Application of MKKS3 as drug resistance marker of intestinal cancer or intestinal cancer drug

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses application of MKKS3 as a drug resistance marker for intestinal cancer or intestinal cancer drugs. Researches show that MKKS3 shows high expression in intestinal cancer tissues and shows low expression in para-carcinoma tissues; meanwhile, the expression condition of the MKKS3 in chemotherapy drug-resistant and sensitive tissues is also detected, the result shows that the MKKS3 is remarkably highly expressed in the intestinal cancer chemotherapy drug-resistant tissues and is low in expression in the chemotherapy sensitive tissues, and most importantly, the Oxaliplatin sensitivity of intestinal cancer cells can be increased by knocking down the MKKS3. In addition, research also finds that high expression of MKKS3 is significantly related to poor prognosis of intestinal cancer patients. In conclusion, the application of the MKKS3 as the marker in preparation of an intestinal cancer detection reagent, a reagent for drug resistance or sensitivity of an intestinal cancer patient or a chemotherapy prognosis evaluation reagent of the intestinal cancer patient has important application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to MKKS3 as a marker for drug resistance of intestinal cancer or intestinal cancer. BACKGROUND

[0002] Currently, surgery, chemotherapy, radiotherapy and even immunotherapy are commonly used in clinical practice to eliminate most tumor cells. On the one hand, most patients are in the middle and late stages when diagnosed, and the effect of surgical resection is limited, and tumors are prone to recurrence, and a part of the patients cannot be treated by surgery, resulting in some patients being unable to undergo surgery. On the other hand, as a treatment method, chemotherapy plays an increasingly important role in the comprehensive treatment of tumors, but tumor cells often develop resistance to chemotherapeutic drugs, resulting in the inability of the drugs to kill tumor cells and causing tumor recurrence, leading to poorer treatment efficacy in the later stages of tumors, resulting in some patients having no available drugs, which is one of the main reasons for the failure of current clinical treatment of tumors. There is still a lack of corresponding countermeasures for the above problems in current clinical practice, which has caused great physical and psychological pain to patients.

[0003] Tumor drug resistance is an important reason for the ultimate lack of available drugs for the treatment of malignant tumors. Studies have shown that 90% of tumor patient deaths are related to tumor drug resistance, so tumor drug resistance is a difficult problem that needs to be solved in current clinical treatment of tumors. In recent years, important discoveries have been made in the study of tumor drug resistance mechanisms at home and abroad, mainly including: the influence of drug transport and efflux by cell membranes, such as multidrug resistance protein (multidrug resistance protein, MRP), lung resistance protein (lung resistance protein, LRP), etc.; changes and increased activity of enzyme systems, such as glutathione reductase (glutathione reductase, GR), etc.; enhanced DNA repair capacity; production of factors and genes related to inhibition of cell apoptosis, etc. However, the mechanism of tumor drug resistance is very complex, and the mechanism of drug resistance of intestinal cancer is still unclear, and there is a lack of effective drug sensitivity prediction molecular markers and corresponding drug resistance reversal treatment measures. SUMMARY

[0004] In order to overcome at least one of the technical problems in the prior art, the present application provides MKKS3 as a marker for drug resistance of intestinal cancer or intestinal cancer.

[0005] The technical solution of the present application is as follows:

[0006] The present application first provides MKKS3 as a marker for drug resistance of intestinal cancer or intestinal cancer.

[0007] The amino acid sequence of the MKKS3 is: MSLRNLWRDYKVLVVMVPLVGLIHLGWYRIKSSPVFQIPKNDDIPEQDSLGLSNLQKSQIQGK.

[0008] Preferably, the drug for intestinal cancer is oxaliplatin.

[0009] The application further provides use of MKKS3 as a marker in the preparation of a detection reagent for intestinal cancer.

[0010] The application further provides use of MKKS3 as a marker in the preparation of a reagent for drug resistance or sensitivity of an intestinal cancer patient.

[0011] Preferably, drug resistance or sensitivity of an intestinal cancer patient is determined by detecting the expression amount of MKKS3 in intestinal cancer tissue of the intestinal cancer patient.

[0012] Preferably, if MKKS3 is highly expressed in intestinal cancer tissue of the intestinal cancer patient, it indicates that the intestinal cancer patient is drug resistant; if MKKS3 is lowly expressed in intestinal cancer tissue of the intestinal cancer patient, it indicates that the intestinal cancer patient is sensitive.

[0013] Preferably, the drug is oxaliplatin.

[0014] The application further provides use of MKKS3 as a marker in the preparation of a reagent for evaluation of chemotherapy prognosis of an intestinal cancer patient.

[0015] Preferably, chemotherapy prognosis of an intestinal cancer patient is determined by detecting the expression amount of MKKS3 in intestinal cancer tissue of the intestinal cancer patient.

[0016] Preferably, if MKKS3 is highly expressed in intestinal cancer tissue of the intestinal cancer patient, it indicates that the intestinal cancer patient has poor chemotherapy prognosis; if MKKS3 is lowly expressed in intestinal cancer tissue of the intestinal cancer patient, it indicates that the intestinal cancer patient has good chemotherapy prognosis.

[0017] Beneficial effects: we found in the research that MKKS3 is highly expressed in intestinal cancer tissue and lowly expressed in paracancerous tissue; meanwhile, we also detected the expression of MKKS3 in chemotherapy resistant and sensitive tissues, and the results showed that MKKS3 is significantly highly expressed in intestinal cancer chemotherapy resistant tissue and lowly expressed in chemotherapy sensitive tissue, and most importantly, knockdown of MKKS3 can increase oxaliplatin sensitivity of intestinal cancer cells. In addition, the research also found that high expression of MKKS3 is significantly related to poor prognosis of an intestinal cancer patient.

[0018] In summary, use of MKKS3 as a marker in the preparation of a detection reagent for intestinal cancer, a reagent for drug resistance or sensitivity of an intestinal cancer patient, or a reagent for evaluation of chemotherapy prognosis of an intestinal cancer patient has important application value. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Figure for the experimental results of detecting the expression of MKKS3 at cell level by anti-MKKS3 antibody.

[0020] Figure 2 Figure for the experimental results of successfully constructing the stable cell strain of knocking down MKKS3, overexpressing MKKS3 and MKKS3mut on the basis of knocking down.

[0021] Figure 3 Figure for the experimental results of knocking down MKKS3 can increase the sensitivity of intestinal cancer cells to oxaliplatin, and re-overexpressing MKKS3 in the form of RNA reverses the regulation of intestinal cancer oxaliplatin sensitivity by knocking down MKKS3.

[0022] Figure 4 Figure for the experimental results that the expression amount of MKKS3 in intestinal cancer tumor tissue is obviously higher than that in para-cancer tissue.

[0023] Figure 5 Figure for the experimental results that the expression amount of MKKS3 in drug-resistant tissue is obviously higher than that in chemotherapeutic sensitive tissue.

[0024] Figure 6 Figure for the experimental results that MKKS3 high expression indicates worse survival rate (patients have received chemotherapy treatment). DETAILED DESCRIPTION

[0025] The present application will be further described in detail below in combination with specific embodiments, but the embodiments do not limit the present application in any form.

[0026] The nucleic acid sequence encoding MKKS3 in the present application is as follows:

[0027] ATGAGCCTTCGGAACTTGTGGAGAGACTACAAAGTTTTGGTTGTTATGGTCCCTTT

[0028] AGTTGGGCTCATACATTTGGGGTGGTACAGAATCAAAAGCAGCCCTGTTTTCCAAATAC

[0029] CTAAAAACGACGACATTCCTGAGCAAGATAGTCTGGGACTTTCAAATCTTCAGAAGAG

[0030] CCAAATCCAGGGGAAGTAA.

[0031] The amino acid sequence of MKKS3 in the present application is as follows:

[0032] MSLRNLWRDYKVLVVMVPLVGLIHLGWYRIKSSPVFQIPKNDDIPEQDSL GLSNLQKSQIQGK.

[0033] In vitro experiment: construct intestinal cancer stable cell line with knockdown of MKKS3, and overexpress MKKS3 and MKKS3mut (mutate the start codon ATG to ATT, so that the protein cannot be translated) on the basis of knockdown to detect the effect of MKKS3 on the oxaliplatin resistance of intestinal cancer cells.

[0034] Stable cell construction: plate 293T cells, and when the density reaches about 50%, prepare for plasmid transfection; according to the aforementioned plasmid transfection method, transfect the knockdown MKKS3 plasmid (purchased from TransheepBio, Shanghai, China, shMKKS3: GCCTTCGGAACTTGTGGAGAG) and pMDLg / pRRE, pRSV-Rev, Pmd2.G plasmids in 293T cells at the same time, and the three packaging plasmids are from Addgene company; after overnight transfection, remove the supernatant, and add 1640 culture medium without double antibodies; collect the cell supernatant twice at 24h and 48h, and at this time the supernatant already contains virus, mix the twice collected supernatant; centrifuge the collected supernatant to remove cell precipitate, and filter with a 0.45 nm filter membrane; f) add polybrene to the filtered supernatant at a ratio of 1:500, and then add it to the intestinal cancer cells plated in advance to infect; 48h after infection, remove the virus-containing culture medium and replace it with normal complete culture medium for continuous culture; when the cell density grows to about 50%, add puromycin to a total concentration of 1 μg / mL for screening; continuously observe cell death, and stop adding drugs when the cells no longer die; collect the screened stable cells for subsequent experiments.

[0035] Cell proliferation detection

[0036] a) Plate the cells into a 96-well plate at a density of 3000 per well;

[0037] b) After the cells adhere, treat them with different concentrations of oxaliplatin for 1-4 days;

[0038] c) Remove the culture medium in the 96-well plate, wash twice with PBS, and add culture medium containing 10% WST-1 and incubate at 37°C for 2h;

[0039] d) Measure the absorbance at 450nm wavelength with a microplate reader, and calculate the cell proliferation inhibition rate.

[0040] Meanwhile, we detected the expression of MKKS3 in the clinical tissues of intestinal cancer. The results showed that MKKS3 was highly expressed in intestinal cancer tissues, but was lowly expressed in paracancerous tissues. Further, it was found that MKKS3 was lowly expressed in chemotherapeutic sensitive tissues of intestinal cancer, but was highly expressed in chemotherapeutic resistant tissues, and was significantly related to the survival prognosis of patients with intestinal cancer.

[0041] Example 1

[0042] (1) Preparation of anti-MKKS3 antibody. (ELISA method was used to detect the titer of anti-MKKS3 antibody, and Western blotting was used for verification Figure 1 ).

[0043] ELISA results:

[0044]

[0045]

[0046] (2) Detection of the function of knocking down MKKS3 (wherein mut is the mutation of the promoter in the ORF region to make it unable to be translated)

[0047] Figure 2 The stable cell strain of knocking down MKKS3 was successfully constructed, and MKKS3 and MKKS3mut were overexpressed on the basis of knocking down. Figure 3 It was proved by cell proliferation experiment that knocking down MKKS3 could increase the sensitivity of intestinal cancer cells to oxaliplatin, and overexpressing MKKS3 but not its RNA form (MKKS3mut: ORF start codon mutation cannot be translated and only acts as RNA form) could restore the increase of the sensitivity of intestinal cancer cells to oxaliplatin caused by knocking down MKKS3.

[0048] (3) Clinical significance of MKKS3

[0049] MKKS3 was highly expressed in intestinal cancer tissues, but was lowly expressed in paracancerous tissues Figure 4 Meanwhile, we also detected the expression of MKKS3 in chemotherapeutic resistant and sensitive tissues. The results showed that MKKS3 was highly expressed in chemotherapeutic resistant tissues of intestinal cancer, but was lowly expressed in chemotherapeutic sensitive tissues Figure 5 Importantly Figure 6 The high expression of MKKS3 was significantly related to the poor prognosis of patients with intestinal cancer.

Claims

1. Application of MKKS3 as a biomarker for colorectal cancer or colorectal cancer drug resistance.

2. The application according to claim 1, characterized in that, The amino acid sequence of MKKS3 is: MSLRNLWRDYKVLVVMVPLVGLIHLGWYRIKSSPVFQIPKNDDIPEQDSLGLS NLQKSQIQGK.

3. The application according to claim 1, characterized in that, The colorectal cancer drug mentioned is oxaliplatin.

4. Application of MKKS3 as a biomarker in the preparation of colorectal cancer detection reagents.

5. Application of MKKS3 as a biomarker in the preparation of reagents for drug resistance or sensitivity in colorectal cancer patients.

6. The application according to claim 5, characterized in that, The expression level of MKKS3 in the colorectal cancer tissue of colorectal cancer patients can be used to determine whether colorectal cancer patients are resistant to or sensitive to drugs.

7. The application according to claim 6, characterized in that, If MKKS3 is highly expressed in the colorectal cancer tissue of a colorectal cancer patient, it indicates that the patient is drug resistant; if MKKS3 is lowly expressed in the colorectal cancer tissue of a colorectal cancer patient, it indicates that the patient is sensitive.

8. The application according to claim 5, characterized in that, The drug in question is oxaliplatin.

9. Application of MKKS3 as a biomarker in the preparation of a reagent for evaluating the prognosis of colorectal cancer patients undergoing chemotherapy.

10. The application according to claim 8, characterized in that, The prognosis of colorectal cancer patients after chemotherapy can be determined by detecting the expression level of MKKS3 in colorectal cancer tissue. Preferably, if MKKS3 is highly expressed in the colorectal cancer tissue of a colorectal cancer patient, it indicates a poor prognosis after chemotherapy; if MKKS3 is low expressed in the colorectal cancer tissue of a colorectal cancer patient, it indicates a good prognosis after chemotherapy.

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