CN111518909B

CN111518909B - Application of METTL2 gene in preparation of kit for detecting treatment sensitivity of colorectal cancer fluorouracil drugs

Info

Publication number: CN111518909B
Application number: CN202010424466.XA
Authority: CN
Inventors: 刘然义; 彭绮华; 岳欣
Original assignee: Sun Yat Sen University Cancer Center
Current assignee: Sun Yat Sen University Cancer Center
Priority date: 2020-05-19
Filing date: 2020-05-19
Publication date: 2022-07-01
Anticipated expiration: 2040-05-19
Also published as: CN111518909A

Abstract

The invention belongs to the technical field of biological medicines, and discloses application of a METTL2 gene in preparation of a kit for detecting treatment sensitivity of colorectal cancer fluorouracil drugs. The application is based on the discovery of the inventor that the METTL2 protein expression can enhance the sensitivity of colorectal cancer cells to 5-FU, and the high expression of METTL2 in the tumor tissues of colorectal cancer patients is in positive correlation with the good prognosis of 5-FU chemotherapy. Therefore, a clinician can determine whether a colorectal cancer patient is suitable for fluorouracil drug treatment or not through the expression condition of the METTL2 gene of the colorectal cancer patient, and a personalized treatment scheme is formulated.

Description

Application of METTL2 gene in preparation of kit for detecting colorectal cancer fluorouracil drug treatment sensitivity

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of a METTL2 gene in preparation of a kit for detecting treatment sensitivity of colorectal cancer fluorouracil drugs.

Background

The incidence and mortality of Colorectal Cancer (CRC) worldwide is third in all types of tumors worldwide. Along with the development of economy and science and technology in China in recent decades, the living standard of people is greatly improved, and the incidence of colorectal cancer is gradually increased along with the popularization of western diet mode. Since the 50 s of the 20 th century, 5-fluorouracil (5-fluorouracil, 5-FU) was used clinically as a basic drug for colorectal cancer treatment, fluorouracil-like drugs (FU) such as 5-FU and Capecitabine (oral drugs) have been an important component of first-line chemotherapy regimens (e.g., CAPEOX, FOLFOX, FOLFIRI, XELOX, etc.) for colorectal cancer patients of different stages to date. However, the single-drug effective rate of 5-FU is only 10-15%, the effective rate of the combined calcium folinate (LV) can be improved from 11% to 25%, but the overall effective rate is still low, and the sensitivity of tumors to FU is one of the most important factors for restricting the curative effect and the prognosis of patients after chemotherapy. Therefore, intensive research is needed to effectively evaluate the sensitivity of colorectal cancer patients to FU, so as to improve the drug effectiveness and control the generation of FU drug resistance. The gene related to specific regulation and control of FU metabolism and sensitivity or drug resistance is searched, so that a clinician can conveniently make a more reasonable personalized treatment scheme according to different colorectal cancer patients, and the method has great significance for optimizing medical resource structures.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides application of a METTL2 gene in preparation of a kit for detecting the treatment sensitivity of colorectal cancer fluorouracil drugs.

The above object of the present invention is achieved by the following technical solutions:

application of METTL2 gene in preparation of kit for detecting colorectal cancer fluorouracil drug treatment sensitivity. Based on the discovery of the inventor, the METTL2 can enhance the sensitivity of colorectal cancer cells to 5-FU, and the high expression of METTL2 in the tumor tissues of colorectal cancer patients is positively correlated with the good prognosis of 5-FU chemotherapy. Therefore, a clinician can determine whether the colorectal cancer patient is suitable for the fluorouracil drug treatment or not through the expression condition of the METTL2 gene in the tumor tissue of the colorectal cancer patient, and a personalized treatment scheme is formulated.

The fluorouracil drug is preferably at least one of 5-FU and Capecitabine; more preferably 5-FU.

The kit comprises one or two of a reagent for detecting the quantity of METTL2mRNA and a reagent for detecting the quantity of METTL2 protein.

The reagent for detecting the quantity of METTL2mRNA is preferably a reagent capable of carrying out reverse transcription-real-time fluorescent quantitative PCR (RT-qPCR).

The reagent capable of carrying out reverse transcription-real-time fluorescence quantitative PCR comprises a primer for real-time fluorescence quantitative PCR designed according to a METTL2mRNA sequence; preferably any one of 20 primer pairs with nucleotide sequences shown as SEQ ID No. 1-40; the first numbers in the primer names with the nucleotide sequences shown as SEQ ID NO. 1-40 are the same as the primers in the same pair.

The reagent capable of carrying out reverse transcription-real-time fluorescence quantitative PCR also comprises a primer which is designed according to the mRNA sequence of the reference gene and is used for real-time fluorescence quantitative PCR; preferably comprises a primer designed according to the mRNA sequence of the reference gene and used for real-time fluorescent quantitative PCR; more preferably, the primer pair comprises nucleotide sequences shown as SEQ ID NO. 41-42.

The reagent capable of carrying out reverse transcription-real-time fluorescence quantitative PCR is preferably at least one of a combined reagent A and a combined reagent B; wherein, the combined reagent A is obtained according to a probe method, and comprises but is not limited to: the kit comprises primers for real-time fluorescent quantitative PCR designed according to a METTL2mRNA sequence, probes for real-time fluorescent quantitative PCR designed according to a METTL2mRNA sequence, primers for real-time fluorescent quantitative PCR designed according to an internal reference gene mRNA sequence, and probes for real-time fluorescent quantitative PCR designed according to an internal reference gene mRNA sequence;

the combined reagent B is obtained by a dye method and comprises but is not limited to the following components: primers for real-time fluorescent quantitative PCR designed according to METTL2mRNA, primers and dye for real-time fluorescent quantitative PCR designed according to reference gene mRNA sequence.

The probe is preferably a Taqman probe.

The dye is preferably SYBR Green.

The reagent for detecting the amount of METTL2 protein is preferably a reagent capable of performing WESTERN BLOT (WB) and a reagent capable of performing immunohistochemical staining (IHC).

Reagents capable of performing WESTERN Black (WB) include, but are not limited to: an anti-METTL 2 antibody, an anti-reference protein antibody, a universal secondary antibody, and a chromogenic or luminescent reagent.

The reference protein is preferably GAPDH.

Reagents capable of immunohistochemical staining (IHC) include, but are not limited to: an anti-METTL 2 antibody, a universal secondary antibody, and a chromogenic reagent.

Compared with the prior art, the invention has the following advantages and effects:

the inventor finds that METTL2 does not affect the proliferative capacity of colorectal cancer cells, but finds that through in vivo and in vitro experiments: METTL2 enhances the sensitivity of colorectal cancer cells to 5-FU both at the cellular level in vitro and in vivo in animals. The 5-FU sensitive gene METTL2 of the colorectal cancer is screened out based on the human genome RNAi technology, the correlation between the expression of the gene and the prognosis of a colorectal cancer patient receiving chemotherapy containing fluorouracil is analyzed, reference evidence is provided for a clinician to formulate a more reasonable chemotherapy scheme according to different individual genetic differences, and the method has great significance for optimizing medical resource structures.

Drawings

FIG. 1 is a flow chart of 5-FU sensitivity related gene screening by whole genome RNAi technology.

FIG. 2 is a graph showing the 5-FU dose-effect curve and the 5-FU resistance change results of HCT116 cell line after stably knocking down different candidate genes.

FIG. 3 is a diagram showing the expression of METTL2 before and after 5-FU treatment of HCT116 cells detected by Western Blot (WB) and reverse transcription-quantitative PCR (RT-qPCR) method.

FIG. 4 is a diagram showing the expression of METTL2 in WB and RT-qPCR detection of HCT-8 and its drug-resistant strain HCT-8-FU.

FIG. 5 is a graph showing the effect of METTL2 on the proliferative capacity of colorectal cancer cells; wherein, the graph A is a CCK-8 experimental result graph; panel B is a graph showing the results of a plate clone formation experiment.

FIG. 6 is a graph of the results of METTL2 enhancing 5-FU sensitivity in colorectal cancer cells; wherein, the graph A is a CCK8 experimental result graph; panel B is a graph showing the results of a plate clone formation experiment.

FIG. 7 is a graph showing the results of METTL2 enhancing 5-FU sensitivity of colorectal cancer cells in nude mouse transplanted tumor model experiments; wherein, the picture A is a picture of the result of the HCT116 transplantation tumor of knocking down METTL 2; panel B is a graph of HT-29 transplantable tumor results overexpressing METTL 2.

FIG. 8 is a graph of the outcome of METTL2 expression versus prognosis for colorectal cancer patients receiving chemotherapy with 5-FU; wherein, panel a is a graph of four intensities of METTL2 expression in colorectal cancer tissue; and the graph B is a survival analysis result graph.

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.

Colorectal cancer cell lines HCT116, SW620, HT-29, DLD1, HCT-15, SW480, HCT-8, normal human intestinal mucosal epithelial cells NCM460 and human embryonic kidney epithelial cell line 293T were all purchased from American Type Culture Collection (ATCC), and 5-FU-resistant strains of HCT-8 (i.e., HCT-8-FU human colon cancer fluorouracil-resistant strains) were purchased from Olympic Biotechnology, Inc., Shanghai. The cell strains used in the research are correctly identified by short tandem repeat column detection of the cells of the China center for type culture collection, and are pollution-free by mycoplasma detection. The cells were cultured in a medium containing 10% fetal bovine serum, and the medium for each cell was: HCT116 cells: McCoy' 5A medium; DLD-1 cells, HCT-15 cells, HCT-8 cells and HCT-8-FU cells: RPMI-1640 medium; SW480 cells and SW620 cells: l-15 medium; NCM460 cells and 293T cells: DMEM medium.

Western Blot (WB) reagents for the experiment: rapid gel preparation kit (friedel organisms, china); primary anti-secondary antibody diluent, primary anti-secondary antibody remover-weak base (bi yun day, china); m2 anti-Flag agarose (Sigma, USA); hypersensitive ECL chemiluminescence kit (Beijing Sizhengbai, China); BCA protein quantitative determination kit (Kaikyi organisms, China); anti-mouse IgG HRP-linked Antibody (Promega, USA); #9803S-IP lysate and anti-rabbitIgG HRP-linked Antibody (Cell Signaling Technology, USA).

The antibodies used were: anti-METTL 2(# SAB1407697, Sigma, USA; for WB dilution 1:250), anti-METTL 2(# TA324450, Origene, USA; for immunohistochemical staining dilution 1:50), anti-GAPDH (# D16H11, Beijing four positive arborvitae, China; for WB dilution 1: 2000); cleavage resistant PARP-1(# D64E10, CST, USA; dilution for WB 1: 1000; dilution for immunohistochemical staining 1:50), cleavage resistant Caspase-3(#9661, CST, USA; dilution for WB 1: 1000; dilution for immunohistochemical staining 1:200), cleavage resistant Caspase-7(# D2Q3L, CST, USA; dilution for WB 1: 1000).

Example 1: 5-FU sensitive gene screening by whole genome RNAi technology

This study utilized a genome-wide shRNA library

LentiPlex^TMPooledshRNA libraries (Sigma, USA) combines the next generation sequencing analysis technology to construct a screening system for screening genes related to 5-FU sensitivity in colorectal cancer in high throughput (the screening process is detailed in figure 1). The library is based on a lentiviral vector pLKO.1, sequences are designed for about 15000 genes of a human genome, 3-5 shRNA sequences are designed for each gene on average, the total library is divided into 10 small libraries (pool1-10), and each small library contains target sequences of about 1500 genes. In the experiment, we used the RNAi library to infect HCT116 cells and HT-29 cells at 0.5MOI (multiplex of infection) in order to ensure that most of the cells infected no more than one viral particle, i.e., each cell successfully infected with the virus received only shRNA targeting one gene, thereby ensuring that the cells did not undergo simultaneous knock-down of multiple genes. The mixed infected cells were then screened for puromycin, and the surviving stably infected HCT116 and HT-29 cells were divided into two groups, one experimental and the other control. The media of the cells of the experimental group were supplemented with 5. mu.M 5-FU at the final concentration, and the media of the control group were supplemented with the same dose of 5-FU as the lytic agent (DMSO). The culture is carried out by changing culture medium every other day, floating cells are removed, and culture medium containing 5-FU (or DMSO) with the same dosage is added continuously for continuous culture. After one week of 5-FU treatment,the sets of cells were harvested and genomic DNA extracted, followed by amplification of specific shRNA sequence fragments using universal primers with tags (see details in the procedures below) and high throughput sequencing analysis. The principle of the screening system is that the copy number of shRNA of a certain gene in the 5-FU treatment group is obviously increased compared with that of a control group, and the capability of resisting 5-FU is proved to be generated by colorectal cancer cells after the gene is knocked down, so that the gene is probably related to the sensitivity of the 5-FU to the colorectal cancer cells.

The specific process is as follows (the flow is shown in figure 1):

1. construction of cellular RNAi library screening System

(1) Cell plating: collecting good cells (purchased from American type culture Collection ATCC) of HCT116 and HT-29 in logarithmic growth phase, digesting, counting, plating to 10cm cell culture dish, plating HCT116 to 1.5 × 10⁶Cell/dish, HT-29 plating of cells 2.5X 10⁶One/one dish; randomly selecting cells in a 10cm culture dish from the two cells for digestion and counting in the next day of plating so as to determine the total cell number of each dish of the two cells;

(2) viral infection: the volume of virus solution required to be added was calculated from the number of cells per dish. Whole genome shRNA library

LentiPlex^TMPooled shRNA viruses were infected at an MOI of 0.5, and polybrene was added to a final concentration of 8. mu.g/ml; meanwhile, the cells of the virus-free infected group are used as a control (equal volume of culture medium is used for replacing virus liquid);

(3) and (3) purine screening: screening is carried out by using a complete culture medium containing puromycin 24 hours after virus infection, wherein the screening concentration of the puromycin in the HCT116 cell culture medium is 1 mu g/ml; the selection concentration of puromycin in HT-29 cell culture medium was 2. mu.g/ml. The puromycin screening time is 2-3 days every time until all cells of the virus-free infected group are killed by puromycin. Changing the liquid, and removing dead cells and floating cell debris;

(4)5-FU screening: dividing the colorectal cancer cells which are stably infected with viruses and are screened by puromycin into two groups respectively; the experimental group was given 5. mu.M 5-FU treatment and the control group was given an equivalent volume of solvent (DMSO) treatment. The culture medium containing the same dose of 5-FU or DMSO was replaced every other day while the floating cells were discarded. Collecting cells for use 1 week after administration;

(5) the groups of cells were collected and genomic DNA was extracted: A) the adherent cells obtained by screening in the step (4) (total number of cells is about 1X 10)⁶Respectively) digesting with pancreatin, centrifuging, washing with PBS for 2 times, centrifuging (rotation speed of 1500rpm, 5min), collecting cell precipitate, and placing in 1.5ml EP tube; B) adding 300 mul of cell lysate for resuspension, and fully lysing in a vortex instrument for 15 s; C) adding 1.5 μ l RNase A, reversing the upper part and the lower part, uniformly mixing for 8-10 times, then putting the mixture into a water bath kettle at 37 ℃ for incubation for 5-10min to remove RNA, and then putting the sample into ice water for rapid cooling; D) adding 100 μ l of protein precipitation solution, mixing uniformly by a vortex apparatus for 30-60s, and centrifuging for 1min at 16000-; E) taking the supernatant, placing in a new EP tube, adding 300. mu.l isopropanol, gently inverting and mixing uniformly, and centrifuging for 1min at 16000-; F) carefully discarding the supernatant, sucking away the residual liquid with a 100 μ l pipette tip as carefully as possible, and air drying at room temperature for 2-5 min; G) adding 30-50 mul of DNA elution buffer solution according to the amount of the DNA, slightly whirling for several seconds, and then placing in a 65 ℃ water bath for 1 hour to fully dissolve the DNA; H) after the DNA concentration and purity were measured in nanodrop 2000, the DNA was stored at-20 ℃.

2. Amplifying target shRNA sequence and performing second-generation sequencing

(1) 80ng of DNA was taken from HCT116 cells or HT-29 cells of the same pool as a template, and nested PCR amplification was performed:

A) first round PCR:

a forward primer: 5'-TACAAAATACGTGACGTAGAAA-3', respectively;

reverse primer: 5'-TTTGTTTTTGTAATTCTTTA-3' are provided.

Reaction system: 80ng of genomic DNA, 2.5. mu.l of 10-fold buffer, 2.5. mu.l of 2mM dNTPs, and 25mM MgSO₄Mu.l, 10. mu.M forward primer 0.75. mu.l, 10. mu.M reverse primer 0.75. mu.l, KOD Plus-Neo enzyme 0.5. mu.l, made up to 25. mu.l with double distilled water.

Reaction conditions are as follows: pre-denaturation at 98 ℃ for 30 s; denaturation at 98 ℃ for 15s, annealing at 68 ℃ and extension for 30s, and 20 cycles; final extension at 72 ℃ for 2 min.

B) Second round PCR:

DNA template: first round PCR product, add ddH₂Diluting with O by 10 times for later use.

The primers of each experimental group are shown in the following table 1:

TABLE 1

Reaction system: DNA template 1. mu.l, 10-fold concentration buffer 5. mu.l, 2mM dNTPs 5. mu.l, 25mM MgSO₄Mu.l of each of 3. mu.l, 10. mu.M of the forward primer, 1.5. mu.l of each of 10. mu.M of the reverse primer, and 1. mu.l of KOD Plus-Neo enzyme were added to make up 50. mu.l with double distilled water.

Reaction conditions are as follows: pre-denaturation at 98 ℃ for 30 s; denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 30s, and extension at 68 ℃ for 20s for 35 cycles; final extension at 72 ℃ for 1 min.

(2) Purification and quantification of PCR products: the second round PCR product was run on a conventional gel, recovered and purified by gel cutting, and the DNA concentration and purity were determined by nanodrop 2000.

(3) Second-generation sequencing and data analysis: the purified and recovered PCR products were mixed in equal proportions and submitted to high-throughput sequencing and analysis by Biometrics. Sequencing results candidate genes were screened according to the following screening conditions: screening sensitive gene (FU treatment group vs. control group), Log₂(Fold Change)>2.0, FDR < 0.05; ② the copy number of at least 2 shRNAs in the FU treatment group is increased by more than 4 times, and the Count is more than 300.

3. Data analysis results: after analyzing the sequencing result, 5-FU sensitivity related genes are screened according to the above screening conditions to obtain 5 candidate genes which are respectively: METTL2, ZNF365, CEP68, IFT122, and BEST 2. The result shows that the copy number of at least 2 shRNAs of a single target gene in two groups of colorectal cancer cells with different genetic backgrounds is obviously increased before and after the HCT116 cells and the HT-29 cells are treated by 5-FU, and the gene is possibly a candidate gene related to 5-FU sensitivity.

4. Cell level verification:

(1) the five gene-stably knocked-down HCT116 cell lines were constructed: after transfecting HCT116 cells with lentiviruses expressing the shRNAs (see Table 2 below) respectively, five HCT116 cell lines with stably knocked-down genes and a control HCT116 cell line are obtained by screening with puromycin.

TABLE 2

(2) Cell 5-FU sensitivity assay: the killing effect of the stable cell line 5-FU (cell viability assay) on the stable cell line in different concentration ranges (0. mu.M, 0.04. mu.M, 0.2. mu.M, 1. mu.M, 5. mu.M, 25. mu.M, 125. mu.M and 1250. mu.M) was tested by using a CCK-8 kit.

(A) Cell plating: inoculating the cells in the log phase with good growth state into a 96-well plate, wherein the number of the cells is 3 multiplied by 10³Each group has 3-5 multiple holes with 5% CO content at 37 deg.C₂Culturing for 24h under the condition of saturated water vapor; (B) adding medicine: after 24h of plating, the original culture medium is discarded, complete culture mediums containing 5-FU (0. mu.M, 0.04. mu.M, 0.2. mu.M, 1. mu.M, 5. mu.M, 25. mu.M, 125. mu.M, 1250. mu.M) with different concentrations are respectively added, and the culture is continued for 72 h; (C) measurement: 1/10 volumes of CCK8 solution were added to each well and incubated for 2-4h under the same conditions as above. Reading an OD value at the wavelength of 458nm by using a microplate reader (in the measurement process, attention is required to be paid to the fact that the measured value cannot be larger than 2, otherwise, the curve is deviated); (D) calculation IC 50: the IC50 of 5-FU and the relative drug resistance coefficient of the cells were calculated by plotting a dose-response curve with the change of 5-FU concentration using Graphpad prism 8.0.

The results are shown in FIG. 2. The results suggest that knocking down METTL2, CEP68 and IFT122 genes all increased IC50 values and drug resistance index of HCT116 cells to 5-FU, with the most significant effect of knocking down METTL 2.

Example 2: detection of expression of METTL2 in HCT116 cells and 5-FU-resistant cell strains treated by 5-FU

1. Expression changes of METTL2 before and after 5-FU treatment of colorectal cancer cells

(1) Treating 5-FU with different concentrations (0, 0.5. mu.M, 1. mu.M, 2. mu.M, 4. mu.M, 8. mu.M) for 24 h; cells were harvested for RNA and protein extraction and METTL2mRNA and protein levels were determined.

(2) mRNA level detection (RT-qPCR method):

(A) extracting total RNA of cells by a TRIzol method (performed according to the kit operating instructions), and measuring the DNA concentration and purity in nanodrop 2000;

(B) reverse transcription is carried out on the RNA according to a conventional method to synthesize corresponding cDNA;

(C) quantitative pcr (qpcr) method to detect mRNA relative content (GAPDH as internal control):

the qPCR primers for METTL2 can be any pair of the following table (table 3):

TABLE 3

Reaction system: 2. mu.l cDNA, 8. mu.l 2 XSSYBR Green PCR Mastermix, 1. mu.l 10. mu.M forward primer, 1. mu.l reverse primer, ddH₂O4μl。

Reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 98 ℃ for 15s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 40s for 40 cycles.

(D) Calculating the delta Ct (Ct value of METTL2 minus Ct value of GAPDH) of each group of cells by taking GAPDH as an internal reference to obtain the relative content 2 of METTL2mRNA^-ΔCtThen, the relative mRAN level of METTL2 was calculated with the relative amount of METTL2mRNA in the shNC group as the reference value 1.

(3) Protein level detection (Western blot method): (A) cracking cells by using a Rica cell lysate mixed with a Cocktail protease inhibitor according to a conventional method, and extracting total cell protein; (B) preparing 10% polyacrylamide gel, and carrying out conventional SDS-polyacrylamide gel electrophoresis, membrane transfer and 5% skim milk confining liquid sealing on the total cell protein; (C) antibody incubation, and protein strip color development (luminescence) by using a hypersensitive ECL chemiluminescence kit (purchased from Beijing Sizhengbai Biotech Co., Ltd.).

(4) As a result: the RT-qPCR method detects that the mRNA level of METTL2 in HCT116 cells treated before and after 5-FU treatment and at different concentrations has no obvious change; according to Western blot detection, the level of METTL2 protein after 5-FU treatment is obviously reduced, and the level of METTL2 protein shows a descending trend along with the increase of the concentration of 5-FU (figure 3).

2. METTL2 expression difference in colorectal cancer 5-FU drug-resistant cell strain and mother strain thereof

(1) Collecting cells of colorectal cancer cell strain HCT-8 and 5-FU-tolerant strain (HCT-8-FU human colon cancer fluorouracil-tolerant strain purchased from Olympic Biotech Co., Ltd., Shanghai) in exponential growth phase, and detecting the expression condition of METTL2 according to the RT-qPCR method and the Western blot method.

(2) As a result: in the 5-FU-tolerant strain (HCT-8-FU), METTL2 was significantly lower in both mRNA and protein levels than the parental cell strain (see FIG. 4).

The experimental results indicate that the colorectal cancer cell line can reduce the content of METTL2 after being stimulated by 5-FU so as to adapt to the adverse stimulation of 5-FU, and reflect that the biological function of METTL2 can be closely related to the sensitivity of 5-FU.

Example 3: construction of colorectal cancer cell line stably knocking down or over expressing METTL2 and influence of colorectal cancer cell line on proliferation capacity.

(1) shRNAs against the target sequences in the following Table (Table 4) were designed and synthesized according to the instructions of pLKO.1 vector (Sigma-Aldrich, USA), inserted into Age I and EcoR I sites of pLKO.1 plasmid, and packaged in 293T cells to obtain lentiviruses expressing the corresponding shRNAs.

TABLE 4

(2) HCT116 and SW620 cells are infected by the lentivirus liquid conventionally, and are screened by puromycin to obtain stable knock-down METTL2 cell strains (HCT116/sh1, HCT116/sh2, SW620/sh1 and SW620/sh2) or negative control cell strains (HCT116/shNC and SW 620/shNC).

(3) Inserting METTL2 cDNA (purchased from Shandong Weizhen Biotech company) into XbaI and NheI sites of pCDH-EF1-MCS-T2A-Puro (System Bioscience, USA) vector to construct a lentivirus METTL2 expression vector; the lentivirus was packaged in 293T cells and then HT-29 cells were infected to construct HT-29/METTL2 cells stably overexpressing METTL2 and empty vector-controlled HT-29/Vec cells.

(4) The effect of METTL2 on the proliferative capacity of intestinal cancer cells was examined: the proliferation potency of the stable cell lines described above was tested in the absence of 5-FU by the CCK8 assay (see example 1 for procedure control) and the plate clone formation assay (see below).

(5) Plate clone formation experiment: (A) colon cancer cells (HCT116 cells 500/well, HT-29 cells 1500/well) in logarithmic growth phase are respectively laid in a 6-well plate, and 3 multiple wells are arranged; (B) culturing the cells with respective complete culture medium, and changing the culture solution once every 3 days; (C) after the cells are cultured for 10-14 days, removing the culture medium, washing with PBS, adding 1 ml/hole of 0.5% crystal violet methanol solution freshly prepared, fixing at room temperature, and dyeing for 30 min; (D) discarding the staining solution of the crystal violet methanol solution, washing the residual staining solution with PBS (phosphate buffer solution) gently, and drying at room temperature; (E) cell clonal populations with cell numbers greater than 50 were calculated by the squared method.

(6) As a result: as shown in FIG. 5, the CCK8 method shows that the proliferation capacity of HCT116 cells is not obviously changed after the METTL2 is knocked down compared with the control group, and the over-expression of METTL2 has no obvious influence on the proliferation capacity of HT-29 cells. The results of the plate clone formation experiments are consistent with the results of the CCK8 method. And (4) conclusion: METTL2 did not affect the proliferative capacity of colorectal cancer cells.

Example 4: effect of METTL2 on 5-FU sensitivity in vitro cultured cells

(1) The sensitivity of cells stably knocking down METTL 2(HCT116/sh 1 and HCT116/sh2, SW620/sh1 and SW620/sh2) or negative control cell strains (HCT116/shNC, SW620/shNC), stably over-expressing cell strains (HT-29/METTL2) and empty vector control cell strains (HT-29/Vec) to 5-FU was tested by the CCK8 method (see example 1) and a plate clone formation experiment (see example 3).

(2) As a result: as shown in FIG. 6, the CCK8 method shows that the IC50 value and the relative drug resistance index of the knock-down METTL2 cell line 5-FU are obviously higher than those of a control cell line, and the IC50 value and the relative drug resistance index of the over-expression METTL2 cell line are obviously lower than those of the control cell line (FIG. 6A); clone formation experimental results showed that the number of clones of the knocked-down METTL2 cell line after 5-FU treatment was greater than that of the control cell line, and the number of clones of the over-expressed cell line was significantly less than that of the vector control cell (FIG. 6B). These results demonstrate that METTL2 enhances the sensitivity of colorectal cancer cells to 5-FU.

Example 5: effect of METTL2 on 5-FU sensitivity in nude mouse graft tumor model

BALB/c nude mice (4-5 weeks old, 14-18g, purchased from the center of Shanghai Slek's laboratory animals) were divided into five groups and subcutaneously tumorigenized by subcutaneously implanting HCT116/sh1, HCT116/sh2(HCT116/shNC is a negative control) that stably knockdown METTL2, and HT-29/METTL2 cells that stably overexpress METTL2 (HT-29/Vec is an empty vector control), respectively. After the tumor mass grows up, taking out the tumor mass and equally dividing the tumor mass into 1-2mm³These small tumors were transplanted into each group of nude mice. After one week, 5-FU was administered at a dose of 0.025 mg/g/day (with an equal volume of saline as a control group) for 5 consecutive days, and then discontinued for 2 days, and repeated for 2 weeks. Tumor volume was measured every 3 days during the administration of 5-FU treatment, mice were sacrificed after 3 weeks of treatment, tumor tissues were dissected and weighed, and tumor inhibition rates of each group were calculated.

The results are shown in FIG. 7. The tumor inhibition rate of 5-FU treatment on METTL2 knockdown HCT116 transplantable tumors (9% and 2%) was much lower than that of the control group (55%), while the tumor inhibition rate of 5-FU on METTL2 overexpressing HT-29 transplantable tumors (78%) was significantly higher than that of the empty vector control group (32%). In contrast, for the stable knockdown and stable overexpression experimental groups without 5-FU treatment, the weights of the tumors were not significantly different between the two groups compared to the respective control groups. The results of the above research show that METTL2 can obviously enhance the sensitivity of 5-FU of colorectal cancer transplantation tumor, but does not influence the growth of colorectal cancer transplantation tumor, and therefore, METTL2 can enhance the sensitivity of colorectal cancer cells to 5-FU in animal bodies.

Example 6: clinical value of METTL2 expression levels in assessing prognosis of colorectal cancer patients receiving 5-FU treatment

The study was approved by ethical review by the ethical review committee of the center for tumor prevention and treatment of university of zhongshan and informed written consent was obtained from the patients. We collected colorectal cancer patients admitted to the center for tumor prevention and treatment at the university of zhongshan in 2007 to 2011. 117 cases of the study were screened under the following conditions. The screening conditions are specifically as follows: firstly, a patient is confirmed to be a colorectal cancer patient at the stage II or III for the first time in a hospital, and does not receive new auxiliary treatment before an operation; adjuvant chemotherapy containing 5-FU medicine is carried out after operation, and concurrent radiotherapy treatment is not carried out; and the patient has complete clinical pathological data. The patient's case follow-up data was obtained from the follow-up department of my hospital. We called clinical information about patients including age, sex, pathology type, clinical stage, tumor index (CEA, CA19-9), and chemotherapy regimen. Wherein the clinical stage of the patient is assessed according to the TNM stage of AJCC (American Joint Committee on cancer) eighth edition. Patients received routine follow-up after treatment. METTL2 expression was detected by immunohistochemistry in surgical tumor specimens from 118 patients with colorectal cancer.

Immunohistochemical experiments: (A) paraffin sections of 4-5. mu.M were routinely dewaxed and hydrated with 0.3% H₂O₂Removing peroxidase from the methanol solution, sealing with instant goat serum sealing solution, and heating 0.01M sodium citrate buffer solution (pH 6.0) in microwave oven to repair antigen; (B) performing METTL2 protein staining and hematoxylin counterstaining by adopting a Dako DAB immunohistochemical kit; (C) differentiation with 1% hydrochloric acid alcohol, dehydration with increasing concentration of ethanol, sealing with neutral resin, air drying in fume hood for 24 hr, and observing under microscope.

After immunohistochemistry was completed, two pathologists performed double-blind scoring on the tissue section staining results. The results were judged by a semi-quantitative method, and the staining ratio and staining intensity were determined for 5 fields (100 ×). Staining intensity was divided into colorless (negative, score 0), pale yellow (weak positive, score 1), yellow (positive, score 2) and tan (strong positive, score 3) (as shown in fig. 8A). The dyeing proportion is less than 5 percent (0 min), 5 to 25 percent (1 min), 26 to 50 percent (2 min), 51 to 75 percent (3 min) and 76 to 100 percent (4 min). The total staining was divided into the average of the product of the staining intensity score and the staining area score for 5 fields.

A one-way Kaplan-Meier Survival assay was performed on the 117 patients described above to determine whether METTL2 expression affected Overall Survival (OS) and Disease-free Survival (DFS) in colorectal cancer patients receiving chemotherapy with 5-FU. The results are shown in fig. 8B, where high expression of METTL2 positively correlated with good prognosis in patients with colorectal cancer receiving fluorouracil-containing adjuvant chemotherapy (overall survival OS, p 0.0056; disease-free survival DFS, p 0.0222). The results of the multifactor COX regression phase analysis are shown in table 5 below, where: n-117, METTL2 expression level is an independent prognostic factor in such patients.

Table 5:

the multifactorial COX regression phase analysis results in table 5 suggest that METTL2 expression levels are independent prognostic factors for colorectal cancer patients receiving fluorouracil-containing adjuvant chemotherapy.

The above embodiments are representative embodiments of the present invention, but the embodiments of the present invention are not limited by 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 regarded as equivalent replacements within the protection scope of the present invention.

Sequence listing

<110> Zhongshan university tumor prevention and treatment center (Zhongshan university affiliated tumor Hospital, Zhongshan university tumor institute)

Application of <120> METTL2 gene in preparation of kit for detecting treatment sensitivity of colorectal cancer fluorouracil drugs

<160> 65

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<223> primer 1-F

<400> 1

gcagtcctcg ccgataagag 20

<210> 2

<223> primer 1-R

cttccgacca ctccacattg t 21

<210> 3

<223> primer 2-F

atgcctggga caatgtggag 20

<210> 4

<223> primer 2-R

cacacccgct ggatactgtt 20

<210> 5

<223> primer 3-F

ccgaatactg gaggttggct 20

<210> 6

<223> primer 3-R

cttgggcact gggtaactct 20

<210> 7

<223> primer 4-F

cgtcttccac cacaatgcct 20

<210> 8

<223> primer 4-R

cctggacttt tctctccgcc 20

<210> 9

<223> primer 5-F

gctggcacct agccaaaatc 20

<210> 10

<223> primer 5-R

attcggtagg tggctgagga 20

<210> 11

<223> primer 6-F

ctgggacaat gtggagtggt 20

<210> 12

<223> primer 6-R

cttgtttctc ctggcacacc 20

<210> 13

<223> primer 7-F

ctggacacgc ttttcaccac 20

<210> 14

<223> primer 7-R

acagaagggg cttgcagtat 20

<210> 15

<223> primer 8-F

gacatggctc agcttcggtt 20

<210> 16

<223> primer 8-R

gtggtgaaaa gcgtgtccag 20

<210> 17

<223> primer 9-F

gccgataaga ggcagcagtt 20

<210> 18

<223> primer 9-R

attgtcccag gcattgtggt 20

<210> 19

<223> primer 10-F

caagaggaac tggacacgct 20

<210> 20

<223> primer 10-R

ggggcttgca gtatttgcac 20

<210> 21

<223> primer 11-F

accgaatact ggaggttggc 20

<210> 22

<223> primer 11-R

acaggtcgtg aacaaaggca 20

<210> 23

<223> primer 12-F

tggacacgct tttcaccact 20

<210> 24

<223> primer 12-R

gacagaaggg gcttgcagta 20

<210> 25

<223> primer 13-F

cctgggacaa tgtggagtgg 20

<210> 26

<223> primer 13-R

cacccgctgg atactgttct 20

<210> 27

<223> primer 14-F

tcctcagcca cctaccgaat 20

<210> 28

<223> primer 14-R

cacaggtcgt gaacaaaggc 20

<210> 29

<223> primer 15-F

atcctcagcc acctaccgaa 20

<210> 30

<223> primer 15-R

ccttgggcac tgggtaactc 20

<210> 31

<223> primer 16-F

tgggacaatg tggagtggtc 20

<210> 32

<223> primer 16-R

acccgctgga tactgttctc 20

<210> 33

<223> primer 17-F

acaagaggaa ctggacacgc 20

<210> 34

<223> primer 17-R

gggcttgcag tatttgcact 20

<210> 35

<223> primer 18-F

caccactgct ggactggaaa 20

<210> 36

<223> primer 18-R

aaggggcttg cagtatttgc 20

<210> 37

<223> primer 19-F

gcagaaggct atcaacaggc 20

<210> 38

<223> primer 19-R

cagcagtggt gaaaagcgtg 20

<210> 39

<223> primer 20-F

gacacgcttt tcaccactgc 20

<210> 40

<223> primer 20-R

gacagaaggg gcttgcagta t 21

<210> 41

<223> primer F

agaaggctgg ggctcatttg 20

<210> 42

<223> primer R

aggggccatc cacagtcttc 20

<210> 43

<223> Forward primer

tacaaaatac gtgacgtaga aa 22

<210> 44

<223> reverse primer

tttgtttttg taattcttta 20

<210> 45

<223> Barcode-1-F

tgaccaaatg gactatcata tgcttaccgt 30

<210> 46

<223> Barcode-1-R

tgaccaacta ttctttcccc tgcactgt 28

<210> 47

<223> Barcode-2-F

gccaataatg gactatcata tgcttaccgt 30

<210> 48

<223> Barcode-2-R

gccaatacta ttctttcccc tgcactgt 28

<210> 49

<223> Barcode-3-F

cttgtaaatg gactatcata tgcttaccgt 30

<210> 50

<223> Barcode-3-R

cttgtaacta ttctttcccc tgcactgt 28

<210> 51

<223> Barcode-4-F

acagtgaatg gactatcata tgcttaccgt 30

<210> 52

<223> Barcode-4-R

acagtgacta ttctttcccc tgcactgt 28

<210> 53

<223> Barcode-5-F

cgatgtaatg gactatcata tgcttaccgt 30

<210> 54

<223> Barcode-5-R

cgatgtacta ttctttcccc tgcactgt 28

<210> 55

<223> Barcode-6-F

cagatcaatg gactatcata tgcttaccgt 30

<210> 56

<223> Barcode-6-R

cagatcacta ttctttcccc tgcactgt 28

<210> 57

<223> targeting sequence for shNC

gcgacgatct gcctaagat 19

<210> 58

<223> targeting sequence of shMETTL2-1

gtccagacaa attcagaata t 21

<210> 59

<223> shZNF365 targeting sequence

ctacgaagaa agaaccctct t 21

<210> 60

<223> targeting sequence of shCEP68

gcagctttac cggcaattta a 21

<210> 61

<223> targeting sequence of shIFT122

ctactttact aaaggcgagt a 21

<210> 62

<223> shBEST2 targeting sequence

ctctttcact atgactggat t 21

<210> 63

<223> target sequence of shNC

gcgacgatct gcctaagat 19

<210> 64

<223> target sequence of shMETTL2-1

gtccagacaa attcagaata t 21

<210> 65

<223> target sequence of shMETTL2-2

gtcagtgtct atctggaaat t 21

Claims

1. Application of a reagent for quantitatively detecting the protein expression level of METTL2 gene in preparation of a kit for detecting the treatment sensitivity of colorectal cancer fluorouracil.

2. The application of the reagent for quantitatively detecting the protein expression level of the METTL2 gene in the preparation of the kit for detecting the fluorouracil treatment sensitivity of colorectal cancer according to claim 1 is characterized in that: the reagent for quantitatively detecting the protein expression level of METTL2 is a WESTERN BLOT detection reagent or an immunohistochemical staining detection reagent.

3. The application of the reagent for quantitatively detecting the protein expression level of the METTL2 gene in the preparation of the kit for detecting the fluorouracil treatment sensitivity of colorectal cancer according to claim 2 is characterized in that:

the WESTERN BLOT detection reagent comprises an anti-METTL 2 antibody, an anti-reference protein antibody, a universal secondary antibody and a color development and luminescence reagent;

the immunohistochemical staining detection reagent comprises an anti-METTL 2 antibody, a universal secondary antibody and a color reagent.