CN106834482B - Primer, kit, detection method and application for detecting miR-874-3p expression - Google Patents
Primer, kit, detection method and application for detecting miR-874-3p expression Download PDFInfo
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Abstract
The invention discloses a primer, a kit, a detection method and application for detecting miR-874-3p expression, and belongs to the technical field of tumor biotherapy. Experiments prove that miR-874-3p is related to the drug resistance of vincristine, and has high expression in vincristine-resistant cells and low expression in sensitive cells, so that the miR-874-3p can be used as a vincristine-resistant marker of tumor cells. The primer, the kit and the detection method designed according to the marker can be used for clinically judging the drug resistance of the tumor cells to vincristine, further guiding clinical medication and realizing accurate treatment.
Description
Technical Field
The invention relates to a primer for detecting miR-874-3p expression, a kit containing the primer, a detection method and application of miR-874-3p in preparation of a medicament for antagonizing vincristine-resistant tumor, and belongs to the technical field of tumor biotherapy.
Background
Colorectal cancer is one of the most common digestive tract malignancies, occurring in the colon, preferably at the junction of the rectum and the sigmoid colon. The incidence rate of colorectal cancer is highest in the age group of 40-50 years, and the ratio of the colorectal cancer to the colorectal cancer is 2-3: 1. The incidence of colorectal cancer is at the 3 rd position in developed western countries. In China, the incidence rate of colorectal cancer is at the 4 th position, and the incidence rate and the fatality rate are gradually increased year by year. Clinical data show that the colorectal cancer has high metastasis rate and low early diagnosis rate, and the colorectal cancer has high mortality rate due to easy recurrence after surgical resection.
Radical resection of colorectal cancer is the most effective treatment, and postoperative chemotherapy has important roles in prolonging patient survival and improving quality of life. Vincristine (VCR) is an anti-tumor chemotherapeutic drug widely applied at present and is used for clinical treatment of colon cancer, but long-term treatment can cause tumor cells to generate drug resistance, so that chemotherapy fails. The data show that 90% of cancer patient deaths are associated with tumor resistance.
Disclosure of Invention
The invention aims to provide a primer for detecting miR-874-3p expression.
Next, the present invention provides a kit comprising the above primer.
Furthermore, the invention provides a method for detecting miR-874-3p expression.
The invention further provides application of the primer and the kit.
Meanwhile, the invention provides application of the miR-874-3p in preparation of a drug resistant to tumor cells.
Finally, the present invention provides a pharmaceutical composition for the treatment of tumors.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the primer for detecting the expression of miR-874-3p is designed and synthesized according to the sequence (shown as SEQ ID NO: 1) of miR-874-3p, and comprises a reverse transcription primer and a fluorescent quantitative PCR primer. The reverse transcription primer is shown as SEQ ID NO 2, 3 or 4. The forward primer of the fluorescent quantitative PCR primer is shown as SEQ ID NO. 5 or 6, and the reverse primer is shown as SEQ ID NO. 7.
The tumor is colorectal cancer, and the drug resistant drug is vincristine.
The kit for detecting miR-874-3p expression comprises the primers, a general miRNA reverse transcription reagent (containing reverse transcriptase and dNTPmix) and a fluorescent quantitative polymerase chain reaction reagent (containing a fluorescent dye, Taq DNA polymerase and dNTPmix).
The kit can also comprise DNA digestion reagents (containing DNase I and RNase-free) and reverse transcription primers and fluorescent quantitative PCR primers of internal references. When the internal reference adopts U6 snRNA housekeeping gene, the reverse transcription primer of the internal reference is shown as SEQ ID NO. 10, and the forward primer and the reverse primer of the fluorescent quantitative PCR of the internal reference are respectively shown as SEQ ID NO. 8 and 9.
A method for detecting expression of miR-874-3p, comprising the steps of:
1) taking total RNA of cells as a template, and carrying out reverse transcription to obtain cDNA;
2) carrying out fluorescence quantitative PCR amplification by taking cDNA as a template, and detecting the expression quantity of miR-874-3 p;
the primers for reverse transcription in the step 1) comprise reverse transcription primers of miR-874-3p and reverse transcription primers of an internal reference, wherein the reverse transcription primers of miR-874-3p are shown as SEQ ID NO 2, 3 or 4;
the primers for the fluorescent quantitative PCR amplification in the step 2) comprise a fluorescent quantitative PCR primer of miR-874-3p and a fluorescent quantitative PCR primer of an internal reference, wherein a forward primer in the fluorescent quantitative PCR primer of miR-874-3p is shown as SEQ ID NO. 5 or 6, and a reverse primer is shown as SEQ ID NO. 7.
The reaction system of reverse transcription in the step 1) is as follows: 5 × RT buffer (250mM Tris-HCl (pH 8.3), 375mM KCl, 15mM MgCl250mM DTT) 4. mu.L, 200U/. mu.L reverse transcriptase 1. mu.L, 10. mu.M reverse transcription primer of miR-874-3p 0.5. mu.L, dNTP (2.5mM each) 2.5. mu.L, 10. mu.M internal reference reverse transcription primer 0.5. mu.L, 1.0. mu.g/. mu.L RNA 1. mu.L, no RNase water make up to 20. mu.L.
The reaction conditions of reverse transcription in the step 1) are as follows: inactivating reverse transcriptase at 16 deg.C for 30min, 42 deg.C for 30min, and 85 deg.C for 5 min; after the reaction is finished, the mixture is put on ice for standby or stored at the temperature of minus 20 ℃.
The reaction system of the fluorescent quantitative PCR amplification in the step 2) is as follows: dNTP (2.5mM each) 2.5. mu.L, 2 XSSYBR Green PCR Master Mix (available from ABI) 10. mu.L, 1.0. mu.g/. mu.L cDNA 1. mu.L, 10. mu.M miR-874-3p forward and reverse primers 0.5. mu.L each, 10. mu.M internal reference forward and reverse primers 0.5. mu.L each, double distilled water to make up to 20. mu.L.
The reaction conditions of the fluorescent quantitative PCR amplification in the step 2) are as follows: denaturation at 95 ℃ for 5min, 40 cycles (95 ℃ 15s, 60 ℃ 15s, 72 ℃ 20s), 95 ℃ 15s, 60 ℃ 30s, 95 ℃ 15 s.
Detecting the expression quantity of miR-874-3p in the step 2), taking U6 snRNA housekeeping gene as internal reference, and adopting a relative quantification method. The reverse transcription primer of the internal reference U6 snRNA is shown as SEQ ID NO. 10, and the forward primer and the reverse primer of the fluorescent quantitative PCR amplification are shown as SEQ ID NO. 8 and 9 respectively.
The primer and the kit are applied to judging the drug resistance of the tumor cells to vincristine.
The primer and the kit are applied to screening of drugs for antagonizing the drug resistance of tumor cells to vincristine.
Application of miR-874-3p in preparing medicine for antagonizing tumor cell drug resistance. The method specifically comprises the following steps: a miR-874-3p inhibitor is designed and synthesized by referring to a miR-874-3p sequence, and a medicament for antagonizing drug resistance of tumor cells is prepared by taking a therapeutically effective amount of the miR-874-3p inhibitor and pharmaceutic adjuvants as raw materials.
A pharmaceutical composition for treating tumors, comprising a therapeutically effective amount of a miR-874-3p inhibitor and a therapeutically effective amount of vincristine.
The invention has the beneficial effects that:
experiments prove that the microRNA molecule miR-874-3p is related to the drug resistance of vincristine, and miR-874-3p is highly expressed in vincristine drug-resistant cells and is lowly expressed in sensitive cells, so that the microRNA molecule miR-874-3p can be used as a vincristine drug-resistant marker of tumor cells. The primer, the kit and the detection method designed according to the marker can be used for clinically judging the drug resistance of the tumor cells to vincristine, further guiding clinical medication and realizing accurate treatment, improving the curative effect and reducing the side effect. The invention provides a new target for designing the vincristine drug resistance drug of the tumor cell, and can be used for directly developing the drug for antagonizing the vincristine drug resistance of the tumor cell. And because the miR-874-3p inhibitor has the biological function of reducing drug resistance of the tumor cells, the cancer chemotherapy effect can be improved by combining the inhibitor with vincristine with a therapeutically effective amount, and an effective way is provided for effectively reversing the drug resistance of the vincristine of the tumor cells and improving the clinical chemotherapy effect of the tumor cells.
Drawings
FIG. 1 shows cell viability under VCR after primary digestion of colon cancer cells;
FIG. 2 is a graph of the relative survival rates of colon cancer cells HCT-8 and HCT-8/VCR at various VCR concentrations;
FIG. 3 is a graph showing the expression levels of hsa-miR-874-3p in HCT-8, HCT-8/VCR cells;
FIG. 4 shows the resistance of HCT-8 cells to vincristine after transfection of hsa-miR-874-3p mimetics (minics);
FIG. 5 shows the resistance of HCT-8/VCR cells transfected with a has-miR-874-3p inhibitor (inhibitor) to vincristine.
Detailed Description
The following examples are intended to illustrate the invention in further detail, but are not to be construed as limiting the invention in any way.
Example 1
The primer for detecting miR-874-3p expression is designed and synthesized according to miR-874-3p sequence (shown as SEQ ID NO: 1), and comprises a reverse transcription primer and a fluorescent quantitative PCR primer, wherein the reverse transcription primer is shown as SEQ ID NO:2, 3 or 4, a forward primer in the fluorescent quantitative PCR primer is shown as SEQ ID NO:5 or 6, and a reverse primer is shown as SEQ ID NO: 7.
Example 2
A kit for detecting miR-874-3p expression comprises the primers in example 1, a reverse transcription primer (shown as SEQ ID NO: 10) of internal reference U6 snRNA, a fluorescent quantitative PCR primer (shown as SEQ ID NO:8 and 9) and universal DNA digestion reagents (DNase I (RNase-free), EDPC and the like), miRNA reverse transcription reagents (reverse transcriptase, 5 xRT buffer, dNTP and the like) and a fluorescent quantitative polymerase chain reaction reagent (dNTP, 2 xSYBR Green PCR Master Mix and the like).
Example 3
A method for detecting expression of miR-874-3p, comprising the steps of:
1) primer design
Designing a primer according to a mature sequence (No: MIMAT0004911) of hsa-miR-874-3p in a miRBase database (http:// www.mirbase.org);
hsa-miR-874-3p：5′-CUGCCCUGGCCCGAGGGACCGA-3′；
reverse transcription primer:
reverse transcription primer 1: 5'-CTCAACTGGTGTCGTGGAGTCGGCAATTCTCGGTCCC-3', respectively;
reverse transcription primer 2: 5'-CTCAACTGGTGTCGTGGAGTCGGCAATTCTCGGTCC-3', respectively;
reverse transcription primer 3: 5'-CTCAACTGGTGTCGTGGAGTCGGCAATTCTCGGTC-3', respectively;
fluorescent quantitative PCR primers:
forward primer 1: 5'-ACACTCCAGCTGGGCTGCCCTGGCCCGAGGG-3', respectively;
forward primer 2: 5'-ACACTCCAGCTGGGCTGCCCTGGCCCGAGG-3', respectively;
reverse primer: 5'-CTCAACTGGTGTCGTGGAGT-3', respectively;
the following primers were designed based on the U6 snRNA housekeeping gene (Genbank: NR-004394.1) in the NCBI database (https:// www.ncbi.nlm.nih.gov /):
reverse transcription primer for U6 snRNA: 5'-CGCTTCACGAATTTGCGTGTCAT-3', respectively;
fluorescent quantitative PCR primers for U6 snRNA:
u6 forward primer: 5'-CTCGCTTCGGCAGCACA-3', respectively;
u6 reverse primer: 5'-AACGCTTCACGAATTTGCGT-3', respectively;
2) preparation of colon cancer tissue cells
Selecting colon cancer tissues of 20 colon cancer patients collected in a certain hospital in the New countryside, determining the pathological typing without any chemotherapy; the selected colon cancer tissue should avoid fibrous connective tissue or necrotic tumor tissue as much as possible and is immediately placed in liquid nitrogen; removing fibrous connective tissue or necrotic tumor tissue on a clean bench, washing the tissue with 10mL PBS for 3 times, removing supernatant, and cutting colon cancer tissue into 1.5mm pieces3Small block (1-2 mm)3All), lightly suspending the tissue mass with 10mL PBS, and removing the supernatant by aspiration; adding 10mL of 0.1% collagenase solution pre-warmed to 37 ℃, and digesting for 45min (30-60 min) at 37 ℃ until complete digestion; stopping digestion, filtering with a 100-mesh screen, centrifuging 500g of filtrate for 9min (8-10 min), and discarding the supernatant; gently blowing and beating with 10mL PBS, suspending and precipitating, centrifuging 500g of filtrate for 9min (8-10 min), and discarding supernatant; repeating the steps once; gently pumping with 2.5mL of RMPI-1640 culture medium (2-3 mL), suspending the precipitate, and adjusting the cell concentration to 5X 105～1×106/mL；
3) MTT agar method in vitro drug sensitivity test
5mL of 2 XRMPI-1640 culture medium pre-warmed to 37 ℃ and 5mL of 1.5% agar which is autoclaved, cooled to 50 ℃ and uniformly mixed are added into a 96-well plate (each well is 100 mu L) and are kept stand for 30min at room temperature for later use; adding vincristine with concentration of 150ng/mL into 100 μ L cell suspension, inoculating into 96-well plate, repeating three wells for each drug concentration, setting background control group (equal volume culture solution without cell), adding culture solution without VCR into blank control group; 37 ℃ and 5% CO2After 72 hours of culture, MTT measures cell activity; adding 20 mu L of 5mg/mL MTT solution into each well of a 96-well plate, and incubating for 4 hours at 37 ℃; adding 100 μ L PBS containing 10% SDS into each well, and incubating for 30min at 60 deg.C; measuring OD value at 560nm wavelength; calculating the average value of the background groups, taking the average value as a zero point to perform zero adjustment, and calculating the survival rate of each group of cells;
cell viability/% (mean (test group mean OD value-background group mean OD value)/(blank control group mean OD value-background group mean OD value) × 100%;
treating 20 cases of primary-cultured colon cancer cells with vincristine with the concentration of 150ng/mL, taking the survival rate of less than 40% as sensitivity and more than 70% as a judgment index of drug resistance, and performing no further analysis on 30-60%; the results showed that of 20 primary cultured colon cancer cells, 14 specimens were judged to be sensitive or resistant, 8 specimens were VCR resistant and 6 specimens were sensitive (see fig. 1);
4) extraction of RNA
Selecting 14 cases of the sensitive or drug-resistant colon cancer primary cells (5-10 multiplied by 10)6one/mL, see Table 1 below), centrifuging, and discarding the supernatant; adding 1mL of pre-cooled Trizol reagent into a pipette, and repeatedly blowing and beating the lysed cells until the cells are uniform and bright; placing the homogenate sample on ice for 5 minutes to ensure that the cells are fully cracked; adding 200 mu L of chloroform, violently whirling for 30 seconds, and standing for 5 minutes at room temperature; centrifuging at 12000 Xg for 15 min at 4 ℃; carefully transferring the supernatant into a 1.5mL RNase-free centrifuge tube, adding isopropanol with the same volume as the volume of the supernatant, and uniformly mixing the mixture; centrifuging at 12000 Xg for 15 min at 4 deg.C, and discarding the supernatant; adding 750 μ L75% ethanol, centrifuging at 4 deg.C and 12000 Xg for 5min, and removing supernatant; after ethanol air drying, adding 45 mu L DEPC treated water, standing at room temperature for 2 minutes to dissolve RNA; after the detection of the denaturation electrophoresis, the determination concentration is 1.8 mug/mug for standby (or preservation at minus 80 ℃);
the extraction process was operated on ice to prevent RNA degradation; the disposable gloves are worn in the whole process; adopting a non-disposable glass ware or a plastic ware without RNase, wherein the glass ware can be baked in an oven at 150 ℃ for 4 hours, the plastic ware can be soaked in 0.5M NaOH solution for 10 minutes, and the plastic ware is thoroughly rinsed with water and then sterilized under high pressure for later use;
5) DNase I treatment
Prepare 10 μ L DNase I treatment system: RNA (1.8. mu.g/. mu.L) 1. mu.g, 10 XBuffer (400mM Tris-HCl (pH7.5), 80mM MgCl250mM DTT)1 μ L, 70U/μ L (60-80U/μ L all) 1 μ L of DNase I (RNase-free), and 0.1% DEPC treated water to 10 μ L;
the treatment conditions were: water bath at 37 ℃ for 30min, and water bath at 65 ℃ for 10min to inactivate DNase I;
6) reverse transcription
Prepare 20 μ L reverse transcription reaction system: 5 × RT buffer (250mM Tris-HCl (pH 8.3), 375mM KCl, 15mM MgCl250mM DTT) 4. mu.L, 200U/. mu.L reverse transcriptase 1. mu.L, 10. mu.L reverse transcription primer for miR-874-3p 10.5. mu.L, 10. mu. M U6 reverse transcription primer for snRNA 0.5. mu.L, dNTP (2.5mM each) 2.5. mu.L, RNA (1.0. mu.g/. mu.L) 1. mu.L, RNase-free water to make up to 20. mu.L;
the reaction conditions are as follows: inactivating reverse transcriptase at 16 deg.C for 30min, 42 deg.C for 30min, and 85 deg.C for 5 min; after the reaction is finished, putting the mixture on ice for standby or storing the mixture at the temperature of minus 20 ℃;
7)Real-time PCR
preparing 20 mu L of fluorescent quantitative PCR reaction system: 2 XSSYBR Green PCR Master Mix (from ABI) 10. mu.L, 1.0. mu.g/. mu.L cDNA 1. mu.L, 10. mu.M miR-874-3p forward primer 1 and reverse primer 0.5. mu.L each, dNTP (2.5mM each) 2.5. mu.L, 10. mu. M U6 snRNA forward and reverse primers 0.5. mu.L each, double distilled water to make up to 20. mu.L;
the reaction conditions are as follows: denaturation at 95 deg.C for 5min, 40 cycles (95 deg.C 15s, 60 deg.C 15s, 72 deg.C 20s), 95 deg.C 15s, 60 deg.C 30s, 95 deg.C 15 s;
8) statistical analysis of hsa-miR-874-3p expression quantity
Calculating the expression level F of the gene by a relative quantification method, wherein the formula is as follows:
F＝2—△△ct；
where Δ ct ═ the average of ct for the target gene in the test sample-the average of ct for the housekeeping gene in the test sample) - (the average of ct for the target gene in the control sample-the average of ct for the housekeeping gene in the control sample); selecting the expression level of the sample hsa-miR-874-3p sensitive to the VCR No. 2 as a control, setting the expression level as 1, and taking the ratio of other sample plates to the sample No. 2 as the expression level of hsa-miR-874-3 p;
the results show that the expression of hsa-miR-874-3p in VCR resistant cells is up-regulated, and the expression amount is obviously up-regulated compared with that of sensitive cell samples (see Table 1 below).
TABLE 1 drug resistance of Primary Colon cancer cells and relative expression levels of hsa-miR-874-3p
Example 4
The application of miR-874-3p in preparing a drug resistant to tumor cells comprises the following specific steps: an antisense oligonucleotide sequence of miR-874-3p is synthesized by a conventional method to be used as a miR-874-3p inhibitor, and a therapeutically effective amount of the miR-874-3p inhibitor and pharmaceutic adjuvants are used as raw materials to prepare tablets.
Example 5
The pharmaceutical composition for treating the tumor comprises vincristine with a therapeutically effective dose, a miR-874-3p inhibitor and pharmaceutic adjuvant, wherein the miR-874-3p inhibitor is synthesized by Shanghai Jiman biotechnology company.
Test example 1
Vincristine plays an important role in the treatment of colon cancer, but the appearance of drug resistance is the main reason limiting the clinical efficacy of vincristine. The biological function and mechanism of miR-874-3p in vincristine resistance of colon cancer cells are determined, and a new biological target can be provided for effectively improving the clinical curative effect of colon cancer.
1. Expression of miR-874-3p in vincristine-resistant cells and sensitive cells
1) Construction of vincristine drug-resistant cell line HCT-8/VCR
The colon cancer cell line HCT-8/VCR was established by stepwise increasing VCR concentrations: culturing the sensitive cell HCT-8 in VCR-containing culture medium at an initial concentration of 5ng/mL, and thereafter gradually increasing the drug concentration at 10ng/mL, 20ng/mL, 100ng/mL, 200ng/mL, 1000ng/mL, 2000ng/mL, 3000ng/mL and 4000ng/mL, respectively; after achieving resistance at each concentration, well-growing cells were cloned by limiting dilution, then placed into culture at the next concentration, and finally HCT-8 was cultured in 2000ng/mL VCR for more than 20 passages, with VCR being stopped 1 week prior to the experiment.
2) Drug sensitivity testing of cells to VCR (MTT method)
Respectively taking the cells of the drug-resistant strain and the sensitive strain in the logarithmic growth phase, and counting the cell density by a counting plate to be 1 multiplied by 10 through a culture medium dilution method5Per mL; at 1 × 104A96-well cell culture plate is inoculated in each well, a background control group (equal volume of culture solution without cells) is set, culture solution without VCR is added in a blank control group, culture solution containing vincristine with 10ng/mL, 20ng/mL, 100ng/mL, 200ng/mL, 1000ng/mL, 2000ng/mL, 3000ng/mL and 4000ng/mL is respectively added in an experimental group, and 100 mu l/well is formed.
Incubating and culturing the cells of each group at 37 ℃ for 48 hours, adding 20 mu L of 5mg/mL MTT solution into each hole, and continuously placing the cells into a 37 ℃ cell culture box for incubation for 4 hours; then, the culture supernatant was aspirated off by a pipette, 150. mu.L of DMSO was added to each well, and after sufficient shaking, the plate was placed in an enzyme-linked immunosorbent assay apparatus, the detection wavelength was set at 570nm, and the absorbance value (OD) of each well was read.
The background group mean was calculated and zero-adjusted at zero, the viability of the cells in each group was calculated, and then the drug concentration at 50% cell survival, i.e., the median inhibitory concentration IC50, was calculated.
Cell viability/% (mean of OD value in test group-mean of OD value in background group)/(mean of OD value in blank control group-mean of OD value in background group) × 100%.
Different concentrations of VCR were used to treat colon cancer cells HCT-8 and HCT-8/VCR, and the inhibition rates are shown in FIG. 2. The half inhibitory concentrations IC50 for HCT-8 cells and HCT-8/VCR cells were 140.265 and 2350.469, respectively.
3) Detecting the expression quantity of hsa-miR-874-3p in HCT-8 and HCT-8/VCR cells
The same procedure as in steps 2) to 6) of example 3 was conducted;
the results show that, compared to HCT-8 cells, hsa-miR-874-3P is upregulated in HCT-8/VCR cells at 4.563-fold greater expression than HCT-8 cells, with statistical differences (P <0.01), as shown in FIG. 3.
2. Cell proliferation-toxicity assay to detect drug resistance of HCT-8 cells transfected with hsa-miR-874-3p mimetics
Inoculating the HCT-8 cell suspension with active growth into a 12-well plate one day before transfection; the next day, hsa-miR-874-3p mimetics (25nM) were transfected, and after 8 hours the cells were digested and plated into 96-well plates; vincristine was added (final concentrations 10ng/mL, 20ng/mL, 100ng/mL, 200ng/mL, 1000ng/mL and 2000ng/mL, respectively); after 48 hours, the 96-well plate was removed and 10 μ L of CCK8 solution was added to each well (taking care not to generate bubbles in the wells in order not to affect the OD readings); the plates were incubated in an incubator for 3 hours and absorbance at 450nm was measured using a microplate reader. If the OD value is not measured temporarily, 10. mu.L of a 1% (w/v) SDS solution may be added to each well and stored at room temperature in the dark, and the absorbance does not change when measured within 24 hours.
The results show that the resistance of HCT-8 cells transfected with the hsa-miR-874-3p mimic to vincristine is significantly higher than that of the untransfected group (see FIG. 4), which indicates that the hsa-miR-874-3p can improve the resistance of colon cancer cells to vincristine.
3. Cell proliferation-toxicity test for detecting drug resistance of HCT-8/VCR cells transfected with hsa-miR-874-3p inhibitor
The HCT-8/VCR cell suspension with active growth is inoculated into a 12-well plate one day before transfection; the next day, hsa-miR-874-3p inhibitor (25nM) was transfected, cells were digested after 8 hours and plated into 96-well plates; vincristine was added (final concentrations of 100ng/mL, 500ng/mL, 1000ng/mL, 2000ng/mL, 3000ng/mL and 4000ng/mL, respectively); after 48 hours the 96-well plate was removed and 10. mu.L of CCK8 solution was added to each well (taking care not to generate bubbles in the wells in order not to affect the OD readings); the plates were incubated in an incubator for 3 hours and absorbance at 450nm was measured using a microplate reader. If the OD value is not measured temporarily, 10. mu.L of a 1% (w/v) SDS solution may be added to each well and stored at room temperature in the dark, and the absorbance does not change when measured within 24 hours.
The results show that the resistance of HCT-8/VCR cells transfected with the hsa-miR-874-3p inhibitor to vincristine is remarkably reduced relative to untransfected cells in a control group (see figure 5), which indicates that the hsa-miR-874-3p inhibitor can reverse the resistance of colon cancer cells to vincristine to a certain extent, and further proves that the hsa-miR-874-3p is closely related to the resistance of colon cancer cells to vincristine.
Test example 2
60 colon cancer cases in 2013-2015 of a certain hospital are selected, miRNA in a paraffin specimen is extracted, and the expression level of miR-874-3p is detected by the method in the embodiment 3. The result shows that the method can specifically and effectively amplify the miR-874-3p sequence; the statistical analysis result shows that the expression quantity of miR-874-3P in 34 cases of specimens is 11.218 +/-2.132 averagely, and the expression quantity of miR-874-3P in the other 26 cases of specimens is 2.412 +/-2.212 averagely, and the difference of the expression quantity is 4.65 times (P < 0.01). By combining with the analysis of clinical treatment effect, the effect of treating colon cancer by using vincristine in the sample with high miR-874-3p expression amount is poor, while the effect of treating colon cancer by using vincristine in the sample with low expression amount is good, which further proves that the expression of miR-874-3p is related to the drug resistance of colon cancer to vincristine.
The cell lines, reagents and the like used in the above examples and test examples are commercially available. HCT-8 cells were purchased from Shanghai cell Bank, Chinese academy of sciences.
The kit and the detection method provided by the invention can be used for clinically judging the drug resistance of colon cancer to vincristine, further guiding clinical medication and realizing accurate treatment, improving the curative effect and reducing the side effect.
It should be noted that the examples listed in the specification are only for understanding the technical solution of the invention, and do not have any limiting effect. In addition to the above embodiments, other embodiments are possible. Any technical solutions formed by equivalent substitutions or equivalent transformations fall within the protection scope of the claims of the present invention.
Sequence listing
SEQUENCE LISTING
<110> New countryside medical college
<120> primer, kit, detection method and application for detecting miR-874-3p expression
<170> PatentIn version 3.5
<211> 22
<212> RNA
<213> sequence
<221> hsa-miR-874-3p sequence
<222> (1)..(22)
<400> 1
CUGCCCUGGC CCGAGGGACC GA 22
<211> 37
<212> DNA
<213> sequence
<221> reverse transcription primer 1
<222> (1)..(37)
<400> 2
CTCAACTGGT GTCGTGGAGT CGGCAATTCT CGGTCCC 37
<211> 36
<212> DNA
<213> sequence
<221> reverse transcription primer 2
<222> (1)..(36)
<400> 3
CTCAACTGGT GTCGTGGAGT CGGCAATTCT CGGTCC 36
<211> 35
<212> DNA
<213> sequence
<221> reverse transcription primer 3
<222> (1)..(35)
<400> 4
CTCAACTGGT GTCGTGGAGT CGGCAATTCT CGGTC 35
<211> 31
<212> DNA
<213> sequence
<221> Forward primer 1 for fluorescent quantitative PCR
<222> (1)..(31)
<400> 5
ACACTCCAGC TGGGCTGCCC TGGCCCGAGG G 31
<211> 30
<212> DNA
<213> sequence
<221> Forward primer 2 for fluorescent quantitative PCR
<222> (1)..(30)
<400> 6
<211> 20
<212> DNA
<213> sequence
<221> reverse primer for fluorescent quantitative PCR
<222> (1)..(20)
<400> 7
<211> 17
<212> DNA
<213> sequence
<221> Forward primer of internal reference U6 snRNA
<222> (1)..(17)
<400> 8
<211> 20
<212> DNA
<213> sequence
<221> reverse primer of internal reference U6 snRNA
<222> (1)..(20)
<400> 9
<211> 23
<212> DNA
<213> sequence
<221> reverse transcription primer of internal reference U6 snRNA
<222> (1)..(23)
<400> 10
CGCTTCACGA ATTTGCGTGT CAT 23
Claims (3)
1. The application of the primer or the kit for detecting miR-874-3p expression in judging the drug resistance of colon cancer cells to vincristine is characterized in that: the kit comprises the primers, wherein the primers comprise a reverse transcription primer and a fluorescent quantitative PCR primer, the reverse transcription primer is shown as SEQ ID NO. 2, 3 or 4, a forward primer in the fluorescent quantitative PCR primer is shown as SEQ ID NO. 5 or 6, and a reverse primer is shown as SEQ ID NO. 7; the use is for non-diagnostic therapeutic purposes.
The application of the miR-874-3p inhibitor in preparing the drug resisting the colon cancer cells to vincristine is characterized in that: the miR-874-3p inhibitor is an antisense oligonucleotide sequence of miR-874-3 p.
3. A pharmaceutical composition for treating tumors, characterized by: comprises a therapeutically effective amount of miR-874-3p inhibitor and a therapeutically effective amount of vincristine, wherein the miR-874-3p inhibitor is an antisense oligonucleotide sequence of miR-874-3 p.
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| WO2012145743A1 (en) * | 2011-04-22 | 2012-10-26 | University Of Houston | Microrna-140-5p as a tumor suppressor and sensitizing agent for chemotherapy |
| CN102973953A (en) * | 2012-12-04 | 2013-03-20 | 中国科学院动物研究所 | MiRNA (micro ribose nucleic acid)-874 and application of miRNA-874antisense nucleotide |
| CN105412140A (en) * | 2015-12-18 | 2016-03-23 | 大连大学 | Application of micromolecule RNA hsa-miR-874 to preparation of drug for curing spongioblastoma |
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| WO2012145743A1 (en) * | 2011-04-22 | 2012-10-26 | University Of Houston | Microrna-140-5p as a tumor suppressor and sensitizing agent for chemotherapy |
| CN102973953A (en) * | 2012-12-04 | 2013-03-20 | 中国科学院动物研究所 | MiRNA (micro ribose nucleic acid)-874 and application of miRNA-874antisense nucleotide |
| CN105412140A (en) * | 2015-12-18 | 2016-03-23 | 大连大学 | Application of micromolecule RNA hsa-miR-874 to preparation of drug for curing spongioblastoma |
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Effective date of registration: 20230522 Address after: 410000 6th floor, building 2, incubation building, 98 XIONGTIAN Road, Longping hi tech park, Furong district, Changsha City, Hunan Province (cluster registration) Patentee after: Changsha Pingjun Intellectual Property Service Co.,Ltd. Address before: 453003 East Section of Jinsui Avenue, Xinxiang City, Henan Province Patentee before: XINXIANG MEDICAL University Effective date of registration: 20230522 Address after: 10th Floor, Zhizhao Building, No. 5255 Beisong Road, Chedun Town, Songjiang District, Shanghai, 200000 Patentee after: Youlian Ruikang (Shanghai) Gene Technology Co.,Ltd. Address before: 410000 6th floor, building 2, incubation building, 98 XIONGTIAN Road, Longping hi tech park, Furong district, Changsha City, Hunan Province (cluster registration) Patentee before: Changsha Pingjun Intellectual Property Service Co.,Ltd. |