CN113717972B

CN113717972B - MYADM targeted siRNA and application thereof

Info

Publication number: CN113717972B
Application number: CN202110940769.1A
Authority: CN
Inventors: 杨秋星; 张金业; 郭丽媛; 沈爱国
Original assignee: Nantong Tumor Hospital
Current assignee: Nantong Tumor Hospital
Priority date: 2021-08-16
Filing date: 2021-08-16
Publication date: 2022-07-01
Anticipated expiration: 2041-08-16
Also published as: CN113717972A

Abstract

The invention discloses MYADM targeted siRNA and application thereof, and belongs to the technical field of molecular biotechnology and genetic engineering. The invention provides a small interfering RNA for inhibiting MYADM gene expression and application thereof, and the small interfering RNA is transfected in esophageal cancer cells, so that MYADM gene expression can be regulated, controlled and interfered, the cell clone formation capacity is reduced, and wound healing and invasion of cells are inhibited; therefore, the small interfering RNA for inhibiting MYADM gene expression can effectively inhibit the proliferation, migration and invasion of esophageal cancer cells by reducing the expression of MYADM. The invention provides a new potential medicine for treating esophageal cancer, and has very high clinical practical application prospect and value.

Description

MYADM targeted siRNA and application thereof

Technical Field

The invention belongs to the technical field of molecular biotechnology and genetic engineering, and particularly relates to MYADM targeted siRNA and application thereof.

Background

Esophageal cancer is a major public health problem worldwide and is one of the most aggressive malignancies. Despite considerable diagnostic and therapeutic advances in recent years, the prognosis of patients with esophageal cancer remains poor due to high rates of metastasis and invasion. The research on the effect of the metastasis and invasion related genes on the proliferation and metastasis of esophageal cancer cells has important theoretical significance and application value for improving the prognosis of patients with esophageal cancer.

RNAi technology can specifically eliminate or close the expression of specific genes, and specifically introduce small interfering RNA into cells of mammals and human by applying RNAi technology to reduce the expression of target genes, thereby causing the expression of target proteins to be reduced, and achieving the efficient and specific gene therapy effect.

Disclosure of Invention

Aiming at the problems in the prior art, the technical problem to be solved by the invention is to provide MYADM targeted siRNA. The invention also aims to solve the technical problem of providing application of the MYADM targeted siRNA in preparation of a medicament for treating esophageal cancer.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a MYADM targeted siRNA molecule is a double-stranded RNA molecule with the following nucleic acid sequence:

a sense sequence: 5 '-CGGCGAGAUCACUGGCUAUdTdT-3',

antisense sequence: 5 '-AUAGCCAGUGAUCUCGCCGdTdT-3'.

The MYADM targeted siRNA molecule is applied to preparation of anti-esophageal cancer drugs.

In the application, the anti-esophageal cancer drug is a drug for reducing the clonogenic capacity of esophageal cancer cells.

In the application, the anti-esophageal cancer drug is a drug for reducing the wound healing capacity of esophageal cancer cells.

In the application, the anti-esophageal cancer drug is a drug for inhibiting the expression of genes related to esophageal cancer cell invasion.

In the application, the cell invasion related gene is a cadherin gene, an N-cadherin gene, a Vimentin gene or a snail gene.

An anti-esophageal cancer drug comprising the MYADM-targeted siRNA molecule.

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

the invention adopts immunohistochemical staining technology to detect the expression of MYADM gene in esophageal cancer, and finds that the expression of MYADM protein in esophageal cancer tissues is obviously higher than that of non-cancer tissues. Then, three small interfering RNAs are designed aiming at the MYADM gene, and experiments show that after one small interfering RNA is transfected into an esophageal cancer cell ECA109, expression of the MYADM gene can be effectively regulated, controlled and interfered, so that the cloning forming capacity of the esophageal cancer cell is reduced, and wound healing and invasion of the esophageal cancer cell are inhibited; therefore, the MYADM gene targeted small interfering RNA has the functions of reducing the expression of MYADM and effectively inhibiting the proliferation, migration and invasion of esophageal cancer cells. The invention provides a new gene target and a medicine for treating esophageal cancer, and has high clinical practical application value.

Drawings

FIG. 1 is a graph showing the results of the expression levels of MYADM in esophageal cancer and paracarcinoma tissues;

FIG. 2 is a graph of the results of western blot experiments for siRNA interfering MYADM efficiency identification;

FIG. 3 is a graph of the results of a cell clonogenic experiment where siRNA interferes with MYADM expression;

FIG. 4 is a graph of the results of cell Transwell experiments with siRNA interfering with MYADM expression;

FIG. 5 is a graph of the results of a cell scratch experiment in which siRNA interferes with MYADM expression;

FIG. 6 is a graph of the results of Western blotting experiments with siRNA interfering with expression of cell-associated genes expressed by MYADM.

Detailed Description

The invention is further described with reference to specific examples. In the following examples, the procedures not described in detail are all routine biological experimental procedures, and can be performed by referring to molecular biology experimental manuals, published journal literature, and the like, or according to the procedures of the kit instructions.

Example 1: obtaining the expression condition of MYADM gene in the tissues of patients with esophageal cancer by immunohistochemical staining

Immunohistochemical staining step: taking esophageal cancer tissues fixed by paraformaldehyde, carrying out paraffin embedding, continuously slicing, and pressing on a glass slide; placing the immunohistochemical sheet in an oven at 80 deg.C, and baking for 30 min; soaking in xylene I and xylene II for 15min respectively; then sequentially adding anhydrous ethanol, 95% ethanol, 80% ethanol, and 70% ethanol for 2min respectively; finally, putting the slices into ultrapure water for soaking and cleaning; placing the tablet in EDTA antigen repairing solution, boiling in a pressure cooker until gas is emitted, heating for 3min, removing heat source, opening the pressure cooker cover, and cooling; after PBST 3 washes, the tissues were circled using an immunohistochemical pen, followed by H₂O₂Incubating at room temperature for 20 min; diluting the primary antibody with an antibody diluent, and incubating overnight at 4 ℃; the next day, re-warming at room temperature for 30 min; after PBST is washed for 3 times, a secondary antibody is incubated for 20 min; after PBST was carefully washed, DAB was used for color development, color change was observed, and when brown was observed, the reaction was terminated with tap water; hematoxylin staining for 30s, tap water rinsing, microscopeObserving, and if the staining is not deep, repeatedly staining with hematoxylin; then sequentially placing into gradient ethanol for soaking for 4min, and then placing into xylene I and xylene II for soaking for 10min respectively. And sealing the neutral resin, airing in a fume hood, and observing and photographing under a microscope. As shown in fig. 1, it can be found that the expression of the MYADM protein in esophageal cancer tissues is significantly higher than that in paracancerous tissues.

Example 2: design of MYADM small interference fragment

Three siRNA sequences were designed for the MYADM gene and synthesized by Happy biotechnology (Shanghai) GmbH.

The base sequence of siRNA-1 is as follows,

sense sequence: 5 '-CGGCGAGAUCACUGGCUAUdTdT-3',

antisense sequence: 5 '-AUAGCCAGUGAUCUCGCCGdTdT-3';

the base sequence of siRNA-2 is as follows:

sense sequence: 5 '-UCUACCAGUUCGAUGAADTT-3',

antisense sequence: 5 '-UUCUCAUCGAACUGGUAGA dTdT-3'

The base sequence of siRNA-3 is as follows:

sense sequence: 5 '-GGCAACUGGUCCAUGUUCAdTdT-3',

antisense sequence: 5 '-UGAACAUGGACCAGUUGCGdCtdT-3'

Example 3: screening of MYADM Small interfering fragments

Cell culture conditions and media: eca109 cell line was cultured in 5% CO with RPMI 1640 containing 10% fetal bovine serum (HyClone SH30809.01)₂In an incubator at 37 ℃.

Cell transfection: respectively diluting siRNA-1, siRNA-2 and siRNA-3 into 20 mu M solution for later use according to the instruction; after digesting the cells, spreading the cells in a 6-well plate, and performing transfection when the cell density is converged to 30-50%; preparing four sterile 1.5mL EP tubes, respectively adding 200. mu.L of OPTI-MEM, corresponding siRNA-1, siRNA-2, siRNA-3 and negative control siRNA solution 5. mu.L into the four EP tubes, rapidly vortexing for 10s to completely mix the tubes; respectively adding 6 mu L of siRNA-Mate transfection reagent into 4 EP tubes, standing for 10min at room temperature to enable the siRNA and the transfection reagent to form a transfection compound, adding the transfection compound into a hole corresponding to the 6-hole plate, and shaking up lightly; after 2 days, immunoblotting was performed to verify the knockdown efficiency and to perform cell function experiments. As shown in fig. 1.

The results of protein expression are shown in fig. 2, and it can be seen that compared with the control group, the expression levels of the MYADM proteins of the experimental group transfected with small interfering siRNA1(siRNA-1), small interfering RNA2(siRNA-2) and small interfering RNA3(siRNA-3) are all reduced, and it is clear from the relative expression levels of the proteins that the interference of the experimental group transfected with small interfering siRNA-1 on the expression of the MYADM proteins is the greatest, and the effect is the best. Therefore, siRNA-1 interfering with MYADM gene expression can be applied to interfering with MYADM gene expression and used in subsequent examples.

Example 4: cytological experiment for detecting inhibition of esophageal cancer metastasis and invasion by MYADM small interference fragment

1. Clone formation experiment proves that siRNA-1 interference with MYADM causes cell-level anti-tumor effect

The cell suspension of 500 cells/well was placed in a 6cm petri dish. Cells were cultured with 3mL of the culture medium in a 37 ℃ incubator. After culturing the cells for 2 weeks, the cells were washed gently with PBS, fixed in formalin, stained with 0.1% crystal violet, and the colony formation rate was measured. As shown in fig. 3. The a picture is the cell state of a clone formation experiment, the proliferation capacity of the esophageal cancer cell ECA109 transfected by the small-interference MYADM is obviously weakened, the b picture is the statistical result of the clone formation experiment, the number of the esophageal cancer cell ECA109 transfected by the small-interference MYADM is obviously reduced, and the clone formation experiment result has statistical significance p < 0.05. The result shows that compared with the NC group, the cell clone number of the MYADM small interfering RNA interference experimental group is remarkably reduced (p is less than 0.05), and the MYADM can promote the rat ECA109 cell clone formation, namely the ECA109 is transfected by the small interfering siRNA-1 to interfere the MYADM gene, and the cell clone formation can be inhibited.

2. Transwell analysis experiment proves the anti-tumor effect of MYADM small interference fragment siRNA-1 cell level

Firstly, preparing a 24-pore plate, and moving the small chamber into the 24-pore plate; in a cell invasion experiment, matrigel needs to be put into a refrigerator at 4 ℃ from minus 20 ℃ one day in advance, a 24-hole plate, a gun head and a small chamber need to be precooled in advance, and all operations need to be carried out on ice; digesting transfected cells from a 6-well plate, centrifuging, removing a supernatant, and leaving a cell precipitate; adding 1mL of PBS for resuspension and centrifuging; adding 1mL of PBS for resuspension, centrifuging, and discarding the supernatant; resuspension was performed by adding a small amount of medium, then 5X 105 cells in a volume of 200. mu.L were placed in a Transwell chamber with a pore size of 8um, with or without Matrigel treatment, and 500. mu.L of medium 1640 containing 10% FBS was placed in the lower compartment. After 24 hours of incubation, the upper chamber cell suspension was removed and the cells on the lower chamber membrane were fixed with 4% paraformaldehyde for 30min and then stained with crystal violet for 5 min. The 5 fields were randomly selected by microscope to count migrating or invading cells. The result is shown in fig. 4, wherein a is a cell state of a migration experiment, the migration ability of the small-interference MYADM-transfected esophageal cancer cell ECA109 is obviously weakened, b is a statistical result of the migration experiment, the number of the small-interference MYADM-transfected esophageal cancer cell ECA109 is obviously reduced, the migration experiment result has a statistical significance p of less than 0.05, c is a cell state of an invasion experiment, the invasion ability of the small-interference MYADM-transfected esophageal cancer cell ECA109 is obviously weakened, d is a statistical result of the invasion experiment, the number of the small-interference MYADM-transfected esophageal cancer cell ECA109 is obviously reduced, and the invasion experiment result has a statistical significance p of less than 0.05.

The result shows that compared with the NC group, the number of migrated and invaded cells of the MYADM small interfering RNA interference experimental group is remarkably reduced (p is less than 0.05), and the MYADM can promote the migration and invasion of rat ECA109 cells, namely the migration and invasion of the cells can be inhibited by transfecting ECA109 genes through small interfering siRNA-1.

3. Scratch experiments prove that the anti-tumor effect of MYADM small interfering fragment siRNA-1 at the cellular level

Each group of cells was seeded in 6-well plates at approximately 90% confluence. Symmetric wounds were made with a 200 μ L pipette tip scratch. After washing twice with PBS, incubation was performed for 24h with serum-free DMEM medium. Transitional photographs were taken 0h and 24h after wound extraction, respectively. The result is shown in fig. 5, wherein a is the cell state of the scratch experiment, the wound of the esophageal cancer cell ECA109 transfected by the small-interference MYADM does not heal obviously after culturing for 48 hours after scratching, the wound healing capability is obviously weakened, b is the statistical result of the scratch experiment, the wound healing rate of the esophageal cancer cell ECA109 transfected by the small-interference MYADM is obviously reduced, and the statistical significance p of the scratch experiment result is less than 0.05.

4. Immunoblotting experiments prove that the anti-tumor effect of MYADM small interference fragment siRNA-1 at the protein level

Two groups of cells from the si-control group and si-MYADM group were aspirated and placed on ice. 1mL of 4 ℃ pre-cooled PBS (0.01M pH 7.2-7.3) was added. The six well plate was then placed on ice and washed twice with pipette. 1mL of lysate plus 10. mu.L of PMSF (100mM) is shaken and placed on ice. After 150. mu.L of lysis solution containing PMSF was added to each well of the cell culture plate and then lysed on ice for 5 minutes, the cells were thoroughly lysed by scraping the cells back and forth using a scraper.

The protein content was determined by BCA quantification using previously labeled extracted cellular proteins, followed by centrifugation at 4 degrees, discarding the underlying pellet and re-labeling each tube. The loading buffer was removed from the minus 20 ℃ freezer, allowed to thaw at room temperature, and then 50 μ L of loading buffer was added to each tube of labeled cellular protein solution. Heating the constant temperature dry protein boiling machine to 100 ℃ in advance, and incubating the mixed cell protein solution in the constant temperature dry protein boiling machine for 10 minutes to denature the cell protein solution. And calculating the concentration of the sample to be measured by using a standard curve, and taking the average value after measuring for three times.

SDS-PAGE electrophoresis, the glass plate used for preparing the gel is cleaned, and then leakage is checked for 5 minutes. And 10% of lower layer glue is prepared according to a certain proportion. Then, the gel was poured using a pipette gun, during which time no air bubbles were observed, and then, the pressure was sealed with ethanol and allowed to gel for 30 minutes. Washing off the sealed ethanol with tap water gently, sucking off the ethanol with absorbent paper which cannot be washed off, preparing the upper layer glue according to a certain proportion, then injecting the upper layer glue with a liquid transfer gun, paying attention to the fact that no air bubbles exist in the process, then inserting a comb, and condensing for 30 minutes. Taking the glue after the glue is solidified, paying attention to the gentle action to prevent the plate from being cracked, adding a small amount of water, and putting the bag into a refrigerator at 4 ℃ for later use, preferably for use in the spot.

The electrophoresis needs to be prepared in advance according to a certain proportion (preferably, the electrophoresis liquid is prepared for use now), and then SDS-PAGE gel is put into an electrophoresis tank, and sufficient electrophoresis liquid is added. And sucking the samples by using a pipette gun to adhere to the wall, and sucking out two groups of samples of the si-control and si-MYADM groups. Adding 200ug of protein into each pore channel, adjusting the pipette to the corresponding volume scale, inserting the needle of the sample injector into the sample, sucking protein, and slowly adding the sample into the sample injection pore. Setting the voltage to 80V, starting to run the gel, increasing the voltage to 120V after the sample is run to the separation gel, stopping electrophoresis until bromophenol blue just runs out, and performing membrane conversion.

Preparing a film transfer liquid according to a certain proportion, and then placing scissors, a scraper blade, a clamp and a sponge mat for film transfer into a tray with the film transfer liquid. Then preparing the PVDF membrane, and putting the PVDF membrane into the methanol and the ddH in sequence₂And O, immersing the filter paper in the membrane transfer liquid. Placing a sponge pad on each of the two sides of the clamp, and then placing two pieces of filter paper on the sponge pads respectively. Putting the plate into a membrane transferring liquid, slightly prying the glass plates on two sides by using a scraper, removing the upper layer of glue, slightly placing the glue on a sponge pad close to the black surface, paying attention to certain gentle movement to prevent the glue from being broken, then placing the PVDF membrane on the glue, making a mark on the front surface of the membrane, aligning the PVDF membrane with the glue, and slightly rolling the scraper to remove bubbles. The clip is placed in a film rotating groove (the film rotating groove generates heat when being rotated, so that the film rotating groove is placed in an ice basin), the black surface of the groove faces the black surface of the clip, and the red surface of the groove faces the white surface of the clip. 300mA was used for 90 minutes.

The transferred PVDF membrane (note that the marker on the membrane is visible at this point) is transferred to a glass dish containing milk and blocked by shaking on a shaker at room temperature for 2 h. TBST was washed 4 times. Each time for 5 minutes. The membrane is cut and marked according to the position of the target protein, the target protein antibody is added, and the membrane is sealed by a sealing membrane and is kept at 4 ℃ overnight. The membrane was removed the next day and incubated at room temperature for 1-2 h, followed by washing with TBST on a decolorization shaker at room temperature for 3 times, 15 minutes each. In the same way, secondary antibodies corresponding to target proteins E-cadherin, N-cadherin, Vimentin and snail are placed in a membrane, beta-tublin is used as an internal reference, and after incubation for 2h at room temperature, TBST is used for decolorizing and washing 3 times on a shaker at room temperature, wherein each time is 15 minutes.

And (3) mixing the solution A and the solution B in equal volume in a centrifuge tube during development, and keeping out of the sun. And (3) taking a proper amount of developing solution on the table top of the developing instrument, clamping the film by using tweezers to ensure that the front and the back are fully contacted with the developing solution, and paying attention to the fact that the cutting angle is on the upper left, namely the front is upward.

The protein experiment result is shown in FIG. 6, the expression level of E-cadherin (E-cad), N-cadherin (N-cad), Vimentin (vim) and snail protein is also obviously reduced; namely, after the MYADM gene is inhibited by the small interfering RNA, the expression levels of translocator E-cadherin, N-cadherin, Vimentin and snail in ECA109 cells are synchronously reduced along with the expression reduction of MYADM. Therefore, the small interfering siRNA-1 can be applied to the preparation of targeted drugs for inhibiting the expression of genes related to cell migration and invasion.

In summary, the small interfering RNA for inhibiting MYADM gene expression provided by the embodiment of the invention can be used as a targeted drug, can regulate and control esophageal cancer cell activity, inhibit cell proliferation, cell migration, invasion and the like by inhibiting MYADM gene expression through the small interfering RNA, and has a good application prospect in clinical development of esophageal cancer targeted drugs.

Sequence listing

<110> tumor hospital in Nantong city

<120> MYADM targeted siRNA and application thereof

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<213> siRNA-1-F(Artificial)

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auagccagug aucucgccg 19

Claims

The application of MYADM targeted siRNA molecules in preparation of anti-esophageal cancer drugs; characterized in that the MYADM-targeted siRNA molecule is a double-stranded RNA molecule with the following nucleic acid sequence:

sense sequence: 5 '-CGGCGAGAUCACUGGCUAUdTdT-3',

antisense sequence: 5 '-AUAGCCAGUGAUCUCGCCGdTdT-3'.
2. The use of claim 1, wherein the anti-esophageal cancer drug is a drug that reduces the clonogenic capacity of esophageal cancer cells.
3. The use of claim 1, wherein the anti-esophageal cancer drug is a drug that inhibits invasion of esophageal cancer cells.