Application of MPP2 gene as prognostic marker and therapeutic target of colon cancer

Abstract

The invention discloses application of an MPP2 gene as a prognostic marker and a therapeutic target of colon cancer, relates to the technical field of biological medicines, and is characterized in that the MPP2 gene is used as the prognostic marker and the therapeutic target of the colon cancer, and a new thought is provided for prognosis and treatment of the colon cancer. Researches in the invention show that the expression quantity of the MPP2 protein in colon cancer organizers is significantly higher than that of normal human colon tissues, and the difference has significant statistical significance (Plt; 0.05) of the method. In addition, research also finds that the expression quantity of the MPP2 protein in low-differentiation colon cancer organizers is obviously smaller than that of high-differentiation colon cancer organizers, and the difference between the MPP2 protein and the high-differentiation colon cancer organizers has obvious statistical significance (Plt; 0.05) of the method.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, more particularly, it relates to the application of MPP2 gene as a prognosis marker and therapeutic target of colon cancer. BACKGROUND

[0002] Colon cancer is a high-incidence malignant tumor worldwide, with the morbidity ranking the third and the mortality ranking the second (data from the World Health Organization). Early diagnosis and accurate prognosis evaluation are the key to improve the survival rate of patients, but the existing means have limitations. Molecular targeted therapy is an important breakthrough in the treatment of colon cancer, but the application of target points (such as EGFR, VEGF) is limited by individual differences and drug resistance of patients.

[0003] Abnormal expression of genes is the core mechanism of tumor occurrence, and finding abnormal expression genes related to colon cancer has become a research hotspot. MPP2 gene is found to be highly expressed in breast cancer, lung cancer and other tumors, but its expression pattern, clinical significance and mechanism in colon cancer have not been clearly defined, and relevant research is lacking.

[0004] The existing technology still has the following disadvantages:

[0005] 1. The existing colon cancer diagnosis relies on colonoscopy and pathological examination, lacks specific molecular markers, and it is difficult to achieve early and accurate diagnosis;

[0006] 2. The prognosis of colon cancer mainly depends on clinical indicators such as TNM staging, which cannot accurately reflect the molecular level differences and long-term survival risk of patients;

[0007] 3. The molecular targeted therapy of colon cancer has limited target points, and the existing drugs have drug resistance problems, so it is urgent to find new potential therapeutic targets. SUMMARY

[0008] The purpose of the present application is to provide the application of MPP2 gene as a prognosis marker and therapeutic target of colon cancer, in order to solve the above-mentioned shortcomings in the background art.

[0009] The above technical purpose of the present application is achieved by the following technical solution: the application of MPP2 gene as a prognosis marker of colon cancer.

[0010] Further, the application of MPP2 gene as a prognosis marker of colon cancer in the preparation of a colon cancer prognosis reagent or kit.

[0011] Further, the application of specific primers for detecting the expression level of MPP2 gene as a prognosis marker of colon cancer, the primer sequence is:

[0012] F-5'-CTGATCGCTACCTGGAC-3',

[0013] R-5'-GTAGTCGTCGCCGTAGA-3'.

[0014] The application also provides an application of the MPP2 gene as a treatment target for colon cancer.

[0015] Further, the application of the MPP2 gene in the preparation of a drug for treating colon cancer.

[0016] The application also provides a detection method for the expression level of the MPP2 gene, and the detection method comprises the following steps:

[0017] (1) Sample processing:

[0018] ①Obtain postoperative cancer tissues and cancer-adjacent normal tissues, and the normal tissues need to be >5 cm away from the tumor;

[0019] ②The collected samples need to be quickly frozen in liquid nitrogen and stored at -80℃ or paraffin-embedded;

[0020] (2) MPP2 detection:

[0021] ① Protein level detection is realized by immunohistochemistry (IHC): an anti-MPP2 monoclonal antibody is selected, a corresponding diluent is prepared, ABC staining is performed, and finally contrast staining is performed to determine the positive signal, and the positive determination method is brown-yellow particles >10% cytoplasm; Western Blot: proteins are extracted using RIPA lysis buffer, 40-50 μg are subjected to SDS-PAGE electrophoresis separation, the membrane is immersed in an anti-MPP2 monoclonal antibody solution diluted to 1:1000, 4℃ incubation overnight, and the signal is visualized by chemiluminescence method, and the image is recorded;

[0022] ② mRNA level:

[0023] RT-PCR: total RNA is extracted using TRIzol reagent to ensure that there is no RNase contamination; the extracted RNA is reverse transcribed into cDNA using a suitable reverse transcriptase and reaction system;

[0024] Primer sequence:

[0025] F-5'-CTGATCGCTACCTGGAC-3',

[0026] R-5'-GTAGTCGTCGCCGTAGA-3';

[0027] (3) Clinical correlation analysis:

[0028] ① Single factor variance analysis: compare the expression level of MPP2 with lymph node metastasis, distant metastasis and TNM staging, and use ANOVA (analysis of variance) method to determine the significant difference between groups;

[0029] ② Kaplan-Meier survival curve: according to the IHC score, it is divided into high expression group (> 3 points) and low expression group (≤ 3 points), the survival curve is drawn by using Kaplan-Meier method, and the survival rates of the two groups are compared by log-rank test;

[0030] ③ COX regression model: the Cox proportional hazards regression model is established, the expression level of MPP2, age, gender and other confounding variables are considered; the influence of each variable on survival is calculated, the hazard ratio (HR) and its 95% confidence interval (CI) are evaluated, and the independence of MPP2 in prognosis is judged.

[0031] In summary, the present application has the following beneficial effects: the present application provides a specific molecular marker for the prognosis and diagnosis of colon cancer, which can more accurately reflect the molecular level difference and long-term survival risk of patients, provides a new treatment target and treatment direction for the treatment of colon cancer, and solves the problem of drug resistance of existing drugs. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 is a representative staining picture (SPx200) of different expressions of MPP2 protein in cancer-adjacent normal colon tissue and colon cancer tissue in the embodiment of the present application; wherein A: negative expression of MPP2 protein in cancer-adjacent normal colon tissue; B: low expression of MPP2 protein in cancer-adjacent normal colon tissue; C: high expression of MPP2 protein in cancer-adjacent normal colon tissue; D: negative expression of MPP2 protein in colon cancer tissue; E: low expression of MPP2 protein in colon cancer tissue; F: high expression of MPP2 protein in colon cancer tissue;

[0033] Figure 2 is that the expression level of MPP2 protein in colon cancer tumor tissue is obviously higher than that in cancer-adjacent normal colon tissue, and the difference is statistically significant (P<0.05);

[0034] Figure 3 A: expression of MPP2 gene in colon cancer tissue and cancer-adjacent normal colon tissue (P<0.01); B: expression of MPP2 gene in colon cancer tissues with different differentiation degrees (P<0.05);

[0035] Figure 4 is the relationship between the expression of MPP2 protein and the overall survival rate of colon cancer patients in the embodiment of the present application: A. Relationship between high expression, low expression of MPP2 protein and overall survival of patients (P<0.01); B. Relationship between positive expression, negative expression of MPP2 protein and overall survival of patients (P<0.01);

[0036] Figure 5The expression intensity of MPP2 mRNA in the colon cancer cell lines HCT-116 and SW480 is obviously higher than that in the normal colon cell line FHC in the embodiment of the application, and the difference is statistically significant (P<0.05).

[0037] Figure 6 The technical roadmap of MPP2 gene expression detection in the embodiment of the application is shown in the figure. DETAILED DESCRIPTION

[0038] The following will be described in detail in combination with the accompanying Figures 1-6 The application will be further described in detail.

[0039] Embodiment:

[0040] 1. MPP2 gene expression detection scheme

[0041] (1) Sample processing

[0042] ① Obtain postoperative cancer tissue and cancer-adjacent normal tissue

[0043] Sample source: Select postoperative tumor tissue and corresponding cancer-adjacent normal tissue (must be more than 5 cm away from the tumor site).

[0044] Collection method: Collect the specimen immediately after surgery, and ensure processing within the shortest time to reduce the risk of sample degradation.

[0045] ② Liquid nitrogen quick freezing and -80℃ storage or paraffin embedding

[0046] Liquid nitrogen quick freezing: The collected sample is quickly frozen in liquid nitrogen as soon as possible to maintain the biological activity and molecular characteristics of the sample. This process should be carried out under sterile conditions to avoid contamination.

[0047] Storage conditions: The frozen sample is transferred to a -80℃ refrigerator for long-term storage. Paraffin embedding can also be selected to facilitate subsequent tissue sectioning and pathological analysis.

[0048] (2) MPP2 detection

[0049] ① Protein level detection:

[0050] Immunohistochemistry (IHC):

[0051] Reagent preparation: Select anti-MPP2 monoclonal antibody and prepare the corresponding diluent. Ensure the specificity and sensitivity of the reagent.

[0052] Staining steps:

[0053] Tissue sections (about 4-5 μm thick) are subjected to deparaffinization and hydration treatment.

[0054] Antigen retrieval is performed, usually by heat retrieval (e.g. in a microwave oven).

[0055] Anti-MPP2 monoclonal antibody is added and incubated at 4°C overnight.

[0056] Secondary antibody is added and visualization is performed (using ABC method), followed by counterstaining to determine positive signals.

[0057] Positive determination: If brownish yellow particles are distributed in the cytoplasm more than 10%, it is determined to be positive.

[0058] Western Blot:

[0059] Sample preparation: Protein is extracted using RIPA lysis buffer. Supernatant is collected and protein concentration is determined.

[0060] SDS-PAGE: Equal amounts of samples (40-50 μg) are subjected to SDS-PAGE electrophoretic separation.

[0061] Membrane transfer: Protein is transferred to PVDF or nitrocellulose membrane according to the standard procedure of membrane transfer.

[0062] Primary antibody incubation: The membrane is immersed in a 1:1000 diluted primary antibody solution and incubated at 4°C overnight.

[0063] Signal detection: Signal visualization is performed using chemiluminescence method and images are recorded.

[0064] ② mRNA level detection:

[0065] RT-PCR:

[0066] RNA extraction: Total RNA is extracted using TRIzol reagent according to the instructions, ensuring no RNase contamination.

[0067] Reverse transcription: Extracted RNA is reverse transcribed into cDNA using appropriate reverse transcriptase and reaction system.

[0068] Primer sequence:

[0069] Forward primer (F): 5'-CTGATCGCTACCTGGAC-3'

[0070] Reverse primer (R): 5'-GTAGTCGTCGCCGTAGA-3'

[0071] PCR reaction: PCR amplification is performed according to the primer design program, using cDNA in solution as template, and setting appropriate cycle number.

[0072] (3) Clinical correlation analysis

[0073] ① One-way ANOVA:

[0074] Variable processing: Compare the expression level of MPP2 with lymph node metastasis, distant metastasis and TNM stage.

[0075] Statistical analysis: Use ANOVA method to determine the significant difference between groups.

[0076] ② Kaplan-Meier survival curve:

[0077] Group division: According to the IHC score, divide into high expression group (> 3 points) and low expression group (≤ 3 points).

[0078] Survival analysis: Use Kaplan-Meier method to draw survival curve, and compare the survival rate of two groups by log-rank test.

[0079] ③ COX regression model:

[0080] Model establishment: Establish Cox proportional hazards regression model, considering the expression level of MPP2, age, gender and other confounding variables.

[0081] Result analysis: Calculate the influence of each variable on survival time, evaluate the hazard ratio (HR) and its 95% confidence interval (CI), and judge the independence of MPP2 in prognosis.

[0082] 2. Clinical and pathological data of patients

[0083] After surgery, we collected all the clinical data of patients. After sorting and analyzing these data, we found that most of the colon cancer patients were elderly (age ≥ 60 years), among which there were 77 male patients (64.2%). Most patients were in the middle and late stages at the time of diagnosis, so the number of patients with postoperative peripheral lymph node metastasis was the majority (68.3%). Through the analysis of the pathological data of patients after surgery, we found that the colon cancer tissue after surgical resection was usually poorly differentiated (63.3%). Table 1 summarizes the clinical data of all patients.

[0084] Table 1 Clinical data of all colon cancer patients

[0085]

[0086]

[0087] 3. Expression difference of MPP2 protein in colon cancer tissue and normal colon tissue

[0088] By immunohistochemical technique, we detected the expression of MPP2 protein in 120 cases of colon cancer and 120 cases of normal colon tissues. The results showed that MPP2 protein mainly existed in the cytoplasm of colon cancer cells, among which 69 cases (57.5%) showed high expression, 37 cases (30.8%) showed low expression, and the remaining 14 cases (11.7%) showed negative expression. Further analysis showed that there was a significant statistical difference between 106 cases of MPP2-positive colon cancer tissue samples and 14 cases of MPP2-negative tissue samples (P < 0.05). In addition, among the 120 cases of normal colon tissues, immunohistochemical results showed that 15 cases (12.5%) of normal colon tissues showed high expression of MPP2, 23 cases (19.2%) of normal tissue samples showed low expression of MPP2, and the remaining 82 cases (68.3%) of normal colon tissues showed negative expression of MPP2. The results showed that the positive expression rate of MPP2 protein in colon cancer tissues was 88.3%, while the positive expression rate in normal colon tissues was only 31.7%, and there was a significant statistical difference between the two (P < 0.05).

[0089] Statistical analysis showed that the expression of MPP2 protein in colon cancer tissues was significantly higher than that in normal colon tissues, and the difference was statistically significant (P < 0.05). In addition, the study also found that the expression of MPP2 protein in low differentiated colon cancer tissues was significantly lower than that in high differentiated colon cancer tissues, and the difference between the two was statistically significant (P < 0.05). Figure 1 Figures A-C show the expression of MPP2 protein in normal colon tissues adjacent to cancer, and the expression level of MPP2 protein in colon cancer tissues is shown in Figure 1 Figures D-F. By Western blot detection, we also found that the expression level of MPP2 protein in colon cancer tissues was significantly higher than that in normal colon tissues, and the difference was statistically significant (P < 0.05) Figure 2 ).

[0090] 4. Relationship between MPP2 expression level and clinical pathological features of colon cancer patients

[0091] The results showed that the expression level of MPP2 protein in colon cancer patients was significantly related to lymph node metastasis, and TNM staging was also closely related to it. It was found that the expression intensity of MPP2 protein in colon cancer tissue with lymph node metastasis was significantly better than that without lymph node metastasis, and the difference was significant (P<0.05). In addition, when the patient had distant metastasis, the expression intensity of MPP2 protein in the tissue was significantly enhanced, and the difference was statistically significant (P<0.05). The expression of MPP2 protein in colon cancer patients with TNM stage III-IV was higher than that in patients with TNM stage I-II, and the difference was statistically significant (P<0.05). After analysis, the expression intensity of MPP2 protein in colon cancer tissue was not significantly related to patient gender, age, tumor size and differentiation degree, and this conclusion was confirmed by the data in Table 2.

[0092] Table 2 Correlation between different expression of MPP2 protein in colon cancer tissue and clinical pathological characteristics of patients

[0093]

[0094]

[0095]

[0096] 5. Expression difference of MPP2 mRNA in colon cancer tissue and normal colon tissue

[0097] Through qRT-PCR technology, it was found that the expression level of MPP2 mRNA in colon cancer tissue was significantly better than that in normal colon tissue, which indicated that the amplified product had specificity, and the difference between them had significant statistical significance (P<0.01) Figure 3 A). Therefore, we can infer that there is a significant difference in the expression level of MPP2 gene in colon cancer tissue and normal colon tissue. Through further research, the expression level of MPP2 mRNA in well-differentiated colon cancer tissue was significantly higher than that in poorly differentiated colon cancer tissue, and the difference had significant statistical value (p<0.01) Figure 3 B), and the difference was statistically significant.

[0098] 6. Expression of MPP2 mRNA in colon cell lines

[0099] The expression level of MPP2 mRNA in HCT-116 and SW480 colon cell lines was significantly higher than that in normal human colon cell line FHC, with expression intensity of 1.16±0.22 and 0.97±0.32, respectively. This result is consistent with the experimental results, indicating that the expression level of MPP2 mRNA in colon cancer tissue and adjacent normal colon tissue is significantly different. Figure 5

[0100] 7. Survival analysis

[0101] (1) The influence of MPP2 expression level on the survival prognosis of colon cancer patients

[0102] In this survey, all patients were followed up, and the deadline was January 1, 2018. We recorded the total survival time of each patient by month, and the average follow-up time was 39.5 days. During the follow-up, 13 patients were lost, and the remaining 107 patients had complete follow-up data, with a patient follow-up rate of 107 / 120 (89.2%). In this follow-up, 31 patients died due to tumor recurrence, of which 19 patients (61.3%) had high expression of MPP2 protein, 8 patients (25.8%) had low expression of MPP2 protein, and only 4 patients (12.9%) had negative expression of MPP2 protein. In addition, the Kaplan-Meier survival curve analysis method was used to analyze the postoperative survival of colon cancer patients with different expression of MPP2 protein, and it was found that colon cancer patients with low expression of MPP2 protein had better prognosis than colon cancer patients with high expression of MPP2 protein, and there was a significant statistical difference (P<0.01, Figure 4 A), further analysis found that colon cancer patients with positive expression of MPP2 protein had significantly worse postoperative survival than colon cancer patients with negative expression of MPP2 protein, and there was a statistical difference (P<0.01, Figure 4 B).

[0103] (2) Single factor COX survival analysis results

[0104] ​For univariate COX regression analysis, we included age, gender, tumor size, lymph node metastasis, tumor differentiation, distant metastasis, TNM stage and MPP2 expression level into the analysis. The statistical analysis results showed that the prognosis of colon cancer patients with higher MPP2 expression level was poorer, and the difference between them was more statistically significant (P<0.05). The results showed that the long-term survival time of patients with lymph node metastasis was significantly shorter than that of colon cancer patients without lymph node metastasis (P<0.05), and the prognosis of patients without distant metastasis before operation was significantly higher than that of patients with distant metastasis. With the continuous increase of TNM stage of patients, the long-term survival time was significantly shortened, and there was a significant statistical difference (P<0.05). After statistical analysis, the prognosis of colon cancer patients was obviously affected by lymph node metastasis, differentiation degree, distant metastasis, TNM stage and MPP2 expression (P<0.05), while the age, gender and tumor size of the patients had no obvious correlation (P≥0.05) (Table 3).

[0105] Table 3 Univariate COX survival analysis of factors affecting the prognosis of colon cancer patients after operation

[0106]

[0107]

[0108] (3) Multivariate COX survival analysis results

[0109] After univariate COX survival analysis, it was found that there was a close relationship between lymph node metastasis, differentiation degree, TNM stage, distant metastasis, MPP2 protein expression and prognosis of colon cancer patients. Multivariate COX regression analysis of these influencing factors found that TNM stage and distant metastasis of tumor were independent factors affecting the prognosis of colon cancer patients, while differentiation degree, MPP2 expression and lymph node metastasis were not independent factors affecting the prognosis of patients. For specific results, see Table 4.

[0110] Table 4 Multivariate COX survival analysis of factors affecting the prognosis of colon cancer patients after operation

[0111]

[0112] This specific embodiment is only an explanation of the present application, which is not a limitation of the present application. Those skilled in the art can make modifications to this embodiment without creative contribution after reading this specification, but as long as it is within the scope of the claims of the present application, it is protected by the patent law.

Claims

1. Application of MPP2 gene as a prognostic marker for colon cancer.

2. The use according to claim 1, characterized in that: The MPP2 gene is used as a colon cancer prognosis marker in the preparation of a colon cancer prognosis reagent or kit.

3. Use of the specific primers for detecting the expression level of the MPP2 gene according to claim 1 as a prognostic marker for colon cancer, characterized in that: The primer sequence: F-5'-CTGATCGCTACCTGGAC-3', R-5′-GTAGTCGTCGCCGTAGA-3′.

4. Application of MPP2 gene as a therapeutic target for colon cancer.

5. The use according to claim 4, characterized in that: The application of the MPP2 gene in preparing drugs for treating colon cancer.

6. The method for detecting the level of MPP2 gene expression according to any one of claims 1 to 5, wherein: The steps include: (1) Sample processing: ① Obtain postoperative cancer tissue and adjacent normal tissue. The normal tissue must be >5 cm away from the tumor. ② The collected samples need to be quickly frozen in liquid nitrogen and stored at -80°C or embedded in paraffin; (2) MPP2 detection: ① Protein level detection was achieved by immunohistochemistry (IHC): anti-MPP2 monoclonal antibody was selected, the corresponding dilution was prepared, and staining was performed according to the ABC method. Finally, contrast staining was performed to determine the positive signal. The positive signal was determined by brown-yellow particles >10% of the cytoplasm. Western Blot: Protein was extracted using RIPA lysis buffer, and 40-50 μg was separated by SDS-PAGE electrophoresis. The membrane was immersed in the primary antibody solution diluted 1:1000 and incubated overnight at 4°C. The signal was visualized using chemiluminescence and the image was recorded. ②mRNA level: RT-PCR: Extract total RNA using TRIzol reagent to ensure the absence of RNase contamination; reverse transcribe the extracted RNA into cDNA using an appropriate reverse transcriptase and reaction system; Primer sequences: F-5'-CTGATCGCTACCTGGAC-3', R-5′-GTAGTCGTCGCCGTAGA-3′; (3) Clinical correlation analysis: ① One-way analysis of variance: The expression level of MPP2 was compared with lymph node metastasis, distant metastasis and TNM stage, and the ANOVA (analysis of variance) method was used to determine the significant differences between the groups; ②Kaplan-Meier survival curve: According to the IHC score, the patients were divided into a high expression group (>3 points) and a low expression group (≤3 points). The Kaplan-Meier method was used to draw the survival curve, and the log-rank test was used to compare the survival rates of the two groups. ③COX regression model: A Cox proportional hazards regression model was established, taking into account MPP2 expression level, age, gender and other confounding variables; The effects of various variables on survival were calculated, the hazard ratio (HR) and its 95% confidence interval (CI) were evaluated, and the independence of MPP2 in prognosis was determined.

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