CN104946653B

CN104946653B - Small interfering RNA sequence of CHRM3 gene and application thereof

Info

Publication number: CN104946653B
Application number: CN201510411371.3A
Authority: CN
Inventors: 于耀清; 王欢
Original assignee: Tangdu Hospital Of Fourth Military Medical University
Current assignee: Tangdu Hospital Of Fourth Military Medical University
Priority date: 2015-07-03
Filing date: 2015-07-03
Publication date: 2021-09-03
Anticipated expiration: 2035-07-03
Also published as: CN104946653A

Abstract

The invention discloses a small interfering RNA sequence of CHRM3 gene and application thereof, wherein the sense strand of the siRNA sequence is SEQ ID NO: 2, the antisense strand is SEQ ID NO: 3, the siRNA can effectively reduce the expression of M3 type mAChR protein, inhibit the proliferation and migration capacity of melanoma cells, and has application value in the aspect of tumor resistance.

Description

Small interfering RNA sequence of CHRM3 gene and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to a small interfering RNA sequence of CHRM3 gene and application thereof.

Background

Malignant melanoma (malignant melanoma) is a malignant tumor originated from melanocytes of neural crest, and has high malignancy degree, easy metastasis and poor prognosis, and the treatment means used at present, such as surgery, radiotherapy, chemotherapy, and the like, have poor effect. The selection of specific and effective targets is the key to treat the disease.

The muscarinic acetylcholine receptor (mAChR) belongs to the family of G protein coupled receptors, is a 7-transmembrane glycoprotein consisting of 460-590 amino acids and has a molecular weight of 51-66 kD. Five mAChR subtypes (i.e., M1-M5) have been cloned. The CHRM3 gene is the coding gene of M3 mAChR, and although M3 mAChR is found to be abnormally expressed in melanoma, the function and mechanism of the receptor in the biological processes of melanoma proliferation, invasion and metastasis and the like are not clear due to the lack of compounds capable of being used as specific antagonists and/or agonists.

RNA interference (RNAi) refers to a highly conserved biological phenomenon in the evolution process, induced by double-stranded RNA (dsRNA), that specifically interferes with the expression of a target gene with high efficiency and specificity of homologous mRNA degradation. The action mechanism is as follows: dicer enzyme of ribonuclease III family binds to dsRNA, cuts it into 21-23nt and Small interfering RNA (siRNA) with 3' end protrusion, then siRNA binds to RNA-induced silencing complex (RISC), unwinds into single strand, activated RISC is guided by the single stranded siRNA, sequence-specifically binds to target messenger RNA (mRNA) and cuts it off, and triggers the specific decomposition of target mRNA, thereby inhibiting the expression of corresponding gene. RNA interference technology brings hope for specific treatment of virus infectious diseases, genetic diseases, tumors and the like, and is an important field competitive in various countries in the world at present. Related patents (e.g., US2011065778a1, US8076472B2, US2010099750a1, etc.) disclose RNA interference sequences of the CHRM3 gene, but the related sequences are directed against CHRM3 gene numbered NM — 000740.1, the sequence comprising 1773 bases. The recent update of CHRM3 gene is encoded as NM-000740.2, the sequence of which comprises 2757 bases. Therefore, the change of the base can directly influence the design, synthesis and even biological function evaluation of the RNA interference sequence.

Disclosure of Invention

The invention aims to provide a novel small interfering RNA sequence for specifically inhibiting CHRM3 gene expression and application thereof by taking the latest CHRM3 gene sequence as a target point. The CHRM3 gene small interfering RNA sequence can generate an interference effect after gene transcription, specifically reduce protein expression of M3 mAChR, inhibit proliferation and invasion of melanoma, and can be used for treating the melanoma in the future.

The specific technical scheme is as follows:

a small interfering RNA sequence of CHRM3 gene, which consists of the following sense strand and antisense strand:

sense strand: 5'-CCUCGCCUUUGUUUCCAAATT-3', as shown in SEQ ID N0: 2 is shown in the specification;

antisense strand: 5'-UUUGGAAACAAAGGCGAGGTT-3', as set forth in SEQ ID N0: 3, respectively.

The invention discloses an application of a small interfering RNA sequence of CHRM3 gene in inhibiting melanoma cell proliferation.

The invention discloses application of a small interfering RNA sequence of CHRM3 gene in reducing the invasion capacity of melanoma cells.

The invention has the beneficial effects that:

the small interfering RNA sequence obtained by the invention is not disclosed in the prior art, can interfere after the transcription of CHRM3 gene, thereby specifically reducing the expression of M3 type mAChR protein, inhibiting the proliferation and invasion of melanoma, and can be used for treating the melanoma in the future.

Drawings

Figure 1 is the down-regulation of melanoma cell line M3 type mAChR protein by siRNA. (Western blot is shown in figure to detect the expression quantity of M3 mAChR protein, and the expression quantity is sequentially a blank group, a control group and an siRNA group; beta-actin is an internal reference; the siRNA is suggested to be capable of obviously reducing the expression of the M3 mAChR protein.

FIG. 2 shows MTT assay to determine the effect of siRNA on melanoma cell proliferation. The results show that: the siRNA can obviously inhibit the proliferation of melanoma A2058 cells, and the inhibition rate is the highest in 24 hours and reaches 48%. After 48 hours, the inhibition rate gradually decreased to 20%. Suggesting that the siRNA has the function of resisting tumor proliferation.

FIG. 3 is a transwell chamber to examine the effect of siRNA on cell migration. A is the typical manifestation of a migrating tumor cell under a light microscope. And B is a statistical result, compared with a control group, the siRNA can obviously inhibit the migration of melanoma A2058 cells. Suggesting that the siRNA has the function of resisting tumor cell invasion.

Detailed Description

The technical solution of the present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

The method used in the present invention is a conventional method unless otherwise specified, the reagents used are purchased from Invitrogen unless otherwise specified, and the interference sequences are synthesized by Shanghai Jima pharmaceutical technology, Inc. The experimental procedures in the examples were carried out according to conventional conditions, with reference to the molecular cloning protocol (third edition, sambrook et al) and the conditions described in the reagent instructions.

The first embodiment is as follows: acquisition of specific interference sequence of targeted human CHRM3 gene

siRNA sequences are designed according to a human CHRM3 gene sequence (Genbank: NM-000740.2) disclosed by NCBI, specifically RNAi online design software provided by the website of the American Invitrogen company (the website of the RNAi is http:// rnaidesigner. Invitrogen. com/rnaiexpress/sort. do) is designed, and target gene sequences containing 19 bases are obtained through alignment screening: 5'-CCTCGCCTTTGTTTCCAAA-3' (SEQ ID NO: 1), the corresponding siRNA interference sequence was synthesized by Shanghai Jima pharmaceutical technology, Inc., as follows:

sense strand: 5'-CCUCGCCUUUGUUUCCAAATT-3' (SEQ ID NO: 2)

Antisense strand: 5'-UUUGGAAACAAAGGCGAGGTT-3' (SEQ ID NO: 3)

Example two: tumor cell culture and Western blot detection of the effect of siRNA on M3 type mAChR protein expression.

1) Melanoma cell line A2058(ATCC, USA) cells seeded 1X 10 in 10% DMEM⁵Culturing in 95% air, 5% CO2, 95% humidity, and 37 deg.C under constant temperature, and changing the culture solution.

2) And (4) siRNA transfection. The experiment was divided into a blank group (normal cell culture without transfection reagent), a control group (negative control purchased from Santa Cruz, sc-37007), and an siRNA group (synthetic small interfering RNA sequence). A single cell suspension (cell number 1X 10) was prepared by digesting the melanoma cell line A2058 in the logarithmic growth phase with 0.125% trypsin⁵) And inoculating the cells in a 6-well plate, and culturing until the cell fusion degree is more than 40%. siRNA (final concentration of transfected cells was 33nM) was diluted in 250. mu.l of serum free medium (Opti-MEM) and gently pipetted into the mix. Mu.l Lipofectamine TM2000 was diluted with 250. mu.l Opti-MEM, gently pipetted 3-5 times and mixed, and allowed to stand at room temperature for 10 min. The transfection reagent and siRNA diluent were mixed, gently aspirated and mixed, and allowed to stand at room temperature for 20 min. Mu.l of the transfection complex was mixed with 1.5ml serum-free DMEM, added to a 24-well cell plate (100. mu.l/well), and mixed well by gentle shaking. The mixture was placed in a 5% CO2 incubator at 37 ℃. After 24h of transfection, the culture solution is changed, and the culture is continued according to the conventional conditions.

3) Western blot detection of M3 type mAChR protein expression. After A2058 cells were transfected for 24h, the medium was aspirated, the cells were washed with PBS for 1-2 times, 100. mu.l of RIPA lysate was added to the cells, the cells were collected into a 1.5ml tube, allowed to stand on ice for 30min, centrifuged at 12000rpm, and the supernatant was taken for 30min to prepare a protein sample for analysis. The prepared protein was added to loading buffer RSB), boiled at 100 ℃ for 5min, immediately transferred to ice to cool, and used as a loading sample for electrophoresis. Separating by electrophoresis with 5% concentrated gel and 12% separation gel, the loading amount is 20 μ g/lane, the electrophoresis voltage is 70V for concentrated gel and 100V for separation gel, and the electrophoresis time is about 2.5 h. The internal reference is beta-actin.

EXAMPLE III MTT assay to examine the Effect of siRNA on cell proliferation

1) A2058 cells were digested with 0.125% trypsin to give single cell suspensions, which were dispensed into 96-well plates at 100. mu.l/well. 37 ℃ and 5% CO₂After 24h incubation in the incubator, the supernatant was discarded and 200. mu.l of serum-free medium was added to each well for transfection for 4h (experiments were divided into blank, control, siRNA as described above).

2) After 24 hours of culture, the supernatant was discarded, 200. mu.110% fetal bovine serum culture medium was added, and after 24 hours, 48 hours, and 72 hours of culture, 20. mu.l of MTT solution (5mg/ml) was added, and the culture was continued for 4 hours.

3) The supernatant was discarded, 150. mu.l of dimethyl sulfoxide was added to each well, and the mixture was shaken on a shaker at a low speed for 10min to dissolve the crystals sufficiently. The absorbance of each well was measured at 490nm of the microplate reader.

4) The percent inhibition of cell growth was calculated as [ < 1-A ]_{(Experimental group)}/A₍To pair_{Light group)}]×100％

Example four migration Chamber to examine the Effect of siRNA on cell migration

1) And (4) pretreatment. A2058 cells were digested with 0.125% trypsin to form a single cell suspension and seeded into migration chambers. 37 ℃ and 5% CO₂After 24h incubation in the incubator, the supernatant was discarded. After 24h of transfection, the medium was changed to normal medium for further use.

2) Cell suspensions and chambers were prepared. Taking out the transfer plate under aseptic condition, standing at room temperature for 10min, and adding 500 μ l of 10% FBS culture medium into lower chamber; adding 200 μ l of low serum culture solution in the upper chamber, standing at room temperature for 10min, and hydrating. Resuspending each group of A2058 cells in a low serum culture medium containing 0.5% FBS at 1X 10⁵cells/mL. 150 μ l of cell suspension was added to the upper chamber and incubated at 37 ℃ for 12 h.

3) Staining and counting. The cotton swab was used to wipe the non-migrating cells from the top of the chamber, the transwell chamber was removed, inverted, air dried, 500. mu.l of 0.1% crystal violet stain was added to the 24-well plate, the chamber was placed in it, the membrane was immersed in the stain, removed after 25min, and washed 3-5 times with PBS. And taking a picture under a light mirror and counting.

The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.

Claims

1. The application of a small interfering RNA sequence of CHRM3 gene in preparing a medicament for inhibiting the proliferation of melanoma cells; the small interfering RNA sequence of the CHRM3 gene consists of the following sense strand and antisense strand:

the sense strand is shown as SEQ ID NO: 2 is shown in the specification;

the antisense strand is shown as SEQ ID NO: 3, respectively.