Uses of human EIF3A gene and related drugs

Abstract

The present invention discloses the use of human EIF3A gene and related drugs. The present invention discloses the use of human EIF3A gene in tumor treatment, tumor diagnosis and drug preparation. The present invention further constructs human EIF3A gene small molecule interfering RNA, human EIF3A gene interfering nucleic acid construct, human EIF3A gene interfering lentivirus and discloses their uses. The siRNA or nucleic acid construct and lentivirus containing the siRNA sequence provided by the present invention can specifically inhibit the expression of human EIF3A gene, especially lentivirus, can efficiently infect target cells, efficiently inhibit the expression of EIF3A gene in target cells, thereby inhibiting the growth of tumor cells and promoting tumor cell apoptosis, which is of great significance in tumor treatment.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically to the use of human EIF3A gene and related drugs. Background Art

[0002] RNA interference (RNAi) is the use of short double-stranded RNA (dsRNA) composed of nucleotides to perform post-transcriptional gene silencing. It can efficiently and specifically block the expression of specific genes in the body, leading to their degradation, thereby causing the silencing of specific genes in the body, causing cells to show the absence of certain gene phenotypes. It is a common laboratory technology that has emerged in recent years to study gene function and find disease treatment methods. Studies have shown that double-stranded RNA with a length of 21-23nt can specifically cause RNAi at the transcriptional and post-transcriptional levels (Tuschl T, Zamore PD, Sharp PA, Bartel DP. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21to23nucleotide intervals. Cell 2000; 101: 25-33.). Although tumor patients undergo chemotherapy, radiotherapy and comprehensive treatment, the five-year survival rate is still very low. If the genes related to tumor onset and progression can be intervened, it will open up new avenues for tumor treatment. In recent years, RNAi has become an effective strategy for gene therapy of tumors. RNAi technology can be used to inhibit the expression of oncogenes, mutated tumor suppressor genes, cell cycle-related genes, anti-apoptosis-related genes, etc. to inhibit tumor progression (Uprichard, Susan L. The therapeutic potential of RNA interference. FEBS Letters 2005; 579: 5996-6007.).

[0003] Eukaryotic protein synthesis includes three stages: initiation, elongation, and termination. This complex physiological process is carried out on the ribosomes in the cytoplasm, and a series of protein factors called eukaryotic translation initiation factors (eifs) are involved. So far, 13 initiation factors have been discovered, among which eIF3, the largest in molecular weight, contains 8-11 subunits and plays an important role in the protein synthesis superinitiation pathway. In the absence of other initiation factors, eIF3 can bind to the ribosomal 40S small subunit to maintain the dissociation state of the 40S and 60S subunits, while also stabilizing and promoting the interaction between the ternary complex eIF2-GTP-methionine and the 5' mRNA [Kolupaeva VG, Unbehaun A, Lomakin IB, Hellen CU, Pestova TV. Binding of eukaryotic initiation factor 3 to ribosomal 40S subunits and its role in ribosomal dissociation and anti-association. RNA. 2005Apr; 11(4): 470-86. Zhou M, Sandercock AM, Fraser CS, Ridlova G, Stephens E, Schenauer MR, Yokoi-Fong T, BarskyD, Leary JA, Hershey JW, Doudna JA, Robinson CV. Mass spectrometry reveals modularity and a complete subunit interaction map of the eukaryotic translation factor eIF3. Proc Natl Acad Sci US A. 2008 Nov 25; 105 (47): 18139-44].

[0004] There are few reports on EIF3A in tumor-related fields. In order to study the correlation between EIF3A and tumors, the present invention selected a colon cancer cell model and used RNAi as a means to study the role of EIF3A in the occurrence and development of colon cancer. Summary of the invention

[0005] The purpose of the present invention is to disclose a treatment method and a drug related to human EIF3A gene, and to study the role of EIF3A gene in the survival and apoptosis of tumor cells by means of RNA interference (RNAi).

[0006] In the first aspect of the present invention, the role of the EIF3A gene in the occurrence and development of tumors is studied by means of RNA interference, and a method for inhibiting or reducing the growth, proliferation, differentiation and / or survival of tumor cells is disclosed. The method comprises: administering to tumor cells a molecule that can specifically inhibit the transcription or translation of the EIF3A gene, or can specifically inhibit the expression or activity of the EIF3A protein, thereby inhibiting the growth, proliferation, differentiation and / or survival of tumor cells.

[0007] The tumor cells are selected from tumor cells whose growth is associated with the expression or activity of EIF3A protein. Preferably, the tumor cells are selected from colon cancer.

[0008] In the method of inhibiting or reducing tumor cell growth, proliferation, differentiation and / or survival, the amount of the molecule administered is sufficient to reduce the transcription or translation of the EIF3A gene, or to reduce the expression or activity of the EIF3A protein. Further, the expression of the EIF3A gene is reduced by at least 50%, 80%, 90%, 95% or 99%.

[0009] The molecule can be selected from, but not limited to, nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs, antibody drugs, polypeptides, proteins or interfering lentiviruses.

[0010] The nucleic acid includes, but is not limited to, antisense oligonucleotides, double-stranded RNA (dsRNA), ribozymes, small interfering RNA (esiRNA) prepared by endoribonuclease III, or short hairpin RNA (shRNA).

[0011] The double-stranded RNA, ribozyme, esiRNA or shRNA contains the promoter sequence of the EIF3A gene or the information sequence of the EIF3A gene.

[0012] Further, the double-stranded RNA is a small interfering RNA (siRNA). The small interfering RNA comprises a first chain and a second chain, the first chain and the second chain complement each other to form an RNA dimer, and the sequence of the first chain is substantially identical to 15-27 consecutive nucleotide sequences in the EIF3A gene. The small interfering RNA can specifically bind to the mRNA fragment encoded by the target sequence and specifically silence the expression of the human EIF3A gene.

[0013] Furthermore, the first strand sequence of the small interfering RNA is substantially identical to the target sequence in the EIF3A gene. Preferably, the target sequence in the EIF3A gene contains a sequence as shown in any one of SEQ ID NO:1.

[0014] The target sequence in the EIF3A gene is the fragment in the EIF3A gene corresponding to the mRNA fragment that complementarily binds to the small interfering RNA when the small interfering RNA specifically silences the expression of the EIF3A gene.

[0015] Preferably, the EIF3A gene is derived from human.

[0016] The first aspect of the present invention also discloses the use of an isolated human EIF3A gene in the preparation or screening of tumor therapeutic drugs, or in the preparation of tumor diagnostic drugs.

[0017] Furthermore, the tumor is selected from colon cancer.

[0018] The use of the isolated EIF3A gene for preparing or screening tumor therapeutic drugs includes two aspects: first, the EIF3A gene is used as a target of drugs or preparations against tumor cells for preparing tumor therapeutic drugs or preparations; second, the EIF3A gene is used as a target of drugs or preparations against tumor cells for screening tumor therapeutic drugs or preparations.

[0019] The use of the EIF3A gene as a drug or preparation target for tumor cells in the preparation of tumor treatment drugs or preparations specifically refers to: using the EIF3A gene as a target for RNA interference to develop drugs or preparations for tumor cells, thereby reducing the expression level of the EIF3A gene in tumor cells.

[0020] The use of the EIF3A gene as a drug or preparation for the target of tumor cells for screening tumor treatment drugs or preparations specifically refers to: using the EIF3A gene as the target, screening drugs or preparations to find drugs that can inhibit or promote the expression of human EIF3A gene as alternative drugs for tumor treatment. The EIF3A gene small molecule interfering RNA (siRNA) described in the present invention is obtained by screening with the human EIF3A gene as the target, and can be used as a drug with the effect of inhibiting tumor cell proliferation. In addition, antibody drugs, small molecule drugs, etc. can also use the EIF3A gene and its protein as the target.

[0021] The use of the EIF3A gene for preparing tumor diagnostic drugs refers to using the EIF3A gene expression product as a tumor diagnostic indicator in the preparation of tumor diagnostic drugs.

[0022] The expression level of EIF3A gene in tumor tissue, normal tissue and normal tissue around the tumor was detected by immunohistochemistry. The study found that the expression level of EIF3A gene in tumor tissue was significantly higher than that in normal tissue and normal tissue around the tumor. This suggests that EIF3A gene may play an important role in the occurrence and development of tumors as an oncogene; the expression level of EIF3A gene may become a marker for tumor diagnosis.

[0023] The tumor therapeutic drug is a molecule that can specifically inhibit the transcription or translation of the EIF3A gene, or can specifically inhibit the expression or activity of the EIF3A protein, thereby reducing the expression level of the EIF3A gene in tumor cells, thereby achieving the purpose of inhibiting the proliferation, growth, differentiation and / or survival of tumor cells.

[0024] The tumor therapeutic drugs or tumor diagnostic drugs prepared or screened by the isolated EIF3A gene include but are not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs, antibody drugs, polypeptides, proteins or interfering lentiviruses.

[0025] The nucleic acid includes, but is not limited to, antisense oligonucleotides, double-stranded RNA (dsRNA), ribozymes, small interfering RNA (esiRNA) prepared by endoribonuclease III, or short hairpin RNA (shRNA).

[0026] The dosage of the tumor therapeutic drug is sufficient to reduce the transcription or translation of the human EIF3A gene, or the expression or activity of the human EIF3A protein, so that the expression of the human EIF3A gene is reduced by at least 50%, 80%, 90%, 95% or 99%.

[0027] The method of treating tumors with the above tumor therapeutic drugs is mainly to achieve the purpose of treatment by reducing the expression level of human EIF3A gene to inhibit the proliferation of tumor cells. Specifically, during treatment, a substance that can effectively reduce the expression level of human EIF3A gene is administered to the patient.

[0028] The second aspect of the present invention discloses an isolated nucleic acid molecule for reducing the expression of the EIF3A gene in tumor cells, the nucleic acid molecule comprising:

[0029] a) double-stranded RNA, wherein the double-stranded RNA contains a nucleotide sequence capable of hybridizing with the EIF3A gene under stringent conditions; or

[0030] b) shRNA, wherein the shRNA contains a nucleotide sequence capable of hybridizing with the EIF3A gene under stringent conditions.

[0031] Further, the double-stranded RNA comprises a first strand and a second strand, the first strand and the second strand are complementary to each other to form an RNA dimer, and the sequence of the first strand is substantially identical to 15-27 consecutive nucleotide sequences in the EIF3A gene. Preferably, the sequence of the first strand is substantially identical to 19-23 consecutive nucleotide sequences in the EIF3A gene; more preferably, the sequence of the first strand is substantially identical to 19, 20 or 21 consecutive nucleotide sequences in the EIF3A gene.

[0032] Furthermore, the double-stranded RNA comprises a first strand and a second strand, the first strand and the second strand are complementary to each other to form an RNA dimer, and the sequence of the first strand is substantially identical to the target sequence in the EIF3A gene.

[0033] The length of the first strand and the second strand of the double-stranded RNA are both 15-27 nucleotides; preferably, the length is both 19-23 nucleotides; most preferably, the length is both 19, 20 or 21 nucleotides.

[0034] Furthermore, the double-stranded RNA is small interfering RNA (siRNA).

[0035] Furthermore, the sequence of the first strand of the small interfering RNA is as shown in SEQ ID NO: 13,

[0036] Specifically, it is 5'-AGGGCAGAAGAACAAAUGCUA-3'.

[0037] The siRNA shown in SEQ ID NO: 13 is one chain of a small interfering RNA targeting the human EIF3A gene, which is designed with the sequence shown in SEQ ID NO: 1 as the RNA interference target sequence. The sequence of the other chain, i.e., the second chain, is complementary to the sequence of the first chain. The siRNA can specifically silence the expression of the endogenous EIF3A gene in tumor cells.

[0038] Further, the shRNA includes a sense strand segment and an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, the sequences of the sense strand segment and the antisense strand segment are complementary, and the sequence of the sense strand segment is substantially identical to 15-27 consecutive nucleotide sequences in the EIF3A gene. The shRNA can be processed into a small interfering RNA (siRNA) to specifically silence the expression of the endogenous EIF3A gene in tumor cells.

[0039] Furthermore, the shRNA includes a sense chain fragment and an antisense chain fragment, and a stem-loop structure connecting the sense chain fragment and the antisense chain fragment, the sequences of the sense chain fragment and the antisense chain fragment are complementary, and the sequence of the sense chain fragment is substantially identical to the target sequence in the EIF3A gene.

[0040] Preferably, the positive strand fragment is substantially identical to 19-23 consecutive nucleotide sequences in the EIF3A gene; more preferably, the positive strand fragment is substantially identical to 19, 20 or 21 consecutive nucleotide sequences in the EIF3A gene.

[0041] Furthermore, 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.

[0042] Furthermore, the sequence of the shRNA is shown in SEQ ID NO: 14, specifically: 5'-AGGGCAGAAGAACAAAUGCUAUUCAAGAGAUAGCAUUUGUUCUUCUGCCCU-3'.

[0043] After enzymatic processing, shRNA can become siRNA, which can specifically silence the expression of endogenous human EIF3A gene in tumor cells.

[0044] The interfering lentiviral vector encoding the gene fragment of the shRNA of the present invention contains the sequence of SEQ ID NO: 1 and its complementary sequence.

[0045] The first strand of the double-stranded RNA or the positive strand fragment of the shRNA is substantially identical to the target sequence in the EIF3A gene. The target sequence of the EIF3A gene is the fragment in the EIF3A gene corresponding to the mRNA fragment recognized and silenced by the siRNA when the siRNA is used to specifically silence the expression of the EIF3A gene.

[0046] Preferably, the target sequence in the EIF3A gene contains the sequence of SEQ ID NO:1.

[0047] Furthermore, the EIF3A gene is derived from human.

[0048] The third aspect of the present invention discloses an EIF3A gene interference nucleic acid construct, which contains a gene fragment encoding the shRNA in the isolated nucleic acid molecule of the present invention and is capable of expressing the shRNA.

[0049] The human EIF3A gene interference nucleic acid construct can be obtained by cloning the gene fragment encoding the aforementioned human EIF3A gene shRNA into a known vector. Further, the EIF3A gene interference nucleic acid construct is an EIF3A gene interference lentiviral vector.

[0050] The EIF3A gene interference lentiviral vector of the present invention is obtained by cloning a DNA fragment encoding the aforementioned EIF3A gene shRNA into a known vector, wherein the known vector is mostly a lentiviral vector. The EIF3A gene interference lentiviral vector is packaged into infectious viral particles, which infect tumor cells and then transcribe the shRNA of the present invention. Through steps such as enzyme cutting and processing, the siRNA is finally obtained, which is used to specifically silence the expression of the EIF3A gene.

[0051] Furthermore, the EIF3A gene interference lentiviral vector also contains a promoter sequence and / or a nucleotide sequence encoding a marker that can be detected in tumor cells; preferably, the detectable marker is such as green fluorescent protein (GFP).

[0052] 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-CMV-TagYFP, pLKO.1-puro-CMV-TagRFP, pLKO.1-puro-CMV-TagFP635, pLKO.1-puro-CMV-Tag KO.1-puro-UbC-TagFP635, pLKO-puro-IPTG-1xLacO, pLKO-puro-IPTG-3xLacO, pLP1, pLP2, pLP / VSV-G, pENTR / U6, pLenti6 / BLOCK-iT-DES T, any of pLenti6-GW / U6-laminshrna, pcDNA1.2 / V5-GW / lacZ, pLenti6.2 / N-Lumio / V5-DEST, pGCSIL-GFP or pLenti6.2 / N-Lumio / V5-GW / lacZ.

[0053] The embodiment of the present invention specifically lists a human EIF3A gene interference lentiviral vector constructed using pGCSIL-GFP as a vector, which is named pGCSIL-GFP-EIF3A-siRNA.

[0054] The isolated nucleic acid molecule of the present invention can be used to prepare a drug for preventing or treating a tumor, wherein the tumor is colon cancer.

[0055] The EIF3A gene siRNA of the present invention can be used to inhibit the proliferation of tumor cells, and further can be used as a drug or preparation for treating tumors. The EIF3A gene interference lentiviral vector can be used to prepare the EIF3A gene siRNA. When used as a drug or preparation for treating tumors, 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 and the patient's health status, which are all within the skill range of skilled physicians.

[0056] In a fourth aspect, the present invention discloses an EIF3A gene interference lentivirus, which is formed by virus packaging of the aforementioned EIF3A gene interference lentivirus vector with the assistance of a lentivirus packaging plasmid and a cell line. The lentivirus can infect tumor cells and produce small interfering RNA targeting the EIF3A gene, thereby inhibiting the proliferation of colon cancer cells. The EIF3A gene interference lentivirus can be used to prepare a drug for preventing or treating tumors.

[0057] In a fifth aspect, the present invention discloses a pharmaceutical composition for preventing or treating tumors, wherein the effective substance comprises a combination of one or more of the aforementioned isolated nucleic acid molecules, EIF3A gene interfering nucleic acid constructs or EIF3A gene interfering lentiviruses.

[0058] Furthermore, the pharmaceutical composition contains 1 to 99 wt % of the double-stranded RNA, shRNA, EIF3A gene interfering nucleic acid construct or EIF3A gene interfering lentivirus, and a pharmaceutically acceptable carrier, diluent or excipient.

[0059] When preparing these compositions, active ingredient is usually mixed with excipient, or diluted with excipient, or wrapped in a carrier that can exist in capsule or sachet form. When excipient acts as a diluent, it can be a solid, semisolid or liquid material as a medium for excipient, carrier or active ingredient. Therefore, the composition can be a tablet, a pill, a powder, a solution, a syrup, a sterile injection solution, etc. The example of suitable excipient includes: lactose, glucose, sucrose, sorbitol, mannitol, starch, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, etc. Preparations can also include: wetting agents, emulsifiers, preservatives (such as methyl hydroxybenzoate and propyl ester), sweeteners, etc.

[0060] The invention also discloses the application of the pharmaceutical composition in preparing a tumor therapeutic drug for treating colon cancer.

[0061] The use of the pharmaceutical composition provides a method for treating tumors, specifically a method for preventing or treating tumors in a subject, comprising administering an effective dose of the pharmaceutical composition to the subject. Further, the tumor is selected from colon cancer.

[0062] When the pharmaceutical composition is used to prevent or treat a tumor in a subject, an effective dose of the pharmaceutical composition needs to be administered to the subject. Using this method, the growth, proliferation, recurrence and / or metastasis of the tumor 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 tumor is inhibited.

[0063] The subject of the method may be a human.

[0064] In a sixth aspect, the present invention discloses a kit for reducing the expression of the EIF3A gene in tumor cells, the kit comprising: the isolated nucleic acid molecule, the EIF3A gene interfering nucleic acid construct, and / or the EIF3A gene interfering lentivirus present in a container.

[0065] In summary, the present invention designs an RNAi target sequence for human EIF3A gene, constructs a corresponding EIF3A RNAi vector, wherein the RNAi vector pGCSIL-GFP-EIF3A-siRNA encoding the sequence SEQ ID NO: 1 can significantly downregulate the expression of EIF3A gene at the mRNA level and protein level. Using lentivirus (abbreviated as Lv) as a gene manipulation tool to carry the RNAi vector pGCSIL-GFP-EIF3A-siRNA can efficiently introduce the RNAi sequence for the EIF3A gene into human colon cancer RKO cells, reduce the expression level of the EIF3A gene, and significantly inhibit the proliferation ability of the above tumor cells. Therefore, lentivirus-mediated EIF3A gene silencing is a potential clinical non-surgical treatment for malignant tumors.

[0066] The siRNA or the nucleic acid construct or lentivirus comprising the siRNA sequence provided by the present invention can specifically inhibit the expression of the human EIF3A gene, especially the lentivirus, which can efficiently infect target cells, efficiently inhibit the expression of the EIF3A gene in target cells, promote cell apoptosis, reduce the invasion and metastasis ability of tumor cells, etc., thereby inhibiting the growth of tumor cells and promoting tumor cell apoptosis, which is of great significance in tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 Represents the pGCSIL-GFP plasmid DNA map.

[0068] Figure 2 It indicates that the EIF3A gene is highly expressed in 5 colon cancer cell lines.

[0069] Figure 3 It indicates that 4 days after EIF3A-RNAi lentivirus infected colon cancer cells, the expression level of EIF3A mRNA in human colon cancer RKO cells was reduced by 80% compared with the control lentivirus infected group.

[0070] Figure 4 This indicates that knocking down the EIF3A gene leads to inhibition of colon cancer cell proliferation. Four days after infection with EIF3A-RNAi lentivirus and control lentivirus, human colon cancer RKO cells were re-plated and cultured for 5 days. Compared with the control group, the number of viable cells decreased by 96.8%.

[0071] Figure 5 Four days after EIF3A-RNAi lentivirus infected human colon cancer RKO cells, the cell cycle of colon cancer cells changed. Compared with the control group, the number of cells in the G1 and S phases increased significantly, while the number of cells in the G2 / M phase decreased significantly.

[0072] Figure 6 Four days after EIF3A-RNAi lentivirus infected human colon cancer RKO cells, it led to increased apoptosis of colon cancer cells. Compared with the control group, the number of apoptotic cells increased significantly, and the proportion of apoptotic cells increased by more than 2 times.

[0073] Figure 7 Ten days after EIF3A-RNAi lentivirus infected human colon cancer RKO cells, the number of colonies of human colon cancer RKO cells was significantly reduced compared with the control group, and the clone formation ability was reduced.

[0074] Figure 8 After EIF3A-RNAi lentivirus infected human colon cancer RKO cells for 4 days, the migration ability of human colon cancer RKO cells decreased by about 45%. DETAILED DESCRIPTION

[0075] The present invention relates to a group of small interfering RNA (siRNA) sequences, RNA interference vectors and RNA interference lentiviruses targeting human EIF3A gene. The human EIF3A mRNA coding region sequence is selected as the target site of siRNA, and the siRNA target sequence is designed according to the continuous 10-30 (preferably 15-27, more preferably 19-23) base sequences in the target site. Through gene cloning, a nucleic acid construct expressing the above siRNA is constructed, and a lentivirus expressing the above siRNA is packaged. Cell experiments have shown that the above siRNA sequence can specifically silence the expression of endogenous EIF3A gene in human tumor cells.

[0076] The inventors of the present invention have found through extensive and in-depth research that the EIF3A gene is significantly highly expressed in tumor tissues; the inventors have found that downregulating the expression of the human EIF3A gene using the RNAi method can effectively inhibit the proliferation of tumor cells, promote cell apoptosis, reduce the invasion and metastasis of tumor cells, etc., and can effectively control the growth process of tumors. This research result shows that the EIF3A gene is a proto-oncogene and can be used as a target for tumor treatment. The inventors further synthesized and tested a variety of siRNAs targeting the EIF3A gene, screened out siRNAs that can effectively inhibit the expression of EIF3A and thus inhibit the proliferation and growth of colon cancer RKO cells, and completed the present invention on this basis.

[0077] The present invention provides a series of small interfering RNA (siRNA) sequences that interfere with the human EIF3A gene, and constructs a lentivirus that can specifically silence the expression of the EIF3A gene. The present invention has found that the small interfering RNA and RNAi lentivirus designed for the human EIF3A gene stably and specifically downregulate the expression of the EIF3A gene, and effectively inhibit the proliferation of human tumor cells. The present invention shows that the EIF3A gene can promote the growth of tumor cells and is expected to become a target for early diagnosis and treatment of tumors. Moreover, silencing the expression of the EIF3A gene by RNAi can be used as an effective means to inhibit tumor development.

[0078] The design idea of ​​the present invention is:

[0079] The invention obtains a human EIF3A gene RNAi lentivirus by screening through the following method: retrieving the human EIF3A gene sequence from Genbank; predicting siRNA sites; synthesizing an effective siRNA sequence for the EIF3A gene and a double-stranded DNA oligo with sticky ends containing restriction sites at both ends; connecting the lentiviral vector with the double-stranded DNA oligo after double restriction digestion to construct an RNAi plasmid expressing the EIF3A gene siRNA sequence; and co-transfecting the RNAi plasmid and an auxiliary vector (Packing Mix, Sigma-aldrich Company) required for lentiviral packaging into human embryonic kidney cells 293T to generate recombinant lentiviral particles, thereby obtaining a lentivirus for efficiently silencing the EIF3A gene.

[0080] Based on the above method, the present invention provides an effective target site for interfering with the EIF3A gene (specifically shown in SEQ ID NO: 1), and constructs a lentivirus that specifically interferes with the human EIF3A gene.

[0081] At the same time, the present invention also discloses a human EIF3A gene RNAi lentivirus (EIF3A-RNAi) and its preparation and application.

[0082] This study found that using the lentivirus-mediated RNAi method, after reducing the expression of the EIF3A gene in tumor cells, the proliferation of tumor cells can be effectively inhibited. This study shows that the EIF3A gene is a proto-oncogene that can promote tumor cell proliferation and has important biological functions in tumor occurrence and development. The EIF3A gene can be a target for tumor treatment, and lentivirus-mediated EIF3A gene-specific silencing can be used as a new means of tumor treatment.

[0083] The present invention is further described below in conjunction with the examples. It should be understood that the examples are only used to illustrate the present invention, and are not intended to limit the scope of the present invention. The experimental methods and reagents without specific conditions in the examples are all carried out or configured according to conventional conditions, such as the conditions described in Sambrook.J et al., Molecular Cloning Experiment Guide, 3rd Edition, Beijing: Science Press, 2002, or the conditions recommended by the manufacturer.

[0084] Example 1 Preparation of RNAi Lentivirus Targeting Human EIF3A Gene

[0085] 1. Screening of effective siRNA targets for human EIF3A gene

[0086] The EIF3A (NM_00375) gene information was retrieved from Genbank; effective siRNA targets for the EIF3A gene were designed. Table 1 lists one of the effective siRNA target sequences for the EIF3A gene.

[0087] Table 1 siRNA target sequences targeting human EIF3A gene

[0088]

[0089] 2. Preparation of Lentiviral Vectors

[0090] A double-stranded DNA oligo sequence (Table 2) containing Age I and EcoR I restriction endonucleases at both ends was synthesized for the siRNA target (taking SEQ ID NO: 1 as an example); Age I and EcoR I restriction endonucleases were used to act on the pGCSIL-GFP vector (provided by Shanghai Jikai Gene Chemistry Technology Co., Ltd. Figure 1 ) to linearize it, and the digested fragments were identified by agarose gel electrophoresis.

[0091] Table 2 Double-stranded DNA oligonucleotides with Age I and EcoR I restriction sites at both ends

[0092]

[0093] The double-digested linearized vector DNA (digestion system as shown in Table 4, 37°C, reaction for 1h) and the purified double-stranded DNA Oligo were connected by T4 DNA ligase, and connected overnight at 16°C in an appropriate buffer system (connection system as shown in Table 5), and the connection product was recovered. The connection product was transformed into fresh Escherichia coli competent cells prepared with calcium chloride (transformation operation reference: Molecular Cloning Experiment Guide Second Edition Page 55-56). The surface of the bacterial clone grown from the connection transformation product was stained, dissolved in 10μl LB culture medium, mixed and taken 1μl as a template; universal PCR primers were designed upstream and downstream of the RNAi sequence in the lentiviral vector, the upstream primer sequence: 5'-CCTATTTCCCATGATTCCTTCATA-3' (SEQ ID NO: 6); downstream primer sequence: 5'-GTAATACGGTTATCCACGCG-3' (SEQ ID NO: 7), and PCR identification experiments were performed (PCR reaction system as shown in Table 6-1, reaction conditions as shown in Table 6-2). The clones identified as positive by PCR were sequenced and compared. The clones with correct comparison were the successfully constructed vectors expressing RNAi against SEQ ID NO: 1, which were named pGCSIL-GFP-EIF3A-siRNA.

[0094] 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 for the Scr siRNA target (Table 3), and the rest of the construction method, identification method and conditions were the same as pGCSIL-GFP-EIF3A-siRNA.

[0095] Table 3 Double-stranded DNA oligonucleotides with Age I and EcoR I restriction sites at both ends

[0096]

[0097] The double-digested linearized vector was ligated with T4 DNA ligase (the digestion system is shown in Table 4, 37°C, reaction for 1 h)

[0098] Table 4 pGCSIL-GFP plasmid restriction enzyme digestion reaction system

[0099] Reagents Volume (μl) pGCSIL-GFP plasmid (1 μg / μl) 2.0 10×buffer 5.0 100×BSA 0.5 Age I (10U / μl) 1.0 EcoRI (10U / μl) 1.0 <![CDATA[dd H2O]]> 40.5 Total 50.0

[0100] Table 5 Vector DNA and double-stranded DNA Oligo ligation reaction system

[0101] Reagents Positive control (μl) Self-ligation control (μl) Connectome (μl) Linearized vector DNA (100 ng / μl) 1.0 1.0 1.0 Annealed double-stranded DNA Oligo (100ng / μl) 1.0 - 1.0 10×T4 phage DNA ligase buffer 1.0 1.0 1.0 T4 bacteriophage DNA ligase 1.0 1.0 1.0 <![CDATA[dd H2O]]> 16.0 17.0 16.0 Total 20.0 20.0 20.0

[0102] Table 6-1 PCR reaction system

[0103] Reagents Volume (μl) 10×buffer 2.0 dNTPs (2.5 mM) 0.8 Upstream primer 0.4 Downstream primer 0.4 Taq polymerase 0.2 template 1.0 <![CDATA[ddH2O]]> 15.2 Total 20.0

[0104] Table 6-2 PCR reaction system program settings

[0105]

[0106] 3. Packaging EIF3A-siRNA Lentivirus

[0107] The DNA of RNAi plasmid pGCSIL-GFP-EIF3A-siRNA was extracted using Qiagen's plasmid extraction kit and prepared into a 100 ng / μl storage solution.

[0108] 24 h before transfection, human embryonic kidney 293T cells in logarithmic growth phase were digested with trypsin and the cell density was adjusted to 1.5×10 5 cells / ml, inoculated into 6-well plates, cultured in a 37°C, 5% CO2 incubator. When the cell density reaches 70%-80%, it can be used for transfection. 2h before transfection, aspirate the original culture medium and add 1.5ml of fresh complete culture medium. According to the instructions of Sigma-aldrich's MISSION Lentiviral Packaging Mix kit, add 20μl of Packing Mix (PVM), 12μl of PEI, and 400μl of serum-free DMEM culture medium to a sterile centrifuge tube, take 20μl of the above-extracted plasmid DNA, and add it to the above-mentioned PVM / PEI / DMEM mixture.

[0109] The transfection mixture was incubated at room temperature for 15 minutes, transferred to the culture medium of human embryonic kidney cell 293T cells, and cultured in a 37°C, 5% CO2 incubator for 16 hours. The culture medium containing the transfection mixture was discarded, washed with PBS solution, and 2 ml of complete culture medium was added. Since the lentiviral vector itself carries a green fluorescent protein reporter gene, the expression of GFP in the two groups of cells can be observed under a fluorescence microscope after 24 hours to determine whether the lentiviral plasmid has been packaged by 293T cells. After 48 hours of continuous culture, the cell supernatant was collected and purified and concentrated using a Centricon Plus-20 centrifugal ultrafiltration device (Millipore) in the following steps: (1) centrifuge at 4000g for 10 minutes at 4°C to remove cell debris; (2) filter the supernatant through a 0.45 μm filter into a 40 ml ultracentrifuge tube; (3) centrifuge at 4000g for 10-15 minutes to the required virus concentration volume; (4) after the centrifugation, separate the filter cup from the filtrate collection cup below, invert the filter cup on the sample collection cup, and centrifuge for 2 minutes at a centrifugal force not exceeding 1000g; (5) remove the centrifuge cup from the sample collection cup, and the sample collection cup contains the virus concentrate. The virus concentrate was aliquoted and stored at -80 degrees Celsius. The sequence of the first strand of siRNA contained in the virus concentrate is shown in SEQ ID NO: 13. The packaging process of the control lentivirus was the same as that of the EIF3A-siRNA lentivirus, except that the pGCSIL-GFP-Scr-siRNA vector was used instead of the pGCSIL-GFP-EIF3A-siRNA vector.

[0110] Example 2 Detection of EIF3A expression abundance in 5 colon cancer cells

[0111] Five colon cancer cell lines HCT116, RKO, SW480, SW620, and LOVO in the logarithmic growth phase were digested with trypsin to prepare cell suspensions (cell number was about 5×10 4 / ml) were inoculated in a 6-well plate and cultured until the cell confluence reached about 30%. According to the infection multiplicity (MOI, RKO: 10), an appropriate amount of the virus prepared in Example 1 was added, and the culture medium was replaced after 24 hours of culture. After the infection time reached 4 days, the cells were collected. According to the Trizol operating instructions of Invitrogen, total RNA was extracted. The specific method is as follows:

[0112] 1) Collect cells, centrifuge at 1000 rpm for 5 min, remove supernatant, add 1 ml Trizol to the cell pellet, quickly pipette and let stand at room temperature for 5 min, then transfer to a new 1.5 ml eppendorf tube;

[0113] 2) Add 200 μl of chloroform to each tube, invert the eppendorf tube by hand for 15 seconds, and let it stand at room temperature for 10 minutes;

[0114] 3) Centrifuge at 4°C, 12,000 rpm for 15 min;

[0115] 4) Pipette the supernatant into a new 1.5 ml eppendorf tube, add an equal volume of pre-cooled isopropanol, mix well and precipitate at 4°C for 10 min;

[0116] 5) Centrifuge at 4°C, 12,000 rpm for 10 min and discard the supernatant;

[0117] 6) Add at least 1 ml of 75% ethanol (freshly prepared with DEPC-treated water) to wash the precipitate;

[0118] 7) Centrifuge at 10,000 rpm for 5 min at 4°C and discard most of the supernatant;

[0119] 8) Centrifuge again at 4°C, 10,000 rpm for 5 min, and remove the supernatant;

[0120] 9) Dry at room temperature;

[0121] 10) When the RNA is basically transparent, add 20 μl of RNase-free water until it is completely dissolved, and determine the concentration of the extracted RNA by ultraviolet analysis.

[0122] Then, according to the M-MLV operating instructions of Promega, RNA was reverse transcribed to obtain cDNA. M-MLV reverse transcriptase and dNTPs were purchased from Promega, Oligo dT was purchased from Shanghai Bioengineering, and RNase-free materials were purchased from Axygen. The specific steps are as follows:

[0123] 1) Add 1 μl Oligo dT (0.5 μg / μl) and 2.0 μg Total RNA to a PCR tube, add DEPC-H2O to 9 μl; mix well and centrifuge, incubate at 70°C for 10 min; then immediately place in a 0°C ice-water mixture for an ice bath to allow OligodT to anneal with the template;

[0124] 2) Prepare the reaction system according to the ratio in Table 7 (on ice), mix well, and centrifuge briefly;

[0125] 3) The above system was reacted at 42°C for 1 h, and then placed in a water bath at 70°C for 10 min to inactivate the RT enzyme;

[0126] 4) The obtained RT product - cDNA was stored at -80°C for future use.

[0127] Finally, a TP800 Real time PCR instrument (TAKARA) was used for real-time quantitative detection. The primers for the EIF3A gene are as follows: upstream primer 5'-GCCAAATAATCACCAACA-3' (SEQ ID NO: 9) and downstream primer 5'-CTGGTTCTGAGCAGGTTC-3' (SEQ ID NO: 10). The housekeeping gene GAPDH was used as an internal reference, and the primer sequences were as follows: upstream primer 5'-TGACTTCAACAGCGACACCCA-3' (SEQ ID NO: 11) and downstream primer 5'-CACCCTGTTGCTGTAGCCAAA-3' (SEQ ID NO: 12). The reaction system was configured according to the ratio in Table 8.

[0128] The program was set to two-step Real-time PCR: pre-denaturation at 95°C for 15s; denaturation at 95°C for 5s in each step; annealing and extension at 60°C for 30s; a total of 45 cycles. The absorbance was read each time during the extension phase. After the PCR was completed, denaturation was performed at 95°C for 1min, and then cooled to 55°C to allow the DNA double strands to fully bind. From 55°C to 95°C, the temperature was increased by 0.5°C in each step, maintained for 4s, and the absorbance was read at the same time to create a melting curve. 2- ΔΔCt The expression abundance of infected EIF3A mRNA was calculated by the analysis method. Figure 2 As shown, the EIF3A gene was highly expressed in 5 colon cancer cell lines.

[0129] Example 3 Real-time fluorescence quantitative RT-PCR method to detect the silencing efficiency of EIF3A gene

[0130] Colon cancer RKO cells in the logarithmic growth phase were digested with trypsin and prepared into a cell suspension (cell number was about 5×10 4 / ml) were inoculated in a 6-well plate and cultured until the cell confluence reached about 30%. According to the infection multiplicity (MOI, RKO: 10), an appropriate amount of the virus prepared in Example 1 was added, and the culture medium was replaced after 24 hours of culture. After the infection time reached 4 days, the cells were collected. According to the Trizol operating instructions of Invitrogen, total RNA was extracted. Then, according to the M-MLV operating instructions of Promega, RNA was reverse transcribed to obtain cDNA (the reverse transcription reaction system is shown in Table 7, 42°C reaction for 1 hour, and then in a water bath at 70°C for 10 minutes to inactivate the reverse transcriptase).

[0131] Real-time quantitative detection was performed using a TP800 Real time PCR instrument (TAKARA). The primers for the EIF3A gene were as follows: upstream primer 5'-GCCAAATAATCACCAACA-3' (SEQ ID NO: 9) and downstream primer 5'-CTGGTTCTGAGCAGGTTC-3' (SEQ ID NO: 10). The housekeeping gene GAPDH was used as an internal reference, and the primer sequences were as follows: upstream primer 5'-TGACTTCAACAGCGACACCCA-3' (SEQ ID NO: 11) and downstream primer 5'-CACCCTGTTGCTGTAGCCAAA-3' (SEQ ID NO: 12). The reaction system was configured according to the ratio in Table 8.

[0132] Table 7 Reverse transcription reaction system

[0133] Reagents Volume (μl) 5×RT buffer 4.0 10mM dNTPs 2.0 RNasin 0.5 M-MLV-RTase 1.0 <![CDATA[DEPC H2O]]> 3.5 Total 11.0

[0134] Table 8 Real-time PCR reaction system

[0135] Reagents Volume (μl) SYBR premix ex taq: 10.0 Upstream primer (2.5 μM): 0.5 Downstream primer (2.5 μM): 0.5 cDNA 1.0 <![CDATA[ddH2O]]> 8.0 Total 20.0

[0136] The program was set to two-step Real-time PCR: pre-denaturation at 95°C for 15s; denaturation at 95°C for 5s in each step; annealing and extension at 60°C for 30s; a total of 45 cycles. The absorbance was read each time during the extension phase. After the PCR was completed, denaturation was performed at 95°C for 1min, and then cooled to 55°C to allow the DNA double strands to fully bind. From 55°C to 95°C, the temperature was increased by 0.5°C in each step, maintained for 4s, and the absorbance was read at the same time to create a melting curve. 2- ΔΔCt The expression abundance of EIF3A mRNA was calculated by the analysis method. Cells infected with the control virus (Lv-Scr-siRNA) were used as controls. Figure 3 , P<0.01) showed that the expression level of mRNA in colon cancer RKO cells was downregulated by 80.0%.

[0137] Example 4 Detection of the proliferation ability of tumor cells infected with EIF3A-siRNA lentivirus

[0138] Colon cancer RKO cells in the logarithmic growth phase were digested with trypsin and prepared into a cell suspension (cell number was about 5×10 4 / ml) were inoculated in 6-well plates and cultured until the cell confluence reached about 30%. According to the infection multiplicity (MOI, RKO: 10), an appropriate amount of virus was added, and the culture medium was replaced after 24 hours of culture. After the infection time reached 4 days, the cells in each experimental group in the logarithmic growth phase were collected. The cells were resuspended in complete medium into a cell suspension (2×10 4 / ml), inoculate 96-well plates at a cell density of about 2000 cells / well. Each group has 5 replicate wells, 100μl per well. After laying the plates, place them in a 37°C, 5% CO2 incubator for culture. Starting from the second day after laying the plates, read the plates once a day using a Cellomics instrument (Thermo Fisher), and continue reading the plates for 5 days. By adjusting the input parameters of the Cellomics arrayscan, the number of cells with green fluorescence in each scan of the well plate can be accurately calculated, and the data can be statistically plotted to draw a cell proliferation curve (the results are shown in Figure 2). Figure 4 The results showed that after 5 days of in vitro culture, the proliferation rate of each tumor in the lentivirus infection group slowed down significantly, which was much lower than that of the tumor cells in the control group, and the number of active cells decreased by more than 90.0%, indicating that EIF3A gene silencing led to the inhibition of tumor cell proliferation ability.

[0139] Example 5 Detection of cell cycle distribution of tumor cells infected with EIF3A-siRNA lentivirus

[0140] According to the steps in Example 3, the cells were infected with viruses, and three duplicate wells / 6cm dish were set up for each group. When the cells in each experimental group of 6cm dish grew to a coverage rate of about 80% (to ensure that the cells have not entered the growth plateau phase): the cell culture supernatant was discarded, the cells were washed once with D-Hanks, the cells were digested with trypsin, the complete culture medium was terminated, and the cells were collected in a 5ml centrifuge tube. After centrifugation to remove the supernatant and washing once with pre-cooled PBS, they were fixed with pre-cooled 70% ethanol overnight. The next day, after centrifugation to remove the fixative and washing once with PBS, the cells were resuspended in the cell staining solution [40×PI mother solution (2mg / ml): 100×RNase mother solution (10mg / ml): 1×PBS=25:10:1000]. The cell suspension was added to a 300-mesh screen and filtered into a flow cytometer tube for detection. By calculating the percentage of each phase, statistical graphs of the three phases of cells G1, S, and G2 / M were drawn (the results are shown in Figure 5 The results showed that the number of human colon cancer RKO cells in the G1 and S phases in the lentivirus infection group increased significantly, and the number of human colon cancer RKO cells in the G2 / M phase decreased significantly, indicating that EIF3A gene silencing led to a significant change in the cycle distribution of human colon cancer RKO cells.

[0141] Example 6 Detection of apoptosis level in tumor cells infected with EIF3A-siRNA lentivirus

[0142] After 4 days of virus infection according to the method in Example 3, the supernatant of cells in each experimental group after infection was collected in a 5 ml centrifuge tube, the cells were washed once with D-Hanks, the cells were digested with trypsin, the culture supernatant was terminated, and the cells were collected in the same 5 ml centrifuge tube. After centrifugation, the supernatant was discarded and the cell pellet was washed once with PBS and 1×binding buffer in sequence, and finally the cell pellet was resuspended with 1×staining buffer (the volume of staining buffer added was determined according to the amount of cell pellet, so that the final density of the cell suspension was 1x 10 6 ―1x10 7 Take 100ul of cell suspension (1x 10 5 —1x 10 6 Cells) were added with 5ulannexin V-APC for staining, and the cells were transferred to the flow cytometry tubes at room temperature in the dark for 10-15min and tested on the flow cytometry machine.

[0143] Perform scatter plot and peak type statistical analysis on flow cytometry data, draw apoptosis peak type and scatter plot (results as shown in Figure 6 The results showed that after 5 days of in vitro culture, the proportion of apoptotic cells in each tumor in the lentivirus infection group increased significantly, which was much higher than the apoptotic proportion of tumor cells in the control group. The proportion of apoptotic cells increased by more than 2 times, indicating that EIF3A gene silencing led to enhanced apoptosis ability of human colon cancer cells.

[0144] Example 7 Detection of the clone-forming ability of tumor cells infected with EIF3A-siRNA lentivirus

[0145] After 4 days of virus infection according to the method in Example 3, the cells of each experimental group in the logarithmic growth phase were trypsinized, resuspended in complete medium, and prepared into cell suspension and counted. In a 6-well culture plate, 800 cells / well were inoculated in each experimental group, and 3 replicate wells were set up for each experimental group. The inoculated cells were cultured in an incubator until 14 days or until the number of cells in most single clones was greater than 50. The medium was changed every 3 days and the cell status was observed. Before the experiment was terminated, the cell clones were photographed under a fluorescence microscope. At the end of the experiment, the cells were washed once with PBS, 1 ml of paraformaldehyde was added to each well, and the cells were fixed for 30 to 60 minutes and then washed once with PBS. 500 μL of clean, impurity-free GIEMSA dye solution was added to each well and the cells were stained for 20 minutes. Finally, the cells were washed several times with ddH2O until the background on the plate was washed clean, dried, and then single clones were photographed under a microscope, and the entire plate was photographed with a digital camera. A statistical graph of the number of cell clones was drawn by clone counting (such as Figure 7 The results showed that the number of cell clones in the lentivirus infection group was significantly reduced, far lower than that in the control group, and the number of cell clones decreased by about 70%, indicating that EIF3A gene silencing led to the inhibition of tumor cell clone formation ability.

[0146] Example 8 Detection of Transwell Migration Ability of Tumor Cells Infected with EIF3A-siRNA Lentivirus

[0147] Take the required number of chambers in an empty 24-well plate, add 100 μL of serum-free medium to the chamber, and place in the incubator for 1-2 hours. Prepare cell suspension: Trypsinize each group of cells in the logarithmic growth phase, resuspend with low serum medium to make a cell suspension, and count the cells of the cell suspension with a hemocytometer. Carefully remove the medium from the chamber and add 600 μL of medium containing 30% FBS (serum concentration can be adjusted as needed) to the lower chamber. Dilute the cells in a certain proportion with serum-free medium, and add 100uL of the cell suspension (containing 100,000-200,000 cells) to each chamber. Use tweezers to transfer the chamber into the lower chamber containing 30% FBS medium and culture in a tissue culture incubator for 4-24 hours. Invert the chamber on absorbent paper to remove the medium, and gently remove non-transferred cells with a cotton swab. Add 400 μl of staining solution to an empty well of a 24-well plate, and then soak the chamber in the staining solution for 20 minutes to stain the transfer cells on the lower surface of the membrane. Soak the chamber in a large cup of water and rinse several times. Air dry. Photograph the membrane under a microscope. Dissolve in 10% acetic acid and measure OD570. The results are as follows: Figure 8 As shown, 4 days after EIF3A-RNAi lentivirus infected human colon cancer RKO cells, the migration ability of human colon cancer RKO cells decreased by about 45%.

[0148] The above is only a preferred embodiment of the present invention, and is not any formal or substantial limitation of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any technician familiar with this profession, without departing from the spirit and scope of the present invention, can make some changes, modifications and evolutions of the technical content disclosed above, which are equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. Use of an isolated nucleic acid molecule in the preparation of a drug for reducing the expression of the EIF3A gene in colon cancer cells, wherein the nucleic acid molecule is shRNA, and the sequence of the shRNA is shown in SEQ ID NO:

14.

2. Use of an EIF3A gene interfering nucleic acid construct in the preparation of a drug for reducing EIF3A gene expression in colon cancer cells, wherein the EIF3A gene interfering nucleic acid construct contains a gene fragment encoding the shRNA in the isolated nucleic acid molecule according to claim 1 and can express the shRNA.

3. The use according to claim 2, characterized in that The EIF3A gene interfering nucleic acid construct is an interfering lentiviral vector.

4. Use of an interfering lentivirus in the preparation of a drug for reducing the expression of the EIF3A gene in colon cancer cells, wherein the interfering lentivirus is formed by viral packaging of the interfering lentiviral vector described in claim 3 with the assistance of a lentiviral packaging plasmid and a cell line.

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