CN110863047B - Application of human CCDC154 gene and related product - Google Patents

Abstract

The invention belongs to the field of biomedical research, and particularly relates to the use of human CCDC154 gene as a target in the preparation of a drug for treating liver cancer. After extensive and in-depth research, the present invention finds that using RNAi method to down-regulate the expression of human CCDC154 gene can effectively inhibit the proliferation of liver cancer cells, promote cell apoptosis, and can effectively control the growth process of liver cancer. The siRNA or the nucleic acid construct comprising the siRNA sequence and the lentivirus provided by the present invention can specifically inhibit the proliferation rate and proliferation activity of liver cancer cells, promote the apoptosis of liver cancer cells, inhibit the cloning of liver cancer cells, and inhibit the growth of liver cancer cells, thereby treating liver cancer. Liver cancer treatment opens up new directions.

Description

Use of human CCDC154 gene and related products

technical field

The invention belongs to the field of biomedical research, and specifically relates to the use of human CCDC154 gene and related products.

Background technique

Coiled-Coil Domain Containing 154 (CCDC154, Coiled-Coil Domain Containing 154) is a novel coding gene that is widely expressed in human and mouse tissues and subcellularly localized mainly in endosomes.

A 5 kb sequence deletion in the exon 1-6 region of the CCDC154 gene is associated with spontaneous autosomal recessive sclerosis mutant mice (ntl mutation). At present, there are few reports on the related functions of this gene.

It has been reported that CCDC154 is involved in the pathogenesis of osteopetrosis. Overexpression of CCDC154 inhibited the proliferation activity and soft agar colony formation ability of HEK293 cells by inducing G2/M cycle arrest, but had no effect on apoptosis. This indicates that CCDC154 is a novel osteosclerosis-related gene involved in cell cycle regulation and cell growth inhibition.

The role of CCDC154 in cancer, especially liver cancer, is still unknown.

SUMMARY OF THE INVENTION

In order to overcome the problems existing in the prior art, the purpose of the present invention is to provide the use of human CCDC154 gene and related products.

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

The first aspect of the present invention provides the use of human CCDC154 gene as a target in preparing a drug for treating liver cancer.

The use of the human CCDC154 gene as a target in preparing a drug for liver cancer treatment specifically refers to: taking the CCDC154 gene as an action object, screening drugs or preparations to find a drug that can inhibit the expression of the human CCDC154 gene as an alternative drug for liver cancer treatment. The CCDC154 gene small interfering RNA (siRNA) according to the present invention is obtained by screening the human CCDC154 gene as the target, and can be used as a drug with the effect of inhibiting the proliferation of liver cancer cells. In addition, such as antibody drugs, small molecule drugs, etc. can also use the CCDC154 gene as the target.

The liver cancer therapeutic drug is a molecule that can specifically inhibit the transcription or translation of the CCDC154 gene, or can specifically inhibit the expression or activity of the CCDC154 protein, thereby reducing the expression level of the CCDC154 gene in the liver cancer cells, thereby inhibiting the proliferation and growth of the liver cancer cells. , differentiation and/or survival purposes.

The liver cancer therapeutic drugs prepared by the CCDC154 gene include but are not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs, antibody drugs, polypeptides, proteins or interfering lentiviruses.

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

The administration amount of the liver cancer therapeutic drug is a dose sufficient to reduce the transcription or translation of the human CCDC154 gene, or to reduce the expression or activity of the human CCDC154 protein. so that the expression of the human CCDC154 gene is reduced by at least 50%, 80%, 90%, 95% or 99%.

The method for treating liver cancer using the aforementioned liver cancer therapeutic drugs mainly achieves the purpose of treatment by reducing the expression level of human CCDC154 gene and inhibiting the proliferation of liver cancer cells. Specifically, during the treatment, a substance that can effectively reduce the expression level of the human CCDC154 gene is administered to the patient.

In one embodiment, the target sequence of the CCDC154 gene is shown in SEQ ID NO:1. Specifically: 5'-ACAAGTGCCTGCTTCATAA-3'.

The second aspect of the present invention provides the use of a CCDC154 inhibitor in the preparation of a product with at least one of the following effects:

treatment of liver cancer;

Inhibit the proliferation rate and proliferation activity of liver cancer cells;

Promote liver cancer cell apoptosis;

Inhibit liver cancer cell clone;

Inhibit the growth of liver cancer.

The product must include a CCDC154 inhibitor, and use the CCDC154 inhibitor as an active ingredient for the aforementioned efficacy.

In the product, the active ingredient that can play the aforementioned function may only be a CCDC154 inhibitor, or may contain other molecules that can play the aforementioned function.

That is, the CCDC154 inhibitor is the only active ingredient or one of the active ingredients of the product.

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

The form of the product is not particularly limited, and can be in the form of various substances such as solid, liquid, gel, semi-liquid, and aerosol.

The product is mainly aimed at mammals. The mammals are preferably rodents, artiodactyls, odd ungulates, lagomorphs, primates and the like. The primate is preferably a monkey, ape or human.

The products include, but are not limited to, medicines, health products, food, and the like.

The CCDC154 inhibitor can be a nucleic acid molecule, an antibody, or a small molecule compound.

As exemplified in the embodiments of the present invention, the CCDC154 inhibitor can be a nucleic acid molecule that reduces the expression of the CCDC154 gene in liver cancer cells. Specifically, it can be double-stranded RNA or shRNA.

The third aspect of the present invention provides a method for treating liver cancer, which is to administer a CCDC154 inhibitor to a subject.

The object can be mammalian or mammalian liver cancer cells. The mammals are preferably rodents, artiodactyls, odd ungulates, lagomorphs, primates and the like. The primate is preferably a monkey, ape or human. The liver cancer cells can be ex vivo liver cancer cells.

The subject may be a patient suffering from liver cancer or an individual for whom treatment of liver cancer is desired. Alternatively, the subject is an isolated liver cancer cell from a liver cancer patient or an individual who is expected to treat liver cancer.

The CCDC154 inhibitor can be administered to the subject before, during and after receiving liver cancer treatment.

The fourth aspect of the present invention discloses a nucleic acid molecule for reducing the expression of CCDC154 gene in liver cancer cells, the nucleic acid molecule comprising double-stranded RNA or shRNA.

Wherein, the double-stranded RNA contains a nucleotide sequence capable of hybridizing with the CCDC154 gene;

The shRNA contains a nucleotide sequence capable of hybridizing with the CCDC154 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 together, and the sequence of the first strand is the same as the target in the CCDC154 gene The sequence is basically the same.

The target sequence in the CCDC154 gene is the segment in the CCDC154 gene corresponding to the mRNA segment recognized and silenced by the nucleic acid molecule when the nucleic acid molecule is used to specifically silence the expression of the CCDC154 gene.

Further, the target sequence of the double-stranded RNA is shown in SEQ ID NO: 1. Specifically: 5'-ACAAGTGCCTGCTTCATAA-3'. Further, the sequence of the first strand of the double-stranded RNA is shown in SEQ ID NO: 2. Specifically, 5'-ACAAAGUGCCUGCUUCAUAA-3'.

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

SEQ ID NO: 2 is one strand of the small interfering RNA against the human CCDC154 gene designed with the sequence shown in SEQ ID NO: 1 as the RNA interference target sequence, and the other strand is the sequence of the second strand and the sequence of the first strand Complementary, the siRNA can specifically silence the expression of endogenous CCDC154 gene in hepatoma 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 sense strand segment and the antisense strand segment are complementary in sequence, and the sense strand segment is complementary to the antisense strand segment. The sequence of the chain fragment is substantially identical to the target sequence in the CCDC154 gene.

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

The shRNA can be converted into a small interfering RNA (siRNA) after being digested with enzymes, thereby specifically silencing the expression of the endogenous CCDC154 gene in liver cancer cells.

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

Further, the sequence of the shRNA is shown in SEQ ID NO:3. Specifically, 5'-CGACAAGUGCCUGCUUCAUAACUCGAGUUAUGAAGCAGGCACUUGUCG-3'.

Further, the CCDC154 gene is derived from human.

The fifth aspect of the present invention discloses a CCDC154 gene interference nucleic acid construct, which contains a gene fragment encoding the shRNA in the aforementioned nucleic acid molecule, and can express the shRNA.

The CCDC154 gene interfering nucleic acid construct can be obtained by cloning the gene fragment encoding the aforementioned human CCDC154 gene shRNA into a known vector.

Further, the CCDC154 gene interference nucleic acid construct is a CCDC154 gene interference lentiviral vector.

The CCDC154 gene interference lentiviral vector disclosed in the present invention is obtained by cloning the DNA fragment encoding the aforementioned CCDC154 gene shRNA into a known vector. Most of the known vectors are lentiviral vectors. The CCDC154 gene interference lentiviral vector is packaged into a virus. After the infectious virus particles are infected with liver cancer cells, the shRNA of the present invention is then transcribed, and the siRNA is finally obtained through steps such as enzymatic cleavage, which is used to specifically silence the expression of the CCDC154 gene.

Further, the CCDC154 gene interference lentiviral vector also contains a promoter sequence and/or a nucleotide sequence encoding a detectable marker in liver cancer cells; preferably, the detectable marker such as green fluorescence protein (GFP).

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

The embodiment of the present invention specifically lists the human CCDC154 gene interference lentiviral vector constructed by using pGCSIL-GFP as a vector, which is named as pGCSIL-GFP-CCDC154-siRNA.

The CCDC154 gene siRNA of the present invention can be used to inhibit the proliferation of liver cancer cells, and further can be used as a drug or preparation for the treatment of liver cancer. The CCDC154 gene interfering lentiviral vector can be used to prepare the CCDC154 gene siRNA. When used as a drug or preparation for treating liver cancer, a safe and effective amount of the nucleic acid molecule is administered to a mammal. The specific dosage should also take into account factors such as the route of administration, the patient's health status, etc., which are all within the skill of the skilled physician.

The sixth aspect of the present invention discloses a CCDC154 gene interference lentivirus, which is formed by viral packaging of the aforementioned CCDC154 gene interference nucleic acid construct with the assistance of lentiviral packaging plasmids and cell lines. The lentivirus can infect liver cancer cells and produce small interfering RNA targeting the CCDC154 gene, thereby inhibiting the proliferation of liver cancer cells. The CCDC154 gene interfering lentivirus can be used to prepare a drug for preventing or treating liver cancer.

The seventh aspect of the present invention provides the use of the aforementioned nucleic acid molecule, or the aforementioned CCDC154 gene interference nucleic acid construct, or the aforementioned CCDC154 gene interference lentivirus, for preparing a drug for preventing or treating liver cancer, or for preparing a drug that reduces liver cancer cells A kit for CCDC154 gene expression.

The application of the drug for preventing or treating liver cancer provides a method for the treatment of liver cancer, specifically a method for preventing or treating liver cancer in a subject, comprising administering an effective dose of the drug to the subject.

Further, when the medicament is used to prevent or treat liver 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 the liver cancer is inhibited. Further, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the growth, proliferation, recurrence and/or metastasis of the liver cancer part is suppressed.

The object of the method may be a human being.

The eighth aspect of the present invention provides a composition for preventing or treating liver cancer, the effective substance containing:

The aforementioned nucleic acid molecule; and/or, the aforementioned CCDC154 gene interference nucleic acid construct; and/or, the aforementioned CCDC154 gene interference lentivirus, and a pharmaceutically acceptable carrier, diluent or excipient.

The composition may be a pharmaceutical composition.

When the composition is used to prevent or treat liver 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 the liver cancer is inhibited. Further, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the growth, proliferation, recurrence and/or metastasis of the liver cancer part is suppressed.

The form of the composition is not particularly limited, and can be in the form of various substances such as solid, liquid, gel, semi-liquid, and aerosol.

The subject of the composition is mainly mammals. The mammals are preferably rodents, artiodactyls, odd ungulates, lagomorphs, primates and the like. The primate is preferably a monkey, ape or human.

To sum up, the present invention designed the RNAi target sequence for the human CCDC154 gene, and constructed the corresponding CCDC154 RNAi vector, wherein the RNAi vector pGCSIL-GFP-CCDC154-siRNA can significantly down-regulate the expression of the CCDC154 gene at the mRNA and protein levels. Using lentivirus (Lv for short) as a genetic manipulation tool to carry the RNAi vector pGCSIL-GFP-CCDC154-siRNA, the RNAi sequence targeting the CCDC154 gene can be efficiently introduced into liver cancer BEL-7404 cells and SMMC-7721 cells, reducing CCDC154 The expression level of the gene significantly inhibited the proliferation ability of the above-mentioned tumor cells. Therefore, lentivirus-mediated gene silencing of CCDC154 is a potential clinical non-surgical treatment for malignant tumors.

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

After extensive and in-depth research, the present invention finds that using RNAi method to down-regulate the expression of human CCDC154 gene can effectively inhibit the proliferation of liver cancer cells, promote cell apoptosis, and can effectively control the growth process of liver cancer. The siRNA or the nucleic acid construct comprising the siRNA sequence and the lentivirus provided by the present invention can specifically inhibit the proliferation rate and proliferation activity of liver cancer cells, promote the apoptosis of liver cancer cells, inhibit the cloning of liver cancer cells, and inhibit the growth of liver cancer cells, thereby treating liver cancer. Liver cancer treatment opens up new directions.

Description of drawings

Figure 1: RT-PCR detection of target gene depletion efficiency at the mRNA level of BEL-7404 cells and SMMC-7721 cells.

Figure 2: Celigo cell automated analysis results reveal that depletion of the CCDC154 gene inhibits the proliferation of hepatoma cells. (The cell lines are BEL-7404 cells on the left and SMMC-7721 cells on the right. The number of cells was counted at 1, 2, 3, 4 and 5 days after virus infection)

Figure 3: The effect of CCDC154 gene on the proliferation ability of BEL-7404 cells was detected by the cell clone formation method. The shRNA lentivirus infected BEL-7404 cells and SMMC-7721 cells. After 14 days of culture, the number of clones was observed. The left side is the digital camera record. The right column results are shown as the mean ± standard deviation of the number of cell clones.

Figure 4: The effect of CCDC154 gene on the proliferation ability of SMMC-7721 cells was detected by the cell clone formation method. The shRNA lentivirus infected BEL-7404 cells and SMMC-7721 cells. After 14 days of culture, the number of clones was observed. The right column results are shown as the mean ± standard deviation of the number of cell clones.

Figure 5: Flow cytometry of Annexin V-APC to detect the effect of sh CCDC154 on apoptosis of BEL-7404 cells, which is a schematic diagram of apoptosis by flow cytometry.

Figure 6: Annexin V-APC flow cytometry to detect the effect of sh CCDC154 on apoptosis of SMMC-7721 cells, and the columnar results are shown as the mean ± standard deviation of the percentage of cells.

Figure 7: BrdU assay for detecting the proliferation activity of lentivirus-infected BEL-7404 cells (left) and SMMC-7721 cells (right).

In the attached drawings,

The bar graph represents the mean of three experiments, and the error bars represent the standard deviation (SD).

**, shCtrl compared with the target gene shRNA lentivirus treatment group, P<0.01.

*, shCtrl compared with the target gene shRNA lentivirus treatment group, 0.01≤P<0.05.

Detailed ways

The inventors of the present invention have found through extensive and in-depth research that the CCDC154 gene is significantly highly expressed in liver cancer tumor tissues; the inventors have found that the use of RNAi to down-regulate the expression of human CCDC154 gene can effectively inhibit the proliferation of tumor cells and promote cell proliferation. Apoptosis can effectively control the clone formation ability of tumor cells. This research result indicates that the CCDC154 gene is a proto-oncogene and can be used as a potential target for tumor therapy. The inventors further synthesized and tested a variety of siRNAs targeting the CCDC154 gene, and screened out siRNAs that can effectively inhibit the expression of CCDC154 and thereby inhibit the proliferation activity and apoptosis of human liver cancer BEL-7404 cells and liver cancer SMMC-7721 cells. The present invention has been completed.

The present invention confirms the role of CCDC154 gene in the occurrence of hepatocellular carcinoma from the perspective of cell function. By constructing the shRNA lentivirus of the target gene and then transfecting the liver cancer cells, compared with the transfection control lentivirus, the expression of the target gene at the mRNA and protein levels in the two groups of liver cancer cell lines was detected; The results showed that compared with the control group, the shRNA group had a significantly higher degree of inhibition of liver cancer cell proliferation and a higher degree of increase in the apoptosis rate than the control group.

CCDC154 inhibitor

Refers to a molecule that has an inhibitory effect on CCDC154. The inhibitory effect on CCDC154 includes, but is not limited to: inhibiting the expression or activity of CCDC154.

Inhibiting the activity of CCDC154 means reducing the activity of CCDC154. Preferably, the activity of CCDC154 is decreased by at least 10% compared to before inhibition, preferably by at least 30%, even better by at least 50%, more preferably by at least 70%, and best by at least 90%.

Inhibiting the expression of CCDC154 may specifically be inhibiting the transcription or translation of the CCDC154 gene, specifically, it may refer to: making the gene of CCDC154 not transcribed, or reducing the transcriptional activity of the gene of CCDC154, or making the gene of CCDC154 not translated, or reducing the expression of CCDC154 gene translation level.

Those skilled in the art can use conventional methods to regulate the gene expression of CCDC154, such as gene knockout, homologous recombination, interfering RNA and the like.

The inhibition of CCDC154 gene expression can be verified by PCR and Western Blot detection of expression levels.

Preferably, the expression of the CCDC154 gene is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, more preferably by at least 70%, and most preferably by at least 90%, compared to the wild type. Jiadi CCDC154 gene was not expressed at all.

small molecule compounds

In the present invention, it refers to a compound consisting of several or dozens of atoms and having a molecular mass below 1000.

Preparation of medicaments for preventing or treating liver cancer

The nucleic acid molecule that reduces the expression of CCDC154 gene in liver cancer cells; and/or the CCDC154 gene interference nucleic acid construct; and/or the CCDC154 gene interference lentivirus can be used as an effective ingredient to prepare a drug for preventing or treating liver cancer. Usually, in addition to the active ingredients, the medicine also includes one or more pharmaceutically acceptable carriers or excipients according to the needs of different dosage forms.

"Pharmaceutically acceptable" means that the molecular entities and compositions do not produce adverse, allergic or other adverse reactions when properly administered to animals or humans.

The "pharmaceutically acceptable carrier or adjuvant" should be compatible with the active ingredient, that is, it can be blended with it without greatly reducing the effect of the drug under normal circumstances. Specific examples of some substances that can be used as pharmaceutically acceptable carriers or excipients are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, Ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oils, corn oil and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycols; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; Flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; and phosphate buffers, among others. These materials are used as needed to aid in the stability of the formulation or to help increase the activity or its bioavailability or to produce an acceptable mouthfeel or odor in the case of oral administration.

In the present invention, unless otherwise specified, the pharmaceutical dosage form is not particularly limited, and can be made into dosage forms such as injections, oral liquids, tablets, capsules, dropping pills, sprays, etc., and can be prepared by conventional methods. The choice of drug dosage form should match the mode of administration.

Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments, It is not intended to limit the protection scope of the present invention. In the following examples, the test methods without specific conditions are usually in accordance with conventional conditions or in accordance with the conditions suggested by various manufacturers.

When numerical ranges are given in the examples, it is to be understood that, unless otherwise indicated herein, both endpoints of each numerical range and any number between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment and materials used in the embodiments, according to the mastery of the prior art by those skilled in the art and the description of the present invention, the methods, equipment and materials described in the embodiments of the present invention can also be used Any methods, devices and materials similar or equivalent to those of the prior art can be used to implement the present invention.

Unless otherwise specified, the experimental methods, detection methods and preparation methods disclosed in the present invention all adopt the conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in the technical field. conventional technology.

Example 1 Preparation of RNAi lentivirus for human CCDC154 gene

1. Screening effective siRNA targets against human CCDC154 gene

The gene information of CCDC154 (NM_001143980) was retrieved from Genbank; an effective siRNA target for the CCDC154 gene was designed. Table 1-1 lists the screened effective siRNA target sequences for the CCDC154 gene.

Table 1-1 siRNA target sequences targeting human CCDC154 gene

SEQ ID NO TargetSeq(5'-3') 1 ACAAGTGCCTGCTTCATAA

2. Preparation of Lentiviral Vectors

Synthesize double-stranded DNA Oligo sequences (Table 1-2) with sticky ends containing Age I and EcoR I restriction sites at both ends against the siRNA target (take SEQ ID NO: 1 as an example); Dicer was applied to the pGCSIL-GFP vector (provided by Shanghai Jikai Gene Chemical Technology Co., Ltd.) to linearize it, and agarose gel electrophoresis was used to identify the digested fragments.

Table 1-2 Double-stranded DNA Oligo with sticky ends containing Age I and EcoR I restriction sites at both ends

Use T4 DNA ligase to linearize the double-enzyme digestion (enzyme digestion system is shown in Table 1-4, 37 ° C, reaction 1h) vector DNA and purified double-stranded DNA Oligo ligation, in an appropriate buffer system (ligation system As shown in Table 1-5), the ligation was performed at 16°C overnight, and the ligation product was recovered. The ligation product was transformed into fresh E. coli competent cells prepared by calcium chloride (transformation operation reference: Molecular Cloning Experiment Guide, Second Edition, pages 55-56). Dip the surface of the clone of the ligated transformation product, dissolve it in 10 μl LB medium, mix well and take 1 μl as a template; design universal PCR primers upstream and downstream of the RNAi sequence in the lentiviral vector, upstream primer sequence: 5'- CCTATTTCCCATGATTCCTTCATA-3' (SEQ ID NO: 6); downstream primer sequence: 5'-GTAATACGGTTATCCACGCG-3' (SEQ ID NO: 7), carry out the PCR identification experiment (the PCR reaction system is shown in Table 1-6, and the reaction conditions are shown in Table 1 -7). The clones identified by PCR were sequenced and compared, and the correct clones were successfully constructed to express the RNAi vector against SEQ ID NO: 1, which was named pGCSIL-GFP-CCDC154-siRNA.

The pGCSIL-GFP-Scr-siRNA negative control plasmid was constructed, and the negative control siRNA target sequence was 5'-TTCTCCGAACGTGTCACGT-3' (SEQ ID NO: 8). When constructing the pGCSIL-GFP-Scr-siRNA negative control plasmid, a double-stranded DNA Oligo sequence containing Age I and EcoR I restriction sites at both ends was synthesized against the Scr siRNA target (Table 1-3). Other construction methods, identification The methods and conditions were the same as those of pGCSIL-GFP-CCDC154-siRNA.

Table 1-3 Double-stranded DNA Oligo with sticky ends containing Age I and EcoR I restriction sites at both ends

Table 1-4 pGCSIL-GFP plasmid digestion reaction system

reagent Volume (μl) pGCSIL-GFP plasmid (1μg/μl) 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 Vector DNA and double-stranded DNA Oligo ligation reaction system

reagent Positive control (μl) Self-ligation control (μl) Connectivity group (μl) Linearized vector DNA (100ng/μl) 1.0 1.0 1.0 Annealed dsDNA Oligo (100ng/μl) 1.0 - 1.0 10 x T4 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 1-6-1 PCR reaction system

Table 1-7 PCR reaction system program settings

3. Packaging CCDC154-siRNA lentivirus

The DNA of the RNAi plasmid pGCSIL-GFP-CCDC154-siRNA was extracted with a plasmid extraction kit from Qiagen, and prepared into a 100 ng/μl stock solution.

24h before transfection, human embryonic kidney cell 293T cells in logarithmic growth phase were digested with trypsin, and the cell density was adjusted to 1.5×10 ⁵ cells/ml in DMEM complete medium containing 10% fetal bovine serum, and seeded in 6-well plates , 37 ° C, 5% CO ₂ incubator. When the cell density reaches 70%-80%, it can be used for transfection. 2h before transfection, the original medium was aspirated, and 1.5 ml of fresh complete medium was added. According to the instructions of the MISSION Lentiviral Packaging Mix kit from Sigma-aldrich, add 20 μl of Packing Mix (PVM), 12 μl of PEI, and 400 μl of serum-free DMEM medium to a sterilized centrifuge tube, take 20 μl of the above-extracted plasmid DNA, add to the above PVM/PEI/DMEM mixture.

The above-mentioned transfection mixture was incubated at room temperature for 15 min, transferred to the medium of human embryonic kidney cells 293T cells, and cultured in a 37° C., 5% CO ₂ incubator for 16 h. The culture medium containing the transfection mixture was discarded, washed with PBS solution, 2 ml of complete medium was added, and the culture was continued for 48 h. The cell supernatant was collected, Centricon Plus-20 centrifugal ultrafiltration device (Millipore) was used to purify and concentrate the lentivirus. The steps were as follows: (1) Centrifuge at 4000g for 10 min at 4°C to remove cell debris; (2) filter the supernatant with a 0.45 μm filter In a 40ml ultracentrifuge tube; (3) Centrifuge at 4000g for 10-15min to the required virus concentration volume; (4) After centrifugation, separate the filter cup from the filtrate collection cup below, and place the filter cup upside down. On the sample collection cup, the centrifugal force of centrifugation for 2 minutes should not exceed 1000g; (5) Remove the centrifuge cup from the sample collection cup, and the virus concentrate in the sample collection cup is. Store the virus concentrate in aliquots at -80°C. The sequence of the first strand of the siRNA contained in the virus concentrate is shown in SEQ ID NO:2. The packaging process of the control lentivirus was the same as that of the CCDC154-siRNA lentivirus, only the pGCSIL-GFP-Scr-siRNA vector was used instead of the pGCSIL-GFP-CCDC154-siRNA vector.

Example 2 Detection of gene silencing efficiency by real-time fluorescence quantitative RT-PCR

Human liver cancer BEL-7404 cells and SMMC-7721 cells in logarithmic growth phase were digested with trypsin, and cell suspensions (about 5×10 ⁴ /ml) were made into 6-well plates and cultured until cell fusion degree of about 30%. According to the multiplicity of infection (MOI, BEL-7404:10, SMMC-7721:10), an appropriate amount of the lentivirus prepared in Example 1 was added, and the medium was replaced after culturing for 24 hours. After the infection time reached 5 days, the cells were collected . Total RNA was extracted according to Invitrogen's Trizol operating instructions. According to the M-MLV operating instructions of Promega company, reverse transcription of RNA to obtain cDNA (reverse transcription reaction system is shown in Table 2-1, 42 °C reaction for 1 h, and then in a 70 °C water bath for 10 minutes to inactivate reverse transcriptase).

Real-time quantitative detection was performed using a TP800 Real time PCR instrument (TAKARA). The primers for the CCDC154 gene were as follows: upstream primer 5'-AGAGTGTCTCCGACAAGTGCCT-3' (SEQ ID NO: 11) and downstream primer 5'-CCCAGGGTTGTCTTCCTTTAGC-3' (SEQ ID NO: 12). Taking housekeeping gene GAPDH as internal reference, the primer sequences are as follows: upstream primer 5'-TGACTTCAACAGCGACACCCA-3' (SEQ ID NO: 13) and downstream primer 5'-CACCCTGTTGCTGTAGCCAAA-3' (SEQ ID NO: 14). The reaction system was configured according to the ratio in Table 2-2.

Table 2-1 Reverse transcription reaction system

reagent Volume (μl) 5×RT buffer 4.0 10mM dNTPs 2.0 RNasin 0.5 M-MLV-RTase 1.0 DEPC H₂O 3.5 Total 11.0

Table 2-2 Real-time PCR reaction system

reagent Volume (μl) SYBR premix ex taq 10.0 Upstream primer (2.5 μM 0.5 Downstream primer (2.5μM) 0.5 cDNA 1.0 ddH₂O 8.0 Total 20.0

The set program was two-step Real-time PCR: pre-denaturation at 95°C for 15s; subsequent denaturation at 95°C for 5s for each step; annealing and extension at 60°C for 30s; a total of 45 cycles were performed. The absorbance was read each time during the extension phase. After PCR, denature at 95°C for 1 min, and then cool to 55°C to fully bind the DNA double strands. Start from 55°C to 95°C, increase 0.5°C in each step, hold for 4s, read the absorbance value at the same time, and make a melting curve. The expression abundance of infected CCDC154 mRNA was calculated by 2- ^ΔΔCt analysis. Cells infected with control virus served as controls. The experimental results are shown in Figure 1, indicating that the expression levels of CCDC154 mRNA in human hepatoma BEL-7404 cells and SMMC-7721 cells were down-regulated by 69.60% and 52.60%, respectively.

Example 3 Detection of proliferation ability of tumor cells infected with CCDC154-siRNA lentivirus

Human liver cancer BEL-7404 cells and SMMC-7721 cells in logarithmic growth phase were digested with trypsin, and cell suspensions (about 5×10 ⁴ /ml) were made into 6-well plates and cultured until cell fusion degree of about 30%. According to the multiplicity of infection (MOI, BEL-7404:10, SMMC-7721:10), an appropriate amount of virus was added, and the medium was replaced after culturing for 24 hours. After the infection time reached 5 days, each experiment in the logarithmic growth phase was collected. group cells. The complete medium was resuspended into a cell suspension (2×10 ⁴ /ml), and the cells were seeded in a 96-well plate at a cell density of about 3000 cells/well. 5 replicate wells in each group, 100 μl per well. After laying the plate, place it in a 37°C, 5% CO ₂ incubator for cultivation. From the second day after plating, the plate was detected and read once a day with Celigo instrument (Nexcelom), and the plate was detected and read continuously for 5 days. By adjusting the input parameters of the analysis settings, the number of cells with green fluorescence in each scan plate was accurately calculated; the data were statistically plotted to draw a 5-day cell proliferation curve. (The results are shown in Figure 2). The results showed that the proliferation rate of each tumor in the lentivirus infection group was significantly slowed down after 5 days of cell culture in vitro, which was much lower than the proliferation rate of tumor cells in the control group. The number of viable cells of human liver cancer BEL-7404 cells and SMMC-7721 cells decreased respectively. 83.54% and 21.47%, indicating that CCDC154 gene silencing can inhibit the proliferation of human hepatoma BEL-7404 cells and SMMC-7721 cells.

Example 4 Detection of clone formation ability of tumor cells infected with CCDC154-siRNA lentivirus

Human hepatoma BEL-7404 cells and SMMC-7721 cells were trypsinized and seeded in 12-well plates at a cell density of 10-15%. Change to fresh medium the next day, containing 5ug/ml polybrene. The CCDC154-siRNA lentivirus was added to the culture plate according to the multiplicity of infection MOI, BEL-7404:10, SMMC-7721:10, and the culture medium was changed after 12-24 hours of infection. After 72 hours of infection, the fluorescence was observed under a fluorescence microscope, and the infection efficiency reached 90%.

After the virus-infected cells in logarithmic growth phase were trypsinized, the complete medium was resuspended to form a cell suspension; after cell counting, the cells were inoculated into a 6-well plate (200 cells/well), and the inoculated cells were placed in a 6-well plate. Continue to culture in the incubator until 14 days or until the number of cells in most single clones is greater than 50, and the medium is changed every 3 days and the cell status is observed; the cell clones are photographed under a fluorescence microscope before the experiment is terminated; The cells were fixed with formaldehyde, washed with PBS, stained with Giemsa, and photographed.

The results are shown in Figures 3 and 4. Compared with the control interference (NC group), after RNA interference reduced gene expression (KD group), the number of clonal plaques formed by BEL-7404 cells and SMMC-7721 cells was significantly reduced, and the number of clones was significantly reduced. The size of the plaques was significantly reduced; indicating that gene silencing resulted in a decrease in the ability of BEL-7404 cells and SMMC-7721 cells to form clones. Plate colony formation assay detected that the clone formation ability of BEL-7404 cells and SMMC-7721 cells decreased after reducing gene expression.

Example 5 Detection of apoptosis level of tumor cells infected with CCDC154-siRNA lentivirus

Human hepatoma BEL-7404 cells and SMMC-7721 cells were trypsinized and seeded in 6-well plates at a cell density of 20-30%. Change to fresh medium the next day. The CCDC154-siRNA lentivirus was added to the culture plate according to the multiplicity of infection MOI, BEL-7404:10, SMMC-7721:10, and the culture medium was changed after 12-24 hours of infection. After 72 hours of infection, the fluorescence was observed under a fluorescence microscope, and the infection efficiency reached 90%.

After trypsinizing the cells in the logarithmic growth phase, the complete medium was resuspended into a cell suspension; the cells were collected in the same 5 mL centrifuge tube with the supernatant cells, and three replicate wells were set in each group (to ensure that the number of cells on the machine was sufficient). , the number of cells≥5×105/treatment). Centrifuge at 1300 rmp for 5 min, discard the supernatant, and wash the cell pellet with pre-cooled PBS at 4°C. The cell pellet was washed once with 1×binding buffer, and the cells were collected by centrifugation at 1300 rmp for 3 min. Resuspend the cell pellet in 200 μL of 1× binding buffer. Add 10 μL Annexin V-APC for staining, and protect from light for 10-15 min at room temperature. According to the amount of cells, 400-800 μL of 1×binding buffer was added and detected by flow cytometer. Analyze the results.

As shown in Figure 5 and Figure 6, Annexin V single staining was used to detect changes in the apoptosis ratio of human hepatoma BEL-7404 cells and SMMC-7721 cells after reducing gene expression. It was found that the apoptotic proportion of tumor cells increased after down-regulation of gene expression. Compared with control interference (NC group), after RNA interference reduced gene expression (KD group), the number of apoptotic tumor cells increased significantly; indicating that gene silencing leads to tumor cell apoptosis.

Example 6 Detection of proliferation activity of lentivirus-infected tumor cells by BrdU method

Determined using Roche Brdu kit. Cells to be tested were seeded at an appropriate density in a 96-well plate. The cells to be tested are human hepatoma BEL-7404 cells and SMMC-7721 cells (multiplicity of infection, BEL-7404:10, SMMC-7721:10) infected with CCDC154-siRNA and Scr-siRNA lentivirus. BrdU reagent was added within the first 2-24h. BrdU was diluted 1:100 with the medium before use, and 10 μL/well of the diluted BrdU was added, and the culture was continued until the set time. After the action of BrdU, the medium was aspirated and discarded, 200 μL/well of FixDenat (Bottle 2) was added, and the cells were fixed in a dark room at room temperature for 30 min. Aspirate and discard the fixative, add 5-10% BSA to block in a dark room at room temperature for half an hour, and aspirate and discard the blocking solution. Add antibody. Add Anti-BrdU-POD working solution 100 μL/well, and react in the dark room at room temperature for 90 min. Anti-BrdU-POD stock solution: Dilute Anti-BrdU-POD (bottle3) with 1.1 mL of double distilled water for 10 min, and mix thoroughly. Anti-BrdU-POD working solution: Dilute Anti-BrdU-POD stock solution 1:100 with antibody dilution solution (bottle 4). Dilute the washingbuffer concentrate (bottle 5) with sterile double-distilled water 1:10, add the diluted washing buffer 200-300 μL/well, wash the plate three times, and pat dry. Finally, sterile water is added to an empty cell plate outer box, and the culture plate to be tested is placed in it. Add 100 μL/well of Substrate solution (bottle 6) and react in a dark room at room temperature for 5-30 min until the reaction solution turns blue. Add 50 μL/well of 10% H ₂ SO ₄ to the culture well, and read the plate at 450 nm single wavelength detection. Finally, the proliferative activity of each group of tumor cells was statistically analyzed on day4 compared to day1.

The results are shown in Figure 7. Compared with control interference (NC group), after RNA interference reduced the expression of CCDC154 gene (KD group), it was found that the day4 cells of tumor BEL-7404 cells and SMMC-7721 cells proliferated after down-regulation of CCDC154 gene expression. slowed down (P<0.05).

The above are only preferred embodiments of the present invention, and are not intended to limit the present invention in any form or substance. It should be pointed out that for those skilled in the art, without departing from the method of the present invention, the Several improvements and supplements can be made, and these improvements and supplements should also be regarded as the protection scope of the present invention. All those skilled in the art, without departing from the spirit and scope of the present invention, can utilize the above-disclosed technical content to make some changes, modifications and equivalent changes of evolution, all belong to the present invention. Equivalent embodiments; at the same time, any modification, modification and evolution of any equivalent changes made to the above embodiments according to the essential technology of the present invention still fall within the scope of the technical solutions of the present invention.

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Claims (7)

1. Use of a CCDC154 inhibitor in the preparation of a product with at least one of the following effects: treatment of liver cancer; Inhibit the proliferation rate and proliferation activity of liver cancer cells; Promote liver cancer cell apoptosis; Inhibit liver cancer cell clone; Inhibit the growth of liver cancer; The CCDC154 inhibitor is selected from double-stranded RNA or shRNA, the shRNA or double-stranded RNA target sequence is shown in SEQ ID NO: 1, the double-stranded RNA comprises a first strand and a second strand, the first strand It is complementary to the second strand to form an RNA dimer, the sequence of the first strand is shown in SEQ ID NO: 2, and the nucleotide sequence of the shRNA is shown in SEQ ID NO: 3. 2. The use of claim 1 , further comprising one or more of the following features: 1) The CCDC154 inhibitor refers to a molecule that has an inhibitory effect on CCDC154; 2) The CCDC154 inhibitor is the only active ingredient or one of the active ingredients of the product. 3. A nucleic acid molecule for reducing CCDC154 gene expression in liver cancer cells, the nucleic acid molecule comprising: a. double-stranded RNA containing a nucleotide sequence capable of hybridizing to the CCDC154 gene; or b. shRNA, containing a nucleotide sequence capable of hybridizing with the CCDC154 gene in the shRNA; Wherein, the double-stranded RNA includes a first strand and a second strand, and the first strand and the second strand complement each other to form an RNA dimer; the shRNA includes a sense strand fragment and an antisense strand fragment, and is connected The stem-loop structure of 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 shRNA or double-stranded RNA target sequence is shown in SEQ ID NO: 1, and the The sequence of the first strand of the double-stranded RNA is shown in SEQ ID NO: 2, and the nucleotide sequence of the shRNA is shown in SEQ ID NO: 3. 4. A CCDC154 gene interference nucleic acid construct, comprising a gene fragment encoding the shRNA in the nucleic acid molecule of claim 3, and capable of expressing the shRNA. 5. A CCDC154 gene interfering lentivirus, which is formed by viral packaging of the interfering nucleic acid construct of claim 4 with the assistance of a lentiviral packaging plasmid and a cell line. 6. The nucleic acid molecule according to claim 3, or the CCDC154 gene interference nucleic acid construct according to claim 4, or the CCDC154 gene interference lentivirus according to claim 5 in the preparation of a drug for the treatment of liver cancer, or in the preparation of reducing liver cancer cells. Application of the kit for CCDC154 gene expression. 7. A composition for treating liver cancer, its effective substance contains: The nucleic acid molecule according to claim 3; and/or, the CCDC154 gene interference nucleic acid construct according to claim 4; and/or, the CCDC154 gene interference lentivirus according to claim 5, and a pharmaceutically acceptable carrier, dilution agent or excipient.

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