutically acceptable carrier or excipient.

8. The medicament of claim 7, wherein the pulmonary fibrosis comprises primary pulmonary fibrosis or secondary pulmonary fibrosis.

9. Medicament according to claim 7, characterized in that the pulmonary fibrosis comprises pulmonary fibrosis induced by drugs, in particular by bleomycin.

10. The use of the siRNA inhibiting CCR4 gene expression according to claim 1 or 2 in the preparation of any preparation for targeted reduction of CCR4 expression, inhibition of CCL17 effect, and reduction of fibroblast activation to produce excessive extracellular matrix.

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