CN111349701B - RSPH14 gene application, RSPH14 inhibitor application, nucleic acid molecule, construct and composition - Google Patents
RSPH14 gene application, RSPH14 inhibitor application, nucleic acid molecule, construct and composition Download PDFInfo
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- CN111349701B CN111349701B CN202010159307.1A CN202010159307A CN111349701B CN 111349701 B CN111349701 B CN 111349701B CN 202010159307 A CN202010159307 A CN 202010159307A CN 111349701 B CN111349701 B CN 111349701B
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Abstract
The invention belongs to the field of biomedical research, and particularly relates to application of a human RSPH14 gene serving as a target in preparation of a lung cancer therapeutic drug or a lung cancer diagnostic drug. The invention is widely and deeply researched, and discovers that after the RNAi method is adopted to down regulate the expression of human RSPH14 gene, the proliferation of lung cancer cells can be effectively inhibited, the apoptosis can be promoted, and the growth process of lung cancer can be effectively controlled. The siRNA or the nucleic acid construct containing the siRNA sequence and the slow virus provided by the invention can specifically inhibit the proliferation rate of lung cancer cells, influence the lung cancer cell cycle, promote the apoptosis of lung cancer cells and inhibit the growth of lung cancer, thereby treating lung cancer and opening up a new direction for treating lung cancer.
Description
Technical Field
The invention belongs to the field of biomedical research, and particularly relates to application of a human RSPH14 gene and related products.
Background
The RSPH14 gene encodes a protein of unknown function that is somewhat similar to yeast vacuolar protein. The gene is located in a chromosome deleted region in rhabdomyosarcoma of brain, kidney, soft tissue and the like of children, but mutation of the gene is irrelevant to diseases (Isolation ofgenes from the rhabdoid tumor deletion region in chromosome band 22q11.2.Zhou J,et al.Gene,2000Jan 4.PMID 10607907). Multiplex ligation-related probe amplification and capillary electrophoresis methods were performed on 354 surgical samples from patients with congenital heart disease, where 11.3% of the patient's genomic 22q11.2 region had a different degree of deletion or amplification, and the RSPH14 gene was located within this region, indicating that it might be associated with congenital heart disease development (Genetic characterisation of 22q11.2 variations and prevalence in patients with congenital heart disease.Arch Dis Child.2019Oct 30.pii:archdischild-2018-316634.Doi:10.1136/archdis child-2018-316634). In addition, the rs4443100SNP near RTDR1 may be associated with serum parathyroid hormone levels.
There is no report on the use of the RSPH14 gene in lung cancer treatment.
Disclosure of Invention
In order to overcome the problems of the prior art, the present invention aims to provide the use of the human RSPH14 gene and related products.
In order to achieve the above and other related objects, the present invention adopts the following technical scheme:
in a first aspect of the invention, there is provided the use of the human RSPH14 gene as a target in the manufacture of a medicament for the treatment of lung cancer or in the manufacture of a medicament for the diagnosis of lung cancer.
The application of the human RSPH14 gene serving as a target in preparing lung cancer therapeutic drugs specifically comprises the following steps: the RSPH14 gene is taken as an acting object, and medicines or preparations are screened to find medicines capable of inhibiting the expression of the human RSPH14 gene to be taken as medicines for treating lung cancer. The small interfering RNA (siRNA) of the RSPH14 gene is obtained by taking the human RSPH14 gene as an action object and can be used as a medicament with the effect of inhibiting the proliferation of lung cancer cells. In addition, RSPH14 gene can be an object of action, such as antibody drugs, small molecule drugs, and the like.
The application of taking the human RSPH14 gene as a target in preparing lung cancer diagnosis medicaments specifically comprises the following steps: the RSPH14 gene expression product is used as a lung cancer diagnosis index to be applied to the preparation of lung cancer diagnosis medicines.
The lung cancer therapeutic drug is a molecule or drug capable of specifically inhibiting the transcription or translation of RSPH14 genes or capable of specifically inhibiting the expression or activity of RSPH14 proteins, so that the expression level of the RSPH14 genes in lung cancer cells is reduced, and the purposes of inhibiting the proliferation, growth, differentiation and/or survival of lung cancer cells are achieved.
The lung cancer therapeutic drug or lung cancer diagnostic drug prepared by using the RSPH14 gene as a target comprises, but is not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemicals, antibody drugs, polypeptides, proteins or interfering lentiviruses.
Such nucleic acid molecules include, but are not limited to: antisense oligonucleotides, double-stranded RNAs (dsRNA), ribozymes, small interfering RNAs prepared by endoribonuclease III, or short hairpin RNAs (shRNA).
The lung cancer therapeutic agent is administered in an amount sufficient to reduce transcription or translation of the human RSPH14 gene, or to reduce expression or activity of the human RSPH14 protein. Such that the expression of the human RSPH14 gene is reduced by at least 40% -100%, such as 50%, 80%, 90%, 95% or 99%.
The method for treating lung cancer by adopting the lung cancer therapeutic drug mainly achieves the aim of treatment by reducing the expression level of human RSPH14 gene and inhibiting the proliferation of lung cancer cells. Specifically, a substance effective to reduce the level of human RSPH14 gene expression is administered to a patient during treatment.
In one embodiment, the RSPH14 gene has a target sequence set forth in SEQ ID NO: 1. The method comprises the following steps: 5'-GATCATCAGCAAAGGTCTGAT-3'.
In a second aspect of the invention, the use of an RSPH14 inhibitor in the manufacture of a medicament for the treatment of lung cancer. Preferably, the RSPH14 inhibitor plays a role in treating lung cancer by at least one of inhibiting proliferation rate of lung cancer cells, affecting lung cancer cell cycle, promoting apoptosis of lung cancer cells or inhibiting growth of lung cancer, thereby realizing efficacy of the RSPH14 inhibitor in medicines for treating lung cancer.
Further, the present invention provides the use of an RSPH14 inhibitor in the preparation of a product having at least one of the following effects:
treating lung cancer;
inhibiting proliferation rate of lung cancer cells;
affecting lung cancer cell cycle;
promoting apoptosis of lung cancer cells;
inhibit lung cancer growth.
The product necessarily comprises an RSPH14 inhibitor and comprises the RSPH14 inhibitor as an active ingredient for the aforementioned efficacy. The RSPH14 inhibitor refers to a molecule with an inhibiting effect on RSPH 14.
In the product, the active ingredient which can play the role can be only the RSPH14 inhibitor, and other molecules which can play the role can also be contained.
That is, the RSPH14 inhibitor is the only active ingredient or one of the active ingredients of the product.
The product can be a single component substance or a multi-component substance.
The form of the product is not particularly limited, and may be solid, liquid, gel, semifluid, aerosol, or the like.
The subject to which the product is primarily directed is a mammal. The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, etc. The primate is preferably a monkey, ape or human.
Such products include, but are not limited to, pharmaceuticals, nutraceuticals, foods, and the like.
The RSPH14 inhibitor may be a nucleic acid molecule (double-stranded RNA, shRNA), an antibody, a small molecule compound.
As exemplified in the examples of the present invention, the RSPH14 inhibitor may be a nucleic acid molecule that reduces the expression of the RSPH14 gene in lung cancer cells. Specifically, it may be a double-stranded RNA or an shRNA.
In a third aspect of the invention, there is provided a method of treating lung cancer by administering an RSPH14 inhibitor to a subject.
The subject may be a mammal or a lung cancer cell of a mammal. The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, etc. The primate is preferably a monkey, ape or human. The lung cancer cell may be an ex vivo lung cancer cell.
The subject may be a patient suffering from lung cancer or an individual desiring treatment for lung cancer. Or the subject is an isolated lung cancer cell of a lung cancer patient or an individual desiring treatment for lung cancer.
The RSPH14 inhibitor may be administered to a subject before, during, or after receiving treatment for lung cancer.
In a fourth aspect, the invention discloses a nucleic acid molecule for reducing RSPH14 gene expression in lung cancer cells, said nucleic acid molecule comprising double stranded RNA or shRNA.
Wherein the double-stranded RNA contains a nucleotide sequence capable of hybridizing with the RSPH14 gene;
the shRNA contains a nucleotide sequence capable of hybridizing with the RSPH14 gene.
Further, the double-stranded RNA comprises a first strand and a second strand that are complementary together to form an RNA dimer, and the sequence of the first strand is substantially identical to a target sequence in the RSPH14 gene.
The target sequence in the RSPH14 gene is a segment in the RSPH14 gene corresponding to the mRNA segment recognized and silenced by the nucleic acid molecule when the nucleic acid molecule is used for specifically silencing the expression of the RSPH14 gene.
Further, the target sequence of the double-stranded RNA is shown as SEQ ID NO: 1. The method comprises the following steps: 5'-GATCATC AGCAAAGGTCTGAT-3'. Further, the sequence of the first strand of the double-stranded RNA is shown in SEQ ID NO: 2. Specifically 5'-GAUCAUCAGCAAAGGUCUGAU-3'.
Further, the double stranded RNA is small interfering RNA (siRNA).
SEQ ID NO:2 is one strand of small interfering RNA which is designed by taking the sequence shown in SEQ ID NO. 1 as an RNA interference target sequence and aims at the human RSPH14 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 the endogenous RSPH14 gene in lung cancer cells.
The shRNA comprises a sense strand segment and an antisense strand segment, and a stem-loop structure linking 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 an RSPH14 gene.
Further, the target sequence of the sh RNA is shown as SEQ ID NO: 1.
The shRNA can become small interfering RNA (siRNA) after enzyme digestion processing, thereby playing a role in specifically silencing the expression of endogenous RSPH14 genes in lung cancer cells.
Further, the sequence of the stem-loop structure of the shRNA may be selected from any one of the following: UUCAAGAGA, AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU and CCACACC.
Further, the sequence of the shRNA is shown as SEQ ID NO: 3. Specifically 5'-GAUCAUCAGCAAAGGUCUGAUCUCGAGAUCAGACCUUUGCUGAUGAUC-3'.
Further, the RSPH14 gene is of human origin.
In a fifth aspect, the invention discloses an RSPH14 gene interfering nucleic acid construct, comprising a gene fragment encoding shRNA in the nucleic acid molecule, capable of expressing the shRNA.
The RSPH14 gene interfering nucleic acid construct can be obtained by cloning a gene fragment encoding the human RSPH14 gene shRNA into a known vector.
Further, the RSPH14 gene interfering nucleic acid construct is an RSPH14 gene interfering lentiviral vector.
The RSPH14 gene interference slow virus vector disclosed by the invention is obtained by cloning a DNA fragment for encoding the RSPH14 gene shRNA into a known vector, wherein most of the known vectors are slow virus vectors, the RSPH14 gene interference slow virus vector is packaged into infectious virus particles, then lung cancer cells are infected, the shRNA is transcribed, and finally the siRNA is obtained through the steps of enzyme digestion processing and the like and is used for specifically silencing the expression of the RSPH14 gene.
Further, the RSPH14 gene interference slow virus vector also contains a promoter sequence and/or a nucleotide sequence for encoding a marker which can be detected in lung cancer cells; preferably, the detectable label is a 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-tGFP, pLKO.1-puro-CMV-TagCFP, pLKO.1-puro-CMV-TagYFP, pLKO.1-puro-CMV-TagRFP, pLKO.1-puro-CMV-TagFP635, pLKO.1-puro-UbC-TurboGFP, pLKO.1-puro-UbC-TagFP635 any one of pLKO-puro-IPTG-1xLacO, pLKO-puro-IPTG-3xLacO, pLP1, pLP2, pLP/VSV-G, pENTR/U6, pLenti6/BLOCK-iT-DEST, pLenti 6-GW/U6-lamrishma, pcDNA1.2/V5-GW/lacZ, pLenti6.2/N-Lumio/V5-DEST, pGCSIL-GFP or pLenti6.2/N-Lumio/V5-GW/lacZ.
The embodiment of the invention specifically enumerates a human RSPH14 gene interference lentiviral vector constructed by taking pGCSIL-GFP as a vector, and is named pGCSIL-GFP-RSPH14-siRNA.
The RSPH14 gene siRNA can be used for inhibiting proliferation of lung cancer cells, and further can be used as a medicine or preparation for treating lung cancer. The RSPH14 gene interference lentiviral vector can be used for preparing the RSPH14 gene siRNA. When used as a medicament or formulation for treating lung cancer, a safe and effective amount of the nucleic acid molecule is administered to a mammal. The particular dosage should 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.
In a sixth aspect of the invention, an RSPH14 gene interfering lentivirus is disclosed, which is obtained by virus packaging the RSPH14 gene interfering nucleic acid construct with the aid of a lentivirus packaging plasmid and a cell line. The lentivirus can infect lung cancer cells and produce small interfering RNA directed against the RSPH14 gene, thereby inhibiting proliferation of lung cancer cells. The RSPH14 gene interference slow virus can be used for preparing medicines for preventing or treating lung cancer.
In a seventh aspect of the invention, there is provided the use of the aforementioned nucleic acid molecule, or the aforementioned RSPH14 gene interfering nucleic acid construct, or the aforementioned RSPH14 gene interfering lentivirus, as follows: can be used for preparing medicines for preventing or treating lung cancer or kits for reducing RSPH14 gene expression in lung cancer cells.
Further, there is provided the use of the aforementioned nucleic acid molecule, or the aforementioned RSPH14 gene interfering nucleic acid construct, or the aforementioned RSPH14 gene interfering lentivirus, for the preparation of a medicament for preventing or treating lung cancer.
Further, the use of the nucleic acid molecule, or the RSPH14 gene interfering nucleic acid construct, or the RSPH14 gene interfering lentivirus is provided for preparing a kit for reducing the expression of the RSPH14 gene in lung cancer cells.
The use of the medicament for preventing or treating lung cancer provides a method for treating lung cancer, in particular for preventing or treating lung cancer in a subject, comprising administering an effective dose of the medicament to the subject.
Further, when the medicament is used for preventing or treating lung cancer in a subject, an effective dose of the medicament is required to be administered to the subject. With this method, the growth, proliferation, recurrence and/or metastasis of the lung cancer is inhibited. Further, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the growth, proliferation, recurrence and/or metastasis of the lung cancer is inhibited.
The object of the method may be a person.
In an eighth aspect of the present invention, there is provided a composition for preventing or treating lung cancer, the active substance comprising:
the nucleic acid molecules as described above; and/or, the aforementioned RSPH14 gene interfering nucleic acid construct; and/or, the RSPH14 gene described above interferes with lentiviruses, as well as pharmaceutically acceptable carriers, diluents or excipients.
Further, the composition may be a pharmaceutical composition.
When the composition is used to prevent or treat lung cancer in a subject, an effective amount of the composition is required to be administered to the subject. With this method, the growth, proliferation, recurrence and/or metastasis of the lung cancer is inhibited. Further, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the growth, proliferation, recurrence and/or metastasis of the lung cancer is inhibited.
The form of the composition is not particularly limited, and may be solid, liquid, gel, semifluid, aerosol, or the like.
The subject to which the composition is primarily directed is a mammal. The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, etc. The primate is preferably a monkey, ape or human.
In summary, the invention designs RNAi target sequences aiming at human RSPH14 genes, and constructs corresponding RSPH14RNAi vectors, wherein RNAi vectors pGCSIL-GFP-RSPH14-siRNA can obviously reduce the expression of the RSPH14 genes at mRNA level and protein level. The RNAi sequence aiming at the RSPH14 gene can be efficiently introduced into lung cancer A549 cells in a targeted manner by using lentiviruses (abbreviated as Lv) as a gene manipulation tool to carry RNAi vectors pGCSIL-GFP-RSPH14-siRNA, so that the expression level of the RSPH14 gene is reduced, and the proliferation capacity of the tumor cells is obviously inhibited. Lentivirus-mediated RSPH14 gene silencing is thus a potential clinical non-surgical treatment modality for malignant tumors.
Compared with the prior art, the invention has the following beneficial effects:
the invention is widely and deeply researched, and discovers that after the RNAi method is adopted to down regulate the expression of human RSPH14 gene, the proliferation of lung cancer cells can be effectively inhibited, the apoptosis can be promoted, and the growth process of lung cancer can be effectively controlled. The siRNA or the nucleic acid construct containing the siRNA sequence and the slow virus provided by the invention can specifically inhibit the proliferation rate of lung cancer cells, influence the lung cancer cell cycle, promote the apoptosis of lung cancer cells and inhibit the growth of lung cancer, thereby treating lung cancer and opening up a new direction for treating lung cancer.
Drawings
Fig. 1: RT-PCR detects the target gene reduction efficiency of the mRNA level of the A549 cells.
Fig. 2: western Blot detects that A549 cell targets reduce the expression condition of RSPH14 gene protein level.
Fig. 3: the results of Celigo cell automatic analysis revealed that depletion of RSPH14 gene inhibited proliferation of lung cancer cells. (cell line A549 cells, 1,2,3,4 and 5 days after virus infection were counted for the number of cells
Fig. 4: the cell OD value statistical result graph of each experimental group is detected by an enzyme labeling instrument 490/570 nm.
Fig. 5-1: the effect of shRSPH14 on A549 cell cycle was detected by PI-FACS method, and the graph shows the results of flow cell cycle peak graph.
Fig. 5-2: the effect of shRSPH14 on a549 cell cycle was examined by PI-FACS, and the graph shows cell histogram statistics for each cycle as a percentage mean ± standard deviation.
Fig. 6-1: annexin V single-dye flow cytometry is used for detecting the influence of shRSPH14 on A549 cell apoptosis, and the graph is a flow cell apoptosis peak graph result.
Fig. 6-2: the effect of shRSPH14 on A549 cell apoptosis is detected by using an Annexin V single-dye flow cytometry, and the graph is a histogram statistical result of apoptotic cells and is shown as a cell percentage average value +/-standard deviation.
In the drawings of which there are shown,
the bar graph represents the average of three experiments and the error bars represent Standard Deviation (SD).
* shCtrl has P <0.01 compared to the target gene shRNA lentivirus treatment group.
* Compared with the target gene shRNA lentivirus treatment group, the shCtrl has the P of more than or equal to 0.01 and less than or equal to 0.05.
Detailed Description
The invention confirms the role of RSPH14 gene in lung cancer occurrence from the aspect of cell function. The expression condition of mRNA and protein level target genes in two groups of lung cancer cell lines is detected by constructing target gene shRNA lentivirus and then transfecting lung cancer cells and comparing the target gene shRNA lentivirus with a transfection control lentivirus; and then, cell proliferation, apoptosis and other detection are carried out through a cell functional experiment, and the result shows that the shRNA group is compared with the control group, the lung cancer cell proliferation inhibition degree of the shRNA group is obviously higher than that of the control group, and the apoptosis rate increase degree is higher than that of the control group.
According to the research results, a new method for diagnosing and treating the gene is further explored and developed, and more choices can be provided for diagnosing and treating lung cancer patients.
RSPH14 inhibitors
Refers to a molecule having an inhibitory effect on RSPH 14. Having inhibitory effects on RSPH14 includes, but is not limited to: inhibit the expression or activity of RSPH 14.
Inhibiting RSPH14 activity refers to decreasing RSPH14 activity. Preferably, RSPH14 activity is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, even more preferably by at least 70%, most preferably by at least 90% compared to that prior to inhibition.
Inhibition of RSPH14 expression may specifically be inhibition of transcription or translation of RSPH14 gene, and specifically may refer to: the gene of RSPH14 is not transcribed, or the transcriptional activity of the gene of RSPH14 is reduced, or the gene of RSPH14 is not translated, or the translation level of the gene of RSPH14 is reduced.
The person skilled in the art can use conventional methods for regulating the gene expression of RSPH14, such as gene knockout, homologous recombination, interfering RNA, etc.
The inhibition of gene expression of RSPH14 can be verified by PCR and Western Blot detection of the expression level.
Preferably, the RSPH14 gene expression is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, even more preferably by at least 70%, yet more preferably by at least 90%, and most preferably the RSPH14 gene is not expressed at all, as compared to the wild type.
Small molecule compounds
The present invention refers to a compound having a molecular mass of 1000 or less, which is composed of several or several tens of atoms.
Preparation of medicine for preventing or treating lung cancer
Nucleic acid molecules that reduce RSPH14 gene expression in lung cancer cells can be utilized; and/or, RSPH14 gene interfering nucleic acid constructs; and/or, the RSPH14 gene interferes with lentivirus, and is used as an active ingredient for preparing a medicament for preventing or treating lung cancer. Typically, the medicament will include, in addition to the active ingredient, one or more pharmaceutically acceptable carriers or excipients, as required by the different dosage forms.
By "pharmaceutically acceptable" is meant that the molecular entity and composition 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. it can be blended therewith without substantially reducing the efficacy of the drug in the usual manner. Specific examples of some substances which may be pharmaceutically acceptable carriers or excipients are sugars, such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methyl cellulose, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifying agents, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting and stabilizing agent; an antioxidant; a preservative; non-thermal raw water; isotonic saline solution; and phosphate buffer, etc. These substances are used as needed to aid stability of the formulation or to aid in enhancing the activity or its bioavailability or to produce an acceptable mouthfeel or odor in the case of oral administration.
In the present invention, the pharmaceutical dosage form is not particularly limited unless otherwise specified, and may be formulated into injection, oral liquid, tablet, capsule, dripping pill, spray, etc., and may be prepared by conventional methods. The choice of the pharmaceutical dosage form should be compatible with the mode of administration.
Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention. The test methods in the following examples, in which specific conditions are not noted, are generally conducted under conventional conditions or under conditions recommended by the respective manufacturers.
Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. 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, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present invention employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and related arts.
Example 1 preparation of RNAi lentivirus against human RSPH14 Gene
1. Screening for effective siRNA targets against human RSPH14 gene
Retrieving RSPH14 (nm_ 014433) gene information from Genbank; an effective siRNA target against the RSPH14 gene was designed. Table 1-1 shows the effective siRNA target sequences screened against the RSPH14 gene.
TABLE 1-1 siRNA target sequences targeting the human RSPH14 Gene
| SEQ ID NO | TargetSeq(5’-3’) |
| 1 | GATCATCAGCAAAGGTCTGAT |
2. Preparation of lentiviral vectors
Double-stranded DNAOligo sequences (tables 1-2) containing the Age I and EcoR I cleavage site sticky ends at the two ends are synthesized aiming at siRNA targets (taking SEQ ID NO:1 as an example); the restriction enzymes AgeI and EcoR I were used to linearize pGCSIL-GFP vector (available from Shanghai Ji Kai Gene chemical technologies Co., ltd.) and the cut fragments were identified by agarose gel electrophoresis.
Tables 1-2 double-stranded DNAOligo containing AgeI and EcoRI cleavage sites at both ends
The vector DNA, which was digested with double enzymes and digested with T4 DNA ligase (cleavage system shown in tables 1 to 4, 37 ℃ C., 1h of reaction) was ligated to the purified double-stranded DNAOligo, and ligated overnight at 16 ℃ in an appropriate buffer system (ligation system shown in tables 1 to 5) to recover the ligation product. The ligation products were transformed into fresh E.coli competent cells prepared from calcium chloride (transformation protocol: see second edition of molecular cloning protocol pages 55-56). Dipping a surface of a clone growing with a transformation product, dissolving in 10 μl of LB culture medium, uniformly mixing, and taking 1 μl as a template; upstream and downstream of the RNAi sequence in the lentiviral vector, universal PCR primers were designed, upstream primer sequences: 5'-CCTATTTCCCATGATTCCTTCATA-3' (SEQ ID NO: 6); downstream primer sequence: 5'-GTAATACGGTTATCCACGCG-3' (SEQ ID NO: 7) and performing PCR identification experiments (the PCR reaction systems are shown in tables 1-6, and the reaction conditions are shown in tables 1-7). Sequencing and comparing the clones positive to the PCR identification, and comparing the correct clones to obtain the sequence of the sequence shown in SEQ ID NO:1, named pGCSIL-GFP-RSPH14-siRNA.
pGCSIL-GFP-Scr-siRNA negative control plasmid was constructed, the negative control siRNA target sequence was 5'-ttctccgaacgtgtcacgt-3' (SEQ ID NO: 8). When constructing pGCSIL-GFP-Scr-siRNA negative control plasmid, double-stranded DNAOligo sequences (tables 1-3) containing AgeI and EcoR I enzyme cutting sites at two ends and sticky ends are synthesized aiming at Scr siRNA targets, and other construction methods, identification methods and conditions are the same as those of pGCSIL-GFP-RSPH14-siRNA.
Tables 1-3 double-stranded DNAOligo containing AgeI and EcoRI cleavage sites at both ends
Table 1-4 pGCSIL-GFP plasmid cleavage reaction System
| Reagent(s) | Volume (mul) |
| pGCSIL-GFP plasmid (1. Mu.g/. Mu.l) | 2.0 |
| 10×buffer | 5.0 |
| 100×BSA | 0.5 |
| AgeI(10U/μl) | 1.0 |
| EcoRI(10U/μl) | 1.0 |
| ddH 2 O | 40.5 |
| Total | 50.0 |
TABLE 1-5 vector DNA and double-stranded DNAOligo ligation reaction System
TABLE 1-6-1 PCR reaction System
| Reagent(s) | Volume (mul) |
| 10×buffer | 2.0 |
| dNTPs(2.5mM) | 0.8 |
| Upstream primer | 0.4 |
| Downstream primer | 0.4 |
| Taq polymerase | 0.2 |
| Template | 1.0 |
| ddH 2 O | 15.2 |
| Total | 20.0 |
TABLE 1-7 Programming of PCR reaction System
3. Packaging RSPH14-siRNA lentiviruses
DNA of RNAi plasmid pGCSIL-GFP-RSPH14-siRNA was extracted with plasmid extraction kit from Qiagen, and 100 ng/. Mu.l of the stock solution was prepared.
24h before transfection, human embryonic kidney 293T cells in logarithmic growth phase were digested with trypsin and cell density was adjusted to 1.5X10% in DMEM complete medium containing 10% fetal bovine serum 5 Cells/ml, seeded in 6-well plates, 37 ℃,5% CO 2 Culturing in an incubator. And the cell density reaches 70-80% and can be used for transfection. 2h before transfection, the original medium was aspirated and 1.5ml of fresh complete medium was added. 20. Mu.l of Packing Mix (PVM), 12. Mu.l of PEI, 400. Mu.l of serum-free DMEM medium, 20. Mu.l of the extracted plasmid DNA were added to the PVM/PEI/DMEM mixture as described in Sigma-aldrich company MISSION Lentiviral Packaging Mix kit.
Incubating the above transfection mixture at room temperature for 15min, transferring into culture medium of human embryo kidney 293T cells, 37 ℃ and 5% CO 2 Culturing in an incubator for 16h. The medium containing the transfection mixture was discarded, washed with PBS solution, and 2ml of complete medium was added to continue the culture for 48 hours. Cell supernatants were collected, and lentiviruses purified and concentrated by a Centricon Plus-20 centrifugal ultrafiltration device (Millipore) as follows: (1) centrifuging at 4 ℃ for 10min at 4000g to remove cell debris; (2) The supernatant was filtered through 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 centrifugation, 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 1000g; (5) The centrifuge cup is removed from the sample collection cup and the virus concentrate is present in the sample collection cup. Packaging the virus concentrate, and storing at-80deg.C. The sequence of the first strand of siRNA contained in the virus concentrate is shown as SEQ ID NO. 2. Control lentivirusesThe packaging process of (2) is the same as that of the RSPH14-siRNA lentivirus, and only pGCSIL-GFP-Scr-siRNA vector is used for replacing pGCSIL-GFP-RSPH14-siRNA vector.
Example 2 detection of silencing efficiency of Gene
Human lung cancer A549 cells in logarithmic growth phase were subjected to pancreatin digestion to prepare cell suspension (cell number about 5X 10) 4 Per ml) was inoculated into 6-well plates and cultured until the cell fusion reached about 30%. According to the complex value of infection (MOI, A549: 10), a suitable amount of lentivirus prepared in example 1 was added, the medium was changed after culturing for 24 hours, and after the infection time reached 5 days, the cells were collected.
a) Real-time fluorescent quantitative RT-PCR
Total RNA was extracted according to Trizol protocol from Invitrogen. RNA was reverse transcribed to obtain cDNA according to the M-MLV protocol from Promega (reverse transcription reaction system see Table 2-1, 42℃for 1h, followed by inactivation of reverse transcriptase in a water bath at 70℃for 10 min).
Real-time quantitative detection was performed using a Real time PCR instrument model TP800 (TAKARA). The primers for the RSPH14 gene were as follows: an upstream primer 5'-GCAATGTGGTGCTTGTCCTGA-3' (SEQ ID NO: 11) and a downstream primer 5'-TCACATGCTCCACTGGGTCTT-3' (SEQ ID NO: 12). The housekeeping gene GAPDH is taken 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
| Reagent(s) | Volume (mul) |
| 5×RTbuffer | 4.0 |
| 10mMdNTPs | 2.0 |
| RNasin | 0.4 |
| M-MLV-RTase | 1.0 |
| DEPCH 2 O | 2.6 |
| Total | 10.0 |
TABLE 2-2 Real-time PCR reaction System
| Reagent(s) | Volume (mul) |
| SYBRpremixextaq | 6.0 |
| Primer MIX (5. Mu.M) | 0.3 |
| cDNA | 0.6 |
| ddH 2 O | 5.1 |
| Total | 12.0 |
The procedure was set as two-step Real-time PCR: pre-denaturation at 95 ℃,15s; then each step is denatured at 95 ℃ for 5s; annealing and extending at 60 ℃ for 30s; a total of 45 cycles were performed. The absorbance was read each time 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 strand to bind sufficiently. Starting from 55 ℃ to 95 ℃, increasing the temperature by 0.5 ℃ in each step, keeping for 4s, and simultaneously reading the absorbance value to prepare a melting curve. By 2 -ΔΔCt The assay calculates the abundance of expression of RSPH14 mRNA that is infected. Cells infected with control virus served as controls. The experimental results are shown in FIG. 1, which demonstrate that the expression level of RSPH14 mRNA in human lung cancer A549 cells was down-regulated by 94.8%.
b) WesternBlotting method
1. Extraction of total cell proteins
(1) Cell samples were received and washed twice with PBS. An appropriate amount of RIPA lysate was taken and PMSF was added over the last few minutes before use to give a final concentration of PMSF of 1mM. ( Using RIPA lysate, instructions link: http:// www.beyotime.com/ripa-lysis-bufferm. Htm )
(2) Adding proper amount of RIPA lysate, and performing ice lysis for 10-15min. Cells were scraped off and transferred into new EP tubes, and then sonicated (20 times 40W total, 1s each, 2s intervals).
( 3) Centrifugation was performed at 12000g at 4℃for 15min, and the protein concentration was measured by the supernatant BCA method (BCA Protein Assay Kit instructions are linked: http:// www.beyotime.com/p0010s.htm )
(4) The addition of fresh lysate adjusts the protein concentration for each sample to be consistent, typically 2. Mu.g/. Mu.L. Then adding 1/5 volume of 6 Xloddingbuffer, mixing, decocting in 100 deg.C metal bath for 10min, centrifuging briefly, and preserving at-80deg.C.
2.SDS-PAGE
(1) And (3) glue preparation: the glue with different concentrations is prepared according to the molecular weight of the target protein, and the specific system is as follows:
TABLE 3-1 8mL System separation gel component
TABLE 3-2 10mL System separation gel Components
TABLE 3-3 concentrated gum compositions
(2) Loading: after the glue 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 80mA for 20min; the gel was separated 120mA for 1h.
3. Immunoblot (Wet turn)
After electrophoresis, the protein was transferred to PVDF membrane by using a transfer electrophoresis apparatus and electroblotting at 4℃for 150min under 300mA constant current.
4. Antibody hybridization:
(1) Closing: the PVDF membrane was blocked with blocking solution (TBST solution containing 5% skimmed milk) at room temperature for 1h or overnight at 4 ℃.
(2) Incubation resistance: the blocking solution diluted antibodies, then with blocked PVDF membrane room temperature incubation 2 hours or 4 degrees overnight, and with TBST membrane washing 4 times, each time 8 minutes.
(3) Secondary antibody incubation: the corresponding secondary antibody was diluted with blocking solution, the PVDF membrane was incubated for 1.5h at room temperature and washed with TBST 4 times for 8min each.
X-ray development: (using CST Co. 20X
Reagent and 20X Peroxide#7003 kit, instruction links: https:// www.cst-c.com.cn/products/wb-ip-reagents/20x-lumiglo-reagent-and-20x-peroxide/7003site-search-type＝Products)/>
(1) The solution A and the solution B in the kit are mixed according to the proportion of 1:1, are inverted and mixed evenly, and can be used after being placed for a plurality of minutes.
(2) Taking out the film, wiping the water absorbing paper, spreading the film into a magazine, dripping a proper amount of well mixed ECL luminous solution, spreading a preservative film (avoiding generating bubbles), putting an X-ray film (avoiding the movement of the X-ray film), closing the magazine, exposing for 1 s-several minutes (the exposure time needs to be more than several times, and properly adjusting the exposure time according to whether the naked eyes can see fluorescence and the intensity of the fluorescence).
(3) Taking out the X-ray film, putting into a developing solution, taking out after the strip appears, rinsing in clear water for a few seconds, and putting into a fixing solution for at least 2min.
(4) Taking out the X-ray film, airing and analyzing.
The results are shown in FIG. 2, and indicate that the target has knockdown effect on the endogenous expression of the target gene, so that the target is an effective target.
Example 3 detection of proliferation Capacity of RSPH14-siRNA lentivirus-infected tumor cells
a) Celigo experiment
Human lung cancer A549 cells in logarithmic growth phase were subjected to pancreatin digestion to prepare cell suspension (cell number about 5X 10) 4 Per ml) was inoculated into 6-well plates and cultured until the cell fusion reached about 30%. According to the complex number of infection (MOI, A549: 10), a proper amount of virus was added, the culture medium was changed after culturing for 24 hours, and after the infection time reached 5 days, the cells of each experimental group in the logarithmic growth phase were collected. Complete medium resuspension of the adult cell suspension (2X 10) 4 Per ml), 96-well plates were seeded at a cell density of about 3000 cells per well. Each group had 5 duplicate wells, 100 μl per well. After being paved, the mixture is placed at 37 ℃ and 5 percent of CO 2 Culturing in an incubator. The plates were read once daily with a Celigo instrument (Nexcelom) starting the next day after plating and were continuously tested for 5 days. Accurately calculating the number of cells with green fluorescence in each scanning hole plate by adjusting the input parameters of analysis settings; statistical plots were made on the data to plot 5 day cell proliferation curves.
The results are shown in fig. 3, and the results show that after each tumor in the lentivirus infected group is cultured in vitro for 5 days, the proliferation speed is obviously slowed down, which is far lower than that of the tumor cells in the control group, and the decrease ratio of the number of viable cells is 91.9%, so that the proliferation capacity of human lung cancer A549 cells is inhibited due to RSPH14 gene silencing.
b) CCK8 experiment
Human lung cancer A549 cells in logarithmic growth phase were subjected to pancreatin digestion to prepare cell suspension (cell number about 5X 10) 4 Per ml) was inoculated into 6-well plates and cultured until the cell fusion reached about 30%. According to the complex number of infection, adding a proper amount of virus, culturing for 24 hours, replacing a culture medium, collecting cells of each experimental group in a logarithmic growth phase, performing pancreatin digestion, completely suspending the culture medium into a cell suspension, and counting. The cell density of the plating is determined according to the growth speed of the cells (2000 cells/well), 3-5 times of each group are repeated, after the cells are uniformly plated, the cell density of each experimental group is observed under a microscope, if the density is uneven, one group is fixed, the cell quantity of other groups is finely adjusted to be plated again (for example, con group cells are found to be more, the cell quantity is reduced to be plated again), and the cells are placed into a cell culture box for culture. Beginning the next day after plating, 20. Mu.L of 5mg/mL MTT was added to the wells 4h before termination of the culture without changing the solution. After 4h, the culture was completely aspirated, and the formazan particles at the bottom of the well plate were not aspirated, and 100. Mu. LDMSO was added to dissolve the formazan particles. The oscillator oscillates for 2-5min, and the OD value is detected by an enzyme labeling instrument 490/570 nm. And (5) data statistics and analysis.
The results are shown in fig. 4, and the results show that after each tumor in the lentivirus infected group is remarkably slowed down, the proliferation speed is far lower than that of the tumor cells in the control group, the number of viable cells is reduced by 68.30%, and the RSPH14 gene silencing results in inhibition of proliferation capacity of human lung cancer A549 cells.
Example 4 tumor cell cycle detection of RSPH14-siRNA lentivirus infection
Human lung cancer A549 cells in logarithmic growth phase were subjected to pancreatin digestion to prepare cell suspension (cell number about 5X 10) 4 Per ml) was inoculated into 6-well plates and cultured until the cell fusion reached about 30%. According to infection complexNumber (MOI, A549: 10), the appropriate amount of virus was added, the medium was changed after culturing for 24 hours, and after the infection time reached 3 days, the cells of each experimental group in the logarithmic growth phase were collected. If the cells are adherent cells, when the 6cm dish cells of each experimental group grow to the coverage rate of about 80% (the cells do not enter the growth platform stage), pancreatin is digested, the complete culture medium is resuspended into a cell suspension, the cells are collected in a 5mL centrifuge tube, and three compound holes are arranged in each group (in order to ensure that the number of the cells on the machine is enough, and the number of the cells is more than or equal to 10) 6 Treatment). In the case of suspension cells, they are collected directly. 1300rmp was centrifuged for 5min, the supernatant was discarded, and the cell pellet was washed 1 time with 4℃pre-chilled D-Hanks (pH=7.2 to 7.4). The cells were fixed with 1300rmp, 5min centrifugation, 75% ethanol pre-chilled at 4℃for at least 1h.1300rmp centrifugation for 5min to remove fixative, D-Hanks washed the cell pellet once, step 2. Preparing a cell staining solution: 40 XPI mother liquor (2 mg/mL): 100X RNase mother liquor (10 mg/mL): 1 xd-hanks=25: 10: staining 1000 cells: according to the Cell quantity, adding a certain volume of Cell staining solution (0.6-1 mL) to re-suspend so that the Cell passing rate is 300-800 Cell/s when the machine is on. And (5) detecting on the machine. And (5) data analysis.
The results are shown in FIGS. 5-1 and 5-2, and show that the RNA interference reduces the expression of the gene (shRSPH 14 group) and then the cells in S phase and G2/M phase decrease and the cells in G1 phase increase, compared with the control interference (shCtrl group).
Example 5 detection of apoptosis level of tumor cells infected with RSPH14-siRNA lentivirus
Human lung cancer A549 cells in logarithmic phase are inoculated into 12-well plate after being digested by pancreatin, the cell density is 10-15%, the next day is changed into fresh culture medium, and 5ug/ml polybrene is contained. Lentiviruses were added to the plates according to the multiplicity of infection (MOI, A549: 10), and fresh medium was changed after 12-24h of infection. After 96h of infection, fluorescence is observed under a fluorescence microscope, and the infection efficiency reaches 90%.
After digestion of the cells in the logarithmic growth phase with pancreatin, the complete medium is resuspended into a cell suspension; collecting the supernatant cells in the same 5mL centrifuge tube, and arranging three compound holes (in order to ensure that the number of the cells on the machine is enough, the number of the cells is more than or equal to 5 multiplied by 10) 5 Treatment). 1300rmp centrifugation for 5min, discarding supernatant, washing with PBS pre-cooled at 4deg.CCell precipitation. The cell pellet was washed once with 1 Xbindingbuffer, centrifuged at 1300rmp for 3min and the cells were collected. 200 μL of 1 Xbinding buffer resuspended cell pellet. Add 10. Mu.LAnnexin V-APC to stain, and keep out light for 10-15min at room temperature. According to the cell amount, 400-800 mu L of 1 Xbindingbuffer is added, and the detection is carried out by an up-flow cytometer. The results were analyzed.
The results are shown in FIG. 6-1 and FIG. 6-2, and indicate that the apoptosis proportion of tumor cells is increased after down-regulating gene expression. The number of apoptotic tumor cells is obviously increased after the expression of the RNA interference reduced gene (shRSPH 14 group) is interfered with by a control (shCtrl group); indicating that gene silencing leads to apoptosis of tumor cells.
While the invention has been described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and additions may be made without departing from the scope of the invention. Equivalent embodiments of the present invention will be apparent to those skilled in the art having the benefit of the teachings disclosed herein, when considered in the light of the foregoing disclosure, and without departing from the spirit and scope of the invention; meanwhile, any equivalent changes, modifications and evolution of the above embodiments according to the essential technology of the present invention still fall within the scope of the technical solution of the present invention.
SEQUENCE LISTING
<110> Sichuan province tumor Hospital
<120> RSPH14 gene use, RSPH14 inhibitor use, nucleic acid molecule, construct and composition
<130> none
<160> 14
<170> PatentIn version 3.5
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Claims (5)
- The use of an rsph14 inhibitor in the manufacture of a product having at least one of the following efficacy,treating lung cancer;inhibiting proliferation rate of lung cancer cells;promoting apoptosis of lung cancer cells;inhibiting lung cancer growth;the method is characterized in that: the lung cancer is non-small cell lung cancer, the RSPH14 inhibitor is siRNA, and the target sequence of the siRNA is shown as SEQ ID NO: 1.
- 2. Use according to claim 1, characterized in that: for the amino acid sequence as set forth in SEQ ID NO:1, and double-stranded DNA Oligo sequences containing Age I and EcoR I enzyme cutting sites at two ends of the target sequence synthesis are respectively shown as SEQ ID NO:4 and SEQ ID NO: shown at 5.
- 3. Use according to claim 1, characterized in that: is used for preparing medicines for preventing or treating lung cancer.
- 4. A kit for reducing RSPH14 gene expression in lung cancer cells, wherein the kit comprises the RSPH14 inhibitor of claim 1, and the lung cancer is non-small cell lung cancer.
- 5. A composition for preventing or treating lung cancer, characterized in that: an active agent comprising the RSPH14 inhibitor of claim 1, and a pharmaceutically acceptable carrier, diluent or excipient.
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