CN110938692A

CN110938692A - Application of human IMPA2 gene and related product

Info

Publication number: CN110938692A
Application number: CN201911227266.9A
Authority: CN
Inventors: 王敏; 李先平; 杨敏; 张衎; 刘蕾
Original assignee: Second Xiangya Hospital of Central South University
Current assignee: Second Xiangya Hospital of Central South University
Priority date: 2019-12-04
Filing date: 2019-12-04
Publication date: 2020-03-31

Abstract

The invention belongs to the field of biomedical research, and particularly relates to application of a human IMPA2 gene as a target in preparation of a cervical cancer treatment drug or a cervical cancer diagnosis drug. The extensive and intensive research shows that the expression of the human IMPA2 gene is down-regulated by adopting an RNAi method, the proliferation of cervical cancer cells can be effectively inhibited, the apoptosis can be promoted, and the growth process of the cervical cancer can be effectively controlled. The siRNA or the nucleic acid construct containing the siRNA sequence and the lentivirus provided by the invention can specifically inhibit the proliferation rate of cervical cancer cells, promote apoptosis of the cervical cancer cells, inhibit cloning of the cervical cancer cells, influence the cycle of the cervical cancer cells and inhibit growth of the cervical cancer cells, thereby treating the cervical cancer and opening up a new direction for treating the cervical cancer.

Description

Application of human IMPA2 gene and related product

Technical Field

The invention belongs to the field of biomedical research, and particularly relates to application of a human IMPA2 gene and a related product.

Background

The IMPA2 gene encodes a phytase enzyme that catalyzes the dephosphorylation of inositol monophosphates to free inositol, an enzyme pathway that is critical in cellular function because inositol is the substrate for the synthesis of membrane lipid Phosphatidylinositol (PI). Dephosphorylation of inositol monophosphates is the rate-limiting step of PI regeneration [ Ohnishi t., Ohba h., Seo k.c., Im j., satoy, Iwayama y.spatial expression patterns and biochemical expression profile a second myo-inositol monophosphomutase IMPA2 from IMPA1.j biolchem.2007; 282(1) 637-646, Harwood A.J. Lithium and bipolar food dissorder, the inositols-removal hy recycled. mol Psychiatry.2005; 10(1):117-126.]. When cells are stimulated, PI 4, 5-diphosphate is hydrolyzed by phospholipase C, producing two second messengers:

inositol

1, 4, 5-trisphosphate (IP3) and diacylglycerol. IP3 triggers Ca (2+) release from intracellular stores and is multi-step dephosphorylated by multiple enzymes for inositol reuse [ Fisher s.k., Novak j.e., agrano ff b.w. inositols and herol phosphate in neural tissues: homeostatis, metabolism and functionalization j neurochem.2002; 82(4) 736-754, Rapoport S.I., Primiani C.T., ChenC.T., Ahn K., Ryan V.H. coordinated expression of phosphorus microorganisms with degradation and alignment of human dorsolateral precursor PLoS one.2015; 10(7)]. There are two genes of IMPase in mammals, the well-characterized myo-inositol monophosphatase 1(IMPA1) and myo-inositol monophosphatase 2(IMPA2) [ Ohnishi T., Ohba H., Seo K.C., Im J., Sato Y., Iwayama Y.spatial expression patterns and biochemical properties diagnosis second myo-inositol monophosphatase IMPA2 from IMP A1.J Biol chem.2007; 282(1):637-646.]. Until now, inositol has important clinical implications in the psychiatric field (e.g. bipolar disorder). Direct inhibition of IMPase has been used as a first-line drug in bipolar disorders in vivo and in vitro for half a century [ Berridge M.J., Downnes C.P., Hanley M.R.neural and translational actions of lithium: a uniform hypothesis.cell.1989; 59(3):411-419.]. The existing studies indicate that the IMPA2 gene is associated with leukemia and renal cancer.

At present, no report about the application of IMPA2 gene in cervical cancer treatment exists.

Disclosure of Invention

In order to overcome the problems in the prior art, the invention aims to provide the application of the human IMPA2 gene and related products.

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

the invention provides an application of a human IMPA2 gene as a target in preparing a cervical cancer treatment drug or a cervical cancer diagnosis drug.

The human IMPA2 gene as a target for preparing the cervical cancer treatment drug specifically comprises the following steps: the IMPA2 gene is used as an action object, and the medicine or preparation is screened to find the medicine which can inhibit the expression of the human IMPA2 gene and is used as the candidate medicine for treating the cervical cancer. The IMPA2 gene small interfering RNA (siRNA) is obtained by screening human IMPA2 gene as an action object and can be used as a medicine for inhibiting the proliferation of cervical cancer cells. In addition to this, for example, antibody drugs, small molecule drugs, etc. may also have the IMPA2 gene as an object of action.

The application of the human IMPA2 gene as a target in preparing the cervical cancer diagnosis medicine specifically comprises the following steps: the IMPA2 gene expression product is used as a cervical cancer diagnosis index to be applied to the preparation of cervical cancer diagnosis drugs.

The cervical cancer treatment drug is a molecule which can specifically inhibit the transcription or translation of an IMPA2 gene or specifically inhibit the expression or activity of an IMPA2 protein, so that the expression level of the IMPA2 gene in a cervical cancer cell is reduced, and the purposes of inhibiting the proliferation, growth, differentiation and/or survival of the cervical cancer cell are achieved.

The cervical cancer treatment drug or cervical cancer diagnosis drug prepared by IMPA2 gene includes but is not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs, antibody drugs, polypeptides, proteins, or interfering lentiviruses.

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

The administration amount of the cervical cancer therapeutic drug is a dose sufficient to reduce transcription or translation of the human IMPA2 gene, or to reduce expression or activity of the human IMPA2 protein. So that the expression of the human IMPA2 gene is reduced by at least 50%, 80%, 90%, 95% or 99%.

The method for treating the cervical cancer by adopting the cervical cancer treatment medicine mainly achieves the purpose of treatment by reducing the expression level of the IMPA2 gene of a human to inhibit the proliferation of cervical cancer cells. In particular, in therapy, a substance effective in reducing the expression level of the human IMPA2 gene is administered to a patient.

In one embodiment, the target sequence of the IMPA2 gene is as set forth in SEQ ID NO:1 is shown. The method specifically comprises the following steps: 5'-CTTACAGACGATTAACTAT-3' are provided.

In a second aspect of the present invention there is provided the use of an IMPA2 inhibitor in the manufacture of a product having at least one of the following effects:

treating cervical cancer;

inhibiting the proliferation rate of cervical cancer cells;

promoting apoptosis of cervical cancer cells;

inhibiting the cloning of cervical cancer cells;

affect the cycle of cervical cancer cells;

inhibiting the growth of cervical cancer.

The product necessarily comprises an IMPA2 inhibitor and an IMPA2 inhibitor as an active ingredient for the aforementioned effects.

In the product, the effective component for the above functions can be only an IMPA2 inhibitor, and can also comprise other molecules for the above functions.

That is, the IMPA2 inhibitor is the only active ingredient or one of the active ingredients of the product.

The product may be a single component material or a multi-component material.

The form of the product is not particularly limited, and can be various substance forms such as solid, liquid, gel, semifluid, aerosol and the like.

The product is primarily directed to mammals. The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, or the like. The primate is preferably a monkey, ape or human.

Such products include, but are not limited to, pharmaceuticals, nutraceuticals, foods, and the like.

The IMPA2 inhibitor can be nucleic acid molecule, antibody, small molecule compound.

As exemplified in the examples herein, the IMPA2 inhibitor may be a nucleic acid molecule that reduces expression of the IMPA2 gene in cervical cancer cells. Specifically, it may be a double-stranded RNA or shRNA.

In a third aspect of the invention there is provided a method of treating cervical cancer by administering to a subject an IMPA2 inhibitor.

The object can be a mammal or a cervical cancer cell of a mammal. The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, or the like. The primate is preferably a monkey, ape or human. The cervical cancer cell may be an isolated cervical cancer cell.

The subject may be a patient suffering from cervical cancer or an individual desiring treatment for cervical cancer. Or the object is an isolated cervical cancer cell of a cervical cancer patient or an individual expected to treat cervical cancer.

The IMPA2 inhibitor may be administered to a subject before, during, or after treatment for cervical cancer.

The fourth aspect of the invention discloses a nucleic acid molecule for reducing IMPA2 gene expression in cervical cancer cells, wherein the nucleic acid molecule comprises double-stranded RNA or shRNA.

Wherein the double-stranded RNA contains a nucleotide sequence capable of hybridizing with IMPA2 gene;

the shRNA contains a nucleotide sequence capable of hybridizing with IMPA2 gene.

Further, the double-stranded RNA comprises a first strand and a second strand, the first strand and the second strand are complementary to form an RNA dimer, and the sequence of the first strand is substantially identical to a target sequence in the IMPA2 gene.

The target sequence in the IMPA2 gene is a fragment in the IMPA2 gene corresponding to an mRNA fragment which is recognized and silenced by the nucleic acid molecule when the nucleic acid molecule is used for specifically silencing the expression of the IMPA2 gene.

Further, the target sequence of the double-stranded RNA is shown as SEQ ID NO:1 is shown. The method specifically comprises the following steps: 5'-CTTACAGACGATTAACTAT-3' are provided. Further, the sequence of the first strand of the double-stranded RNA is shown as SEQ ID NO:2, respectively. Specifically 5'-CUUACAGACGAUUAACUAU-3'.

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

SEQ ID NO:2 is designed by taking the sequence shown in SEQ ID NO. 1 as an RNA interference target sequence and aiming at one strand of small interfering RNA of the human IMPA2 gene, the sequence of the other strand, namely the second strand, is complementary with the sequence of the first strand, and the siRNA can play a role in specifically silencing the expression of endogenous IMPA2 gene in cervical cancer cells.

The shRNA includes a sense strand segment and an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, the sequences of the sense strand segment and the antisense strand segment are complementary, and the sequence of the sense strand segment is substantially identical to a target sequence in the IMPA2 gene.

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

The shRNA can become small interfering RNA (siRNA) after enzyme digestion and processing, and further plays a role in specifically silencing endogenous IMPA2 gene expression in cervical cancer cells.

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

Further, the sequence of the shRNA is shown as SEQ ID NO: 3, respectively. Specifically 5'-GCCUUACAGACGAUUAACUAUCUCGAGAUAGUUAAUCGUCUGUAAGGC-3'.

Further, the IMPA2 gene is derived from human.

In the fifth aspect of the invention, the invention discloses an IMPA2 gene interfering nucleic acid construct which contains a gene segment for coding shRNA in the nucleic acid molecule and can express the shRNA.

The IMPA2 gene interfering nucleic acid construct can be obtained by cloning a gene fragment coding the human IMPA2 gene shRNA into a known vector.

Further, the IMPA2 gene interference nucleic acid construct is an IMPA2 gene interference lentiviral vector.

The IMPA2 gene interference lentiviral vector disclosed by the invention is obtained by cloning a DNA fragment for coding the IMPA2 gene shRNA into a known vector, wherein the known vector is mostly a lentiviral vector, the IMPA2 gene interference lentiviral vector is packaged into infectious viral particles by viruses, then the cervical cancer cells are infected, the shRNA is further transcribed, and the siRNA is finally obtained through the steps of enzyme digestion processing and the like and is used for specifically silencing the expression of the IMPA2 gene.

Further, the IMPA2 gene interference lentiviral vector also contains a promoter sequence and/or a nucleotide sequence for encoding a marker which can be detected in the cervical cancer cell; preferably, the detectable label is Green Fluorescent Protein (GFP).

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

The embodiment of the invention specifically discloses a human IMPA2 gene interference lentiviral vector constructed by taking pGCSIL-GFP as a vector, which is named as pGCSIL-GFP-IMPA 2-siRNA.

The IMPA2 gene siRNA can be used for inhibiting the proliferation of cervical cancer cells, and further can be used as a medicament or a preparation for treating cervical cancer. The IMPA2 gene interference lentiviral vector can be used for preparing the IMPA2 gene siRNA. When used as a medicament or formulation for treating cervical cancer, a safe and effective amount of the nucleic acid molecule is administered to a mammal. The particular dosage will also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

The sixth aspect of the invention discloses an IMPA2 gene interference lentivirus, which is formed by virus packaging of the IMPA2 gene interference nucleic acid construct under the assistance of lentivirus packaging plasmids and cell lines. The lentivirus can infect cervical cancer cells and generate small interfering RNA aiming at IMPA2 gene, thereby inhibiting the proliferation of the cervical cancer cells. The IMPA2 gene interference lentivirus can be used for preparing medicines for preventing or treating cervical cancer.

In a seventh aspect of the present invention, there is provided a use of the aforementioned nucleic acid molecule, or the aforementioned IMPA2 gene interfering nucleic acid construct, or the aforementioned IMPA2 gene interfering lentivirus, wherein: is used for preparing a medicine for preventing or treating cervical cancer or a kit for reducing the expression of IMPA2 gene in cervical cancer cells.

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

Further, when the drug is used for preventing or treating cervical cancer in a subject, an effective dose of the drug needs to be administered to the subject. Using this method, the growth, proliferation, recurrence and/or metastasis of cervical cancer is inhibited. Further, at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% fraction of the growth, proliferation, recurrence and/or metastasis of cervical cancer is inhibited.

The subject of the method may be a human.

In an eighth aspect of the present invention, there is provided a composition for preventing or treating cervical cancer, which comprises the following effective substances:

the aforementioned nucleic acid molecules; and/or, the aforementioned IMPA2 gene interfering nucleic acid construct; and/or the aforementioned IMPA2 gene interfering lentivirus, and a pharmaceutically acceptable carrier, diluent or excipient.

The composition may be a pharmaceutical composition.

When the composition is used for preventing or treating cervical 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 cervical cancer is inhibited. Further, at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% fraction of the growth, proliferation, recurrence and/or metastasis of cervical cancer is inhibited.

The form of the composition is not particularly limited, and may be in the form of various substances such as solid, liquid, gel, semifluid, aerosol, etc.

The subject to which the composition is primarily directed is a mammal. The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, or the like. The primate is preferably a monkey, ape or human.

In conclusion, the invention designs an RNAi target sequence aiming at the human IMPA2 gene and constructs a corresponding IMPA2RNAi vector, wherein the RNAi vector pGCSIL-GFP-IMPA2-siRNA can obviously reduce the expression of the IMPA2 gene at the mRNA level and the protein level. The lentivirus (lentivirus, abbreviated as Lv) is used as a gene operation tool to carry an RNAi vector pGCSIL-GFP-IMPA2-siRNA, so that the RNAi sequence aiming at the IMPA2 gene can be efficiently introduced into the cervical cancer SIHA cell in a targeted manner, the expression level of the IMPA2 gene is reduced, and the proliferation capacity of the tumor cell is obviously inhibited. Lentivirus-mediated silencing of IMPA2 gene is therefore a potential clinical non-surgical treatment modality for malignancies.

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

the extensive and intensive research shows that the expression of the human IMPA2 gene is down-regulated by adopting an RNAi method, the proliferation of cervical cancer cells can be effectively inhibited, the apoptosis can be promoted, and the growth process of the cervical cancer can be effectively controlled. The siRNA or the nucleic acid construct containing the siRNA sequence and the lentivirus provided by the invention can specifically inhibit the proliferation rate of cervical cancer cells, promote apoptosis of the cervical cancer cells, inhibit cloning of the cervical cancer cells, influence the cycle of the cervical cancer cells and inhibit growth of the cervical cancer cells, thereby treating the cervical cancer and opening up a new direction for treating the cervical cancer.

Drawings

FIG. 1: RT-PCR detects the target gene reduction efficiency of SiHa cell mRNA level.

FIG. 2: western Blot detection shows that SiHa cell target reduces the protein level expression of IMPA2 gene.

FIG. 3: the results of the Celigo cell automated analysis revealed that the subtraction of the IMPA2 gene suppressed the proliferation of cervical cancer cells. (cell lines were SiHa cells, and cell numbers were counted 1, 2, 3, 4 and 5 days after viral infection)

FIG. 4: the influence of IMPA2 gene on SiHa cell proliferation capacity is detected by a cell clone formation method, shRNA lentivirus infects SiHa cells, the number of clones is observed after the SiHa cells are cultured for 16 days, a digital camera record chart is arranged on the left side, and a right side column result is displayed by the average value +/-standard deviation of the number of cell clones.

FIG. 5 a: annexin V-APC flow apoptosis assay the effect of sh IMPA2 on SiHa cell cycle (flow cell cycle schematic).

FIG. 5b Annexin V-APC flow apoptosis assay the effect of sh IMPA2 on SiHa cell cycle (shown as percent cell mean. + -. standard deviation).

FIG. 6 a: annexin V-APC flow apoptosis assay the effect of sh IMPA2 on SiHa apoptosis (flow apoptosis schematic).

FIG. 6 b: annexin V-APC flow apoptosis assay the effect of sh IMPA2 on SiHa apoptosis (shown as the percent cell mean ± standard deviation).

In the drawings, there is shown in the drawings,

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

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

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

Detailed Description

The invention proves the function of IMPA2 gene in cervical carcinoma generation from the aspect of cell function. Transfecting cervical cancer cells after constructing a target gene shRNA lentivirus, and comparing the target gene shRNA lentivirus with a transfection control lentivirus to detect the expression conditions of mRNA and protein level target genes in two groups of cervical cancer cell lines; and then cell proliferation, apoptosis and other detection are carried out through cytofunctional experiments, and the results show that the cervical cancer cell proliferation inhibition degree of the shRNA group is obviously higher than that of the control group and the increase degree of the apoptosis rate of the shRNA group is higher than that of the control group compared with the shRNA group and the control group.

According to the research results, a new method for diagnosing and treating the gene is further explored and developed, so that more choices can be provided for diagnosing and treating the cervical cancer patients.

IMPA2 inhibitors

Refers to a molecule having inhibitory effect on IMPA 2. Having inhibitory effects on IMPA2 include, but are not limited to: inhibiting the expression or activity of IMPA 2.

Inhibition of IMPA2 activity refers to a decrease in IMPA2 activity. Preferably, the activity of IMPA2 is reduced by at least 10%, more preferably by at least 30%, still more preferably by at least 50%, even more preferably by at least 70%, and most preferably by at least 90% compared to that before inhibition.

The inhibition of the expression of IMPA2 may specifically be inhibition of the transcription or translation of IMPA2 gene, and specifically may refer to: to make the gene of IMPA2 non-transcribed or to reduce the transcriptional activity of the gene of IMPA2, or to make the gene of IMPA2 non-translated or to reduce the level of translation of the gene of IMPA 2.

The regulation of gene expression of IMPA2 can be performed by one skilled in the art using conventional methods, such as gene knock-out, homologous recombination, interfering RNA, etc.

Inhibition of gene expression of IMPA2 was confirmed by PCR and Western Blot detection of expression levels.

Preferably, the expression of the IMPA2 gene is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, even more preferably by at least 70%, still more preferably by at least 90%, most preferably the IMPA2 gene is not expressed at all, compared to the wild type.

Small molecule compounds

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

Preparation of medicine for preventing or treating cervical cancer

Nucleic acid molecules that reduce expression of the IMPA2 gene in cervical cancer cells can be utilized; and/or, an IMPA2 gene interfering nucleic acid construct; and/or, the IMPA2 gene interferes with lentivirus to be used as an effective component for preparing a medicament for preventing or treating cervical cancer. Generally, the medicament can comprise one or more pharmaceutically acceptable carriers or auxiliary materials besides the effective components according to the requirements of different dosage forms.

By "pharmaceutically acceptable" is meant that the molecular entities and compositions do not produce adverse, allergic, or other untoward reactions when properly administered to an animal or human.

The "pharmaceutically acceptable carrier or adjuvant" should be compatible with the active ingredient, i.e., capable of being blended therewith without substantially diminishing the effectiveness of the drug under ordinary circumstances. Specific examples of some substances that can serve as pharmaceutically acceptable carriers or adjuvants are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium methylcellulose, ethylcellulose and methylcellulose; powdered gum tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyhydric alcohols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting agents, stabilizers; an antioxidant; a preservative; pyrogen-free water; isotonic saline solution; and phosphate buffer, and the like. These materials are used as needed to aid in the stability of the formulation or to aid in the enhancement of the activity or its bioavailability or to produce an acceptable mouthfeel or odor upon oral administration.

In the present invention, unless otherwise specified, the pharmaceutical dosage form is not particularly limited, and may be prepared into injection, oral liquid, tablet, capsule, dripping pill, spray, etc., and may be prepared by a conventional method. The choice of the pharmaceutical dosage form should be matched to the mode of administration.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the respective manufacturers.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts.

Example 1 preparation of RNAi lentivirus against human IMPA2 Gene

1. Screening effective siRNA target point aiming at human IMPA2 gene

Calling IMPA2 (NM-014214) gene information from Genbank; designing effective siRNA target point aiming at IMPA2 gene. The effective siRNA target sequences screened against IMPA2 gene are listed in Table 1-1.

TABLE 1-1 siRNA target sequences targeting the human IMPA2 gene

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

2. Preparation of Lentiviral vectors

Synthesizing double-stranded DNA Oligo sequences (Table 1-2) containing Age I and EcoR I enzyme cutting sites at two ends aiming at siRNA targets (taking SEQ ID NO:1 as an example); the restriction enzymes Age I and EcoR I act on pGCSIL-GFP vector (provided by Shanghai Jikai Gene chemistry Co., Ltd.), linearize it, and identify the enzyme-cleaved fragments by agarose gel electrophoresis.

TABLE 1-2 double-stranded DNA Oligo with Age I and EcoR I cleavage sites at both ends

The vector DNA linearized by double digestion (digestion system shown in tables 1-4, 37 ℃ C., reaction 1h) and the purified double-stranded DNA Oligo were ligated by T4 DNA ligase at 16 ℃ C. overnight in an appropriate buffer system (ligation system shown in tables 1-5), and the ligation product was recovered. The ligation product was transformed into calcium chloride prepared fresh E.coli competent cells (transformation protocol reference: molecular cloning protocols second edition, pages 55-56). Dipping the surface of the clone of the strain growing out of the connected transformation product, dissolving the surface in 10 mul LB culture medium, uniformly mixing and taking 1 mul as a template; designing universal PCR primers at the upstream and downstream of RNAi sequence in the lentiviral vector, wherein the upstream primer sequence: 5'-CCTATTTCCCATGATTCCTTCATA-3' (SEQ ID NO: 6); the sequence of the downstream primer is as follows: 5'-GTAATACGGTTATCCACGCG-3' (SEQ ID NO: 7), and PCR identification experiments were performed (PCR reaction system shown in tables 1-6, reaction conditions shown in tables 1-7). Sequencing and comparing the clones which are identified to be positive by the PCR, wherein the correctly compared clones are the clones which are successfully constructed and are directed at the nucleotide sequence shown in SEQ ID NO:1, named pGCSIL-GFP-IMPA 2-siRNA.

pGCSIL-GFP-Scr-siRNA negative control plasmid was constructed with negative control siRNA target sequence 5'-TTCTCCGAACGTGTCACGT-3' (SEQ ID NO: 8). When pGCSIL-GFP-Scr-siRNA negative control plasmids are constructed, double-stranded DNA Oligo sequences (tables 1-3) containing adhesive ends of Age I and EcoR I enzyme cutting sites at two ends are synthesized aiming at the Scr siRNA target spots, and the rest construction methods, identification methods and conditions are the same as pGCSIL-GFP-IMPA 2-siRNA.

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

TABLE 1-4 pGCSIL-GFP plasmid digestion reaction System

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

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

TABLE 1-6-1 PCR reaction System

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

TABLE 1-7 PCR reaction System Programming

3. Packaging IMPA2-siRNA lentivirus

The DNA of RNAi plasmid pGCSIL-GFP-IMPA2-siRNA was extracted using a plasmid extraction kit from Qiagen corporation to prepare 100 ng/. mu.l of stock solution.

24h before transfection, human embryonic kidney cell 293T cells in logarithmic growth phase were trypsinized and cell density was adjusted to 1.5X 10 in DMEM complete medium containing 10% fetal bovine serum⁵Cells/ml, seeded in 6-well plates at 37 ℃ with 5% CO₂Culturing in an incubator. The cell density can reach 70-80% to be used for transfection. 2h before transfection, the original medium was aspirated and 1.5ml of fresh complete medium was added. Mu.l of Packing Mix (PVM), 12. mu.l of PEI, and 400. mu.l of serum-free DMEM medium were added to a sterilized centrifuge tube according to the instructions of the MISSION Lentiviral Packing Mix kit from Sigma-aldrich, and 20. mu.l of the above-extracted plasmid DNA was added to the above-mentioned PVM ionPEI/DMEM mixed solution.

The transfection mixture was incubated at room temperature for 15min, transferred to medium of human embryonic kidney 293T cells at 37 ℃ with 5% CO₂Culturing for 16h in an incubator. The medium containing the transfection mixture was discarded, washed with PBS solution, 2ml of complete medium was added and incubation continued for 48 h. The cell supernatant was collected, and the lentivirus was purified and concentrated by a Centricon Plus-20 centrifugal ultrafiltration device (Millipore) according to the following steps: (1) centrifuging at 4 deg.C and 4000g for 10min to remove cell debris; (2) filtering the supernatant with a 0.45 μm filter in a 40ml ultracentrifuge tube; (3) centrifuging at 4000g for 10-15min to obtain the required virus concentration volume; (4) after the centrifugation is finished, separating the filter cup from the lower filtrate collecting cup, reversely buckling the filter cup on the sample collecting cup, and centrifuging for 2min until the centrifugal force is not more than 1000 g; (5) the centrifuge cup is removed from the sample collection cup, and the virus concentrate is obtained. Subpackaging the virus concentrated solution and storing at-80 ℃. The sequence of the first strand of siRNA contained in the virus concentrated solution is shown in SEQ ID NO. 2. The packaging process of the control lentivirus was the same as that of the IMPA2-siRNA lentivirus, except that the pGCSIL-GFP-Scr-siRNA vector was used in place of the pGCSIL-GFP-IMPA2-siRNA vector.

Example 2 testing of silencing efficiency of Gene infected with IMPA2-siRNA lentivirus

Human cervical carcinoma SiHa cells in logarithmic growth phase are trypsinized to prepare cell suspension (the cell number is about 5X 10)⁴/ml) were inoculated in 6-well plates and cultured until the degree of cell confluence reached about 30%. According to the complex infection value (MOI, SiHa: 20), an appropriate amount of the lentivirus prepared in example 1 was added, the medium was changed after 24 hours of culture, and cells were collected after the infection time reached 5 days.

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

Total RNA was extracted according to the Trizol protocol of Invitrogen corporation. The RNA was reverse-transcribed to obtain cDNA according to the M-MLV protocol of Promega (reverse transcription reaction system shown in Table 2-1, reaction at 42 ℃ for 1 hour, and then reverse transcriptase was inactivated by water bath for 10min at 70 ℃ in a water bath).

Real-time quantitative detection was carried out using a TP800 Real time PCR instrument (TAKARA). The primers for IMPA2 gene are as follows: an upstream primer 5'-GGGCAGGACAGATCATCAGAA-3' (SEQ ID NO: 11) and a downstream primer 5'-GAAACCTCTCTCGCAACTCAG-3' (SEQ ID NO: 12). The housekeeping gene GAPDH is used as an internal reference, and the primer sequences are as follows: an upstream primer 5'-TGACTTCAACAGCGACACCCA-3' (SEQ ID NO: 13) and a downstream primer 5'-CACCCTGTTGCTGTAGCCAAA-3' (SEQ ID NO: 14). The reaction system was prepared in the proportions shown in Table 2-2.

TABLE 2-1 reverse transcription reaction System

TABLE 2-2 Real-time PCR reaction System

Reagent	Volume (μ l)
		SYBR premix ex taq	6.0
Primer MIX (5. mu.M)	0.3
		cDNA	0.6
ddH₂O	5.1
		Total	12.0

The program was a two-step Real-time PCR: pre-denaturation at 95 ℃ for 15 s; then, denaturation is carried out at 95 ℃ for 5s in each step; annealing and extending for 30s at 60 ℃; a total of 45 cycles were performed. Each time reading the absorbance value during the extension phase. After the PCR was completed, the DNA was denatured at 95 ℃ for 1min, and then cooled to 55 ℃ to allow the DNA double strands to be sufficiently bound. Melting curves were prepared by increasing the temperature from 55 ℃ to 95 ℃ by 0.5 ℃ for 4 seconds and reading the absorbance. By using 2^-ΔΔCtThe assay calculates the abundance of expression of IMPA 2-infected mRNA. Cells infected with the control virus served as controls. The experimental results are shown in figure 1, and show that the expression level of IMPA2 mRNA in SiHa cells of human cervical cancer is down-regulated by 84.20%.

b) Western Blotting method for detecting gene silencing efficiency

1. Extraction of Total cellular proteins

(1) Cell samples were received and washed twice with PBS. An appropriate amount of RIPA lysate was taken and PMSF was added to a final concentration of 1mM within a few minutes before use. (using RIPA lysate, instruction chain: http:// www.beyotime.com/RIPA-lysine-buffer. htm)

(2) Adding appropriate amount of RIPA lysate, and lysing on ice for 10-15 min. Cells were scraped off and transferred to a new EP tube, and then cells were sonicated (20 times at 40W, 1s each, 2s apart).

(3) Centrifugation was carried out at 12000g for 15min at 4 ℃ and the supernatant BCA method was used to determine the Protein concentration (BCA Protein Assay Kit instruction: http:// www.beyotime.com/p0010s. htm)

(4) The protein concentration of each sample was adjusted to be consistent by adding fresh lysate, typically 2. mu.g/. mu.L. Then 6X padding buffer with the volume of 1/5 is added and mixed evenly, the mixture is boiled for 10min in a metal bath with the temperature of 100 ℃, and the mixture is stored for standby at the temperature of 80 ℃ after being centrifuged for a short time.

2.SDS-PAGE

(1) Preparing glue: according to the molecular weight of the target protein, glue with different concentrations is prepared, and the specific system is as follows:

tables 3-18 mL fractions of separation gel

TABLE 3-210 mL fractions of the separation gel

Tables 3-3 different volume 5% concentrated gum compositions

(2) Loading: after the gel is solidified, the comb is pulled out, the electrophoresis buffer solution is used for cleaning the sample loading hole, and the prepared sample is loaded.

(3) Electrophoresis: concentrating the gel at 80mA for 20 min; the separation gel was 120mA, 1 h.

3. Immunoblotting (Wet transfer)

After the electrophoresis is finished, the protein is transferred to the PVDF membrane by using a transfer electrophoresis device and electrotransfer for 150min under the constant current condition of 300mA at 4 ℃.

4. Antibody hybridization:

(1) and (3) sealing: PVDF membrane was blocked with blocking solution (TBST solution containing 5% skim milk) at room temperature for 1h or overnight at 4 ℃.

(2) Primary antibody incubation: the antibody was diluted with blocking solution and incubated with the blocked PVDF membrane at room temperature for 2h or overnight at 4 ℃ and the membrane was washed 4 times with TBST for 8min each.

(3) And (3) secondary antibody incubation: the corresponding secondary antibody was diluted with blocking solution, the PVDF membrane was incubated at room temperature for 1.5h, and the membrane was washed 4 times with TBST, 8min each.

X-ray development: (use of 20X from CST Co., Ltd.)

Reagent and 20X Peroxide #7003 kit, instruction linked:

https://www.cst-c.com.cn/products/wb-ip-reagents/20x-lumiglo-reagent-and-20x-peroxide/7003？site-search-type＝Products)

(1) the solution A and the solution B in the kit are mixed according to the proportion of 1:1, inverted and mixed evenly, and can be used after being placed for a plurality of minutes.

(2) Taking out the film, wiping the absorbent paper dry, spreading into a cassette, dripping a proper amount of uniformly mixed ECL luminous liquid, spreading a preservative film (avoiding generating bubbles), putting an X-ray film (avoiding the movement of the X-ray film), closing the cassette, and exposing for 1 s-several min (the exposure time needs to be tried for several times, and the exposure time is properly adjusted according to whether the naked eye can see fluorescence and the strength of the fluorescence.

(3) Taking out the X-ray film, placing in developing solution, taking out after banding occurs, rinsing in clear water for several seconds, and placing in fixing solution for at least 2 min.

(4) Taking out the X-ray film, drying and analyzing.

As shown in FIG. 2, it can be seen that the target has a knockdown effect on the exogenous expression of the target gene, and thus is an effective target.

Example 3 examination of the proliferative Capacity of tumor cells infected with IMPA2-siRNA lentivirus

Human cervical carcinoma SiHa cells in logarithmic growth phase are trypsinized to prepare cell suspension (the cell number is about 5X 10)⁴/ml) were inoculated in 6-well plates and cultured until the degree of cell confluence reached about 30%. According to the infection complex number (MOI, SiHa: 20), adding a proper amount of virus, culturing for 24h, then replacing the culture medium, and collecting cells of each experimental group in the logarithmic growth phase after the infection time reaches 5 days. Complete medium resuspension into cell suspension (2X 10)⁴Per ml) at a cell density of about 1000 per well, 96-well plates were seeded. Each set of 5 duplicate wells, 100. mu.l per well. After the plate is laid, the plate is placed at 37 ℃ and 5% CO₂Culturing in an incubator. The plate reading was performed once a day with Celigo instrument (Nexcelom) starting the next day after plating, and the plate reading was performed continuously for 5 days. The number of green fluorescent cells in the well plate of each scanning was accurately calculated by adjusting the input parameters of analysis settings, and the data was statistically plotted to generate a cell proliferation curve (the result is shown in fig. 3).

The results show that after each tumor of the lentivirus infection group is cultured in vitro for 5 days, the proliferation speed is obviously slowed down and is far lower than that of the tumor cells of the control group, the number of viable cells is reduced by 55.29 percent, and the SiHa cell proliferation capacity of the human cervical carcinoma caused by the silencing of IMPA2 gene is inhibited.

Example 4 examination of the clonogenic Capacity of tumor cells infected with IMPA2-siRNA lentivirus

The human cervical carcinoma SiHa cells are digested by trypsin and then inoculated into a 12-hole plate, and the cell density is 10-15%. The next day, the medium was changed to fresh medium containing 5ug/ml polybrene. IMPA2-siRNA lentivirus was added to the plates at MOI, SiHa:20, and the medium was replaced with fresh medium 12-24h after infection. After infection for 72h, fluorescence is observed under a fluorescence microscope, and the infection efficiency reaches 80%.

After the cells infected with the virus in the logarithmic growth phase are digested by pancreatin, the complete culture medium is re-suspended into cell suspension; after counting the cells, inoculating the cells into a 6-well plate (800 cells/well), continuously culturing the inoculated cells in an incubator for 16 days, changing the liquid every 3 days in the middle, and observing the cell state; photographing the cell clone under a fluorescent microscope before the experiment is terminated; at the end of the experiment, cells were fixed with paraformaldehyde, washed with PBS, Giemsa stained, and photographed.

The results are shown in fig. 4, compared with the control interference (NC group), after the RNA interference reduces the expression of the gene (KD group), the number of the clones formed by the SiHa cells of the human cervical cancer is obviously reduced, and the volume of the clone spots is obviously reduced; the silencing of IMPA2 gene is shown to cause the reduction of the capability of forming clone of SiHa cells of human cervical carcinoma. The plate cloning test detects that after the expression of the gene is reduced, the cloning capacity of the tumor cells is reduced.

Example 5 tumor cell cycle assay for IMPA2-siRNA lentivirus infection

Cervical cancer SiHa cells in logarithmic growth phase are trypsinized to prepare cell suspension (the cell number is about 5X 10)⁴/ml) were inoculated in 6-well plates and cultured until the degree of cell confluence reached about 30%. According to the infection complex number MOI, SiHa:20, adding a proper amount of virus, culturing for 24h, then replacing the culture medium, and collecting the cells of each experimental group in the logarithmic growth phase after the infection time reaches 5 days. In case of adherent cells, when 6cm dish cells of each experimental group were grown to a coverage of about 80% (cells did not enter the growth plateau), pancreatin digestion was performed and the total culture medium was usedSuspending into cell suspension, collecting cells in 5mL centrifuge tube, each group having three multiple holes (cell number is more than or equal to 10 to ensure enough cells on machine)⁶Treatment). And directly collecting the suspension cells. 1300rmp was centrifuged for 5min, the supernatant was discarded, and the cell pellet was washed 1 time with 4 ℃ pre-cooled D-Hanks (pH 7.2-7.4). 1300rmp, 5min centrifugation, 4 ℃ pre-cooled 75% ethanol fixed cells for at least 1 h. 1300rmp centrifugation for 5min, removal of fixative, D-Hanks washing of cell precipitation. 1300rmp was centrifuged for 5min, the supernatant was discarded, and the cell pellet was washed 1 time with 4 ℃ pre-cooled D-Hanks (pH 7.2-7.4). . Preparing a cell staining solution: 40 XPI stock (2 mg/mL): 100 XRNase stock (10 mg/mL): 1 × D-Hanks ═ 25: 10: 1000 cell staining: according to the Cell amount, adding a certain volume of Cell staining solution (0.6-1mL) for resuspension, so that the Cell passing rate during loading is 300-800 cells/s. Adding a flow cytometer for detection.

As shown in FIGS. 5a and 5b, the periodic distribution of tumor cells was changed by the PI-FACS method, which detected the decrease of IMPA2 gene expression. After RNA interference reduced gene expression (KD group) compared to control interference (NC group), the percentage of cells in G1 phase did not change significantly, the percentage of cells in S phase increased, and the percentage of cells in G2/M phase decreased, indicating that IMPA2 affected the cycle of cervical cancer cells.

Example 6 detection of apoptosis levels in tumor cells infected with IMPA2-siRNA lentivirus

Human cervical carcinoma SiHa cells are inoculated in a 6-well plate after being digested by trypsin, and the cell density is 10-15%. The next day was changed to fresh medium. IMPA2-siRNA lentivirus was administered according to MOI, SiHa:20 were added to the plates and the medium was changed fresh 12-24h after infection. After 5 days of infection, fluorescence was observed under a fluorescence microscope, and the infection efficiency reached 90%.

After trypsinizing the cells in logarithmic growth phase, resuspending the complete medium into a cell suspension; collecting the supernatant in the same 5mL centrifuge tube, each group having three multiple holes (the number of cells is not less than 5 × 10 to ensure enough cells on the machine⁵Treatment). 1300rmp for 5min, discard the supernatant and wash the cell pellet with 4 ℃ pre-cooled PBS. The cell pellet was washed once with 1 Xbinding buffer, centrifuged at 1300rmp for 3min, and the cells were collected. 200 muL1 × binding buffer resuspended the cell pellet. Add 10. mu.L Annexin V-APC staining, and keep away from light for 10-15min at room temperature. According to the cell amount, 400-800. mu.L of 1 XBindingbuffer is added, and detection is carried out by an up-flow cytometer. The results were analyzed.

As shown in FIGS. 6a and 6b, the change of apoptosis ratio of tumor cells after the Annexin V single staining method detects the expression of the reduced gene. It was found that the apoptosis rate of tumor cells increases after down-regulating the expression of IMPA2 gene. Following reduced gene expression by RNA interference (KD group), the number of apoptotic tumor cells increased significantly compared to control interference (NC group); indicating that gene silencing leads to apoptosis of tumor cells.

While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.

Sequence listing

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Claims

1. The application of the human IMPA2 gene as a target in preparing a cervical cancer treatment drug or a cervical cancer diagnosis drug.

Use of an IMPA2 inhibitor for the manufacture of a product having at least one of the following effects:

treating cervical cancer;

inhibiting the proliferation rate of cervical cancer cells;

promoting apoptosis of cervical cancer cells;

inhibiting the cloning of cervical cancer cells;

affect the cycle of cervical cancer cells;

inhibiting the growth of cervical cancer.

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

1) the IMPA2 inhibitor is a molecule having inhibitory effect on IMPA 2;

2) the IMPA2 inhibitor is the only effective component or one of the effective components of the product;

3) the IMPA2 inhibitor is selected from double-stranded RNA, shRNA, antibody or small molecule compound.

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

1) the shRNA or double-stranded RNA target sequence is shown as SEQ ID NO:1 is shown in the specification;

2) the double-stranded RNA comprises a first strand and a second strand, wherein the first strand and the second strand are complementary to form an RNA dimer, and the sequence of the first strand is shown as SEQ ID NO:2 is shown in the specification;

3) the nucleotide sequence of the shRNA is shown as SEQ ID NO: 3, respectively.

5. A nucleic acid molecule for reducing expression of the IMPA2 gene in a cervical cancer cell, the nucleic acid molecule comprising:

a. a double-stranded RNA containing a nucleotide sequence capable of hybridizing with IMPA2 gene; or

shRNA containing a nucleotide sequence capable of hybridizing with IMPA2 gene;

wherein the double-stranded RNA comprises a first strand and a second strand, the first strand and the second strand are complementary to form an RNA dimer, and the sequence of the first strand is substantially identical to a target sequence in the IMPA2 gene; the shRNA includes a sense strand segment and an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, the sequences of the sense strand segment and the antisense strand segment are complementary, and the sequence of the sense strand segment is substantially identical to a target sequence in the IMPA2 gene.

6. The nucleic acid molecule for reducing expression of an IMPA2 gene in a cervical cancer cell according to claim 5, further comprising one or more of the following characteristics:

2) the double-stranded RNA is siRNA, and the sequence of the first strand of the siRNA is shown as SEQ ID NO:2 is shown in the specification;

3) the nucleotide sequence of the shRNA is shown as SEQ ID NO: 3, respectively.

7. An IMPA2 gene interfering nucleic acid construct containing a gene fragment encoding the shRNA in the nucleic acid molecule according to any one of claims 5 to 6, capable of expressing the shRNA.

8. An IMPA2 gene interfering lentivirus, which is prepared by virus packaging the interfering nucleic acid construct of claim 7 with the help of lentivirus packaging plasmid and cell line.

9. The nucleic acid molecule of any one of claims 5 to 6, or the IMPA2 gene interfering nucleic acid construct of claim 7, or the IMPA2 gene interfering lentivirus of claim 8, for use in: is used for preparing a medicine for preventing or treating cervical cancer or a kit for reducing the expression of IMPA2 gene in cervical cancer cells.

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

the nucleic acid molecule of any one of claims 5-6; and/or, the IMPA2 gene interfering nucleic acid construct of claim 7; and/or the IMPA2 gene interfering lentivirus of claim 8, and a pharmaceutically acceptable carrier, diluent or excipient.