Application of miR-196b-5p as colorectal cancer diagnosis or treatment marker

Abstract

The invention discloses application of microRNA (Ribonucleic Acid) as a diagnostic marker in an advanced colorectal cancer drug resistance diagnostic kit and application of microRNA in preparation of drugs for treating advanced colorectal cancer. The invention discovers that miR-196b-5p is highly expressed in a cetuximab-sensitive intestinal cancer cell line, and the expression quantity of miR-196b-5p in a drug-resistant intestinal cancer cell line is generally low. The miR-196b-5p is subjected to an overexpression or knockout experiment, which proves that the miR-196b-5p can effectively influence the sensitivity of intestinal cancer cells to cetuximab. The miR-196b-5p is expected to become a novel biomarker for early prediction and screening of drug resistance of colorectal cancer patients using cetuximab, and assists in dynamic monitoring of curative effect and overcoming of drug resistance.

Description

Technical Field

[0001] The invention discloses a microRNA and belongs to the technical field of nucleic acid. Background Art

[0002] Colorectal cancer (CRC) is one of the most common malignant tumors, with high morbidity and mortality. The prevention and treatment of CRC has always been a research hotspot in the medical field. With the continuous development of medical technology, its treatment-related research has made great progress. Due to the promotion of individualized treatment in recent years, the research on precision therapeutic drugs (such as targeted therapy, immunotherapy, etc.) in the field of tumor treatment has become the main direction. Cetuximab (trade name: ERBITUX, Merck Kleon Pharmaceuticals, Germany) is a major breakthrough in the treatment of patients with advanced colorectal cancer (metastatic colorectal cancer, mCRC). Its target is the epidermal growth factor receptor (EGFR), which can inhibit tumor development by blocking the downstream signaling pathway of EGFR. Its clinical application has significantly improved the prognosis of mCRC patients and has become the standard treatment for stage IV RAS / RAF wild-type mCRC patients. Despite the existence of such powerful new treatment methods, the five-year survival rate of mCRC is still as low as about 15%, one of the important reasons for which is the existence of primary or secondary drug resistance. Therefore, exploring the resistance mechanism of cetuximab and finding solutions are of key significance for improving the prognosis of mCRC patients and achieving individualized and precise treatment. Currently, many studies have confirmed that changes in the expression or mutation of molecules such as EGFR, KRAS, NRAS, BRAF, HER2 / HER3, MAPK, PIK3CA, and PRSS1 can lead to resistance to cetuximab. These studies are all focused on the genetic level, but there are relatively few studies on resistance at the non-genetic level. About 30% of cetuximab resistance is due to unknown, obvious non-genetic resistance mechanisms.

[0003] In the past decade, the progress of high-throughput transcriptome sequencing technology (RNA sequencing, RNA-Seq) technology and bioinformatics methods has gradually led researchers to discover that non-coding RNA (ncRNA) plays an important role in the occurrence of EGFR monoclonal antibody resistance. MicroRNA (miRNA) is a component of ncRNA, which mainly regulates the expression of target genes by participating in post-transcriptional regulation of genes. When it is fully complementary to its target gene messenger RNA (mRNA), it can cut mRNA and degrade it, while when it is not fully complementary, it will inhibit translation and the gene will be silent. MiRNA post-transcriptional regulation is mostly to downregulate the expression of protein-coding genes. A single miRNA can regulate multiple target mRNAs at the same time. Abnormal expression of miRNA is common in tumors, and these abnormal expressions of miRNA are closely related to tumor progression and drug resistance. The purpose of the present invention is to provide a microRNA as a diagnostic or therapeutic marker for advanced colorectal cancer. Summary of the invention

[0004] Based on the above purpose, the present invention first provides a microRNA, the sequence of which is shown in SEQ ID NO. 1. In the present invention, the microRNA is named "miR-196b-5p".

[0005] The present invention has found that miR-196b-5p is highly expressed in cetuximab-sensitive colorectal cancer cell lines, but is lowly expressed in cetuximab-resistant cell lines. Moreover, patients with low serum miR-196b-5p copies have significantly shorter progression-free survival (PFS) than patients with high serum miR-196b-5p. Moreover, patients with high levels of miR-196b-5p are more likely to have a partial improvement (PR) or complete improvement (CR) in the first efficacy evaluation of cetuximab, while patients with low levels of miR-196b-5p are more likely to have a progressive disease (PD) in the first efficacy evaluation of cetuximab. During long-term cetuximab treatment, when most patients develop PD, the level of miR-196b in the blood shows a relatively obvious downward trend. During the efficacy evaluation process, when the level of miR-196b in the blood decreases significantly, it indicates that the patient's disease may progress greatly.

[0006] Secondly, based on the above findings, the present invention provides the above-mentioned microRNA, i.e., the use of miR-196b-5p as a diagnostic marker in a diagnostic kit for advanced colorectal cancer drug resistance, wherein the kit contains a reagent for detecting the content or expression level of the microRNA in the sample to be tested.

[0007] A person skilled in the art can detect miR-196b-5p in the body fluids or solid tissue specimens of colorectal cancer patients to predict or combine with other technical means to assist in prediction, screen or combine with other technical means to assist in screening whether the colorectal tumor suffered by the patient is a cetuximab-resistant tumor; to provide guidance for clinical treatment strategies, and during the treatment process, by detecting the patient's miR-196b-5p level, to dynamically monitor the efficacy of cetuximab in colorectal tumor patients, evaluate the patient's treatment status, for example, complete improvement, partial improvement or progressive condition, and adjust the clinical treatment strategy in time.

[0008] In a specific embodiment of the present invention, the above-mentioned body fluid or solid tissue specimens of colorectal cancer patients include patient plasma, lymph fluid, puncture fluid, tissue biopsy specimens, and in situ detection.

[0009] In a preferred embodiment, the advanced colorectal cancer drug resistance diagnostic kit is a gene detection kit.

[0010] In a more preferred embodiment, the gene detection kit is a PCR detection kit, and the sequences of the upstream and downstream primers of the PCR are shown in SEQ ID NO. 2 and 3, respectively. In a specific embodiment of the present invention, the PCR is qRT-PCR. The technical solution provided by the present invention identifies colorectal cancer cell lines that are sensitive or resistant to cetuximab after detecting the expression of miR-196b-5p in 8 colorectal cancer cell lines. Therefore, miR-196b-5p can be used to screen whether the colorectal tumor suffered by the patient is a cetuximab-resistant tumor.

[0011] In another preferred technical solution of the present invention, the gene detection kit is a droplet digital PCR, and the kit further contains a probe, the sequence of the probe is shown in SEQ ID NO.4.

[0012] In the detection of plasma RNA of mCRC patients using cetuximab in the present invention, it was found that the progression-free survival (PFS) of patients with low serum miR-196b-5p was significantly shorter than that of patients with high serum miR-196b-5p.

[0013] Patients with high miR-196b-5p levels were more likely to have partial remission / complete remission as the first efficacy evaluation, while patients with low miR-196b-5p levels were more likely to have PD as the first efficacy evaluation. Therefore, miR-196b-5p can be used to predict the first efficacy of cetuximab in CRC patients.

[0014] During long-term cetuximab treatment, when most patients developed PD, the level of miR-196b in the blood showed a relatively obvious downward trend. That is, during the efficacy evaluation process, a decrease of more than 26.8% in the level of miR-196b in the blood compared with the baseline indicated that the patient's disease may progress greatly. Therefore, miR-196b can be used to dynamically monitor the efficacy of cetuximab in CRC patients.

[0015] Third, the present invention provides the use of the above-mentioned microRNA in the preparation of a drug for treating advanced colorectal cancer.

[0016] In the research of the present invention, it was found that the cell proliferation activity of the colon cancer cell LIM1215 cells overexpressing miR-196b-5p was reduced, the cell migration ability was reduced, and the cell apoptosis ability was increased in the presence of cetuximab. Therefore, miR-196b-5p in the body can be used as a target and its expression level can be regulated to achieve the purpose of colorectal cancer treatment. According to the understanding of those skilled in the art, any technical means that can regulate the expression of miR-196b-5p, especially to increase the expression of miR-196b-5p, can become the application of the present invention.

[0017] In a preferred embodiment of the present invention, the drug is prepared as a vector containing microRNA.

[0018] In a more preferred embodiment, the vector is a lentiviral vector.

[0019] In a specific embodiment of the present invention, the target sequence of the lentiviral vector is shown as SEQ ID NO.7.

[0020] In a specific embodiment of the present invention, the template sequence of the lentiviral vector is shown as SEQ ID NO.8.

[0021] Finally, the present invention provides a composition comprising a vector expressing the above-mentioned microRNA, ie, miR-196b-5p.

[0022] Based on the findings of the present invention, overexpressed miR-196b-5p helps to reduce the proliferation and migration activity of intestinal cancer cells and enhance their apoptosis. This is also the conventional technical effect pursued in tumor cell therapy. Therefore, any combination of the overexpressed miR-196b-5p proposed by the present invention and other tumor treatment techniques, such as chemotherapy, radiotherapy, and immunological treatment methods, can become a component of the composition of the present invention.

[0023] In a preferred embodiment, the composition also contains cetuximab. The research of the present invention shows that miR-196b-5p is overexpressed or inhibited, which proves that it can effectively rescue cetuximab acquired resistance and has a synergistic sensitization effect on cetuximab; overexpression of miR-196b-5p combined with cetuximab treatment can inhibit CRC cell proliferation, inhibit CRC cell migration, and promote CRC cell apoptosis. Therefore, the present invention provides a vector expressing miR-196b-5p and cetuximab, and the composition can be used to prepare colorectal cancer drugs.

[0024] In summary, miR-196b-5p discovered in the present invention is expected to become a new biomarker for early screening and prediction of cetuximab resistance in CRC and a new target for rescue treatment after resistance occurs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The differential miRNA heat map of liver metastatic tissue specimens from 2 mCRC patients before and after cetuximab treatment resistance; Figure 2 The figure shows the results of the sensitivity test of 8 colorectal cancer cell lines to cetuximab; Figure 3 The results of the expression of miR-196b-5p in different colorectal cancer cell lines are shown in Figure 2. Figure 4 The result graph is a comparison of the PFS curves of the high absolute quantitative group of baseline plasma miR-196b-5p and the low absolute quantitative group of baseline miR-196b-5p in CRC patients; Figure 5 The results of the relationship between the baseline miR-196b-5p level in CRC patients and the first efficacy of cetuximab (***P<0.001, **P<0.01, *P<0.05); Figure 6 The result graph of the receiver operating characteristic (ROC) curve for the prediction of the baseline miR-196b-5p level for the first partial response (PR) of cetuximab in CRC patients; Figure 7 The ROC curve results of the baseline miR-196b-5p level predicting the first partial response (Progressive disease, PD) of CRC patients using cetuximab; Figure 8 This is a result diagram of using miR-196b-5p to dynamically monitor the efficacy of cetuximab in mCRC patients; Fig. 9 The result diagram of the ROC curve of PD for predicting the efficacy of treatment during the course of treatment is shown in the decrease of miR-196b-5p compared with the baseline; Fig.10 Plasmid maps for silencing lentivirus and overexpression lentivirus; Fig.11 This is the expression detection result diagram after overexpression or knockdown of miR-196b-5p; Fig.12 High expression of miR-196b-5p significantly enhanced the sensitivity of tumor cells to cetuximab, while low expression of miR-196b-5p significantly reduced the sensitivity of tumor cells to cetuximab; Fig.13 This is the result diagram showing that high expression of miR-196b-5p significantly inhibits the proliferation of tumor cells in the presence of cetuximab; Fig.14 This is a result diagram showing that knocking down miR-196b-5p significantly accelerated the proliferation of tumor cells in the presence of cetuximab; Fig.15 This is the result diagram showing that high expression of miR-196b-5p significantly slows down the migration of tumor cells; Fig.16 This is the result diagram showing that low expression of miR-196b-5p significantly accelerates the migration of tumor cells; Fig.17 In the presence of cetuximab, high expression of miR-196b-5p significantly promoted the apoptosis of tumor cells; Fig.18 In the presence of cetuximab, low expression of miR-196b-5p significantly inhibited the apoptosis of tumor cells. DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0027] Example 1. Low expression of miR-196b-5p in liver metastatic tumor tissues of patients resistant to cetuximab Source of tissue specimens: This research group cooperated with the hospital's oncology department and selected 2 patients with colorectal cancer who were treated with cetuximab as research subjects. The patients were fully informed and signed informed consent before admission. Subsequently, liver metastatic tissue specimens of colon cancer patients before and after cetuximab resistance were collected for second-generation high-throughput sequencing.

[0028] Deep sequencing of the transcriptome of liver metastasis tissue specimens from colorectal cancer patients treated with cetuximab before and after drug resistance revealed that the level of miR-196b in liver metastasis tissue after drug resistance was significantly lower than that in liver metastasis tissue before treatment (Figure 1).

[0029] Example 2: miR-196b-5p is underexpressed in cetuximab-resistant cell lines 1. Determination of the sensitivity of eight colorectal cancer cell lines to cetuximab A total of 8 CRC cell lines were involved, namely: DiFi, LIM1215, Caco-2, LS174T, HT-29, COLO320DM, HCT-8, and LoVo, which were purchased from the ATCC cell bank in the United States.

[0030] 1. Spread 2800 cells in each well of a 96-well plate, with 3 sets of wells for each cell line, 10 concentration gradients, and test after 72 hours of cetuximab treatment.

[0031] 2. Perform the experiment according to the instructions of CellTiter-Glo® Luminescent Cell Viability Assay (Promega #G7571). CellTiter-Glo is a rapid cell viability assay based on ATP detection, a recognized high-sensitivity luminescent assay, and the gold standard for cell viability detection, which is widely used at home and abroad. Therefore, the present invention uses CellTiter-Glo to detect cell viability.

[0032] 1) Add a volume of CellTiter Glo® Reagent equal to the volume of cell culture medium present in each well. For a 96-well plate, add 100µl of reagent to 100µl of culture medium containing cells.

[0033] 2) Mix the contents on a shaker for 2 minutes to induce cell lysis.

[0034] 3) Allow the plate to stand at room temperature for 10 minutes to stabilize the luminescent signal.

[0035] 4) The measurement time for each well is 0.25–1 s, and the luminescence is recorded.

[0036] The results are as follows Figure 2 As shown, eight colorectal cancer cell lines have different sensitivities to cetuximab.

[0037] 2. Expression of miR-196b-5p in colorectal cancer cell lines with different sensitivities to cetuximab 1. Primer design Primers were designed based on the gene sequences of miR-196b-5p and U6 (as internal reference gene) for real-time fluorescence quantitative PCR reaction. The specific primer names and sequences are shown in Table 1: Table 1. Primer sequence information used in PCR reaction

[0038] 2. qRT-PCR reaction system and conditions Extract RNA from the cell line to be tested, reverse transcribe to obtain cDNA as a template, and perform qRT-PCR using the following system and procedure: According to the primer synthesis instructions, add an appropriate amount of RNase-free-water to prepare a primer solution with a concentration of 10 μM. Use TOYOBO's THUNDERBIRDTM SYBR qPCR Mix Without ROX kit and prepare the reaction system according to the instructions as follows (Table 2): Table 2. qRT-PCR reaction system

[0039] Add the prepared qRT-PCR reaction system shown in Table 2 to a 96-well PCR plate, add 18 μL of the qRT-PCR reaction system shown in Table 5 to each reaction well, then add 2 μL of cDNA to make a 20 μL reaction system, and place in a real-time fluorescence quantitative PCR instrument for reaction. The PCR reaction conditions are as follows (Table 3): Table 3. qRT-PCR reaction conditions

[0040] Three replicate wells were set for each sample, and U6 was used as the internal reference gene. After the qPCR reaction was completed, the average Ct value of the three replicate wells of the target gene in each sample was used as the final result. The relative expression of each target gene was calculated using the formula 2^-△Ct, where △Ct = Ct (target gene) - Ct (internal reference gene).

[0041] qRT-PCR was used to detect the expression of miR-196b-5p in eight colorectal cancer cell lines.

[0042] The results are as follows Figure 3 As shown, miR-196b-5p was highly expressed in cetuximab-sensitive cell lines, for example, DiFi and LIM1215, with relative expression levels of 1.0 and 0.85, respectively, and was lowly expressed in cetuximab-resistant cell lines, for example, Caco-2, LS174T, HT-29, COLO320DM, HCT-8, and LoVo, with relative expression levels of 0.2, 0.23, 0.05, 0.15, 0.15, and 031, respectively.

[0043] Example 3: ddPCR detection of plasma RNA Source of blood specimens in the following embodiments: This research group cooperated with the hospital oncology department to select all colorectal cancer patients admitted to the hospital for cetuximab treatment as research subjects. All patients were fully informed and signed informed consent before admission. Subsequently, blood specimens were collected from patients, and the blood specimens were separated into three parts: plasma, PBMC and RBC, and packaged into RNase-free EP tubes for frozen storage. While collecting the specimens, the patient's detailed medical records were recorded and retained, including age, gender, body mass index (BMI), smoking time, drinking history, number of metastatic organs before using cetuximab, whether there was liver metastasis, neutrophil to lymphocyte ratio (NLR), lactate dehydrogenase (LDH) level, tumor markers CA19-9, CEA, CA724 levels, etc., and telephone follow-up or outpatient medical records were followed up for patient PFS.

[0044] The sample selection criteria and grouping basis in the following embodiments are: Patient inclusion and exclusion criteria (1) Inclusion criteria: ①Age ≥ 18 years old; ② Patients with stage IV mCRC confirmed by clinical and pathological examinations; ③ During cetuximab treatment, imaging examination data are available for efficacy evaluation, and there are measurable target lesions that meet the RECIST criteria; ④RAS / RAF gene status is wild type (2) Exclusion criteria: ① The patient's general condition is poor and he cannot tolerate the treatment; ②Having received anti-EGFR treatment before the first blood specimen collection; ③The overall survival time after diagnosis is less than 6 weeks; ④Others who do not meet the inclusion criteria.

[0045] The baseline data of the patients are shown in Table 4 . Plasma samples of patients who met the inclusion criteria were used as experimental subjects for screening and detection of differentially expressed genes.

[0046] Table 4. Clinical data of patients

[0047] BMI, Body mass index; NLR, Neutrophil to lymphocyte ratio, neutrophil to lymphocyte ratio; LDH, Lactate dehydrogenase, lactate dehydrogenase.

[0048] Some of the experimental methods in the following examples are as follows: 1. Separation of plasma

[0049] 10 mL of fasting venous blood was collected from the patient before surgery and placed in a blood collection tube containing EDTA as an anticoagulant. The blood was separated within 2 hours after collection.

[0050] 1) Add 5 volumes of QIAzol Lysis Reagent to the sample (e.g., for 200 μl of sample, add 1 ml of QIAzol Lysis Reagent). Vortex to mix.

[0051] 2) Incubate at room temperature (15-25°C) for 5 min.

[0052] 3) Add an equal volume of chloroform (e.g., for 200 μl sample, add 200 μl chloroform). Shake vigorously for 15 seconds.

[0053] 4) Leave at room temperature for 2-3 minutes.

[0054] 5) Centrifuge at 12000g, 4°C for 15 min.

[0055] 6) Transfer the upper aqueous phase to a new collection tube (prepare your own). As quickly as possible, add 1.5 volumes of 100% ethanol (e.g., for 600 μl of aqueous phase, add 900 μl of ethanol) and mix by pipetting.

[0056] 7) Place 700 μl of the sample including the pellet and RNeasy MinElute spin column in a 2 ml collection tube. Close the lid and centrifuge at ≥8000g for 15 s at room temperature. Discard the waste liquid at the bottom.

[0057] 8) Repeat step 8 with the remaining liquid from step 7.

[0058] 9) Add 700 μl Buffer RWT to the RNeasy MinElute spin column. Close the lid and centrifuge at ≥8000g for 15 seconds. Discard the waste liquid at the bottom.

[0059] 10) Pipette 500 μl Buffer RPE onto the RNeasy MinElute spin column. Close the lid and centrifuge at ≥8000g for 15 seconds. Discard the waste liquid at the bottom.

[0060] 11) Add 500 μl of 80% ethanol to the RNeasy MinElute spin column. Close the lid and centrifuge at ≥8000 g for 2 min. Discard the waste liquid and collection tube at the bottom.

[0061] 12) Place the RNeasy MinElute spin column in a new 2 ml collection tube (provided in the kit). Open the cap of the spin column and centrifuge at top speed for 5 min to dry the membrane. Discard the waste liquid and collection tube at the bottom.

[0062] 13) Place the RNeasy MinElute spin column in a new 1.5 ml collection tube (provided in the kit). Add 14 μl of RNase-free water directly to the center of the spin column membrane. Gently close the lid and centrifuge at maximum speed for 1 min to elute the RNA.

[0063] 2. Reverse transcription of plasma total RNA

[0064] 1) Prepare reverse transcription mixture (Table 5) and keep on ice for later use; Table 5. Reverse transcription reaction system

[0065] 2) After preparing the reverse transcription mixture, add it to 8-tube strips, mix it by gently pipetting, cover it tightly, and centrifuge it briefly; 3) Place in a 96-well PCR instrument and set the reaction conditions as follows (Table 6): Table 6. Reverse transcription PCR conditions

[0066] 3. Droplet digital PCR (ddPCR) is a third-generation PCR technology. ddPCR can accurately quantify the copy number of specific nucleotides in plasma without internal reference genes. It has very high sensitivity and requires only a small amount of template. It is particularly suitable for trace and difficult-to-obtain samples. Since there is no suitable reference gene in plasma for standardization of miR-196b-5p expression levels in real-time quantitative PCR experiments, the present invention uses ddPCR to detect the expression of plasma miR-196b-5p.

[0067] (1) Primer design (primers are the same as SEQ ID NO. 2 or 3 in Example 1) Probes were designed based on the microRNA gene sequences for ddPCR reactions. The specific probe names and sequences are shown in Table 7: Table 7. Probe sequence information used in PCR reactions

[0068] (2) ddPCR experimental steps 1) Prepare the reaction system as follows (Table 8): Table 8. ddPCR reaction system

[0069] 2) Place a new DG8 droplet generator card into the card slot, paying attention to the direction of the notch; 3) Add 20 μL of sample reaction system to the 8 wells in the middle row of the DG8 droplet generator card. If there are less than 8 samples, add 20 μL of 1× control buffer diluted by one time. When adding samples, place the tip of the gun close to the bottom of one side of the well, at an angle of about 15° to the side wall, and slowly eject the liquid. After ejecting a part of the liquid, slowly raise the tip of the gun and eject the remaining liquid to avoid bubbles. 4) Add 70 μL of droplet generating oil (DG Oil) to each of the 8 wells in the bottom row of the DG8 droplet generating card; 5) Cover with rubber pad, making sure to hook the small holes on both sides firmly; 6) Gently and steadily place the above card slot in the droplet generator and start generating droplets for about 2-3 minutes; 7) Droplets are generated in the top row of wells on the droplet generation card. Place the card slot flat, insert the gun tip at an angle of 30-45° to the well wall, lightly touch the bottom of the well, carefully and slowly draw 40μL of the mixture, and then slowly inject it into the well at the corresponding position of the 96-well plate. Keep the gun tip close to the well wall and the bottom of the well. Be sure to seal the cover to prevent oil evaporation. Discard the used droplet generation card and rubber pad each time. 8) After the oil droplets are transferred into the 96-well plate, place the heat seal film with the red line (the reflective bright side) facing up on the 96-well plate and fix it. Use the preheated PX1 heat sealer to seal the film. The operating program is: 180°C, 10 seconds; 9) After sealing the membrane, the PCR reaction should be carried out within 30 minutes, or placed in a 4°C refrigerator for 4 hours and completed on a T100 96-well PCR instrument. Note that the heating and cooling speed should be ≤2.5°C / s; 10) PCR reaction conditions are shown in Table 9: Table 9. ddPCR amplification reaction conditions

[0070] 11) Place the 96-well plate that has completed the PCR reaction into the reading slot and assemble it. Pay attention to the angle of the plate. After assembly, gently and steadily place it into the droplet reader. 12) Open the QuantaSoft software on the computer connected to the droplet reader. If it has not been used for more than a week, run the Prime and then the Flush System program. Then set and run the sample information in the 96-well plate. Finally, you can directly read the absolute copy number of miRNA in each sample.

[0071] like Figure 4 The results showed that baseline miR-196b-5p levels were associated with PFS (progression-free survival: the period from the start of treatment to the observation of disease progression or death due to any cause) in mCRC patients using cetuximab. Taking the median of 31850 copies / mL as the boundary, the PFS of patients with serum miR-196b-5p lower than the median 31850 copies / mL was significantly shorter than that of patients with serum miR-196b-5p higher than 31850 copies / mL, with mPFS of 19.6 months and 9.8 months, respectively, P﹤0.01.

[0072] like Figure 5 The results showed that the baseline miR-196b-5p level was associated with the first efficacy of cetuximab. The baseline miR-196b-5p level was closely related to the first efficacy of mCRC patients treated with cetuximab. Patients with high miR-196b-5p levels were more likely to have a first efficacy evaluation of partial response (PR, partial response means that the tumor volume is reduced but not completely disappeared) / complete response (CR), while patients with low miR-196b-5p levels were more likely to have a first efficacy evaluation of progressive disease (PD). Therefore, miR-196b-5p can be used to predict the first efficacy of cetuximab in CRC patients.

[0073] like Figure 6 As shown in the results, ROC curve analysis was used to explore the cut-off value of its level to predict the first efficacy PR of cetuximab. The results showed that when the cut-off value = 42262.5 copies / mL, the maximum Youden index = 0.695 was reached, at which time the sensitivity = 86.2% and the specificity = 83.3%, that is, when the baseline miR-196b level was higher than 42262.5 copies / mL, the patient was more likely to have a PR in the first evaluation.

[0074] like Figure 7 As shown in the results, ROC curve analysis was used to explore the cut-off value of its level to predict the first efficacy of cetuximab PD. The results showed that when the cut-off value = 23800.0 copies / mL, the maximum Youden index = 0.561 was reached, at which time the sensitivity = 83.3% and the specificity = 72.7%, that is, when the baseline miR-196b level was lower than 23800.0 copies / mL, the patient was more likely to be evaluated for PD in the first evaluation.

[0075] like Figure 8 The results showed that miR-196b levels can dynamically monitor the efficacy of cetuximab in mCRC patients. During long-term cetuximab treatment, when most patients developed PD, the level of miR-196b in the blood showed a relatively obvious downward trend.

[0076] like Fig. 9 The results showed that after ROC curve analysis, when the cut-off value = -26.8%, the maximum Youden index = 0.770 was reached, at this time the sensitivity = 90.6%, the specificity = 86.4%, P < 0.01, that is, during the efficacy evaluation process, the change in the level of miR-196b in the blood decreased by more than 26.8% compared with the baseline, indicating that the patient's disease progression is likely to be large. Therefore, miR-196b can be used to dynamically monitor the efficacy of cetuximab in CRC patients.

[0077] Example 4. Application of miR-196b-5p in rescuing CRC cetuximab resistance 1. Construction of miR-196b-5p overexpression cells and knockdown cells The silencing lentivirus (Suzhou Genetech Co., Ltd., target sequence see SEQ ID NO. 5, template sequence see SEQ ID NO. 6, plasmid map see Fig.10 ), control lentivirus, overexpression lentivirus (Suzhou Genema Gene Co., Ltd., target sequence, i.e. target site sequence, see SEQ ID NO.7, template sequence, i.e. miRNA precursor sequence, see SEQ ID NO.8, plasmid map see Fig.10 ), the miRNA precursor sequence was constructed into the lentiviral shuttle plasmid LV2 (U6 / puro), that is, U6 was used as the promoter to drive transcription, "-TTTTTTT-" was used as the transcription termination signal, and mature miRNA was produced by intracellular enzyme processing. The colorectal cancer cell LIM1215 was transfected with lentivirus, and Puro pressure screening was used to obtain stably infected miR-196b-5p overexpression cells, control cells and knockdown cells.

[0078] RNA was extracted from miR-196b-5p overexpressing cells, control cells and knockdown cells, and reverse transcribed according to the above method to obtain cDNA as a template for the above RT-PCR amplification.

[0079] The expression results of miR-196b-5p overexpressing cells and control cells are shown in Fig.11 As shown in the left figure, it can be seen that compared with the control cells, the expression level of miR-196b-5p in the overexpressing cells was significantly upregulated (42 times that of the control) (P<0.0001), indicating that cells overexpressing miR-196b-5p were obtained.

[0080] The expression results of miR-196b-5p knockdown cells and control cells are shown in Fig.11 As shown in the right figure, it can be seen that compared with the control cells, the expression level of miR-196b-5p in the overexpressing cells was significantly downregulated (60% of the control) (P<0.0001), indicating that cells with miR-196b-5p knockdown were obtained. 2. In the presence of cetuximab, miR-196b-5p is associated with the proliferation of colorectal cancer cells Cell Viability Assay (CTG) and EdU assays were used to detect the sensitivity and proliferation of colorectal cancer cells to cetuximab. The treatment time of cetuximab (20 μg / mL) was 72 h.

[0081] 1. CTG assay to detect cell proliferation The miR-196b-5p overexpressing cells, control cells and knockdown cells obtained above were counted using a cell counting plate from Bio-Rad, and a cell suspension (density of about 4×10 4 / mL), 2800 cells were inoculated into 96-well plates per well, and 3 replicates were set up per group. After the cells adhered to the wall, the culture medium containing multi-concentration gradient cetuximab was replaced. After 72 hours of treatment, CTG detection was performed according to the above method. The optical density of cells in each group was measured using the same method, and the values ​​were recorded and the curves were drawn.

[0082] The results are as follows Fig.12 As shown, the upper figure is the silent group, and the lower figure is the overexpression group. It can be seen that in the CTG experiment, after 72 hours of cetuximab treatment, under the same concentration of cetuximab, the activity of the overexpression group was lower than that of the control group, and the trend line IC 50 Left shift, IC 50 decline( Fig.12 After silencing miR-196b, the activity of the knockdown group was higher than that of the control group, and the trend line IC 50 Shift right, IC50 improve( Fig.12 The results confirmed that miR-196b-5p can affect the sensitivity of colorectal cancer cells to cetuximab. The effects of overexpression and silencing of miR-196b-5p on cell viability in the presence of cetuximab are shown in Table 10: Table 10. Effects of overexpression and silencing of miR-196b-5p on cell viability (%) in the presence of cetuximab

[0083] 2. EdU experiment The experiment was performed according to the instructions of the BeyoClick EdU-594 Cell Proliferation Detection Kit (Biyuntian, Cat. No. C0078L). EdU is a thymidine nucleoside analog that can replace thymidine and be incorporated into the replicating DNA molecule during the cell proliferation period. Then, by detecting fluorescently labeled EdU, cell proliferation can be accurately reflected. The miR-196b-5p overexpressing cells, control cells and knockdown cells were EdU labeled, fixed with 4% paraformaldehyde for 15 min, washed twice with PBS for 5 min each, permeabilized with 1% Triton-X100 at room temperature for 10 min, the permeabilization solution was removed, and washed twice with PBS for 5 min each. 1mL of staining solution was prepared, and the formula was as follows: 860μL-Click Reaction Buffer, 40 μL-CuSO4, 2 μL-Azide 594, 100μL-Click Additive Solution. The cells were stained with the prepared staining solution for 30 minutes under light-proof conditions, and washed twice with PBS for 5 minutes each. The cells were stained for DNA using mounting medium with DAPI (Abcam, catalog number ab104139) in the dark and then photographed under a fluorescence microscope.

[0084] The results are as follows Figure 13-14 As shown, Fig.13 For the overexpression group, Fig.14 The knockdown group was divided into two groups. No less than 6 fields of view were collected. In the presence of cetuximab, the positive cell rate in the overexpression group was significantly lower than that in the control group, the proportion of positive cells was significantly reduced, and the cell proliferation activity was significantly decreased (32% vs 48%) (P<0.01, Fig.13 ); In the presence of cetuximab, the positive cell rate in the knockdown group was significantly higher than that in the control group, the positive cell rate was significantly increased, and the cell proliferation activity was significantly increased (57% vs 48%) (P < 0.01, Fig.14 ).

[0085] The above results indicate that in the presence of cetuximab, overexpression of miR-196b can inhibit the proliferation of colorectal cancer cells, while inhibition of miR-196b promotes the proliferation of colorectal cancer cells.

[0086] 3. miR-196b-5p is associated with the migration ability of colorectal cancer cells The Transwell chamber migration assay was used to detect the migration ability of colorectal cancer cells. The Transwell assay uses a special porous membrane to separate cells into two chambers, upper and lower, and simulates in vivo cell migration through in vitro experimental methods.

[0087] LIM1215 cell lines were infected with miR-196b-5p overexpression, silencing and control lentivirus, respectively. The treatment time of cetuximab was 72h.

[0088] 1) Preparation of cell suspension Digest the cells, centrifuge and discard the culture medium after terminating the digestion (wash 1-2 times with PBS), and resuspend in serum-free medium. Adjust the cell density to 5×10 5 / ml.

[0089] 2) Seeding cells ① Take 100 μl of cell suspension and add it into the Transwell chamber.

[0090] ② Generally, 600 μl of culture medium containing 20% ​​serum is added to the lower chamber of the 24-well plate, and no cells are produced.

[0091] ③Cultivate cells: routinely culture for 10-48 hours.

[0092] 3) Result statistics Take out the Transwell chamber, discard the culture medium in the well, wash twice with PBS, fix with 90% ethanol for 30 minutes, and air-dry the chamber appropriately.

[0093] Stain with 0.1% crystal violet for 30 min, gently wipe off the upper layer of non-migrated cells with a cotton swab, and wash three times with PBS.

[0094] The cells were observed in five fields of view at random under a 400x microscope and counted.

[0095] The results of the overexpression group were as follows Fig.15 The results of the knockdown group are shown in Fig.16 As shown, Fig.15 and Fig.16In the figure, the upper row is PBS control, and the lower row is cetuximab (20μg / mL). It can be seen that in the Transwell chamber migration experiment, the overexpression group and the control group were treated with PBS / cetuximab (20μg / mL) for 24h and photographed and counted. Regardless of the presence of cetuximab, the cell migration rate of the miR-196b-5p overexpression group was significantly reduced compared with the control group (the control group decreased to 60%, and the cetuximab group decreased to 40%) (P<0.001, Fig.15 ). The above results indicate that overexpression of miR-196b-5p can slow down the migration of colorectal cancer cells; in the Transwell chamber migration experiment, the knockdown group and the control group were photographed and counted at 24 hours. The results showed that regardless of the presence of cetuximab, the migration rate of the knockdown group cells was significantly increased compared with the control group cells (the control group increased to 1.4 times, and the cetuximab group increased to 1.86 times) (P<0.001, Fig.16 ). The above results indicate that silencing the expression of miR-196b-5p can promote the migration of colorectal cancer cells.

[0096] 4. miR-196b-5p is associated with apoptosis of colorectal cancer cells in the presence of cetuximab The apoptosis rate of colorectal cancer cells was detected using Annexin V-FITC apoptosis detection kit (Beyotime, catalog number C1062L).

[0097] LIM1215 cell lines were infected with miR-196b-5p overexpression, silencing and control lentivirus, respectively. The treatment time of cetuximab was 72h.

[0098] 1) Aspirate the culture medium into a centrifuge tube, wash with PBS, and add an appropriate amount of trypsin to digest the cells.

[0099] 2) Transfer the cells to a centrifuge tube and centrifuge at 1200 rpm for 3 min. Discard the supernatant, resuspend the cells in PBS and count them.

[0100] 3) Take 10×10^4 resuspended cells, centrifuge at 1200 rpm for 3 min, discard the supernatant, add 195 μL Annexin V-FITC and gently resuspend.

[0101] 4) Add 5 μL Annexin V-FITC and 10 μL PI staining solution.

[0102] 5) Place the sample away from light at room temperature for 10 minutes before testing.

[0103] The results of the overexpression group were as follows Fig.17 The results of the knockdown group are shown in Fig.18 As shown, Fig.17 and Fig.18 In the figure, the left column is PBS control, and the right column is cetuximab (20μg / mL). It can be seen that in the cell apoptosis experiment, the overexpression group and the control group were treated with PBS / cetuximab (20μg / mL) for 72h and photographed and counted. In the presence of cetuximab, the apoptosis rate of the miR-196b-5p overexpression group was significantly increased compared with the control group (22.4% vs 15.4%, P<0.01, Fig.17 ). The above results indicate that overexpression of miR-196b-5p can increase the apoptosis of colorectal cancer cells in the presence of cetuximab. In the apoptosis experiment, the knockdown group and the control group were treated with PBS / cetuximab (20 μg / mL) for 72 h, and then photographed and counted. In the presence of cetuximab, the apoptosis rate of cells in the miR-196b-5p knockdown group was significantly reduced compared with that in the control group (10.7% vs 15.4, P<0.05, Fig.18 The above results indicate that knocking down miR-196b-5p can reduce the apoptosis of colorectal cancer cells in the presence of cetuximab.

[0104] The above results indicate that in the presence of cetuximab, exogenous high expression of miR-196b-5p significantly increased the apoptosis of colorectal cancer cells, while low expression could significantly inhibit the apoptosis of colorectal cancer cells.

Claims

1. A method for using microRNA as a diagnostic marker in a diagnostic kit for drug resistance of advanced colorectal cancer, characterized in that: The sequence of the microRNA is shown as SEQ ID NO.1, and the kit contains a reagent for detecting the content or expression amount of the microRNA in the sample to be detected.

2. The use according to claim 1, characterized in that: The advanced colorectal cancer drug resistance diagnostic kit is a gene detection kit.

3. The use according to claim 2, characterized in that: The gene detection kit is a PCR detection kit, and the sequences of the upstream primer and the downstream primer of the PCR are shown as SEQ ID NO. 2 and 3 respectively.

4. The use according to claim 3, characterized in that The gene detection kit also contains a probe, the sequence of which is shown in SEQ ID NO.

4.

5. Use of a microRNA in the preparation of a drug for treating advanced colorectal cancer, characterized in that: The sequence of the microRNA is shown in SEQ ID NO.

1.

6. The use according to claim 5, characterized in that: The drug is prepared as a vector containing microRNA.

7. The use according to claim 6, characterized in that: The vector is a lentiviral vector.

8. The use according to claim 7, characterized in that The target sequence of the lentiviral vector is shown in SEQ ID NO.

7.

9. The use according to claim 8, characterized in that The template sequence of the lentiviral vector is shown as SEQ ID NO.

8.

10. A composition, characterized in that The composition contains a vector expressing a microRNA whose sequence is shown in SEQ ID NO.

1.

11. The composition according to claim 10, characterized in that The composition also contains cetuximab.

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