CN113281516A - Application of CUL9 as marker in colorectal cancer prognosis evaluation - Google Patents
Application of CUL9 as marker in colorectal cancer prognosis evaluation Download PDFInfo
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Abstract
The invention relates to application of CUL9 as a marker in colorectal cancer prognosis evaluation. And (3) evaluating the clinical characteristics and prognosis of the colorectal cancer patient based on the positive expression of CUL9 cytoplasm by taking the CUL9 as a marker. Compared with the prior art, the method for evaluating the clinical characteristics and the overall prognosis of the patient with colorectal cancer through the positive expression condition of the CUL9 cytoplasm of the colorectal cancer can directly determine the clinical characteristics and the prognosis information of the patient according to the expression condition of the CUL9 cytoplasm in the tumor tissue of the patient with the colorectal cancer.
Description
Technical Field
The invention belongs to the technical field of colorectal cancer prognosis evaluation, and particularly relates to application of CUL9 as a marker in colorectal cancer prognosis evaluation.
Background
Colorectal cancer is a common malignant tumor of the digestive tract, and has no small threat to the health of people. According to global epidemiological data (GLOBOCAN 2020) issued by the International agency for research on cancer (IARC) of the world health organization, 193.16 ten thousand new cases and 93.52 ten thousand cases of colorectal cancer were estimated to be located at the third and second sites of all malignant tumors in 2020. In recent decades, the incidence of colorectal cancer has increased with the improvement of people's living standard and the change of dietary structure.
With the continuous and intensive research on colorectal cancer, the prognosis of colorectal cancer is on the trend of improvement over the past decades. However, for the clinical diagnosis and treatment of colorectal cancer, the tumor stage is still an important prognostic factor, and there is a large heterogeneity in the prognosis of early colorectal cancer and middle and late colorectal cancer.
Chinese patent CN107622800A discloses a mathematical model for colorectal cancer liver metastasis curative effect prediction, which provides a formula, wherein the factors related to the formula comprise genotypes of six genes, namely ATP6V1B1, CUL9, ERBB2, LY6G6D, RBMXL3 and PTCH1, and the patent shows that after a cetuximab-sensitive population is screened, the objective response rate is remarkably improved, so that the prediction of a treatment mode can be better performed.
Chinese patent CN103954757B discloses a method for evaluating colorectal cancer through the positive expression condition of TEAD4 cell nucleus, namely, firstly preparing a tumor tissue chip, then carrying out immunohistochemical experiment and immune scoring, grouping according to the immune scoring, finally dividing patients into a TEAD4 cell nucleus positive expression group and a TEAD4 cell nucleus negative expression group according to the cell nucleus staining condition of TEAD4, and then analyzing the clinical characteristics and the difference of the total survival period of the two groups of patients by using a statistical method. The method for evaluating the clinical characteristics and the overall prognosis of the colorectal cancer patient through the positive expression condition of the nucleus of the colorectal cancer immunological index TEAD4 is characterized in that the clinical characteristics and the prognosis information of the patient can be directly determined according to the positive expression condition of the nucleus of TEAD4 in the tumor tissue of the colorectal cancer patient.
However, in the prior art, no method for evaluating the clinical characteristics and prognosis of colorectal cancer patients through positive expression of a colorectal cancer marker CUL9 cytoplasm is available.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides an application of CUL9 as a marker in colorectal cancer prognosis evaluation.
The purpose of the invention can be realized by the following technical scheme:
the invention provides an application of CUL9 as a marker in colorectal cancer prognosis evaluation, wherein CUL9 is used as a marker to evaluate clinical characteristics and prognosis of a colorectal cancer patient based on CUL9 cytoplasm positive expression.
The invention provides an application of CUL9 as a marker in colorectal cancer prognosis evaluation, which comprises the following steps:
a. manufacturing a wax block and a tissue chip;
b. embedding the tissue chip;
c. dewaxing and hydrating;
d. repairing and sealing the antigen;
overnight incubation of cull 9 primary antibody;
f. incubating the second antibody at room temperature after washing, and developing a color and amplifying the solution;
g. performing DAB coloration after incubation;
h. staining nuclei with hematoxylin, and fixing;
i. dehydrating to wax, and keeping the slices;
j. reading the result;
k. statistical analysis: all data were statistically analyzed using the SPSS statistical software package (22.0; SPSS inc., Chicago, Illinois, USA), calculation and analysis of OS was performed using the Kaplan-Meier method, risk factors were expressed as hazard ratio [ HR, 95% Confidence Interval (CI) ], and a P value less than 0.05 was defined as statistically significant.
In one embodiment of the invention, the method for antigen retrieval and blocking is: immersing tissue slices in an antigen repairing solution prepared from EDTA, heating, taking out, cooling at room temperature, washing, immersing the tissue slices in an inactivating solution for inactivation, washing, scrubbing the tissue slices, dripping 5% BSA (bovine serum albumin) aqueous solution on each tissue slice as a sealing solution, incubating, and sealing non-specific antigens.
In one embodiment of the present invention, the method for reading the result in step j comprises:
the positive staining of CUL9 is that the cytoplasm of the cell presents brown yellow particles, two persons independently perform immunohistochemical scoring respectively, the scoring result is reevaluated when objection exists, and the staining result is scored, and the positive staining comprises two indexes: 1) dyeing strength: 0 part (-): 0% cell staining; 1 part (+): 0-10% incomplete staining of cells; 2 parts (++): 10% cells stained weakly to moderately completely; 3 points (+++): moderate to intense intact staining of 10% cells; 2) positive staining is proportional to the total field;
the product of the two indexes is the final immunohistochemical score, namely the expression quantity, the Cut-off value is the median of the expression quantity, the expression is high when the Cut-off value is larger than the Cut-off value, and the expression is low when the Cut-off value is smaller than or equal to the Cut-off value.
The invention also provides a method for verifying the relationship between the high expression and low expression of CUL9 cytoplasm and clinical characteristics and prognostic survival rate of colorectal cancer patients,
firstly, preparing a tumor tissue chip, then carrying out immunohistochemical experiments and immune scoring, grouping according to the immune scoring, finally dividing patients into a CUL9 cytoplasm high expression group and a CUL9 cytoplasm low expression group according to the cytoplasm staining condition of CUL9, and then analyzing the clinical characteristics and the difference of the total survival period of the two groups of patients by using a statistical method.
In one embodiment of the present invention, after surgical excision of the specimen, a paraffin section-based tissue chip is first prepared and the expression level of CUL9 is experimentally verified by immunohistochemistry for colorectal cancer patients.
In one embodiment of the present invention, the specific method is:
a. manufacturing a wax block and a tissue chip;
b. embedding the tissue chip;
c. dewaxing and hydrating;
d. repairing and sealing the antigen;
overnight incubation of cull 9 primary antibody;
f. incubating the second antibody at room temperature after washing, and developing a color and amplifying the solution;
g. performing DAB coloration after incubation;
h. staining nuclei with hematoxylin, and fixing;
i. dehydrating to wax, and keeping the slices;
j. reading the result;
k. statistical analysis: all data were statistically analyzed using the SPSS statistical software package, OS calculation and analysis were performed using the Kaplan-Meier method, risk factors were expressed as hazard ratio [ HR, 95% Confidence Interval (CI) ], P-value less than 0.05 was defined as statistically significant, and statistical analysis data were CUL9 immunohistochemical score data and correlation analysis of colorectal cancer clinical pathology.
In one embodiment of the invention, the method for antigen retrieval and blocking is: immersing tissue slices in an antigen repairing solution prepared from EDTA, heating, taking out, cooling at room temperature, washing, immersing the tissue slices in an inactivating solution for inactivation, washing, scrubbing the tissue slices, dripping 5% BSA (bovine serum albumin) aqueous solution on each tissue slice as a sealing solution, incubating, and sealing non-specific antigens.
In one embodiment of the present invention, the method for reading the result in step j comprises:
the positive staining of CUL9 is that the cytoplasm of the cell presents brown yellow particles, two persons independently perform immunohistochemical scoring respectively, the scoring result is reevaluated when objection exists, and the staining result is scored, and the positive staining comprises two indexes: 1) dyeing strength: 0 part (-): 0% cell staining; 1 part (+): 0-10% incomplete staining of cells; 2 parts (++): 10% cells stained weakly to moderately completely; 3 points (+++): moderate to intense intact staining of 10% cells; 2) positive staining is proportional to the total field;
the product of the two indexes is the final immunohistochemical score, namely the expression quantity, the Cut-off value is the median of the expression quantity, the expression is high when the Cut-off value is larger than the Cut-off value, and the expression is low when the Cut-off value is smaller than or equal to the Cut-off value.
In one embodiment of the present invention, the present invention data suggests: CUL9 high expression was positively correlated with tumor diameter >4cm (P ═ 0.001), tumor low differentiation (P ═ 0.037), N1/2(P ═ 0.028) and M1(P ═ 0.001); high expression of CUL9 protein (P0.001, HR 1.71, 95% CI: 1.45-2.34) was poorly associated with OS.
The results of the multifactor regression analysis show: T3/T4 staging (P0.001, HR 1.23, 95% CI: 1.03-1.67), N1/2 staging (P0.001, HR 1.51, 95% CI: 1.47-2.57), M1 staging (P0.001, HR 3.75, 95% CI: 2.83-4.56) and CUL9 protein high expression (P0.001, HR 1.55, 95% CI: 1.38-2.04) are all independent risk factors affecting overall CRC survival.
The invention provides a method for evaluating clinical characteristics and overall prognosis of a patient with colorectal cancer through positive expression of CUL9 cytoplasm of the colorectal cancer, namely, clinical characteristics and prognosis information of the patient can be directly determined according to expression of CUL9 cytoplasm in tumor tissues of the patient with the colorectal cancer.
Immunohistochemistry is a well-established experimental approach for assessing tumor tissue expression. Based on the previous experiments and the accumulation of samples, the invention aims to provide a method for evaluating colorectal cancer prognosis through immunohistochemical experiments. Experiments prove that the expression of CUL9 in colorectal cancer cytoplasm can obviously reflect the prognosis of colorectal cancer patients and has statistical difference by the statistics and analysis of the method.
Drawings
FIG. 1: drawing the colorectal cancer specimen;
FIG. 2: representative pictures of different expression levels of CUL 9;
FIG. 3: representative pictures of high and low expression of CUL 9;
FIG. 4: correlation of CUL9 expression level and prognosis of colorectal cancer patients.
Detailed Description
The invention is described in detail below with reference to the figures and specific embodiments.
Example 1
The method of the embodiment comprises the following steps:
a. the wax blocks of the 911 colorectal cancer tissues used in this example were all from the general surgery of the subsidiary zhongshan hospital of the university of fudan. The experiment passes through the ethical committee of Zhongshan hospital, and each patient signs informed consent.
b. Material taking: the wax block preparation is carried out on 911 samples required by the experiment, the 911 colorectal cancer tissue sampling positions are shown in figure 1, and the specific preparation mode of the wax block is as follows: the material is fresh; the size of the tissue block taken is about 2.0cm × 2.0cm × 0.3 cm; tissue blocks of approximately 0.8cm by 0.3cm were cut with a razor blade. Placing the tissue-containing plastic embedding frame into a disposable plastic embedding frame, marking by a marking pen, and placing the tissue-containing plastic embedding frame into ethanol with different concentration gradients in sequence in a dehydration step; generally, the tissue is soaked in a mixed solution of pure alcohol and a transparent agent for 1-2 hours, and then is soaked in the pure transparent agent; the process of immersing the tissue in molten paraffin after the tissue is transparent is called wax immersion. The plastic embedding frame is placed into a container containing molten paraffin in an oven. Pouring the melted paraffin into a stainless steel embedding frame, opening the plastic embedding frame, putting the tissue material block with the section facing downwards after being soaked with the paraffin into the stainless steel embedding frame by using heated tweezers, quickly cooling the tissue material block when the surface layer of the paraffin liquid is solidified, and preparing the paraffin block containing the tissue block after the paraffin liquid is completely solidified. The wax block should be flat and finished.
c. Preparing a tissue chip: before constructing the tissue chip, how many samples to test should be planned in advance, and then design should be performed accordingly (generally, it is enough to place 60-100 samples on a conventional slide glass); case and related wax lumps were collected: selecting tumor tissue wax blocks with follow-up data at different development stages, and marking representative points in the paraffin specimen according to the HE section, wherein the representative points comprise typical tumors and corresponding normal tissues, so as to construct a tumor tissue chip; TMA receptor wax block preparation: 97.5 g of lycra wax +2.5 g of beeswax (2.5%) were mixed to produce a blank wax block 36mm long by 26mm wide by 17mm high, and a 10X 7 dot tissue array was designed within 20mm by 16mm of the block. Reserving a space of 0.5cm-0.7cm around the tissue, and punching the tissue by using a tissue analyzer to prepare a TMA wax block; and (3) punching holes on the receptor wax block by using a fine needle on a tissue chip manufacturing machine, wherein the aperture is proper to be 1-1.5 mm, and punching holes at the corresponding part marked on the donor wax block to collect tissue cores. The aperture is also 1-1.5 mm; transferring the tissue cores into the wells of the receptor module, preferably with a spacing of 0.2mm between each tissue core; in order to prevent the phenomena of missing dots, slide sheets and slide sheets in the dotting, slicing, dyeing or immunohistochemical process, each sample can be loaded with 1-2 dots; the constructed TMA chip wax block was placed in a suitable plastic box and held tightly against displacement. Placing the receptor module in a 55 ℃ incubator for about 10 minutes, taking out the receptor module before the wax is completely dissolved, cooling the receptor module at room temperature to integrate the wax of the receptor module with the newly inserted small cylindrical tissue, taking off the wax block, and storing the receptor module in a 4 ℃ refrigerator for later use; before slicing, the wax block needs to be precooled for about 4h at 4 ℃, then clamped on a microtome for correction, and the correction is carried out until all tissues are complete. Sticking pre-cooled ice bag at-20 deg.C on the wax block for 5-10min, quickly and continuously slicing 30-50 sheets, and freezing the tissue block with ice bag or directly in freezing microtome until the tissue is completely cut. Respectively rinsing 4 μm continuous slices in cold water, naturally spreading, sequentially transferring the slices to 45 deg.C warm water, spreading for about 2min, air drying, baking at 60 deg.C for about 3min, and baking at 58 deg.C for 18h and-20 deg.C.
d. The sections were dewaxed and hydrated using xylene, the hydration being performed by gradient alcohol, specifically: washing the tissue slices with dimethylbenzene for 2 times, 10 minutes each time, 2 times with absolute ethyl alcohol, 5 minutes each time, 2 times with 95% alcohol, 5 minutes each time, 2 times with 75% alcohol, 5 minutes each time, so that paraffin on the surfaces of the tissue slices is removed and fully hydrated, washing the slices with tap water for 1min, and washing the slices with PBS buffer solution for 1 minute multiplied by 3 times;
e. EDTA antigen retrieval and sealing, immersing the tissue slice in an antigen retrieval solution (cargo number: P0085, Biyun, the antigen retrieval solution is configured according to a kit, is mainly used for performing antigen retrieval on formalin fixed paraffin embedded tissue slices, removing cross-linking among proteins caused by aldehyde fixing reagents, and fully exposing antigen epitopes in samples such as paraffin slices and the like so as to greatly improve the immunostaining effect), heating for 3 minutes at a high-fire level in a microwave oven, then heating for 4 minutes multiplied by 2 times at a low-fire level, cooling at room temperature after taking out, washing with PBS buffer solution for 5 minutes multiplied by 3 times; soaking the tissue slices in an inactivation solution prepared from 30% hydrogen peroxide and methanol for 30 minutes to inactivate endogenous peroxidase, and washing with PBS (phosphate buffer solution) for 5 minutes and 3 times; scrubbing tissue slices, dripping 50 microliters of confining liquid, namely 5% BSA (bovine serum albumin) aqueous solution on each tissue slice, incubating for 30 minutes, and confining nonspecific antigens;
f. using an anti-human CUL9 monoclonal antibody (cat # NBP1-02783, NOVUS antibody Co., Ltd.) in the following manner of 1: 200 was diluted in PBS (phosphate buffered saline) and incubated overnight at 4 ℃ and a secondary antibody (rabbit secondary antibody: cat # ab 6721; Abcam antibody Co., Ltd., diluted at a ratio of 1: 1000) was incubated at room temperature for 2 hours after washing with PBS at 4 ℃;
g. incubating for 30min at room temperature, developing with DAB, staining with hematoxylin, and fixing;
h. washing the tissue slice with 75% alcohol for 2 times, 5 minutes each time, 95% alcohol for 2 times, 5 minutes each time, absolute ethyl alcohol for 2 times, 5 minutes each time, and xylene for 2 times, 10 minutes each time; placing in a fume hood for 5min to volatilize xylene on the tissue slice, sealing with neutral gum (Catalina) (product number: S30509, Yuanyeau), and placing in an oven for 1 hr to solidify the gum as soon as possible;
i. and (4) analyzing results: positive staining of CUL9 was a brownish yellow particle on the cytoplasm of the cells, which were scored separately by immunohistochemistry in two individuals and re-assessed when the scoring was incongruous. And (3) scoring the dyeing result, wherein the scoring comprises two indexes: 1) dyeing strength: 0 part (-): 0% cell staining; 1 part (+): 0-10% incomplete staining of cells; 2 parts (++): 10% cells stained weakly to moderately completely; 3 points (+++): moderate to intense intact staining of 10% cells; 2) positive staining accounts for the proportion of the total field. The product of the two indices is the final immunohistochemical score (expression level) (fig. 2 is a representative picture). Cut-off values were median expressed (immunohistochemical score). High expression when the Cut-off value is larger than the Cut-off value, and low expression when the Cut-off value is smaller than or equal to the Cut-off value.
j. Statistical analysis: all data from this study were statistically analyzed using the SPSS statistical software package (22.0; SPSS inc., Chicago, Illinois, USA). Calculation and analysis of OS was performed using the Kaplan-Meier method. The risk factor is expressed as a hazard ratio [ HR, 95% Confidence Interval (CI) ]. A P value less than 0.05 is defined as statistically significant. The statistical analysis data is CUL9 immunohistochemical score data and correlation analysis of colorectal cancer clinical pathological characteristics.
k. Clinical relevance of CUL9 expression to colorectal cancer patients was analyzed and the results are shown in table 1.
Table 1: correlation analysis of CUL9 expression and colorectal cancer clinical characteristics
Table 1 appendix: CEA (carcinoembryonic antigen) detection is performed by detecting blood prior to tumor surgery. Histological type, tumor size, T stage, N stage, and M stage were determined based on patient surgical specimen examination according to AJCC international staging standard.
The chi square test analyzed the correlation of CUL9 expression with colorectal cancer clinical characteristics in 911 colorectal cancer specimens. The results show that CUL9 high expression is positively correlated with tumor diameter >4cm (P ═ 0.001), tumor low differentiation (P ═ 0.037), N1/2(P ═ 0.028) and M1(P ═ 0.001) (tumor size is the size of the surgical specimen, longest diameter is taken, degree of differentiation, T stage, N stage, M stage are statistically analyzed according to the AJCC international staging standard). FIG. 3 shows representative pictures of low expression and high expression of CUL 9. Continuing to analyze the effect of CUL9 expression on the assessment of prognosis in patients with colorectal cancer, table 2 and fig. 4 show that survival analysis shows a poorer prognosis in patients with CRC with high CUL9 expression.
TABLE 2 Single-and Multi-factor analysis of overall survival for CRC
Table 2 appendix: survival analysis adopts Kaplan-Meier method to draw survival curve, and Log-Rank is adopted to compare difference. Single/multifactor analysis Risk ratios (OR) and corresponding 95% Confidence Intervals (CI) were calculated using Logistic regression.
Single factor analysis found that tumor hypodifferentiation (P0.001, HR 1.24, 95% CI: 1.05-1.46), T3/T4 (P0.001, HR 1.98, 95% CI: 1.62-2.24), N1/2 (P0.001, HR 1.83, 95% CI: 1.32-2.54), M1 (P0.001, HR 4.55, 95% CI: 3.69-5.66) and CUL9 protein overexpression (P0.001, HR 1.71, 95% CI: 1.45-2.34) were all factors that led to OS shortening. The results of the above statistical differences, the multifactor regression analysis results show: T3/T4 staging (P0.001, HR 1.23, 95% CI: 1.03-1.67), N1/2 staging (P0.001, HR 1.51, 95% CI: 1.47-2.57), M1 staging (P0.001, HR 3.75, 95% CI: 2.83-4.56) and CUL9 protein high expression (P0.001, HR 1.55, 95% CI: 1.38-2.04) are all independent risk factors affecting overall CRC survival.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (10)
1. The use of CUL9 as a marker for the prognostic evaluation of colorectal cancer, wherein CUL9 as a marker is used for evaluating the clinical characteristics and prognosis of patients with colorectal cancer based on the positive expression of CUL9 cytoplasm.
2. The use of CUL9 as a marker for the prognostic evaluation of colorectal cancer according to claim 1, wherein the specific method comprises:
a. manufacturing a wax block and a tissue chip;
b. embedding the tissue chip;
c. dewaxing and hydrating;
d. repairing and sealing the antigen;
overnight incubation of cull 9 primary antibody;
f. incubating the second antibody at room temperature after washing, and developing a color and amplifying the solution;
g. performing DAB coloration after incubation;
h. staining nuclei with hematoxylin, and fixing;
i. dehydrating to wax, and keeping the slices;
j. reading the result;
k. statistical analysis: all data were statistically analyzed using the SPSS statistical software package, OS calculation and analysis were performed using the Kaplan-Meier method, risk factors were expressed as hazard ratio [ HR, 95% Confidence Interval (CI) ], P-value less than 0.05 was defined as statistically significant, and statistical analysis data were CUL9 immunohistochemical score data and correlation analysis of colorectal cancer clinical pathology.
3. The use of CUL9 as a marker for the prognostic evaluation of colorectal cancer according to claim 1, wherein the antigen recovery and blocking method comprises: immersing tissue slices in an antigen repairing solution prepared from EDTA, heating, taking out, cooling at room temperature, washing, immersing the tissue slices in an inactivating solution for inactivation, washing, scrubbing the tissue slices, dripping 5% BSA (bovine serum albumin) aqueous solution on each tissue slice as a sealing solution, incubating, and sealing non-specific antigens.
4. The use of CUL9 as a marker for the prognostic evaluation of colorectal cancer according to claim 1, wherein the result reading method in step j comprises:
the positive staining of CUL9 is that the cytoplasm of the cell presents brown yellow particles, two persons independently perform immunohistochemical scoring respectively, the scoring result is reevaluated when objection exists, and the staining result is scored, and the positive staining comprises two indexes: 1) dyeing strength: 0 part (-): 0% cell staining; 1 part (+): 0-10% incomplete staining of cells; 2 parts (++): 10% cells stained weakly to moderately completely; 3 points (+++): moderate to intense intact staining of 10% cells; 2) positive staining is proportional to the total field;
the product of the two indexes is the final immunohistochemical score, namely the expression quantity, the Cut-off value is the median of the expression quantity, the expression is high when the Cut-off value is larger than the Cut-off value, and the expression is low when the Cut-off value is smaller than or equal to the Cut-off value.
5. A method for verifying the relation between the high expression and low expression of CUL9 cytoplasm and the clinical characteristics and prognosis survival rate of colorectal cancer patients is characterized in that firstly, a tumor tissue chip is prepared, then an immunohistochemical experiment and an immune score are carried out, grouping is carried out according to the immune score, finally, the patients are divided into a CUL9 cytoplasm high expression group and a CUL9 cytoplasm low expression group according to the cytoplasm staining condition of CUL9, and then the clinical characteristics and the difference of the total survival rate of the two groups of patients are analyzed by a statistical method.
6. The method for verifying the relationship between the high expression and low expression of CUL9 cytoplasm and clinical characteristics and prognostic survival rate of patients with colorectal cancer according to claim 5, wherein the method comprises:
a. manufacturing a wax block and a tissue chip;
b. embedding the tissue chip;
c. dewaxing and hydrating;
d. repairing and sealing the antigen;
overnight incubation of cull 9 primary antibody;
f. incubating the second antibody at room temperature after washing, and developing a color and amplifying the solution;
g. performing DAB coloration after incubation;
h. staining nuclei with hematoxylin, and fixing;
i. dehydrating to wax, and keeping the slices;
j. reading the result;
k. statistical analysis: all data were statistically analyzed using the SPSS statistical software package, OS calculation and analysis were performed using the Kaplan-Meier method, risk factors were expressed as hazard ratio [ HR, 95% Confidence Interval (CI) ], P-value less than 0.05 was defined as statistically significant, and statistical analysis data were CUL9 immunohistochemical score data and correlation analysis of colorectal cancer clinical pathology.
7. The method for verifying the relationship between the high and low expression levels of CUL9 cytoplasm and clinical characteristics and prognostic survival rate of patients with colorectal cancer according to claim 6, wherein the antigen is repaired and blocked by: immersing tissue slices in an antigen repairing solution prepared from EDTA, heating, taking out, cooling at room temperature, washing, immersing the tissue slices in an inactivating solution for inactivation, washing, scrubbing the tissue slices, dripping 5% BSA (bovine serum albumin) aqueous solution on each tissue slice as a sealing solution, incubating, and sealing non-specific antigens.
8. The method for verifying the relationship between the high and low expression levels of CUL9 cytoplasm and clinical characteristics and prognostic survival rate of patients with colorectal cancer according to claim 6, wherein the result reading in step j comprises:
the positive staining of CUL9 is that the cytoplasm of the cell presents brown yellow particles, two persons independently perform immunohistochemical scoring respectively, the scoring result is reevaluated when objection exists, and the staining result is scored, and the positive staining comprises two indexes: 1) dyeing strength: 0 part (-): 0% cell staining; 1 part (+): 0-10% incomplete staining of cells; 2 parts (++): 10% cells stained weakly to moderately completely; 3 points (+++): moderate to intense intact staining of 10% cells; 2) positive staining is proportional to the total field;
the product of the two indexes is the final immunohistochemical score, namely the expression quantity, the Cut-off value is the median of the expression quantity, the expression is high when the Cut-off value is larger than the Cut-off value, and the expression is low when the Cut-off value is smaller than or equal to the Cut-off value.
9. The method for verifying the relationship between the high and low expression of CUL9 cytoplasm and clinical characteristics and prognostic survival rate of patients with colorectal cancer according to claim 5, wherein CUL9 high expression positively correlates with tumor diameter >4cm, tumor hypodifferentiation, N1/2 and M1; low differentiation of tumors, high expression of T3/T4, N1/2, M1 and CUL9 proteins are all factors that cause OS shortening.
10. The method for verifying the relation between the high and low expression levels of CUL9 cytoplasm and clinical characteristics and prognostic survival rate of patients with colorectal cancer according to claim 5, wherein the T3/T4 stage, N1/2 stage, M1 stage and the high expression level of CUL9 protein are independent risk factors affecting the overall survival rate of CRC.
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