CN111218510A

CN111218510A - Application of Smurf1 gene, expression product and derivative thereof or inhibitor thereof in colorectal cancer chemotherapy

Info

Publication number: CN111218510A
Application number: CN202010079654.3A
Authority: CN
Inventors: 韦荣飞; 郭静
Original assignee: Institute of Laboratory Animal Science of CAMS
Current assignee: Institute of Laboratory Animal Science of CAMS
Priority date: 2020-02-04
Filing date: 2020-02-04
Publication date: 2020-06-02

Abstract

The application belongs to the field of medicines and discloses a new application of Smurf1, in particular a new application of Smurf1 gene, expression product and derivatives thereof or inhibitors thereof. The influence of the Smurf1 gene and the expression product on the sensitivity of the colorectal cancer cells to the chemotherapeutic drugs is detected by a cell, a tumor-bearing mouse and a human tumor xenograft model (PDX model), and the result shows that the colorectal cancer cells with low Smurf1 expression have higher sensitivity to the chemotherapeutic drugs. Therefore, the invention provides the application of the Smurf1 gene, the expression product and the derivatives thereof in preparing the marker of the sensitivity of tumor cells to chemotherapeutic drugs. The invention also provides application of the Smurf1 inhibitor in preparing a sensitizer for tumor cells to chemotherapeutic drugs.

Description

Application of Smurf1 gene, expression product and derivative thereof or inhibitor thereof in colorectal cancer chemotherapy

Technical Field

The invention belongs to the field of medicines, and particularly relates to a novel application of Smurf1, in particular to a novel application of Smurf1 gene, an expression product, a derivative thereof or an inhibitor thereof.

Background

The balance of protein synthesis and degradation in eukaryotic cells plays an important role in the maintenance of cellular homeostasis. In eukaryotic cells there are two protein degradation pathways, the lysosome pathway and the ubiquitin proteasome pathway, which function in different ways. The lysosome pathway mainly degrades foreign proteins entering the cell through endocytosis or pinocytosis, while the ubiquitin proteasome pathway mainly degrades intracellular proteins, and the specific action mode is as follows: the protein is labeled with ubiquitin called a "death tag" under the cascade action of ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligase (E3), and thus recognized by proteasome and finally degraded, in which the most critical enzyme is ubiquitin ligase E3. Approximately 1000E 3 were found in the human genome, and E3 determined the specificity of the substrate, enabling targeted ubiquitination degradation of the substrate proteins. The ubiquitin proteasome pathway is involved in numerous biological processes, including regulation of cell cycle, cell differentiation and growth, apoptosis, etc., and abnormalities of which can lead to the development of cancer, neurodegenerative and autoimmune diseases, etc. According to the structural characteristics and the mode of action, E3 is mainly classified into a RING (Ring intersecting new gene) zinc finger domain class accounting for 95% of the total amount of E3 and a HECT (homologus to E6AP C-terminus) domain class accounting for less than 5%.

Earlier studies on HECT ubiquitin-like ligases revealed that the new 4 (neutral prefrosor cells-expressed degraded down-regulated 4) family includes nine members of Smurf1, Smurf2, new 4-1, new 4-2, NEDL1, NEDL2, WWP1, WWP2, AIP 4/Itch. Smurf1 is a well studied member of this family. Research shows that Smurf1 participates in regulation of a plurality of biological processes by targeting and degrading a plurality of proteins in a plurality of signal paths, including targeting and degrading MEKK2 protein in a BMP signal path to block BMP path signal transduction, thereby inhibiting the activity of osteoblasts; the double knockout of Smurf1 and Smurf2 leads to the lethal developmental block of the embryonic stage of mice; smurf1 promotes the proliferation of colorectal cancer cells and the growth of tumors, etc. Smurf1 is highly expressed in a variety of tumors, including colorectal, ovarian, breast, and the like.

Chinese patent ZL 201410335052.4 discloses a method for screening an anti-tumor drug taking Smurf1 ubiquitination degradation RhoB as a target. Wherein after the candidate compound is determined to be a potential anti-tumor drug, a compound capable of increasing the content of RhoB protein in cancer cells is further selected: contacting a candidate compound identified as a potential drug with a different type of tumor cell; observing the effect of a candidate compound identified as a potential drug on the amount of RhoB protein in different types of tumor cells; and carrying out further apoptosis experiments and nude mouse tumorigenicity model experiments on the screened potential compounds capable of improving the RhoB content in different tumor cells so as to select effective antitumor drugs. However, the method does not screen the anti-tumor drugs aiming at specific tumor types, and has poor specificity and low efficiency.

Colorectal cancer is a common malignancy of the digestive tract, with global morbidity and mortality rates third and second, respectively. In recent years, new or improved chemotherapeutic drugs for colorectal cancer have been emerging, however, some patients are not sensitive to chemotherapeutic drugs and the chemotherapeutic effect is poor. Research shows that the sensitivity of tumor cells to chemotherapeutic drugs can be predicted by detecting molecular markers. In order to improve the chemotherapy strategy of colorectal cancer, including effective selection of chemotherapeutic drugs, etc., the combination of biomarkers and chemotherapy sensitivity is necessary.

Disclosure of Invention

In view of the above, the present invention aims to provide the application of Smurf1 gene, its expression product and its derivatives or its inhibitor in colorectal cancer chemotherapy.

Previous research reports reveal that Smurf1 can promote the occurrence and development of colorectal cancer and play the function of oncogene. However, the effect of Smurf1 on the sensitivity of colorectal cancer cells to chemotherapeutic drugs is unclear. The sensitivity of colorectal cancer cells to chemotherapeutic drugs is predicted or judged by detecting the levels of Smurf1 genes and expression products. The results show that colorectal cancer cells highly expressed by Smurf1 are less sensitive to chemotherapeutic drugs, and vice versa.

Further, in some embodiments, the influence of Smurf1 gene and expression product on the sensitivity of colorectal cancer cells to chemotherapeutic drugs is detected by using a cell, a tumor-bearing mouse and a human tumor xenograft model (PDX model), and the result shows that the colorectal cancer cells with low Smurf1 expression have higher sensitivity to the chemotherapeutic drugs.

Therefore, the invention provides the application of the Smurf1 gene, the expression product and the derivatives thereof in preparing the marker of the sensitivity of tumor cells to chemotherapeutic drugs.

In the present invention, the Smurf1 gene may be DNA encoding Smurf1 or RNA encoding Smurf 1.

In the invention, the Smurf1 derivative is different isomer proteins generated by expression of Smurf1 gene, including but not limited to isomer proteins with different amino acid sequence lengths generated by encoding Smurf1 gene, such as isomer proteins with lengths of 757aa, 731aa and 728 aa.

The invention also provides a kit for detecting the sensitivity of tumor cells to chemotherapeutic drugs, which comprises a reagent for detecting the Smurf1 gene, an expression product or a derivative thereof.

In some embodiments, the reagents for detecting Smurf1 gene in the kit comprise Smurf1 gene detection primers. The primer can be designed and synthesized according to a method known to those skilled in the art.

In some embodiments, the reagents of the kit for detecting the expression product of Smurf1 gene include antibodies to the expression product of Smurf1 gene. The antibody can be polyclonal antibody of Smurf1 gene expression product, and can also be monoclonal antibody of Smurf1 gene expression product, and can be prepared according to the method known by the technicians in the field.

In some embodiments, the reagents for detecting Smurf1 gene derivatives in the kit comprise detection primers required for detecting mRNA levels and detection antibodies required for detecting protein levels when Smurf1 gene expression produces different isoforms.

Due to the fact that colorectal cancer cells with low expression of Smurf1 are more sensitive to chemotherapeutic drugs, the Smurf1 inhibitor is used, so that the activity of Smurf1 in the colorectal cancer cells is inhibited, namely the biological function of Smurf1 is weakened, the sensitivity of tumor cells to the chemotherapeutic drugs is improved, and the chemotherapeutic effect of patients is enhanced. Therefore, the invention also provides the application of the Smurf1 inhibitor in preparing a sensitizer for tumor cells to chemotherapeutic drugs.

In the invention, the Smurf1 inhibitor is a protein, a small molecule compound or an antibody which antagonizes the function of Smurf 1. The small molecule compounds include, but are not limited to, MLN 4924. The inhibitor also comprises siRNA, namely the expression of the Smurf1 can be knocked down, so that the effect of reducing the Smurf1 protein level and weakening the function of the Smurf1 protein is achieved.

In the present invention, the tumor cell is a colorectal cancer cell.

In the present invention, the chemotherapeutic agent is Gemcitabine (Gemcitabine) and/or Cisplatin (Cisplatin). Namely, the chemotherapeutic drug is gemcitabine or cisplatin which is used alone, or can be gemcitabine and cisplatin which are used together.

According to the technical scheme, the invention provides a novel application of Smurf1, in particular a novel application of Smurf1 gene, an expression product and a derivative or an inhibitor thereof. The influence of the Smurf1 gene and the expression product on the sensitivity of the colorectal cancer cells to the chemotherapeutic drugs is detected by a cell, a tumor-bearing mouse and a human tumor xenograft model (PDX model), and the result shows that the colorectal cancer cells with low Smurf1 expression have higher sensitivity to the chemotherapeutic drugs. Therefore, the invention provides the application of the Smurf1 gene, the expression product and the derivatives thereof in preparing the marker of the sensitivity of tumor cells to chemotherapeutic drugs. The invention also provides application of the Smurf1 inhibitor in preparing a sensitizer for tumor cells to chemotherapeutic drugs.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows that knockdown of Smurf1 in HCT116 cells promotes Gemcitabine and Cisplatin-induced apoptosis in example 1; (A) after Gemcitabine with different concentrations (0.5, 1, 2, 5 and 10 mu M) acts on HCT116 cells for 48 hours, detecting apoptosis of the cells in a flow mode and carrying out statistical analysis on the proportion of apoptotic cells; (B) after Cisplatin with different concentrations (12.5, 25, 50, 100 and 200 mu M) acts on HCT116 cells for 48h, the apoptosis of the cells is detected by flow and the proportion of apoptotic cells is analyzed statistically; GEM: gemcitabine; CIS: cisplatin;

FIG. 2 shows that knockdown of Smurf1 in HCT116 cells significantly enhances the sensitivity of the cells to Gemcitabine and Cisplatin in example 2; after Gemcitabine (A) and Cisplatin (B) at different concentrations (0, 0.5, 1, 2, 5, 10, 20, 50. mu.M) and a combination of both (C) at different concentrations (0, 0.1, 0.2, 0.5, 1, 2, 5, 10. mu.M) were applied to HCT116 cells for 48h, CCK8 detected cell proliferation and statistically analyzed for cell viability,%;

FIG. 3 shows that example 3HCT116 cell-derived tumor-bearing mice demonstrate that knockdown of Smurf1 enhances the sensitivity of tumor cells to Gemcitabine and Cisplatin; tumor size (a) and tumor weight (B) in mice;

FIG. 4 shows the results of immunohistochemical detection of antibodies against the marker proteins PCNA (A) and Cyclin D1(B) of example 3;

FIG. 5 shows RNA-seq and immunohistochemical measurements of the levels of Smurf1RNA and protein in tumor tissue derived from four patients with colorectal cancer in example 4;

FIG. 6 shows example 4 construction of the four colorectal cancer patient-derived tumor PDX model of FIG. 5, which detects statistics of Gemcitabine and Cisplatin tumor size (A) and tumor volume (B) for two Smurf1 low-expressing and two Smurf1 high-expressing PDX tumor cells;

FIG. 7 shows the statistics of the number of proliferating cells in low-and high-expressing tumor tissues of Smurf1, in example 4, after the action of Gemcitabine, Cisplatin and a combination of both.

Detailed Description

The invention discloses application of Smurf1 gene, expression product and derivatives thereof or inhibitors thereof in colorectal cancer chemotherapy. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the techniques of the invention can be implemented and applied by modifying or appropriately combining the methods described herein without departing from the spirit, scope and spirit of the invention.

The invention relates to application of Smurf1 gene, expression product and inhibitor thereof in sensitivity of colorectal cancer cells to chemotherapeutic drugs. The invention discloses a detection method and a judgment index of Smurf1 gene and expression product in prediction or judgment of sensitivity of colorectal cancer to chemotherapeutic drugs.

The invention relates to and discloses a method for detecting the influence of Smurf1 gene and expression product level on the sensitivity of colorectal cancer cells to chemotherapeutic drugs, wherein the involved or used chemotherapeutic drugs are Gemcitabine and Cisplatin, and other chemotherapeutic drugs can be used for verification.

In addition, the mouse models (tumor-bearing mice and PDX mouse models) used in the invention can also be used for verifying the influence of the Smurf1 gene expression product (RNA or protein) level on the sensitivity of colorectal cancer cells to chemotherapeutic drugs by using in vitro 3D culture or organoid models.

The nucleotide sequence of human Smurf1 (NCBI Gene ID:57154) related to the invention is: gaggc ggcggcagcggcggaagcggcgagggcggcgggcgtccggctctgaggtggtggaggcggcggaggcggcggcggaggcggcggcggctcgggactgggctcggctggaagcagcgagggtcagagcgccgcagcaagcgccg atctcccggctcgaccatccgcctgccgcccggacgcctgggccgcggagtttgtgtcccggctcggaccccggcgcccagcccggagccgtaaccttgaggcggcggcggcggggccgggccgggccgggctggggggcggtggcgctggatccgcggctgcccgatcgttggcgggagatgtcgaaccccgggacacgcaggaacggctccagcatcaagatccgtctgacagtgttatgtgccaagaaccttgcaaagaaagacttcttcaggctccctgacccttttgcaaagattgtcgtggatgggtctgggcagtgccactcaaccgacactgtgaaaaacacattggacccaaagtggaaccagcactatgatctatatgttgggaaaacggattcgataaccattagcgtgtggaaccataagaaaattcacaagaaacagggagctggcttcctgggctgtgtgcggctgctctccaatgccatcagcagattaaaagataccggataccagcgtttggatctatgcaaactaaacccctcagatactgatgcagttcgtggccagatagtggtcagtttacagacacgagacagaataggaaccggcggctcggtggtggactgcagaggactgttagaaaatgaaggaacggtgtatgaagactccgggcctgggaggccgctcagctgcttcatggaggaaccagccccttacacagatagcaccggtgctgctgctggaggagggaattgcaggttcgtggagtccccaagtcaagatcaaagacttcaggcacagcggcttcgaaaccctgatgtgcgaggttcactacagacgccccagaaccgaccacacggccaccagtccccggaactgcccgaaggctacgaacaaagaacaacagtccagggccaagtttactttttgcatacacagactggagttagcacgtggcacgaccccaggataccaagtccctcggggaccattcctgggggagatgcagcttttctatacgaattccttctacaaggccatacatctgagcccagagaccttaacagtgtgaactgtgatgaacttggaccactgccgccaggctgggaagtcagaagtacagtttctgggaggatatattttgtagatcataataaccgaacaacccagtttacagacccaaggttacaccacatcatgaatcaccagtgccaactcaaggagcccagccagccgctgccactgcccagtgagggctctctggaggacgaggagcttcctgcccagagatacgaaagagatctagtccagaagctgaaagtcctcagacacgaactgtcgcttcagcagccccaagctggtcattgccgcatcgaagtgtccagagaagaaatctttgaggagtcttaccgccagataatgaagatgcgaccgaaagacttgaaaaaacggctgatggtgaaattccgtggggaagaaggtttggattacggtggtgtggccagggagtggctttacttgctgtgccatgaaatgctgaatccttattacgggctcttccagtattctacggacaatatttacatgttgcaaataaatccggattcttcaatcaaccccgaccacttgtcttatttccactttgtggggcggatcatggggctggctgtgttccatggacactacatcaacgggggcttcacagtgcccttctacaagcagctgctggggaagcccatccagctctcagatctggaatctgtggacccagagctgcataagagcttggtgtggatcctagagaacgacatcacgcctgtactggaccacaccttctgcgtggaacacaacgccttcgggcggatcctgcagcatgaactgaaacccaatggcagaaatgtgccagtcacagaggagaataagaaagaatacgtccggttgtatgtaaactggaggtttatgagaggaatcgaagcccagttcttagctctgcagaaggggttcaatgagctcatccctcaacatctgctgaagccttttgaccagaaggaactggagctgatcataggcggcctggataaaatagacttgaacgactggaagtcgaacacgcggctgaagcactgtgtggccgacagcaacatcgtgcggtggttctggcaagcggtggagacgttcgatgaagaaaggagggccaggctcctgcagtttgtgactgggtccacgcgagtcccgctccaaggcttcaaggctttgcaaggttctacaggcgcggcagggccccggctgttcaccatccacctgatagacgcgaacacagacaaccttc cgaaggcccatacctgctttaaccggatcgacattccaccatatgagtcctatgagaagctctacgagaagctgctgacagccgtggaggagacctgcgggtttgctgtggagtgaaaagcaaccaaaggcaacagagtctagctcatggccaccagaccaaaagcatccagcttctgtgcacctcctgcaaagctggcagaggccctggaattccagatcacctgaggggaaagggttgtctctctcctttctgttgggggagggggatgggggacttttgttggtggctcccacccatatatccctcctttaccatagtactcccacccacttccatcacccatccaataaaatgcagccaggtttagcctttggctttggtcacacaggatattctgctgtgttgcaacccatgtggtgataaggctcacagccctgagctctttacgggagcatcaactcacagttaggggactgggcgtggctgattgagggtttggaactggtggctatgccagctattccatctcaaaacagccttgaggccccttttcaatttgagcagctgctagatatcttatcagagctcagattccagatttcacatcccagcagccggttctgggtagcagatcaatttccaactggaaaataactatataatgtatgcttattggaattctgccacagcaggaagcttgagtcaaaatgtgtttcccctttgaaaggagaaggaattggagcagcttttcctggaggcccaggatatttcttttctgggtatcttggctgaaaattttgttttacatagagaaaaacgatcttttaagggtcccttttgctgcattatctgtccagtttgacttttttttcagtgaaaacaccatgtcatggagtgtaggaaagagcagaccaaaatcagccctagagccaaccagtcagtcccaaagctgtgacctctgtgccactgttgtccatagaagagcatcgactgtgtcacttaaaatattagtaaaccatgatgcagcaactgctaagagctaaactaacaaaattgtgtcatcatagctgctggcttggtgtgaactcgcttaaaagcaatggtgaaaggataacctcgatgatgtaaatccacccaaagatactgttctacaaaaagtagggtgtggacgcaaacctgtgacagcagagggggacgacttcacactcactgcctcatgtggcccctttcccagtggcagctggtgacactaacgattgctactcggttcacttgcccagatgtcttcatatgatgagcaaggccagaagcaaggctagattcgaagtttctgacaccatttccagtttgcacaaaagtcagtattttatcttaaagtggcttgatttccaatagctgaacttgggcagaaaacagcaggccaatgttcctatgtggtttctttgttgttgtttttgtttggggtgggggcaagtacagggtaattcatgagcaagacatttcactgctgtcgaagtctctgggatcccgctgtgggtctgagatggcctgggaaggaccttgtggacaatggttttatctgttctttttgtcactgttaatttctgggctgctgaggttctagaatagaagggctgccaaatgaggtttgctgcaggaggaaagtttaatcccccattccaaaagtccaggccaaatggtgggcttagcctctttgaaaagttctgccttgcccccacaggtgggcacatcctgtgtctcattcaccatgatgcttcctgagggtgttctagaagcccgttccccagtggctgtatccagcctttccttgcatcatcttcctcttgaaggtgaggaagtgaaaactacagacctcccccggacagcccactctctatcacgagcctaacccgcgggaggcggaagagacatccattcgagaactgaagcggcctccgggatgaggtcagaggccccacctgattttcctggtggtggtatccaaaatcttcagtaactaggaaggaaaccagggtctcatggtttaaaagactttgaagcaggaatgttgcatttgacgcctttaaaactacctttttgctgttgggaggagtcgggggcgagccttagcagctgcaccgccatccccatgctggttggtgctgccctgcctctcgtgccgggtgttgcttcagcccagagccagagggctgggtcccgggtcctccacaggtgaccccggtggacacacgcgttcccatcctggcctccgtctctgcttttccacttctacctgcgtgtgggtttgccgccttgtcatcggttgtgtgagtgtcgcagacctttccagagctccggttcactctttccaaacaggcctccctgtcggtggcactgcactc ctagaaccttcagtttctacgatggtttgtttggtccttttgaaccaccccaaagaactcaacatggcaaagcaaatggtaaaagcttcccgactgttctactttgggtccgcgcgaagcccactcacgtgtgatctgtgttgcccctctcggtggtcccaggcgatccagccatgccccctgcccctctgcccagatgcttcaggggcccggcttttcaggcttgccctcaccagcggccgtcagccgacactcagggatgtagctaacaccactccgccagtgctttcagtaggaagagctgaggctgcctgggaggcccggggcgaccggaaaagggctctctcaagttctgaaaagagaatctgccaccagatcgaatttcgacccctgagcttgttcggacgtatggtccaaattcagattaaggtggtcacccaacccgagatgtcaggaaaggccttctgcagagaaaatgtccccccacccgccatctgcagccaggtgtgtgccacacggcagccttcccgaaacatagtatggattttaaaaatgtgtttatttttgtttctcaaccactttataacgtattttttaatttattttgtaatgtcttgttttgaagtattgctgctatccttgttatccttcccactgtttttatcactgatttattttgtgaaagttgtacactaatgttctatgtcaaaatcaaaagtatttaatgaaatactagttctatttaatgtggttatggaaccagctggaaacacaaaacaaacagtgattgtacagcaggctgggcccaggaggtcaggttcattttgttacatatgcaataaactcacgacttta are provided.

The amino acid sequence of Smurf1 is as follows: MSNPGTRRNGSSIKIRLTVLCAKNLAKKDFFRL PDPFAKIVVDGSGQCHSTDTVKNTLDPKWNQHYDLYVGKTDSITISVWNHKKIHKKQGAGFLGCVRLLSNAISRLKDTGYQRLDLCKLNPSDTDAVRGQIVVSLQTRDRIGTGGSVVDCRGLLENEGTVYEDSGPGRPLSCFMEEPAPYTDSTGAAAGGGNCRFVESPSQDQRLQAQRLRNPDVRGSLQTPQNRPHGHQSPELPEGYEQRTTVQGQVYFLHTQTGVSTWHDPRIPSPSGTIPGGDAAFLYEFLLQGHTSEPRDLNSVNCDELGPLPPGWEVRSTVSGRIYFVDHNNRTTQFTDPRLHHIMNHQCQLKEPSQPLPLPSEGSLEDEELPAQRYERDLVQKLKVLRHELSLQQPQAGHCRIEVSREEIFEESYRQIMKMRPKDLKKRLMVKFRGEEGLDYGGVAREWLYLLCHEMLNPYYGLFQYSTDNIYMLQINPDSSINPDHLSYFHFVGRIMGLAVFHGHYINGGFTVPFYKQLLGKPIQLSDLESVDPELHKSLVWILENDITPVLDHTFCVEHNAFGRILQHELKPNGRNVPVTEENKKEYVRLYVNWRFMRGIEAQFLALQKGFNELIPQHLLKPFDQKELELIIGGLDKIDLNDWKSNTRLKHCVADSNIVRWFWQAVETFDEERRARLLQFVTGSTRVPLQGFKALQGSTGAAGPRLFTIHLIDANTDNLPKAHTCFNRIDIPPYESYEKLYEKLLTAVEETCGFAVE are provided.

In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available.

The construction method of the stable HCT116 cell line for knocking down Smurf1 comprises the following steps:

(1) smurf1 shRNA sequence (GGGCUCUUCCAGUAUUCUATT) is inserted into hU6-MCS-Ubiquitin-EGFP-IRES-puromycin vector to construct Smurf1 lentivirus knock-down vector, and then packaging of lentivirus particles is carried out. The step is completed by the Shanghai Jikai gene.

(2) HCT116 cells plated 6 well plates (8X 10)⁴cells/well), lentiviral infection was performed 18-24 hours later: lentivirus (control and Smurf1 knockdown) infected with MOI 10 in 6 × 10 virus doses⁵TU/well/2 ml1640 complete medium;

(3) after the lentivirus infects HCT116 cells for 8 hours, replacing a fresh 1640 complete culture medium, culturing for 3-4 days, observing the expression condition of Green Fluorescent Protein (GFP) by a fluorescence microscope, and judging the virus infection efficiency;

(4)4 mu g/ml puromycin is used for screening the positive cells infected by the lentivirus for 2 to 3 days, and the liquid is changed once every day;

(5) and continuously culturing the cells in a common 1640 complete culture medium, and freezing and storing after passage and expansion or using for experiments.

Example 1: knockdown of Smurf1 in HCT116 cells promotes Gemcitabine and Cisplatin-induced apoptosis

Selecting stable HCT116 cells with knockdown Smurf1 to be plated in a 12-well plate, 24h later, after Gemcitabine (0.5, 1, 2, 5 and 10 mu M) or Cisplatin (12.5, 25, 50, 100 and 200 mu M) with different concentrations acts on the HCT116 cells for 48h, detecting apoptosis of the cells in a flow mode, and carrying out statistical analysis on the proportion of apoptotic cells.

The results show that HCT116 apoptotic cell numbers gradually increased with increasing working concentrations of Gemcitabine following Smurf1 knockdown (fig. 1A). Also following Smurf1 knockdown, HCT116 apoptotic cell numbers increased with increasing Cisplatin working concentrations (FIG. 1B). The above results indicate that knockdown of Smurf1 in HCT116 cells promotes Gemcitabine and cissplatin-induced apoptosis.

Example 2: knockdown of Smurf1 in HCT116 cells significantly enhanced the sensitivity of the cells to Gemcitabine and Cisplatin (FIG. 4)

Selecting stable HCT116 cells with knockdown Smurf1, plating the cells in a 96-well plate, after 24 hours, acting Gemcitabine and Cisplatin (0, 0.5, 1, 2, 5, 10, 20, 50 mu M) with different concentrations and combination drug (0, 0.1, 0.2, 0.5, 1, 2, 5, 10 mu M) with different concentrations on the HCT116 cells for 48 hours, after incubating the cells for 2 hours with CCK8, detecting OD450nm by an enzyme labeling instrument, and statistically analyzing the survival rate of the cells (cellthiabilty,%),

the results show that Gemcitabine (FIG. 2A), Cisplatin (FIG. 2B) alone and the combination of both (FIG. 2C) significantly reduced the survival of Smurf 1-knockdown HCT116 cells, indicating that knocking-down Smurf1 significantly enhanced the sensitivity of HCT116 cells to Gemcitabine and Cisplatin.

Example 3: HCT116 cell-derived tumor-bearing mice demonstrated that knockdown of Smurf1 enhances the sensitivity of tumor cells to Gemcitabine and Cisplatin

To further confirm the effect of Smurf1 on the sensitivity of colorectal cancer cells HCT116 to the chemotherapeutic drugs Gemcitabine and cissplatin, in vivo experiments were performed. 6-8 weeks old nude mice, each nude mouse injected with 8X 10 subcutaneous injections⁵Control (HCT 116 cells without Smurf1 knockdown) and Smurf1 knockdown HCT116 cells until tumors grew to 100mm³In this case, Gemcitabine (50mg/kg), Cisplatin (2.5mg/kg) and combinations of both (25mg/kg Gemcitabine +1.25mg/kg Cisplatin) were administered intraperitoneally, and mice were sacrificed once a week after 4 weeks and tumor sizes were photographed (FIG. 3A) and tumors were weighed and statistically analyzed (FIG. 3B).

The results show that Gemcitabine, Cisplatin and the combination of Gemcitabine and Cisplatin can obviously inhibit the growth of tumors, and particularly the combination has the strongest inhibition effect. Compared with a control group, after the Smurf1 is knocked down, the sensitivity of tumor cells to chemotherapeutic drugs is obviously increased, and the inhibition effect of the chemotherapeutic drugs on the tumor growth is stronger.

Meanwhile, dissected tumor tissues were fixed and paraffin sections were cut, and immunohistochemical staining was performed on the sections using cell proliferation-associated marker protein antibodies (PCNA and CyclinD1), and statistical analysis was performed on positive cells.

The results show that Gemcitabine and Cisplatin both can obviously inhibit the proliferation of tumor cells in tumor tissues, and the inhibition effect of Gemcitabine and Cisplatin on the proliferation of tumor cells is increased after the Smurf1 is knocked down (FIG. 4).

The above results demonstrate that knocking down Smurf1 enhances the sensitivity of tumor cells to Gemcitabine and cissplatin in a tumor-bearing mouse model derived from HC116 cells.

Example 4: a human-derived tumor xenograft (PDX) model proves that colorectal cancer cells with low expression of Smurf1RNA or protein have enhanced drug sensitivity to Gemcitabine and Cisplatin

In order to further confirm the influence of the expression level of Smurf1 on the sensitivity of tumor cells to chemotherapeutic drugs, a human-derived tumor xenograft (PDX) model is constructed, and the growth condition of the tumor is statistically analyzed after the model is used. The specific experimental method is as follows:

(1) smurf1RNA and protein level assays in clinical colorectal cancer tumor tissues (FIG. 7)

1) Smurf1RNA level detection

Colorectal cancer tissues taken out of clinical surgery were frozen in liquid nitrogen, followed by RNA extraction and sequencing analysis, which was completed in huada gene corporation. The results showed that Smurf1 showed higher levels of RNA expression in both tissues of CRC1 and CRC2 compared to CRC3 and CRC4 (fig. 5A).

2) Smurf1 protein level assay

Colorectal cancer tissues clinically surgically removed were fixed in 10% formalin, followed by paraffin sectioning, followed by immunohistochemical staining using Smurf1 antibody, and the protein expression level of Smurf1 in colorectal cancer tissues was examined. The results showed that, consistent with the results of fig. 5A, Smurf1 showed higher levels of protein expression in both tissues of CRC1 and CRC2 compared to CRC3 and CRC4 (fig. 5B).

(2) Construction and medication of colorectal cancer PDX model

A.2-3% isoflurane induced anesthesia of mice (or 1% pentobarbital, 60 μ l/10g), maintenance of anesthesia, supine placement of mice, abdominal skin preparation and sterilization, and cutting a small opening of about 2mm in the center of the abdomen with an ophthalmic scissors.

b. The tissue was washed with RPMI-1640 medium (containing 1% penicillin streptomycin double antibody), and connective tissue and necrotic tissue were removedWeaving and cutting the tissue specimen into 1 × 1 × 1mm³The tumor tissue small blocks are ready for use;

c. selecting 1-2 pieces of proper tumor tissues, and sucking into a puncture needle;

d. the puncture needle enters into the subcutaneous part from the incision, gently reaches the armpit of the upper limb and the leg pit of the lower limb under the skin, quickly pushes out the tumor tissue block, lightly presses the lower part of the puncture needle by hand, and rotationally pulls out to finish inoculation;

e. suturing 1-2 needles at the incision with absorbable thread, dripping a drop of penicillin at the incision to prevent wound infection, wrapping the operation part with autoclaved gauze, and laterally placing in a newly replaced cage;

f. filling a newly added cage label according to the experimental requirements, and placing the newly added cage label on a corresponding cage;

g. penicillin was administered 2 ten thousand U/mouse (10 ten thousand U/mL) for three consecutive days after surgery, and the condition of the mice, diet and wound recovery were observed;

h. when the tumor grows to 100mm³In this case, Gemcitabine (50mg/kg), Cisplatin (2.5mg/kg) and combinations thereof (25mg/kg Gemcitabine +1.25mg/kg Cisplatin) were administered intraperitoneally, and mice were sacrificed once a week, 4 weeks later, and tumor sizes were photographed and tumor volumes were analyzed statistically.

The results are shown in FIG. 6, Gemcitabine, Cisplatin and the combination of the two can obviously inhibit the growth of tumors, and especially the combination has the strongest inhibition effect. Gemcitabine, Cisplatin and the combination of Gemcitabine and Cisplatin have stronger tumor growth inhibition effect on the Smurf1 low-expression group (CRC3 and CRC4) compared with the Smurf1 high-expression group (CRC1 and CRC 2).

Meanwhile, immunohistochemical staining is carried out on tumor tissues by using PCNA (cell proliferation associated marker protein) and CyclinD1 antibodies, and statistical analysis is carried out, so that the results show that Gemcitabine, Cisplatin and the combination of Gemcitabine and Cisplatin can obviously inhibit the proliferation of tumor cells in the tumor tissues, and the Gemcitabine, Cisplatin and the combination of Gemcitabine and Cisplatin have stronger inhibition effect on the proliferation of tumor cells with low Smurf1 expression (FIG. 7).

The above results demonstrate that colorectal cancer cells that under-express Smurf1RNA or protein have enhanced drug sensitivity to Gemcitabine and Cisplatin.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

Application of Smurf1 gene, expression product and derivative thereof in preparing marker of sensitivity of tumor cells to chemotherapeutic drugs.
2. The use of claim 1, wherein the tumor cell is a colorectal cancer cell.
3. The use of claim 1, wherein the chemotherapeutic agent is gemcitabine and/or cisplatin.
4. The use according to claim 1, wherein the Smurf1 gene derivative produces different isoform proteins when the Smurf1 gene is expressed.
5. A kit for detecting the sensitivity of tumor cells to chemotherapeutic drugs comprises a reagent for detecting the Smurf1 gene, expression product or derivative thereof.
6. The kit according to claim 5, wherein the reagent for detecting the Smurf1 gene comprises a Smurf1 gene detection primer; the reagent for detecting the Smurf1 gene expression product comprises an antibody of the Smurf1 gene expression product.
The application of the Smurf1 inhibitor in preparing the sensitizer of tumor cells for chemotherapeutic drugs.
8. The use according to claim 7, wherein the inhibitor of Smurf1 is a protein, small molecule compound, antibody that antagonizes Smurf1 function.
9. The use of claim 7 or 8, wherein the tumor cell is a colorectal cancer cell.
10. The use of claim 7 or 8, wherein the chemotherapeutic agent is gemcitabine and/or cisplatin.