阅读说明：本技术 人ddx10基因的用途及相关产品 (Application of human DDX10 gene and related product ) 是由 杨晓东 浦玉伟 吴勇 邵乐宁 赵奎 邢春根 于 2020-03-25 设计创作，主要内容包括：本发明属于生物医药研究领域,具体涉及人DDX10基因作为靶标在制备结直肠癌治疗药物或者在制备结直肠癌诊断药物中的用途。本发明经过广泛而深入的研究发现,采用RNAi方法下调人DDX10基因的表达后可有效地抑制结直肠癌细胞的增殖、促进细胞凋亡,可以有效地控制结直肠癌的生长进程。本发明提供的siRNA或者包含该siRNA序列的核酸构建体、慢病毒能够特异性抑制结直肠癌细胞的增殖速率、促进结直肠癌细胞凋亡、抑制结直肠癌细胞克隆、抑制结直肠癌细胞侵袭、抑制结直肠癌细胞转移、抑制结直肠癌生长,从而治疗结直肠癌,为结直肠癌治疗开辟新的方向。(The invention belongs to the field of biomedical research, and particularly relates to application of a human DDX10 gene as a target in preparation of a colorectal cancer treatment drug or a colorectal cancer diagnosis drug. The invention discovers that the proliferation of colorectal cancer cells can be effectively inhibited and the apoptosis can be promoted after the expression of the human DDX10 gene is down regulated by adopting an RNAi method, and the growth process of colorectal 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 colorectal cancer cells, promote apoptosis of the colorectal cancer cells, inhibit cloning of the colorectal cancer cells, inhibit invasion of the colorectal cancer cells, inhibit metastasis of the colorectal cancer cells and inhibit growth of the colorectal cancer cells, so that the colorectal cancer is treated, and a new direction is opened for treatment of the colorectal cancer.)

1. The application of human DDX10 gene as target in preparing medicine for treating colorectal cancer or medicine for diagnosing colorectal cancer.

Use of an inhibitor of DDX10 in the manufacture of a product having at least one of the following effects:

treating colorectal cancer;

inhibiting the rate of proliferation of colorectal cancer cells;

promoting apoptosis of colorectal cancer cells;

inhibiting colorectal cancer cell cloning;

inhibiting colorectal cancer cell invasion;

inhibiting colorectal cancer cell metastasis;

inhibiting colorectal cancer growth.

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

1) the DDX10 inhibitor refers to a molecule having inhibitory effect on DDX 10;

2) the DDX10 inhibitor is the only effective component or one of the effective components of the product;

3) the DDX10 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 the expression of DDX10 gene in a colorectal cancer cell, the nucleic acid molecule comprising:

a. double-stranded RNA containing a nucleotide sequence capable of hybridizing with the DDX10 gene; or

shRNA containing a nucleotide sequence capable of hybridizing with the DDX10 gene;

wherein the double-stranded RNA comprises a first strand and a second strand, the first strand and the second strand are complementary to form an RNA dimer, and the sequence of the first strand is substantially identical to a target sequence in a DDX10 gene; the shRNA comprises a sense strand segment and an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, wherein the sequences of the sense strand segment and the antisense strand segment are complementary, and the sequence of the sense strand segment is basically identical to a target sequence in a DDX10 gene.

6. The nucleic acid molecule for reducing the expression of DDX10 gene in a colorectal cancer cell according to claim 5, further comprising one or more of the following characteristics:

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 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 DDX10 gene interfering nucleic acid construct containing a gene segment encoding shRNA in the nucleic acid molecule according to any one of claims 5 to 6, capable of expressing the shRNA.

8. A DDX10 gene interference lentivirus, which is prepared by virus packaging the interference nucleic acid construct of claim 7 with the help of lentivirus packaging plasmid and cell line.

9. The nucleic acid molecule of any one of claims 5-6, or the DDX10 gene interfering nucleic acid construct of claim 7, or the DDX10 gene interfering lentivirus of claim 8, for use in: for the preparation of a medicament for the prevention or treatment of colorectal cancer, or for the preparation of a kit for reducing the expression of DDX10 gene in colorectal cancer cells.

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

the nucleic acid molecule of any one of claims 5-6; and/or, the DDX10 gene interfering nucleic acid construct of claim 7; and/or, the DDX10 gene interfering lentivirus of claim 8, and a pharmaceutically acceptable carrier, diluent or excipient.

Technical Field

The invention belongs to the field of biomedical research, and particularly relates to application of a human DDX10 gene and a related product.

Background

DDX10((DEAD-Box Helicase 10) encodes a DEAD Box protein, possibly involved in ribosome assembly.A DEAD Box protein characterized by the conserved motif Asp-Glu-Ala-Asp (DEAD) is an RNA Helicase, which is involved in many cellular processes, including alterations in RNA secondary structure, such as translation initiation, nuclear and mitochondrial splicing, and ribosome and spliceosome assembly.based on their distribution patterns, some members of this family are thought to be involved in embryogenesis, spermatogenesis and cell growth and division.A chromosomal translocation by inversion of the 11(p15q22) is found in hematological malignancies and is fused to the nucleoporin gene NUP 98.

Colorectal cancer, hereinafter referred to as colorectal cancer, is a common malignant tumor in gastrointestinal tracts, has unobvious early symptoms, shows symptoms such as defecation habit change, hematochezia, diarrhea and constipation alternation, local abdominal pain and the like along with the increase of cancer, and shows general symptoms such as anemia, weight loss and the like at late stage. The incidence and fatality rate of the cancer are only second to those of gastric cancer, esophageal cancer and primary liver cancer in digestive system malignant tumors.

There is no report on the use of DDX10 gene in colorectal cancer treatment.

Disclosure of Invention

In order to overcome the problems in the prior art, the invention aims to provide the application of the human DDX10 gene and related products.

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

in a first aspect of the invention, the use of human DDX10 gene as a target for the manufacture of a medicament for the treatment of colorectal cancer or for the manufacture of a medicament for the diagnosis of colorectal cancer is provided.

The human DDX10 gene as a target for preparing the colorectal cancer treatment drug specifically comprises the following steps: the DDX10 gene is used as an action object, and the drug or the preparation is screened to find the drug which can inhibit the expression of the human DDX10 gene and is used as a candidate drug for treating the colorectal cancer. The DDX10 gene small interfering RNA (siRNA) is obtained by screening human DDX10 gene serving as an action object and can be used as a medicine for inhibiting the proliferation of colorectal cancer cells. In addition, for example, antibody drugs, small molecule drugs, etc. may also have the DDX10 gene as an object of action.

The application of the human DDX10 gene as a target in preparing a colorectal cancer diagnosis medicament specifically comprises the following steps: the DDX10 gene expression product is used as a colorectal cancer diagnosis index to be applied to the preparation of colorectal cancer diagnosis medicines.

The colorectal cancer treatment drug is a molecule which can specifically inhibit the transcription or translation of DDX10 gene, or can specifically inhibit the expression or activity of DDX10 protein, so that the expression level of DDX10 gene in colorectal cancer cells is reduced, and the purpose of inhibiting the proliferation, growth, differentiation and/or survival of the colorectal cancer cells is achieved.

The colorectal cancer treatment drug or colorectal cancer diagnosis drug prepared by the DDX10 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 colorectal cancer treatment drug is administered in an amount sufficient to reduce transcription or translation of the human DDX10 gene, or to reduce expression or activity of the human DDX10 protein. Such that the expression of the human DDX10 gene is reduced by at least 50%, 80%, 90%, 95%, or 99%.

The method for treating the colorectal cancer by adopting the colorectal cancer treatment medicine mainly achieves the aim of treating the colorectal cancer by reducing the expression level of human DDX10 gene to inhibit the proliferation of colorectal cancer cells. In particular, in therapy, a substance effective to reduce the expression level of human DDX10 gene is administered to a patient.

In one embodiment, the target sequence of the DDX10 gene is set forth in SEQ ID NO:1 is shown. The method specifically comprises the following steps: 5'-GATGTGAGCAAGTTACCTATA-3' are provided.

In a second aspect of the invention, there is provided the use of an inhibitor of DDX10 in the manufacture of a product having at least one of the following effects:

treating colorectal cancer;

inhibiting the rate of proliferation of colorectal cancer cells;

promoting apoptosis of colorectal cancer cells;

inhibiting colorectal cancer cell cloning;

inhibiting colorectal cancer cell invasion;

inhibiting colorectal cancer cell metastasis;

inhibiting colorectal cancer growth.

The product necessarily comprises the DDX10 inhibitor and takes the DDX10 inhibitor as an effective component of the aforementioned effects.

In the product, the effective component for the above functions can be only DDX10 inhibitor, and can also comprise other molecules for the above functions.

That is, the DDX10 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 DDX10 inhibitor can be a nucleic acid molecule, an antibody, a small molecule compound.

As exemplified in the examples herein, the DDX10 inhibitor can be a nucleic acid molecule that reduces the expression of the DDX10 gene in a colorectal 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 colorectal cancer by administering to a subject a DDX10 inhibitor.

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

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

The DDX10 inhibitor may be administered to a subject before, during, or after treatment for colorectal cancer.

In a fourth aspect, the invention discloses a nucleic acid molecule for reducing the expression of DDX10 gene in colorectal cancer cells, wherein the nucleic acid molecule comprises double-stranded RNA or shRNA.

Wherein, the double-stranded RNA contains a nucleotide sequence capable of hybridizing with DDX10 gene;

the shRNA contains a nucleotide sequence capable of hybridizing with a DDX10 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 DDX10 gene.

The target sequence in the DDX10 gene is a fragment in the DDX10 gene corresponding to an mRNA fragment which is recognized and silenced by the nucleic acid molecule when the nucleic acid molecule is used for specifically silencing the expression of the DDX10 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'-GATGTGAGCAAGTTACCTATA-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'-GAUGUGAGCAAGUUACCUAUA-3'.

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

SEQ ID NO:2 is designed by taking the sequence shown in SEQ ID NO. 1 as an RNA interference target sequence and aiming at one strand of small interfering RNA of the human DDX10 gene, and the sequence of the other strand, namely the second strand, is complementary with the sequence of the first strand, and the siRNA can play a role in specifically silencing the expression of endogenous DDX10 gene in colorectal cancer cells.

The shRNA comprises a sense strand segment and an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, wherein the sequences of the sense strand segment and the antisense strand segment are complementary, and the sequence of the sense strand segment is basically identical to a target sequence in a DDX10 gene.

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

The shRNA can become small interfering RNA (siRNA) after enzyme digestion and processing, and further plays a role in specifically silencing the expression of endogenous DDX10 genes in colorectal 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'-GAUGUGAGCAAGUUACCUAUACUCGAGUAUAGGUAACUUGCUCACAUC-3'.

Further, the DDX10 gene is derived from a human.

In the fifth aspect of the invention, the DDX10 gene interference nucleic acid construct contains a gene segment for coding shRNA in the nucleic acid molecule and can express the shRNA.

The DDX10 gene interfering nucleic acid construct can be obtained by cloning a gene segment for coding the human DDX10 gene shRNA into a known vector.

Further, the DDX10 gene interference nucleic acid construct is a DDX10 gene interference lentiviral vector.

The DDX10 gene interference lentiviral vector disclosed by the invention is obtained by cloning a DNA fragment for coding the DDX10 gene shRNA into a known vector, wherein the known vector is mostly a lentiviral vector, the DDX10 gene interference lentiviral vector is packaged into infectious viral particles by viruses, and then infects colorectal cancer cells to transcribe the shRNA, and the siRNA is finally obtained by the steps of enzyme digestion processing and the like and is used for specifically silencing the expression of the DDX10 gene.

Further, the DDX10 gene interference lentiviral vector also contains a promoter sequence and/or a nucleotide sequence encoding a marker detectable in colorectal cancer cells; preferably, the detectable label is Green Fluorescent Protein (GFP).

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

The embodiment of the invention specifically lists a human DDX10 gene interference lentiviral vector constructed by taking pGCSIL-GFP as a vector, and is named as pGCSIL-GFP-DDX 10-siRNA.

The DDX10 gene siRNA can be used for inhibiting the proliferation of colorectal cancer cells, and further can be used as a medicine or a preparation for treating colorectal cancer. DDX10 gene interference lentiviral vector can be used for preparing the DDX10 gene siRNA. When used as a medicament or formulation for treating colorectal 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 invention also discloses a method for preparing the DDX10 gene interference lentivirus, which is characterized in that the DDX10 gene interference nucleic acid construct is packaged by the virus under the assistance of lentivirus packaging plasmids and cell lines. The lentivirus can infect the colorectal cancer cells and produce small interfering RNA aiming at DDX10 gene, thereby inhibiting the proliferation of the colorectal cancer cells. The DDX10 gene interference lentivirus can be used for preparing a medicine for preventing or treating colorectal cancer.

In a seventh aspect of the present invention, there is provided a use of the aforementioned nucleic acid molecule, or the aforementioned DDX10 gene interfering nucleic acid construct, or the aforementioned DDX10 gene interfering lentivirus, wherein: for the preparation of a medicament for the prevention or treatment of colorectal cancer, or for the preparation of a kit for reducing the expression of DDX10 gene in colorectal cancer cells.

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

Further, when the medicament is used for preventing or treating colorectal 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 colorectal 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 said colorectal 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 colorectal cancer, which comprises the following effective substances:

the aforementioned nucleic acid molecules; and/or, the aforementioned DDX10 gene interfering nucleic acid construct; and/or, the aforementioned DDX10 gene interfering lentivirus, and a pharmaceutically acceptable carrier, diluent or excipient.

The composition may be a pharmaceutical composition.

When the composition is used for preventing or treating colorectal cancer in a subject, an effective dose of the composition needs to be administered to the subject. Using this method, the growth, proliferation, recurrence and/or metastasis of colorectal 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 said colorectal 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 DDX10 gene and constructs a corresponding DDX10RNAi vector, wherein the RNAi vector pGCSIL-GFP-DDX10-siRNA can obviously reduce the expression of the DDX10 gene at the mRNA level and the protein level. The slow virus (lentivirus, abbreviated as Lv) is used as a gene operation tool to carry an RNAi vector pGCSIL-GFP-DDX10-siRNA, so that the RNAi sequence aiming at the DDX10 gene can be efficiently introduced into colorectal cancer HCT116 and RKO cells in a targeted manner, the expression level of the DDX10 gene is reduced, and the proliferation capacity of the tumor cells is remarkably inhibited. Lentivirus-mediated DDX10 gene silencing is therefore a potential clinical non-surgical treatment modality for malignancies.

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

the invention discovers that the proliferation of colorectal cancer cells can be effectively inhibited and the apoptosis can be promoted after the expression of the human DDX10 gene is down regulated by adopting an RNAi method, and the growth process of colorectal 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 colorectal cancer cells, promote apoptosis of the colorectal cancer cells, inhibit cloning of the colorectal cancer cells, inhibit invasion of the colorectal cancer cells and inhibit metastasis of the colorectal cancer cells; the growth of the colorectal cancer is inhibited, so that the colorectal cancer is treated, and a new direction is opened for the treatment of the colorectal cancer.

Drawings

FIG. 1 a: RT-PCR measures the efficiency of target gene depletion at HCT116 cell mRNA levels.

FIG. 1 b: RT-PCR detects the target gene reduction efficiency of mRNA level of RKO cells.

FIG. 2 a: western Blotting examined the efficiency of target gene depletion of HCT116 cell protein level.

FIG. 2 b: western Blotting examined the RKO cell protein level target gene depletion efficiency.

FIG. 3 a: results of automatic analysis of Celigo cells revealed that depletion of DDX10 gene inhibited proliferation of colorectal cancer cells. (cell line is HCT116 cell, cell number was counted 1, 2, 3, 4 and 5 days after viral infection)

FIG. 3 b: results of automatic analysis of Celigo cells revealed that depletion of DDX10 gene inhibited proliferation of colorectal cancer cells. (cell lines are RKO cells, cell numbers were counted 1, 2, 3, 4 and 5 days after viral infection)

FIG. 4 a: MTT assay revealed that depletion of DDX10 gene inhibited proliferation of HCT116, colorectal cancer cells.

FIG. 4 b: MTT method revealed that depletion of DDX10 gene inhibited proliferation of colorectal cancer cells RKO.

FIG. 5 a: the influence of inhibiting DDX10 gene on HCT116 cell proliferation capacity is detected by a cell clone formation method, shRNA lentivirus infects HCT116 cells, the number of clones is observed after 8 days of culture, the upper graph is a digital camera recording graph, and the lower graph column results are shown by the average value +/-standard deviation of the number of cell clones.

FIG. 5 b: the cell clone formation method is used for detecting the influence of the inhibition DDX10 gene on the proliferation capacity of the RKO cells, the shRNA lentivirus infects the RKO cells, the cloning number is observed after the RKO cells are cultured for 8 days, the upper graph is a digital camera recording graph, and the lower graph column result is displayed by the average value +/-standard deviation of the cell cloning number.

FIG. 6 a: the Annexin V-APC flow apoptosis test shows the effect of sh DDX10 on HCT116 apoptosis and is a schematic diagram of flow apoptosis.

FIG. 6 b: annexin V-APC flow apoptosis assay the effect of sh DDX10 on HCT116 apoptosis, and bar results are shown as the mean of cell percentages. + -. standard deviation.

FIG. 7 a: annexin V-APC flow apoptosis test sh DDX10 influence on RKO apoptosis is a schematic diagram of flow apoptosis.

FIG. 7 b: annexin V-APC flow apoptosis assay sh DDX10 effect on RKO apoptosis, bar results are shown as percent cell mean. + -. standard deviation.

FIG. 8 a: transwell invasion experimental photographs revealed that silencing of DDX10 gene inhibited the invasive ability of colorectal cancer HCT116 cells.

FIG. 8 b: is a histogram of the results of fig. 8 a.

FIG. 9 a: transwell invasion experimental photographs revealed that silencing of DDX10 gene inhibited the invasive ability of colorectal cancer RKO cells.

FIG. 9 b: is a histogram of the results of fig. 9 a.

FIG. 10 a: transwell transfer experimental photographs revealed that silencing of DDX10 gene inhibited the invasive ability of colorectal cancer HCT116 cells.

FIG. 10 b: is a histogram of the results of fig. 10 a.

FIG. 11 a: transwell metastasis experimental photographs revealed that silencing of DDX10 gene inhibited the metastatic ability of colorectal cancer RKO cells.

FIG. 11 b: is a histogram of the results of FIG. 11 a.

FIG. 12 a: the scratch healing experiments revealed that silencing of DDX10 gene inhibited the metastatic ability of colorectal cancer HCT116 cells (upper panel is a fluorescence micrograph, lower panel is a bar chart of the results of 72h mobility).

FIG. 12 b: the scratch healing experiments revealed that silencing of DDX10 gene inhibited the metastatic ability of colorectal cancer RKO cells (upper panel is a fluorescence micrograph, lower panel is a bar chart of the results of 72h mobility).

In the drawings, there is shown in the drawings,

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

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

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

Detailed Description

The invention proves the function of DDX10 gene in colorectal cancer generation from the viewpoint of cell function. Transfecting colorectal cancer cells after constructing a target gene shRNA lentivirus, and comparing the transfected colorectal cancer cells with a transfection control lentivirus to detect the expression conditions of mRNA and protein level target genes in two groups of colorectal cancer cell lines; and then cell proliferation, apoptosis and other detection are carried out through a cytofunctional experiment, and the result shows that the shRNA group is compared with a control group, the colorectal 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 is higher than that of 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 diagnosing and treating the colorectal cancer patient.

DDX10 inhibitors

Refers to a molecule having inhibitory effect on DDX 10. Having inhibitory effects on DDX10 include, but are not limited to: inhibiting the expression or activity of DDX 10.

Inhibiting DDX10 activity refers to a decrease in DDX10 activity. Preferably, DDX10 activity is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, even more preferably by at least 70%, and most preferably by at least 90% as compared to its activity prior to inhibition.

The inhibition of the expression of DDX10 specifically can be the inhibition of the transcription or translation of DDX10 gene, and specifically can be the inhibition of the expression of DDX 10: making the gene of DDX10 non-transcribed, or reducing the transcriptional activity of the gene of DDX10, or making the gene of DDX10 non-translated, or reducing the level of translation of the gene of DDX 10.

The regulation of gene expression of DDX10 can be accomplished by one skilled in the art using conventional methods, such as gene knock-out, homologous recombination, interfering RNA, and the like.

The inhibition of gene expression of DDX10 was confirmed by detecting the expression level by PCR and Western Blot.

Preferably, the DDX10 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 the DDX10 gene is not expressed at all, compared to the wild type.

Small molecule compounds

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

Preparation of medicine for preventing or treating colorectal cancer

Nucleic acid molecules that reduce the expression of DDX10 gene in colorectal cancer cells can be utilized; and/or, a DDX10 gene interfering nucleic acid construct; and/or, DDX10 gene interferes lentivirus, and is used as an effective component for preparing a medicament for preventing or treating colorectal 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.