CN112057465A

CN112057465A - Application of 3' UTR of GSN mRNA in preparing antitumor drugs

Info

Publication number: CN112057465A
Application number: CN202010765354.0A
Authority: CN
Inventors: 刘朗夏; 刘小会; 黄秀珠; 马洁; 高学娟; 李璐
Original assignee: Jinan University
Current assignee: Jinan University; University of Jinan
Priority date: 2020-08-03
Filing date: 2020-08-03
Publication date: 2020-12-11

Abstract

The invention discloses application of 3' UTR of GSN mRNA in preparing an anti-tumor medicament. The invention discovers for the first time that the 3'UTR of the GSN mRNA can inhibit the migration, invasion and proliferation of tumor cells through mediating an epithelial cell-mesenchymal transition signal pathway, and the 3' UTR of the GSN mRNA has the function of independent of exogenous coexpression or endogenous expression of GSN protein. The 3' UTR of the GSN mRNA has small molecular weight, is easy to produce and purify in vitro, is not easy to be recognized and rejected by the immune system of a host, can obviously inhibit lung adenocarcinoma cells from generating EMT, and has the potential of becoming a novel RNA medicament different from the existing lung adenocarcinoma proteins or antibody medicaments.

Description

Application of 3' UTR of GSN mRNA in preparing antitumor drugs

Technical Field

The invention belongs to the field of genetic engineering, relates to a new discovery of a function of a non-coding region at a specific 3 'end, and particularly relates to an application of a 3' UTR of GSN mRNA in preparation of an anti-tumor medicament.

Background

The cytoplasmic Gelsolin (GSN) is actin regulatory protein with the strongest effect in the currently known F-actin cutting proteins, can regulate and control the structure and metabolic functions of actin by cutting and blocking actin filaments or aggregating actin and the like, influences the processes of tumor growth, metastasis, apoptosis, proliferation, differentiation and the like, and has great relation with the prognosis condition of cancer. A large body of clinical data suggests that GSN proteins may play an important role in the metastatic process of non-small cell lung cancer. Shieh et al studied 229 patients with stage I non-small cell lung cancer and found that patients with high expression of GSN protein often had lower survival rates. The Jun Yang et al study indicated that high GSN protein expression was associated with poor prognosis in stage II non-small cell lung cancer patients and squamous cell carcinoma patients. The GSN protein is mainly researched on the coding region at present, and the research on the non-coding region is rarely reported. With the intensive research on mechanism exploration, the important role of the non-coding region is gradually shown.

For many years, it has been thought that the transfer of information from DNA to proteins is entirely accomplished by translating the coding regions of mRNAs into amino acids of the protein. Although it is well known that the 5 'and 3' ends of mrnas also contain non-coding regions (5 'UTR, 3' UTR), the prevailing view is that these regions regulate protein abundance primarily by regulating translation or stability of the mRNA. However, some researchers have discovered that 3'UTR of certain genes plays an important role in the regulation of biological complexity, and 3' UTR can play a biological function by regulating gene expression, positioning mRNA, and competitively binding endogenous miRNA or protein.

Epithelial-mesenchymal transition (epithelial-mesenchymal transition, EMT), refers to the biological process by which epithelial cells are transformed by a specific procedure into cells with a mesenchymal phenotype. Plays an important role in embryonic development, chronic inflammation, tissue reconstruction, cancer metastasis and various fibrotic diseases, and is mainly characterized by the reduction of expression of cell adhesion molecules (such as E-cadherin (E-cadherin)), the transformation of a cytokeratin cytoskeleton into a cytoskeleton mainly comprising Vimentin and the morphological characteristics of mesenchymal cells, and the like. Through EMT, epithelial cells lose cell polarity, lose epithelial phenotypes such as connection with a basement membrane and the like, and obtain interstitial phenotypes such as higher migration and invasion, apoptosis resistance, extracellular matrix degradation capability and the like. EMT is an important biological process for malignant cells of epithelial origin to acquire the ability to migrate and invade.

Disclosure of Invention

Aiming at the defects and shortcomings in the prior art, the invention aims to provide the application of the 3' UTR of the GSN mRNA in preparing anti-tumor drugs. The invention is based on that the inventor detects the influence of 3' UTR of GSN mRNA on migration, invasion, proliferation and clone forming capability of lung adenocarcinoma H1299 and A549 cells and the expression of EMT related protein E-cadherin and vimentin in cancer cells by experimental methods such as Transwell, MTT, clone forming and Western blot. The results found the effect of the 3' UTR of GSN on the migration, invasion, proliferation and clonogenic capacity of lung adenocarcinoma H1299 and a549 cells, and this effect was able to reverse the effect of GSN proteins on cancer cell transformation.

The purpose of the invention is realized by the following technical scheme:

application of 3' UTR of GSN mRNA in preparing antitumor drugs.

The nucleotide sequence of the 3' UTR of the GSN mRNA is shown in SEQ ID NO. 1.

Application of 3' UTR of GSN mRNA in preparing medicine for inhibiting proliferation, migration, invasion and EMT of tumor cell.

Application of 3' UTR of GSN mRNA in preparing medicine for reversing the promoting effect of GSN protein on proliferation, migration, invasion and EMT of tumor cell.

Use of the 3' UTR of GSN mRNA in the regulation of expression of E-cadherin and/or vimentin in a tumour cell. The application is the application in research of non-disease treatment or diagnosis.

In the application, E-cadherin is up regulated and vimentin is down regulated.

In the above application, the tumor is preferably lung adenocarcinoma; the tumor cell preferably refers to lung adenocarcinoma cell, more preferably to lung adenocarcinoma cell H1299 and/or lung adenocarcinoma cell A549.

In the application, the medicine contains pharmaceutically acceptable auxiliary materials.

In the application, the medicament is in the dosage form of tablets, granules, capsules, dripping pills, sustained release preparations, oral preparations or injections.

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

the inventor firstly discovers that the 3' UTR of the GSN mRNA can inhibit the migration, invasion and proliferation of tumor cells through mediating an epithelial cell-mesenchymal transition signal channel; and the 3' UTR of the GSN mRNA has a function independent of the exogenously co-expressed or endogenously expressed GSN protein.

The traditional treatment method of lung cancer has various defects, and the strong metastatic property and the unlimited proliferation capacity of lung cancer cells are always important reasons for hindering the treatment. Therefore, exploring the pathogenesis of lung cancer, searching the action target of the drug and developing a novel drug become an irresistible task. The current biological treatment scheme of lung adenocarcinoma is mainly EGFR targeted therapy or pan-HER inhibitor (such as erlotinib, gefitinib or gefitinib) combined with targeted PD-1/PDL-1 immunotherapy scheme, and the anti-tumor protein medicament indeed brings good progress to the treatment of lung cancer. Because these proteins are large, some toxic side effects and dose dependence also occur. The invention shows that the 3' end non-coding region of the GSN mRNA can reverse the influence of the coding region on the transformation of cancer cells, can obviously inhibit the proliferation and metastasis of the cancer cells, and is a potential drug for a new type of lung adenocarcinoma. The 3' UTR of the GSN mRNA has small molecular weight, is easy to produce and purify in vitro, is not easy to be recognized and rejected by the immune system of a host, and can obviously inhibit lung adenocarcinoma cells from generating EMT, so the GSN mRNA has the potential of becoming a novel RNA medicament different from the existing protein or antibody medicaments.

Drawings

FIG. 1 is a graph showing the results of experiments in which the 3' UTR of GSN mRNA was investigated to reverse the promoting effect of GSN protein on migration and invasion of H1299 cells and A549 cells; wherein A is H1299 cells, and B is A549 cells.

FIG. 2 is a graph showing the results of experiments in which the 3' UTR of GSN mRNA was investigated to reverse the promoting effect of GSN protein on the proliferation of H1299 cells and A549 cells; wherein A is H1299 cells, and B is A549 cells.

FIG. 3 is a graph showing the results of experiments in which the 3' UTR of GSN mRNA reverses the promoting effect of GSN protein on epithelial mesenchymal transition of H1299 cells and A549 cells; wherein A is H1299 cells, and B is A549 cells.

FIG. 4 is a graph of experimental results investigating that the 3' UTR of GSN mRNA inhibits migration, invasion, EMT and proliferation of H1299 cells and A549 cells; wherein A is H1299 cells, and B is A549 cells.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents and the like used are, unless otherwise specified, reagents and materials obtained from commercial sources.

The cells used in the following examples were lung adenocarcinoma cell H1299 cells and lung adenocarcinoma cell a549 cells, both purchased from ATCC.

Plasmids used for transfection in the following examples:

the gene sequence (NM-000177.4) of the GSN protein was analyzed, and the multiple cloning site was determined by analyzing the vector information by Primer5 software. The primers were designed by adding restriction sites (underlined) to the upstream and downstream primers, respectively (Table 1). The primers were synthesized by Shanghai Biotech Co., Ltd.

TABLE 1 plasmid construction of related primers

Extracting genome RNA of H1299 cells, carrying out reverse transcription to generate cDNA serving as a template, carrying out PCR by using the primer pair, and respectively connecting gene segments obtained by amplification with a vector to obtain recombinant plasmids for transfection, wherein the specific operation is as follows:

using cDNA as a template and pCMV-N-Flag-GSN-F and pCMV-N-Flag-GSN-R as primers, carrying out PCR amplification on GSN fragments with BamHI and XhoI enzyme cutting sites on two sides, carrying out double enzyme cutting on a purified PCR product by using BamHI and XhoI, and then connecting the purified PCR product with a pCMV-N-Flag vector (namely Flag-vector) subjected to the same double enzyme cutting to obtain a recombinant plasmid pCMV-N-Flag-GSN, namely Flag-GSN for short;

using cDNA as a template, using pCMV-N-Flag-GSN-3 ' UTR-F and pCMV-N-Flag-GSN-3 ' UTR-R as primers, carrying out PCR amplification on GSN-3 ' UTR fragments with XhoI and SpeI enzyme cutting sites at two sides, carrying out double enzyme cutting on a purified PCR product by using XhoI and SpeI, and connecting the purified PCR product with a pCMV-N-Flag carrier subjected to the same double enzyme cutting to obtain a recombinant plasmid pCMV-N-Flag-GSN-3 ' UTR, namely Flag-GSN-3 ' UTR;

using commercial pGL3-Basic Vector (promega) as a template, using Luc-Vector-F and Luc-Vector-R as primers, carrying out PCR amplification on Luc fragments with HindIII and XhoI enzyme cutting sites on two sides, carrying out double enzyme cutting on a purified PCR product by using HindIII and XhoI, and then connecting the purified PCR product with a pcDNA3.1 Vector subjected to the same double enzyme cutting to obtain a recombinant plasmid pcDNA3.1-Luc, which is called Luc-Vector for short;

using cDNA as a template, using Luc-3' UTR-F and Luc-3' UTR-R as primers, carrying out PCR amplification on GSN-3 ' UTR fragments with EcoRI and XhoI enzyme cutting sites on two sides, carrying out double enzyme cutting on a purified PCR product by using EcoRI and XhoI, and then connecting the purified PCR product with Luc-vector subjected to the same double enzyme cutting to obtain a recombinant plasmid pcDNA3.1-Luc-3 ' UTR, which is called Luc-3' UTR for short;

using cDNA as a template and pCMV-N-EGFP-3 ' UTR-F and pCMV-N-EGFP-3 ' UTR-R as primers, carrying out PCR amplification on GSN-3 ' UTR fragments with XhoI and SpeI enzyme cutting sites on two sides, carrying out double enzyme cutting on a purified PCR product by using XhoI and SpeI, and then connecting the purified PCR product with a pCMV-N-EGFP vector (namely eGFP-vector) subjected to the same double enzyme cutting to obtain a recombinant plasmid pcDNA3.1-Luc-3 ' UTR, which is called Luc-3' UTR for short;

flag-vector (no-load, control), Flag-GSN-3' UTR;

luc-vector (empty, control), Luc-3' UTR;

eGFP-vector (empty, control), eGFP-3' UTR.

Example 1

Experiment 1: liposome transfection technique

1. This example used the Invitrogen Lipofectamine2000 reagent to transfect adherent cells in monolayer.

2. Cell culture: cells in logarithmic growth phase were plated at 4X 10 cells 1 day before transfection⁵Cells/well were seeded in 6-well culture plates and 1ml of complete medium without antibiotics (complete medium formulation: DMEM with 10% FBS) was added to ensure that the cells were confluent at transfection to 80-90%.

3. Preparation of transfection solution: the following two solutions (amounts used to transfect 1 empty cell) were prepared in polystyrene tubes:

solution A: 250 μ l Optimen +2 μ g plasmid or no load, gently and evenly, and incubating at room temperature for 5 min;

and B, liquid B: 250 μ l Optimen +3.5 μ g Lipofectamine2000, gently mixed, incubated at room temperature for 5 min;

mixing solution A and solution B, and standing at room temperature for 20 min;

note: plasmid: lipofectamine2000 is preferably 1:1 to 1: 2. Six hole plate 2 u g plasmid/hole.

4. Preparation of transfection: the plates are aspirated and the cells are washed 2 times with PBS or serum-free medium (best).

5. Transfection: add solution A and solution B complex (total volume 100. mu.l) to the wells and gently shake the plate back and forth to distribute it evenly. After the cells are placed in an incubator and incubated for 4-6 h, serum-containing culture solution (DMEM containing 10% FBS) can be replaced to remove the compound.

6. Expression identification: the expression condition of the transferred gene can be observed after 24-48 h. The cells can be subjected to migration invasion, MTT, clone formation and Western blot experiment.

Experiment 2: transwell cell migration experiment

1. Cells in logarithmic growth phase are expressed by 4 x 10⁵Cells/well were seeded in 6-well plates and cultured overnight, and transient transfection was performed on cells using liposome-encapsulated plasmids as described in example 1.

2. And after transient transfection for 4h, replacing the cell with a complete culture medium, and continuously culturing for 36-48 h.

3. Add 500. mu.L DMEM with 10% FBS to the lower chamber of a 24-well plate.

4. After completion of the culture, each well cell was digested with 0.25% trypsin. Centrifuging at room temperature for 3min at 220 Xg, completely discarding supernatant, suspending cells in 500 μ L complete medium, counting cells, and adjusting cell density to 5X 10⁵one/mL.

5. Taking 100 μ L of density of 5 × 10⁵one/mL of cell suspension was added to the upper layer of the Transwell chamber.

6. The cells are continuously placed in an incubator to be cultured for 8-10 hours.

7. After completion of the incubation, the 24-well plate was removed from the incubator, the Transwell chamber was carefully removed, and the original culture was discarded and washed 2 times with 1 × PBS.

8. And (3) fixing the lower layer cells of the small chamber in the lower chamber for half an hour by taking 500 mu L of methanol, and after the fixation is finished, reversely buckling the small chamber on absorbent paper and airing for 5-10 min.

9. 0.1% crystal violet was stained in the lower chamber for 20min for the cells in the lower chamber.

10. Gently and gently rubbing the upper layer of the chamber with cotton to remove non-migrated cells, washing excess crystal violet with 1 × PBS, and then air drying the chamber upside down on absorbent paper for 5-10 min.

11. The cells were photographed in an inverted microscope 400-fold microscope and at least 5 fields of view were counted per sample.

Experiment 3: transwell cell invasion assay

1. Will logCells in the growth phase are at a ratio of 4X 10⁵Cells/well were seeded in 6-well plates and cultured overnight, and transient transfection was performed on cells using liposome-encapsulated plasmids as described in example 1.

3. Will be provided with

BioCoat^TMThe matrigel is diluted by DMEM without FBS according to the proportion of 1:20, 100 mu L of the matrigel is added into a Transwell chamber, and the mixture is placed in an incubator for standing for 40min to 1 hour to uniformly solidify the matrigel for later use.

4. Add 500. mu.L DMEM with 10% FBS to the lower chamber of a 24-well plate.

5. The cells after 36 to 48 hours of culture were taken out, and the cells in each well were digested with 0.25% trypsin. Centrifuging at room temperature for 3min at 220 Xg, completely discarding supernatant, suspending cells in 500 μ L complete medium, counting cells, and adjusting cell density to 6X 10⁵～8×10⁵one/mL.

6. Taking 100 μ L of density 6 × 10⁵～8×10⁵The cells are placed in the incubator for 8-10 hours after the cell suspension is added into the upper layer of the Transwell chamber.

7. The subsequent experiment is the same as the migration step, which is described in detail in the migration experiment step.

Experiment 4: western blot experiment

1. Cell lysis

(1) And taking out the cells which are transiently transfected for 36-48 h by using the liposome-encapsulated plasmid, removing the culture medium, and adding 1 XPBS to wash the cells for 3 times.

(2) 50ml of a solution containing PMSF (final concentration: 1mmol/L) and Na was prepared₃VO₄(final concentration 1mmol/L), NaF (final concentration 1mmol/L) and (Roche) Protease Inhibitor cocktail tablet (Protease Inhibitor cocktail, Roche, Cat. No.04693132001) (one tablet). Adding appropriate amount of EBC lysate containing protease inhibitor according to the size of each culture dish, performing ice lysis for 30min, transferring the cell lysate to an EP tube, and performing ice bath for 30min, shaking 2 times during; followed by centrifugation at 12000 Xg for 30min at 4 ℃. Centrifuging, taking out the EP tube, and transferring the supernatant to a new EP tube

2. Protein concentration determination

(1) To 25mg of protein standard BSA was added 1mL of the standard dilution, and the mixture was shaken and mixed by a shaker so that the concentration of the standard was 25 mg/mL. Subpackaging the prepared standard substance, wherein each tube has 20 μ L, and storing at-20 deg.C for use.

(2) Adding 980 mu L of EBC lysate into 20 mu L of standard substance with the concentration of 25mg/mL, uniformly mixing by vortex oscillation, storing the uniformly mixed protein standard substance with the final concentration of 0.5mg/mL at-20 ℃ for later use.

(3) Mixing the BCA reagent solution A and the solution B according to the volume ratio of 50:1, fully and uniformly mixing by vortex, and keeping at room temperature for later use.

(4) Detecting the protein concentration in a 96-well plate, adding a corresponding protein sample or standard substance according to the table 2, supplementing 20 mu L with EBC lysate, finally adding 200 mu L of the mixed BCA working solution, gently shaking and mixing, and standing in an incubator at 37 ℃ for 30 min.

(5) After incubation for 30min, changing light green BCA working solution into purple, detecting absorbance value at 570nm by using an enzyme-labeling instrument, and drawing a standard curve, wherein R of the standard curve²When the value is 0.995 or more, the standard curve is acceptable.

(6) And substituting the absorbance value of the 570nm position of the sample to be detected into a standard curve equation, and calculating the protein concentration of the sample corresponding to the corresponding absorbance value.

TABLE 2 BCA protein concentration assay

Western blot experiment

(1) The amount of protein loaded (typically 30. mu.g) was determined, and the corresponding volume of protein solution was taken and an appropriate amount of SDS Loading Buffer was added to give a final concentration of 1X SDS Loading Buffer, according to the protein sample concentration measured by BCA.

(2) And (3) vortexing and uniformly mixing the protein sample, centrifuging the liquid on the tube wall, boiling the sample in boiling water for 10min to ensure that the protein is fully denatured under the action of SDS, centrifuging the sample at 3000rpm at room temperature for 3min, and storing the sample which is not used for the moment at-80 ℃.

(3) A10% SDS-PAGE polyacrylamide gel was prepared.

(4) And (3) Loading according to experimental requirements, ensuring that the final volume of the liquid added into each hole is the same, and filling the holes with insufficient volume by using 1 xSDS Loading Bufffer.

(5) The polyacrylamide gel is divided into upper layer concentrated gel and lower layer separation gel, the electrophoresis is carried out for 30min by using a constant voltage of 80V, the voltage is changed to 100V after the sample completely enters the separation gel, and the electrophoresis time is determined according to the molecular weight of the minimum protein required.

(6) A1 × Trans buffer containing 20% methanol for membrane transfer was prepared and pre-cooled at 4 deg.C. According to the number of electrophoresis loading holes and the spanning range of the molecular weight of the target protein, the length and the width of the needed PVDF membrane with the diameter of 0.22 mu m are determined, the corresponding PVDF membrane is intercepted, and the mark is made at the fixed position, so that the front side and the back side of the PVDF membrane are distinguished. Before use, the PVDF membrane needs to be soaked in methanol for 2min for activation. The activated PVDF membrane, the membrane transfer filter paper, the sponge and the SDS-PAGE polyacrylamide gel are soaked in a precooled 1 × Trans buffer for 5min in advance.

(7) A sandwich structure of the transfer membrane was formed in the order "sponge, filter paper, SDS-PAGE gel, PVDF membrane, filter paper, sponge", and placed in a transfer cassette. When the PAGE gel and PVDF membrane of the rotating membrane are placed, air bubbles are prevented from being generated.

(8) And (3) putting the assembled transfer printing clamp box into a film transferring groove, inserting an electrode to place the whole film transferring device on ice, and keeping the film transferring process under a low-temperature condition all the time. The membrane transfer time is determined according to the molecular weight of the target protein (about 40min for the protein of 20-50kD, about 70min for the protein of 50-80 kD).

(9) A plastic box containing 1 XTSS-T was prepared and the membrane was washed once by quickly placing it in 1 XTSS-T after the membrane transfer was completed.

(10) Skimmed milk powder was prepared using 1 × TBS-T, and 5% skimmed milk powder was prepared as a blocking solution. The PVDF membrane is completely immersed in the sealing solution, and the sealing solution is sealed for 1 hour at a low speed by a shaking table at room temperature.

(11) Antibodies (Flag (A2220, Sigma), Gelsolin (11644-2-AP, ProteinTech Group), GAPDH (ZS-25778, Zsgb-Bio, China), Visentin Rabbit polymeric antibody (10366-1-AP, ProteinTech Group), E-cadherin Rabbit polymeric antibody (20874-1-AP, ProteinTech Group)) were diluted with 1 XBTS-T according to the different antibody specifications and mixed by vortexing to add the different antibodies to the corresponding capsules. The PVDF membrane is then transferred to a membrane cassette, immersed in the antibody solution, and incubated overnight on a shaker at 4 ℃ or for 2 hours on a shaker at room temperature.

(12) The primary antibody after incubation was recovered and the PVDF membrane was washed three times with 1 XBSS-T, 5min each time.

(13) According to the primary antibody attribute, preparing a secondary antibody of the corresponding species, and diluting the secondary antibody by using 1 xTBS-T according to a proper dilution ratio. The antibody was vortexed and mixed, different secondary antibodies were added to the corresponding capsules and incubated at room temperature for 2 hours.

(14) After the secondary antibody incubation was complete, 1 × TBS-T was added to wash the PVDF membrane three times, 5min each time.

And (5) developing by a fluorescence imager.

Experiment 5: MTT assay

2. 4h after transfection treatment, each well cell was digested with 0.25% pancreatin. Centrifuging at 220 Xg room temperature for 3min, removing supernatant, suspending cells in 1mL of complete culture medium, counting cells, and collecting final concentration of 0.02X 10⁵～0.04×10⁵Adding the cell amount of the cells/hole into a 96-well plate, wherein each group of samples at least comprises 3 multiple holes in the same 96-well plate, designing the number of the 96-well plates (at least three times of detection) according to the detection times required by the experiment, laying the rest cells back into a 6-well plate, and collecting samples after 48 hours to perform WB detection transfection efficiency.

3. mu.L of MTT was added to 180. mu.L of complete medium to prepare MTT working solution with a final concentration of 5mg/mL, 200. mu.L was added to a 96-well plate, and the incubator was incubated for 4 hours in the dark.

4. The MTT working solution was carefully aspirated, 150. mu.L of DMSO was added to each well under the exclusion of light, the mixture was incubated at 20rpm for 10min in a shaker at room temperature, and the absorbance was measured at 570 nm.

Experiment 6: clone formation

2. 4h after transfection treatment, each well cell was digested with 0.25% pancreatin. Centrifuging at 220 Xg room temperature for 3min, removing supernatant, suspending cells in 1mL of complete culture medium, counting cells, and collecting final concentration of 0.02X 10⁵Cell/well cell amount was added to 6-well plates and cells were shaken up. The remaining cells were plated back in 6-well plates and harvested 48 hours later for WB assay transfection efficiency.

3. Observing cell cloning spots by using a microscope every two days, changing the liquid, and continuously culturing for 8-10 days.

4. After the culture was completed, the medium was aspirated and the cells were washed 2 times with 1 × PBS to avoid blowing out the colony spots.

5. 4% paraformaldehyde was added to the 6-well plate and the cells were fixed for 30min at room temperature.

6. After removing 4% paraformaldehyde by pipetting, the cells were washed 3 times with 1 XPBS, 1mL of crystal violet was added and the mixture was allowed to stand at room temperature for 20min for staining.

7. And after dyeing is finished, recovering the crystal violet, slowly washing by tap water, washing off the non-specifically bound crystal violet, and airing at room temperature.

8. The scanner scans the clones to form results.

Wherein, plasmids Flag-vector, Flag-GSN and Flag-GSN-3' UTR are respectively transfected into H1299 cells and A549 cells, and the results of the Transwell experiment are shown in figure 1. The results show that the 3' UTR of the GSN mRNA reverses the promotion effect of the GSN protein on the migration and invasion of tumor cells.

The results of the MTT and colony formation experiments in which the plasmids Flag-vector, Flag-GSN-3' UTR were transfected into H1299 cells and A549 cells, respectively, are shown in FIG. 2. The results show that the 3' UTR of the GSN mRNA reverses the promotion effect of the GSN protein on the proliferation of tumor cells.

The plasmids Flag-vector, Flag-GSN and Flag-GSN-3' UTR are respectively transfected into H1299 cells and A549 cells, and the result of Western Blot experiment is shown in FIG. 3. The result shows that the 3' UTR of the GSN mRNA can reverse the promotion effect of GSN protein on the epithelial mesenchymal transition of tumor cells.

The plasmids Luc-vector, Luc-3 'UTR/eGFP-vector, eGFP-3' UTR were transfected into H1299 cells and A549 cells, respectively, and the results of the Transwell, MTT and clone formation experiments and Western Blot experiments are shown in FIG. 4. The results indicate that the 3' UTR of GSN mRNA inhibits tumor cell migration, invasion, EMT and proliferation.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> river-south university

Application of <120> 3' UTR of GSN mRNA in preparation of antitumor drugs

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 240

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence encoding 3' UTR of GSN mRNA

<400> 1

ggaggggcag ggcccaccca tgtcaccggt cagtgccttt tggaactgtc cttccctcaa 60

agaggcctta gagcgagcag agcagctctg ctatgagtgt gtgtgtgtgt gtgtgttgtt 120

tctttttttt ttttttacag tatccaaaaa tagccctgca aaaattcaga gtccttgcaa 180

aattgtctaa aatgtcagtg tttgggaaat taaatccaat aaaaacattt tgaagtgtga 240

<210> 2

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pCMV-N-Flag-GSN-F

<400> 2

gctggatcca tggctccgca ccgccccgcg cccg 34

<210> 3

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pCMV-N-Flag-GSN-R

<400> 3

gccctcgagt caggcagcca gctcagccat ggcc 34

<210> 4

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pCMV-N-Flag-GSN-3’UTR-F

<400> 4

aatctcgagg gaggggcag 19

<210> 5

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pCMV-N-Flag-GSN-3’UTR-R

<400> 5

ccgactagtc acacttcaaa atg 23

<210> 6

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Luc-vector-F

<400> 6

cgaagcttat ggaagacgcc aaaaa 25

<210> 7

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Luc-vector-R

<400> 7

aaactcgagc cgaattctta cacggcgat 29

<210> 8

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Luc-3' UTR-F

<400> 8

ccggaattcg gaggggcagg gcccacccat gtc 33

<210> 9

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Luc-3' UTR-R

<400> 9

ccgctcgagt cacacttcaa aatgttttta ttgg 34

<210> 10

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pCMV-N-EGFP-3’UTR-F

<400> 10

aatctcgagg gaggggcag 19

<210> 11

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pCMV-N-EGFP-3’UTR-R

<400> 11

ccgactagtc acacttcaaa atg 23

Claims

Application of 3' UTR of GSN mRNA in preparing antitumor medicine.
2. Use of the 3' UTR of GSN mRNA according to claim 1 in the preparation of an anti-tumor medicament, wherein:

the nucleotide sequence of the 3' UTR of the GSN mRNA is shown in SEQ ID NO. 1.
Use of the 3' UTR of GSN mRNA in the manufacture of a medicament for inhibiting proliferation, migration, invasion and EMT of tumour cells.
Application of 3' UTR of GSN mRNA in preparing medicine for reversing the promoting action of GSN protein on proliferation, migration, invasion and EMT of tumor cell.
Use of the 3' UTR of GSN mRNA for modulating expression of E-cadherin and/or vimentin in tumour cells for research purposes other than disease treatment or diagnosis.
6. Use of the 3' UTR of a GSN mRNA according to claim 5 for modulating E-cadherin and/or vimentin expression in a tumour cell, wherein:

e-cadherin is up regulated and vimentin is down regulated.
7. Use according to any one of claims 1 to 6, wherein:

the tumor is lung adenocarcinoma.
8. Use according to any one of claims 3 to 6, wherein:

the tumor cell is lung adenocarcinoma cell H1299 and/or lung adenocarcinoma cell A549.
9. Use according to any one of claims 1 to 3, characterized in that:

the medicine contains pharmaceutically acceptable auxiliary materials.
10. Use according to any one of claims 1 to 3, characterized in that:

the dosage form of the medicine is tablets, granules, capsules, dripping pills, sustained release preparations, oral preparations or injections.