CN110882390B

CN110882390B - Application of human LSM5 gene and related product

Info

Publication number: CN110882390B
Application number: CN201911117856.6A
Authority: CN
Inventors: 朱琨; 李康
Original assignee: First Affiliated Hospital of Medical College of Xian Jiaotong University
Current assignee: First Affiliated Hospital of Medical College of Xian Jiaotong University
Priority date: 2019-11-15
Filing date: 2019-11-15
Publication date: 2022-03-15
Anticipated expiration: 2039-11-15
Also published as: CN110882390A

Abstract

The invention belongs to the field of biomedical research, and particularly relates to application of a human LSM5 gene as a target in preparation of a colorectal cancer treatment drug. The invention is widely and deeply researched to discover that the proliferation of colorectal cancer cells can be effectively inhibited and the apoptosis can be promoted after the expression of the human LSM5 gene is down regulated by adopting an RNAi method, and the growth process of colorectal cancer can be effectively controlled. The siRNA or the nucleic acid construct containing the siRNA sequence and the lentivirus provided by the invention can specifically inhibit the proliferation rate and the proliferation capacity of colorectal cancer cells, promote the apoptosis of the colorectal cancer cells, inhibit the cloning of the colorectal cancer cells and inhibit the growth of the colorectal cancer cells, thereby treating the colorectal cancer and opening up a new direction for the treatment of the colorectal cancer.

Description

Application of human LSM5 gene and related product

Technical Field

The invention belongs to the field of biomedical research, and particularly relates to application of a human LSM5 gene and a related product.

Background

LSM5(LSM5 Homolog, U6 Small Nuclear RNA And MRNA Degradation Association; U6 SnRNA-Associated Sm-Like Protein LSm5) is located at 7p14.3 And encodes a U6 microRNA-Associated Sm-Like Protein found in many species And believed to have sequence homology with the Sm Protein family (see SNRPD 2; MIM 601061). The Sm-like protein comprises 2 domains of the Sm sequence motif, which consists of 2 domains separated by variable length linkers, which can form a circular fold. Sm-like protein is known to form a stable heteromer of 3-snRNA (U4/U6-U5), and the heptameric LSM2-8 complex specifically binds to the 3' terminal U-region of U6 snRNA, participates in the assembly process of spliceosome and plays an important role in the splicing process of precursor mRNA.

At present, LSM5 is reported to be related to the circadian rhythm of human and plants, while the function of LSM5 in cancer is not reported.

Disclosure of Invention

In order to overcome the problems in the prior art, the invention aims to provide the application of the human LSM5 gene and related products.

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

in a first aspect of the invention, there is provided a use of human LSM5 gene as a target for the preparation of a medicament for the treatment of colorectal cancer.

The human LSM5 gene as a target for preparing the colorectal cancer treatment drug specifically comprises the following steps: the LSM5 gene is used as an action object, and the medicine or the preparation is screened to find the medicine which can inhibit the expression of the human LSM5 gene and is used as a candidate medicine for treating the colorectal cancer. The LSM5 gene small interfering RNA (siRNA) is obtained by screening human LSM5 gene as an action object and can be used as a medicine for inhibiting the proliferation of colorectal cancer cells. In addition, the LSM5 gene can be used as an object of action, for example, an antibody drug, a small molecule drug, or the like.

The colorectal cancer treatment drug is a molecule which can specifically inhibit the transcription or translation of the LSM5 gene or specifically inhibit the expression or activity of the LSM5 protein, so that the expression level of the LSM5 gene in colorectal cancer cells is reduced, and the purpose of inhibiting the proliferation, growth, differentiation and/or survival of the colorectal cancer cells is achieved.

The colorectal cancer treatment drug prepared by the LSM5 gene includes but is not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs, antibody drugs, polypeptides, proteins, or interfering lentiviruses.

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

The colorectal cancer treatment drug is administered in an amount sufficient to reduce transcription or translation of the human LSM5 gene, or to reduce expression or activity of the human LSM5 protein. Such that the expression of the human LSM5 gene is reduced by at least 50%, 80%, 90%, 95%, or 99%.

The method for treating the colorectal cancer by adopting the colorectal cancer treatment medicine mainly achieves the aim of treating the colorectal cancer by reducing the expression level of human LSM5 gene and inhibiting the proliferation of colorectal cancer cells. In particular, in therapy, a substance effective to reduce the expression level of human LSM5 gene is administered to a patient.

In one embodiment, the target sequence of the LSM5 gene is set forth in SEQ ID NO:1 is shown. The method specifically comprises the following steps: 5'-TGGTACTCTTCTAGGATTT-3' are provided.

In a second aspect of the invention, there is provided the use of an LSM5 inhibitor in the manufacture of a product having at least one of the following effects:

treating colorectal cancer;

inhibiting the rate and capacity of proliferation of colorectal cancer cells;

promoting apoptosis of colorectal cancer cells;

inhibiting colorectal cancer cell cloning;

inhibiting colorectal cancer growth.

The product necessarily comprises an LSM5 inhibitor, and an LSM5 inhibitor as an active ingredient for the aforementioned effects.

In the product, the effective component for the above functions may be only the LSM5 inhibitor, and may also include other molecules for the above functions.

That is, the LSM5 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 LSM5 inhibitor may be a nucleic acid molecule, an antibody, a small molecule compound.

As exemplified in the examples herein, the LSM5 inhibitor may be a nucleic acid molecule that reduces the expression of LSM5 gene in colorectal 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 colorectal cancer by administering to a subject an LSM5 inhibitor.

The subject may be a mammal or a mammalian colorectal cancer cell. The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, or the like. The primate is preferably a monkey, ape or human. The colorectal cancer cell can be an ex vivo colorectal cancer cell.

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

The LSM5 inhibitor may be administered to a subject before, during, or after treatment for colorectal cancer.

In a fourth aspect, the invention discloses a nucleic acid molecule for reducing the expression of the LSM5 gene in colorectal cancer cells, the nucleic acid molecule comprising a double-stranded RNA or shRNA.

Wherein the double-stranded RNA contains a nucleotide sequence capable of hybridizing with the LSM5 gene;

the shRNA contains a nucleotide sequence capable of hybridizing with the LSM5 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 LSM5 gene.

The target sequence in the LSM5 gene is a segment in the LSM5 gene corresponding to an mRNA segment which is recognized and silenced by the nucleic acid molecule when the nucleic acid molecule is used for specifically silencing the expression of the LSM5 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'-TGGTACTCTTCTAGGATTT-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'-UGGUACUCUUCUAGGAUUU-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 human LSM5 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 LSM5 gene in colorectal 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 LSM5 gene.

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

The shRNA can become small interfering RNA (siRNA) after enzyme digestion and processing, and further plays a role in specifically silencing the expression of endogenous LSM5 gene in colorectal cancer cells.

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

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

Further, the LSM5 gene is derived from human.

In the fifth aspect of the invention, the LSM5 gene interference nucleic acid construct contains a gene segment for coding shRNA in the nucleic acid molecule and can express the shRNA.

The LSM5 gene interfering nucleic acid construct can be obtained by cloning a gene segment for coding the human LSM5 gene shRNA into a known vector.

Further, the LSM5 gene interference nucleic acid construct is an LSM5 gene interference lentiviral vector.

The LSM5 gene interference lentiviral vector disclosed by the invention is obtained by cloning a DNA fragment for coding the LSM5 gene shRNA into a known vector, wherein most of the known vectors are lentiviral vectors, the LSM5 gene interference lentiviral vector is packaged into infectious viral particles by viruses, and then infects colorectal cancer cells to transcribe the shRNA, and the siRNA is finally obtained through the steps of enzyme digestion processing and the like and is used for specifically silencing the expression of the LSM5 gene.

Further, the LSM5 gene interference lentiviral vector further comprises a promoter sequence and/or a nucleotide sequence encoding a marker detectable in colorectal cancer cells; preferably, the detectable label is Green Fluorescent Protein (GFP).

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

The embodiment of the invention specifically lists a human LSM5 gene interference lentiviral vector constructed by taking pGCSIL-GFP as a vector, and is named as pGCSIL-GFP-LSM 5-siRNA.

The LSM5 gene siRNA can be used for inhibiting the proliferation of colorectal cancer cells, and further can be used as a medicine or a preparation for treating colorectal cancer. The LSM5 gene interference lentiviral vector can be used for preparing the LSM5 gene siRNA. When used as a medicament or formulation for treating colorectal cancer, a safe and effective amount of the nucleic acid molecule is administered to a mammal. The particular dosage will also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

The invention also discloses a LSM5 gene interference lentivirus, which is formed by virus packaging of the LSM5 gene interference nucleic acid construct under the assistance of lentivirus packaging plasmids and cell lines. The lentivirus can infect the colorectal cancer cells and generate small interfering RNA aiming at the LSM5 gene, thereby inhibiting the proliferation of the colorectal cancer cells. The LSM5 gene interference lentivirus can be used for preparing medicines for preventing or treating colorectal cancer.

In a seventh aspect of the present invention, there is provided a use of the aforementioned nucleic acid molecule, or the aforementioned LSM5 gene interfering nucleic acid construct, or the aforementioned LSM5 gene interfering lentivirus, wherein: used for preparing a medicine for preventing or treating colorectal cancer or used for preparing a kit for reducing the expression of LSM5 gene in colorectal cancer cells.

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

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

The subject of the method may be a human.

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

the aforementioned nucleic acid molecules; and/or, the aforementioned LSM5 gene interfering nucleic acid construct; and/or the aforementioned LSM5 gene interfering lentivirus, and a pharmaceutically acceptable carrier, diluent or excipient.

The composition may be a pharmaceutical composition.

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

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

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

In conclusion, the invention designs an RNAi target sequence aiming at the human LSM5 gene and constructs a corresponding LSM5 RNAi vector, wherein the RNAi vector pGCSIL-GFP-LSM5-siRNA can obviously reduce the expression of the LSM5 gene at the mRNA level and the protein level. The slow virus (lentivirus, abbreviated as Lv) is used as a gene operation tool to carry an RNAi vector pGCSIL-GFP-LSM5-siRNA, so that the RNAi sequence aiming at the LSM5 gene can be efficiently introduced into colorectal cancer cells in a targeted manner, the expression level of the LSM5 gene is reduced, and the proliferation capacity of the tumor cells is obviously inhibited. Lentivirus-mediated silencing of the LSM5 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 invention is widely and deeply researched to discover that the proliferation of colorectal cancer cells can be effectively inhibited and the apoptosis can be promoted after the expression of the human LSM5 gene is down regulated by adopting an RNAi method, and the growth process of colorectal cancer can be effectively controlled. The siRNA or the nucleic acid construct containing the siRNA sequence and the lentivirus provided by the invention can specifically inhibit the proliferation rate and the proliferation capacity of colorectal cancer cells, promote the apoptosis of the colorectal cancer cells, inhibit the cloning of the colorectal cancer cells and inhibit the growth of the colorectal cancer cells, thereby treating the colorectal cancer and opening up a new direction for the treatment of the colorectal cancer.

Drawings

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

FIG. 2: RT-PCR measures the efficiency of target gene depletion at HCT116 cell mRNA levels.

FIG. 3: analysis of the results using a Tecan infiinite enzyme labelling instrument revealed that depletion of the LSM5 gene inhibited proliferation of colorectal cancer cells RKO.

FIG. 4: analysis of the results using a Tecan infiinite enzyme labelling instrument revealed that depletion of the LSM5 gene inhibited proliferation of the colorectal cancer cells HCT 116.

FIG. 5: a digital camera record of the effect of LSM5 gene on the proliferation potency of RKO cells was examined by cell clonogenic assay.

FIG. 6: cell clonogenic assay the effect of LSM5 gene on RKO cell proliferation potency was plotted and the bar results are presented as mean cell clone number. + -. standard deviation.

FIG. 7: a digital camera record of the effect of LSM5 gene on the proliferation potency of HCT116 cells was examined by cell clonogenic assay.

FIG. 8: the influence of LSM5 gene on the proliferation capacity of HCT116 cells was examined by cell clonogenic assay, and the bar results are shown as the mean value of the number of cell clones. + -. standard deviation.

FIG. 9: schematic flow cytoapoptosis assay for the effect of shLSM5 on RKO apoptosis in Annexin V-APC flow cytoapoptosis.

FIG. 10: annexin V-APC flow apoptosis assay shLSM5 effect on RKO apoptosis, bar results are shown as percent cell mean. + -. standard deviation.

FIG. 11: schematic flow apoptosis of Annexin V-APC flow apoptosis assay shLSM5 for its effect on HCT116 apoptosis.

FIG. 12: annexin V-APC flow apoptosis assay shLSM5 effect on HCT116 apoptosis, bar results are shown as the mean of cell percentage ± standard deviation.

FIG. 13: celigo cell counting validated the effect of LSM5 gene on RKO cell proliferation, and cell pictures were recorded for 5 consecutive days by Celigo.

FIG. 14: celigo cell counting demonstrated the effect of LSM5 gene on RKO cell proliferation, a plot of cell number versus time for shLSM5 and shCtrl control.

FIG. 15: the Celigo cell counting method verifies the effect of the LSM5 gene on HCT116 cell proliferation, and cell pictures are recorded by Celigo for 5 consecutive days.

FIG. 16: the Celigo cell counting method verifies the effect of LSM5 gene on HCT116 cell proliferation, and the time-dependent cell number curves of shLSM5 group and shCtrl control group.

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 inventor of the invention has found through extensive and intensive research that the LSM5 gene is significantly highly expressed in colorectal cancer tumor tissues; the inventor finds that after the expression of the human LSM5 gene is down-regulated by an RNAi method, the proliferation of tumor cells can be effectively inhibited, the apoptosis can be promoted, and the cloning forming capability of the tumor cells can be effectively controlled, so that the research result shows that the LSM5 gene is a protooncogene and can be used as a target point for tumor treatment. The inventor further synthesizes and tests a plurality of siRNAs aiming at the LSM5 gene, screens out the siRNA which can effectively inhibit the expression of the LSM5 and further inhibit the proliferation of human colon cancer RKO cells and colon cancer HCT116 cells, and completes the invention on the basis.

The invention proves the function of the LSM5 gene in colorectal cancer generation from the viewpoint of cell function. Transfecting colorectal cancer cells after constructing a target gene shRNA lentivirus, and comparing the transfected colorectal cancer cells with a transfection control lentivirus to detect the expression conditions of mRNA and protein level target genes in two groups of colorectal cancer cell lines; and then cell proliferation, apoptosis and other detection are carried out through a cytofunctional experiment, and the result shows that the shRNA group is compared with a control group, the colorectal cancer cell proliferation inhibition degree of the shRNA group is obviously higher than that of the control group, and the increase degree of the cell apoptosis rate is higher than that of the control group.

LSM5 inhibitor

Refers to a molecule having an inhibitory effect on LSM 5. Having inhibitory effects on LSM5 include, but are not limited to: inhibiting the expression or activity of LSM 5.

Inhibiting LSM5 activity refers to a decrease in LSM5 activity. Preferably, the activity of LSM5 is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, even more preferably by at least 70%, and most preferably by at least 90% as compared to its activity prior to inhibition.

The inhibition of the expression of LSM5 may specifically be the inhibition of the transcription or translation of LSM5 gene, and specifically may refer to: the gene of LSM5 is not transcribed, or the transcriptional activity of the gene of LSM5 is reduced, or the gene of LSM5 is not translated, or the level of translation of the gene of LSM5 is reduced.

The regulation of LSM5 gene expression can be performed by one skilled in the art using conventional methods, such as gene knock-out, homologous recombination, interfering RNA, and the like.

The inhibition of LSM5 gene expression was confirmed by PCR and Western Blot detection of expression level.

Preferably, the LSM5 gene expression is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, more preferably by at least 70%, still more preferably by at least 90%, most preferably completely absent of the LSM5 gene, as compared to the wild type.

Small molecule compounds

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

Preparation of medicine for preventing or treating colorectal cancer

Nucleic acid molecules that reduce expression of the LSM5 gene in colorectal cancer cells can be utilized; and/or, a LSM5 gene interfering nucleic acid construct; and/or LSM5 gene interferes with lentivirus, and is used as an effective component for preparing a medicament for preventing or treating colorectal cancer. Generally, the medicament can comprise one or more pharmaceutically acceptable carriers or auxiliary materials besides the effective components according to the requirements of different dosage forms.

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

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

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

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

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

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

Example 1 preparation of RNAi lentivirus against human LSM5 Gene

1. Screening of effective siRNA target against human LSM5 gene

Calling LSM5 (NM-012322) gene information from Genbank; designing effective siRNA target point aiming at LSM5 gene. Table 1-1 lists the effective siRNA target sequences screened against LSM5 gene.

TABLE 1-1 siRNA target sequences targeting the human LSM5 gene

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

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. And (3) converting the ligation product into calcium chloride to prepare fresh escherichia coli competent cells (conversion operation, namely adding 10 mu L of exchange reaction product into 100 mu L of competent cells, flicking the tube wall, uniformly mixing, placing on ice for 30min, carrying out heat shock at 42 ℃ for 90s, carrying out ice water bath incubation for 2min, adding 500 mu L of LB culture medium, placing on a 37 ℃ shaking table for shaking culture for 1h, taking a proper amount of bacterial liquid, uniformly coating the bacterial liquid on a flat plate containing corresponding antibiotics, and carrying out inverted culture in a constant-temperature incubator for 12-16 h). 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 performing PCR identification experiment (the PCR reaction system is shown in tables 1-6, the reaction conditions are shown in tables 1-7, shaking and mixing, and centrifuging briefly, picking single colony from a sterile gun head into a 20 μ L identification system in an ultraclean bench, blowing and mixing, and placing in a PCR instrument for reaction). 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-LSM 5-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 (shown in tables 1-3) containing adhesive ends of Age I and EcoR I enzyme cutting sites at two ends are synthesized aiming at Scr siRNA targets, and the rest construction methods, identification methods and conditions are the same as pGCSIL-GFP-LSM 5-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

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

TABLE 1-6-1 PCR reaction System

TABLE 1-7 PCR reaction System Programming

3. Packaging of LSM5-siRNA lentiviruses

The DNA of RNAi plasmid pGCSIL-GFP-LSM5-siRNA is extracted by a small-extraction medium-amount kit of Tiangen endotoxin-free plasmid, and 100 ng/mul stock solution is prepared.

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 serum⁶Cells/15 ml, seeded in 10cm dishes at 37 ℃ with 5% CO₂Culturing in an incubator. To be treatedThe cell density can reach 70-80% to be used for transfection. 2h before transfection, the original culture medium is sucked out and replaced by a serum-free culture medium. Each of the prepared DNA solutions (20. mu.g of GV vector plasmid, 15. mu.g of pHelper 1.0 vector plasmid, 10. mu.g of pHelper 2.0 vector plasmid) was added to a sterilized centrifuge tube according to the instructions of the MISSION Lentiviral Packaging Mix kit from Sigma-aldrich Co., Ltd. and mixed uniformly with the corresponding volume of the Gecky transfection reagent to adjust the total volume to 1 ml.

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 concentrate is shown in SEQ ID NO. 2. The packaging procedure for the control lentivirus was identical to LSM5-siRNA lentivirus except that pGCSIL-GFP-Scr-siRNA vector was used instead of pGCSIL-GFP-LSM5-siRNA vector.

Example 2 detection of Gene silencing efficiency by real-time fluorescent quantitative RT-PCR

Human colorectal cancer RKO cells and HCT116 cells in logarithmic growth phase are trypsinized to prepare cell suspension (the number of cells is about 2X 10)⁵/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, RKO: 10; MOI, HCT 116: 10), an appropriate amount of the lentivirus prepared in example 1 was added, the medium was changed after 16h of culture, and cells were collected after the infection time reached 3 days. According to the Trizol instructions of Invitrogen corporationTotal RNA was extracted. 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). Primers for the LSM5 gene were as follows: an upstream primer 5'-CTAACGCTACTACCAACCCGT-3' (SEQ ID NO: 11) and a downstream primer 5'-TCCTTCTCCTCCAGGAACCAG-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

Reagent	Volume (μ l)
		5×RT buffer	4.0
10mM dNTPs	2.0
		RNasin	0.4
M-MLV-RTase	1.0
		DEPC H₂O	2.6
Total	10.0

TABLE 2-2 Real-time PCR reaction System

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 adopting 2-^ΔΔCtThe assay calculated the expression abundance of LSM 5-infected mRNA. Cells infected with the control virus served as controls. The results of the experiments are shown in FIG. 1 and FIG. 2, respectively, which indicate that the expression level of LSM5mRNA in human colorectal cancer RKO cells is down-regulated by 97.00% and the expression level of LSM5mRNA in human colorectal cancer HCT116 cells is down-regulated by 93.80%.

Example 3 examination of the proliferative Capacity of tumor cells infected with LSM5-siRNA lentivirus

Human colorectal cancer RKO cells and HCT116 cells in logarithmic growth phase are trypsinized to prepare cell suspension (the number of cells is about 2X 10)⁵/ml) were inoculated in 6-well plates and cultured until the degree of cell confluence reached about 30%. According to the number of infection (MOI, RKO: 10; MOI, HCT 116: 10), a proper amount of virus is added, the culture medium is replaced after 16h of culture, and after the infection time reaches 3 days, cells of each experimental group in the logarithmic growth phase are collected. Complete medium resuspension into cell suspension (RKO: 2.5X 10)⁴/ml，HCT116：2×10⁴Per ml) at a cell density of about RKO: 2500/hole; HCT 116: 2000/well, 96-well plates were inoculated. Each set of 3 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 reading plate is detected once every day by a Tecan infinite enzyme labeling instrument from the next day after the plate laying, and the detection is continuously carried outRead plate for 5 days. The data were statistically plotted by adjusting the OD 490nm of the Tecan infinite assay to generate a cell proliferation curve. (the results are shown in FIGS. 3 and 4). The results show that after 5 days of in vitro cell culture of each tumor of the lentivirus infection group, the proliferation speed is remarkably slowed down and is far lower than that of tumor cells of a control group, the number of human RKO cell viable cells is reduced by 81.66%, and the number of human HCT116 cell viable cells is reduced by 56.60%, which indicates that the proliferation capacity of human colorectal cancer RKO cells and HCT116 cells is inhibited due to LSM5 gene silencing.

Example 4 examination of the clonogenic Capacity of tumor cells infected with LSM5-siRNA lentivirus

Human colorectal cancer RKO cells and HCT116 cells are trypsinized and then inoculated into a 12-well plate, and the cell density is 10-15%. The next day, the medium was changed to fresh medium containing 5ug/ml polybrene. LSM5-siRNA lentiviruses were added to the plates according to the multiplicity of infection (MOI, RKO: 10; MOI, HCT 116: 10) and replaced with fresh medium 16h after infection. After infection for 72h, fluorescence is observed under a fluorescence microscope, and the infection efficiency reaches 90%.

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 until the number of the cells in 14 days or most of single clones is more than 50, changing the liquid at intervals of 3day, and observing the cell state; photographing the cell clone under a fluorescent microscope before the experiment is terminated; at the end of the experiment, the cells were fixed with paraformaldehyde, washed with PBS, stained with crystal violet, and photographed.

As shown in fig. 5 and fig. 6, in human colorectal cancer RKO cell experiments, the number of clone spots formed by tumor cells is significantly reduced and the volume of the clone spots is significantly reduced after the expression of LSM5 gene is reduced by RNA interference (KD group) compared with the control (NC group); indicating that gene silencing results in a reduction in the ability of tumor cells to form clones. The plate cloning test detects that after the expression of the gene is reduced, the cloning capacity of the tumor cells is reduced.

As shown in fig. 7 and fig. 8, in the human colorectal cancer HCT116 cell experiment, the number of clonal plaques formed by tumor cells is significantly reduced and the volume of the clonal plaques is significantly reduced after the expression of LSM5 gene is reduced by RNA interference (KD group) compared with the control (NC group); indicating that gene silencing results in a reduction in the ability of tumor cells to form clones. 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 detection of apoptosis levels in tumor cells infected with LSM5-siRNA lentivirus

Human colorectal cancer RKO cells and HCT116 cells are inoculated in a 6-well plate after being digested by trypsin, and the cell density is 10-15%. The next day, the medium was changed to fresh medium containing 5ug/ml polybrene. Lentiviruses were added to the plates according to multiplicity of infection (MOI, RKO: 10; MOI, HCT 116: 10) and replaced with fresh medium 16h after infection. After infection for 72h, fluorescence is observed under a fluorescence microscope, and the infection efficiency reaches 90%. Continuously culturing for 2 days after passage, digesting the pancreatin of cells in logarithmic growth phase, and re-suspending the complete culture medium into cell suspension; annexin V-APC staining was performed for 15min in the absence of light. And adding the suspension into a 96-well plate, detecting on a Guava flow cytometer, and analyzing by using Guava InCyte software to obtain a result.

As shown in FIGS. 9 and 10, in human colorectal cancer RKO cell experiments, the Annexin V-APC method detects the change of the early apoptosis cell ratio of tumor cells after the expression of genes is reduced. It was found that the apoptosis rate of tumor cells increases after down-regulating gene expression. Compared with the control (NC group), the early apoptotic cell proportion is remarkably increased after the expression of the RNA interference reduction gene (KD group); indicating that gene silencing leads to apoptosis of tumor cells.

As shown in FIGS. 11 and 12, in the human colorectal cancer HCT116 cell experiment, the Annexin V-APC method detects the change of the early apoptosis cell ratio of the tumor cells after the expression of the genes is reduced. It was found that the apoptosis rate of tumor cells increases after down-regulating gene expression. Compared with the control (NC group), the early apoptotic cell proportion is remarkably increased after the expression of the RNA interference reduction gene (KD group); indicating that gene silencing leads to apoptosis of tumor cells.

Example 6 examination of the proliferation Capacity of tumor cells celgo infected with LSM5-siRNA lentivirus

After trypsinizing the virus-infected cells in logarithmic growth phase (MOI, RKO: 10; MOI, HCT 116: 10), the complete medium was resuspended into a cell suspension and counted; the plating cell density was determined according to the growth rate of the cells (most of the cell plating numbers were RKO: 2000 cells/well; HCT 116: 1000 cells/well). Each group has 3 multiple wells, the culture system is 100 μ L/well, the number of cells added into each well is consistent in the plating process, the temperature is 37 ℃, and the CO content is 5%₂(ii) a Detecting and reading the plate once by Celigo (Nexcelom) every day from the next day after the plate is paved, and continuously detecting and reading the plate for 5 days; culturing in an incubator; accurately calculating the number of cells with green fluorescence in each scanning pore plate by adjusting input parameters of analysis settings; the data were statistically plotted and cell proliferation curves were plotted for 5 days.

As shown in fig. 13 and 14, in human colorectal cancer RKO cell experiments, when the expression of LSM5 gene was reduced by RNA interference (KD group) compared to the control (NC group), it was found that the proliferation ability of tumor cells having green fluorescence was reduced by down-regulating LSM5 gene expression.

As shown in fig. 15 and 16, in the human colorectal cancer HCT116 cell experiment, when the expression of LSM5 gene was reduced by RNA interference (KD group) compared to the control (NC group), it was found that the proliferation ability of the tumor cells having green fluorescence was reduced by down-regulating the expression of LSM5 gene.

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

<110> first subsidiary Hospital of medical college of Western-Ann transportation university

Application of <120> human LSM5 gene and related product

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tggtactctt ctaggattt 19

<210> 2

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ugguacucuu cuaggauuu 19

<210> 3

<211> 46

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ugguacucuu cuaggauuuc ucgagaaauc cuagaagagu accaac 46

<210> 4

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ccgggttggt actcttctag gatttctcga gaaatcctag aagagtacca actttttg 58

<210> 5

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

aattcaaaaa gttggtactc ttctaggatt tctcgagaaa tcctagaaga gtaccaac 58

<210> 6

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cctatttccc atgattcctt cata 24

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gtaatacggt tatccacgcg 20

<210> 8

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ttctccgaac gtgtcacgt 19

<210> 9

<211> 54

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ccggttctcc gaacgtgtca cgtctcgaga cgtgacacgt tcggagaatt tttg 54

<210> 10

<211> 54

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

aattcaaaaa ttctccgaac gtgtcacgtc tcgagacgtg acacgttcgg agaa 54

<210> 11

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ctaacgctac taccaacccg t 21

<210> 12

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tccttctcct ccaggaacca g 21

<210> 13

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tgacttcaac agcgacaccc a 21

<210> 14

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

caccctgttg ctgtagccaa a 21

Claims

Use of an LSM5 inhibitor for the preparation of a product having at least one of the following effects:

treating colorectal cancer;

inhibiting the rate and capacity of proliferation of colorectal cancer cells;

promoting apoptosis of colorectal cancer cells;

inhibiting colorectal cancer cell cloning;

inhibiting the growth of the colorectal cancer,

the LSM5 inhibitor is selected from double-stranded RNA or shRNA, and the target sequence of the shRNA or the double-stranded RNA is shown as SEQ ID NO:1, 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 shown as SEQ ID NO:2, the nucleotide sequence of the shRNA is shown as SEQ ID NO: 3, respectively.
2. The use of claim 1, further comprising one or more of the following features:

1) the LSM5 inhibitor refers to a molecule having inhibitory effect on LSM 5;

2) the LSM5 inhibitor is the only effective component or one of the effective components of the product.
3. A nucleic acid molecule that reduces LSM5 gene expression in colorectal cancer cells, the nucleic acid molecule comprising:

a. double-stranded RNA containing a nucleotide sequence capable of hybridizing with the LSM5 gene; or

shRNA containing a nucleotide sequence capable of hybridizing with the LSM5 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 identical to a target sequence in the LSM5 gene; the shRNA comprises a sense strand segment and an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, wherein the sequences of the sense strand segment and the antisense strand segment are complementary, the sequence of the sense strand segment is identical to a target sequence in an LSM5 gene, and the shRNA or double-stranded RNA target sequence is shown in SEQ ID NO:1, the double-stranded RNA comprises a first strand and a second strand, the first strand 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, the nucleotide sequence of the shRNA is shown as SEQ ID NO: 3, respectively.
4. An LSM5 gene interfering nucleic acid construct comprising a gene segment encoding the shRNA in the nucleic acid molecule of claim 3, capable of expressing the shRNA.
5. An LSM5 gene interfering lentivirus, which is formed by virus packaging of the interfering nucleic acid construct of claim 4 with the help of lentivirus packaging plasmid and cell line.
6. The nucleic acid molecule of claim 3, or the LSM5 gene-interfering nucleic acid construct of claim 4, or the use of the LSM5 gene-interfering lentivirus of claim 5, which is: used for preparing a medicine for treating colorectal cancer or a kit for reducing the expression of LSM5 gene in colorectal cancer cells.
7. A composition for treating colorectal cancer, which comprises the effective components:

the nucleic acid molecule of claim 3; and/or, the LSM5 gene interfering nucleic acid construct of claim 4; and/or the LSM5 gene interfering lentivirus of claim 5, together with a pharmaceutically acceptable carrier, diluent or excipient.