CN110609141A - GLTSCR1 prostate cancer prognosis detection reagent and kit thereof - Google Patents
GLTSCR1 prostate cancer prognosis detection reagent and kit thereof Download PDFInfo
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Abstract
The invention discloses a GLTSCR1 prostate cancer prognosis detection reagent and a kit thereof, wherein the reagent comprises the following components (20 parts): h2O21ml, non-immune sheep serum working solution 1ml, primary antibody: rabbit anti-GLTSCR 1 polyclonal antibody 10 μ l, secondary antibody: 1ml of biotin-labeled goat anti-rabbit IgG working solution, 1ml of streptavidin-peroxidase solution and 2-3ml of DAB color developing agent. The GLTSCR1 prostate cancer prognosis detection kit comprises a detection reagent for detecting GLTSCR 1. The reagent of the invention can accurately predict the recurrence of the prostate cancer.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a reagent for GLTSCR1 prostate cancer prognosis detection, and also relates to a GLTSCR1 prostate cancer prognosis detection kit.
Background
Prostate cancer is the most common malignant disease of the male reproductive system worldwide, with the incidence of secondary malignancy among all malignancies in older men. The incidence of prostate cancer varies greatly from country to country. Prostate cancer has been more prevalent in the united states than lung cancer, and is the highest incidence of male malignancies. The incidence of prostate cancer in our country is far lower than that in the countries in Europe and America, but the incidence of prostate cancer in our country is on a remarkable rising trend in recent years. The current main treatment for localized prostate cancer is surgical treatment. However, there are still some patients who experience local recurrence and/or distant metastasis after surgical treatment. For patients with clinical relapse, biochemical relapse may occur in its early stages. Biochemical recurrence as prostate cancer can now be predicted by PSA. Despite the strong sensitivity of PSA, there are deficiencies in that it is not accurate in predicting clinical recurrence and in that it is not predictive of whether a patient will develop metastases. Moreover, the specificity is insufficient, and researches show that the PSA in blood plasma can be increased due to various diseases such as prostatic hyperplasia, prostatitis, acute urinary retention and the like. Therefore, the development of tumor markers with high sensitivity, strong specificity and stable results is an important research direction for predicting the recurrence of prostate cancer.
Glioma tumor suppressor candidate region gene 1 (GLTSCR candidate gene 1, GLTSCR1) has a relationship with the development and progression of a variety of cancers. The promotion of autophagy in a variety of cancers has been demonstrated. A key protein in the autophagy process is named MAP1S (Microtub μ le-associated protein1S), is a member of microtubule-associated protein family 1, and can regulate the mitotic process. No studies on the expression and biological effects of GLTSCR1 in prostate cancer have been found.
Disclosure of Invention
The invention aims to provide a GLTSCR1 prostate cancer prognosis detection reagent capable of accurately detecting the recurrence of prostate cancer.
The invention also aims to provide a kit containing the GLTSCR1 prostate cancer prognosis detection reagent.
The invention relates to a GLTSCR1 prostate cancer prognosis detection reagent, which comprises the following components (20 parts):
the invention discloses a GLTSCR1 prostate cancer prognosis detection kit, which comprises a detection reagent for detecting GLTSCR 1.
The invention discloses a GLTSCR1 prostate cancer prognosis detection kit, which comprises the following use methods:
(1) placing the slices subjected to dewaxing, hydration and antigen restoration in an incubation box, enclosing the tissues by a water-blocking pen to prevent the dropwise added reagent from diffusing, slightly absorbing water on the slices by absorbent paper without touching the tissues, and adding H2O250 μ l of the tissue was covered and incubated for 30min at room temperature to block the activity of endogenous peroxidase;
(2) washing with PBS for 3min, and repeating for 3 times;
(3) slightly absorbing PBS on the section by absorbent paper, dropwise adding 50 mu l of non-immune goat serum working solution, and incubating for 15 minutes at room temperature;
(4) gently removing serum without rinsing, adding 50 μ l of primary antibody dilution of rabbit-derived anti-GLTSCR 1 polyclonal antibody PBS 1:100, and incubating at room temperature for 60 min or overnight at 4 deg.C;
(5) washing with PBS for 5min, and repeating for 3 times;
(6) the section was slightly blotted with absorbent paper, and 50. mu.l of secondary antibody: incubating goat anti-rabbit IgG working solution labeled by biotin for 20 minutes at room temperature;
(7) washing with PBS for 5min, and repeating for 3 times;
(8) slightly absorbing PBS on the slices by absorbent paper, dropwise adding 50 mu l of streptavidin-peroxidase solution to each slice, and incubating for 15 minutes at room temperature;
(9) washing with PBS for 3min, and repeating for 3 times;
(10) slightly absorbing PBS liquid on the slices by absorbent paper, dropwise adding 100 mul of freshly prepared DAB color developing agent (50 mul of each of 20 XDAB buffer solution, 20 XDAB substrate and 20 XDAB chromogen is dropwise added into 850 mul of double distilled water to prepare 1ml of fresh color developing solution, the effective use time is 30min) for color development, fully washing by tap water, re-dyeing, dehydrating and transparentizing, and sealing the slices;
(11) judging standard of immunohistochemical detection result: under a Leica fluorescence microscope, five high-power lens fields are randomly selected for each section to observe cells, positive cells are obtained when brown or brownish particles appear in cytoplasm and/or nucleus, and the experimental result is judged according to the staining intensity of the positive cells and the number proportion of the positive cells in the total cells. Scoring by staining intensity: colorless is 0 minutes, yellowish is 1 minute, tan is 2 minutes, and tan is 3 minutes; then scored by positive cell rate: the number of the patients without positive staining in the tumor cells is 0, the positive cell rate is less than or equal to 10%, 1, 11-50% 2, 51-75% 3 and more than 75% 4, and the product of the two integrals is used as the judgment standard of the staining result: 0 is determined as-negative; 1-4 are divided into plus and weak positive; 5-8 are divided into + +, positive; 9-12 are divided into +++, strong positive.
Compared with the prior art, the invention has obvious beneficial effects, and the technical scheme can show that: according to the invention, MAP1S related genes are analyzed through a TCGA database, and screening shows that GLTSCR1 is moderately related to MAP1S in the mRNA expression level in prostate cancer (r is 0.460, P is less than 0.001), GLTSCR1 can promote the progression of the prostate cancer by regulating autophagy, and the expression of GLTSCR1 in prostate cancer tissues is higher than that of paracarcinoma, so that GLTSCR1 plays an important role in the proliferation and metastasis of the prostate cancer, therefore, GLTSCR1 is used as a biomarker for predicting the recurrence of the prostate cancer, and a feasible method is provided for the diagnosis of the prostate cancer and the prediction of the recurrence metastasis rate, the recurrence metastasis time and the survival time of a prostate cancer patient.
Drawings
FIG. 1: expression of GLTSCR1 in prostate cancer tissues and paracarcinoma tissues.
FIG. 2: GLTSCR1 expression in prostate cancer is correlated with the prognosis of biochemical relapse survival.
FIG. 3: GLTSCT1 affects the proliferative capacity of prostate cancer cells.
FIG. 4: GLTSCR1 affects prostate cancer cell invasiveness.
FIG. 5: GLTSCR1 affects prostate cancer cell migration ability.
Detailed Description
Example 1:
the reagent for detecting the prognosis of the prostate cancer by GLTSCR1 comprises the following components (20 parts):
the use method of the kit containing the formula comprises the following steps:
(1) placing the slices subjected to dewaxing, hydration and antigen restoration in an incubation box, enclosing the tissues by a water-blocking pen to prevent the dropwise added reagent from diffusing, slightly absorbing water on the slices by absorbent paper without touching the tissues, and adding H2O250 μ l of the tissue was covered and incubated for 30min at room temperature to block the activity of endogenous peroxidase;
(2) washing with PBS for 3min, and repeating for 3 times;
(3) slightly absorbing PBS on the section by absorbent paper, dropwise adding 50 mu l of non-immune goat serum working solution, and incubating for 15 minutes at room temperature;
(4) gently removing serum without rinsing, adding 50 μ l of primary antibody dilution of rabbit-derived anti-GLTSCR 1 polyclonal antibody PBS 1:100, and incubating at room temperature for 60 min or overnight at 4 deg.C;
(5) washing with PBS for 5min, and repeating for 3 times;
(6) the section was slightly blotted with absorbent paper, and 50. mu.l of secondary antibody: incubating goat anti-rabbit IgG working solution labeled by biotin for 20 minutes at room temperature;
(7) washing with PBS for 5min, and repeating for 3 times;
(8) slightly absorbing PBS on the slices by absorbent paper, dropwise adding 50 mu l of streptavidin-peroxidase solution to each slice, and incubating for 15 minutes at room temperature;
(9) washing with PBS for 3min, and repeating for 3 times;
(10) slightly absorbing PBS liquid on the slices by absorbent paper, dropwise adding 100 mul of freshly prepared DAB color developing agent (50 mul of each of 20 XDAB buffer solution, 20 XDAB substrate and 20 XDAB chromogen is dropwise added into 850 mul of double distilled water to prepare 1ml of fresh color developing solution, the effective use time is 30min) for color development, fully washing by tap water, re-dyeing, dehydrating and transparentizing, and sealing the slices;
(11) judging standard of immunohistochemical detection result: under a Leica fluorescence microscope, five high-power lens fields are randomly selected for each section to observe cells, positive cells are obtained when brown or brownish particles appear in cytoplasm and/or nucleus, and the experimental result is judged according to the staining intensity of the positive cells and the number proportion of the positive cells in the total cells. Scoring by staining intensity: colorless is 0 minutes, yellowish is 1 minute, tan is 2 minutes, and tan is 3 minutes; then scored by positive cell rate: the number of the patients without positive staining in the tumor cells is 0, the positive cell rate is less than or equal to 10%, 1, 11-50% 2, 51-75% 3 and more than 75% 4, and the product of the two integrals is used as the judgment standard of the staining result: 0 is determined as-negative; 1-4 are divided into plus and weak positive; 5-8 are divided into + +, positive; 9-12 are divided into +++, strong positive.
Example 2:
the reagent for detecting the prognosis of the prostate cancer by GLTSCR1 comprises the following components (20 parts):
the use method of the kit containing the formula comprises the following steps:
(1) placing the slices subjected to dewaxing, hydration and antigen restoration in an incubation box, enclosing the tissues by a water-blocking pen to prevent the dropwise added reagent from diffusing, slightly absorbing water on the slices by absorbent paper without touching the tissues, and adding H2O250 μ l of the tissue was covered and incubated for 30min at room temperature to block the activity of endogenous peroxidase;
(2) washing with PBS for 3min, and repeating for 3 times;
(3) slightly absorbing PBS on the section by absorbent paper, dropwise adding 50 mu l of non-immune goat serum working solution, and incubating for 15 minutes at room temperature;
(4) gently removing serum without rinsing, adding 50 μ l of primary antibody dilution of rabbit-derived anti-GLTSCR 1 polyclonal antibody PBS 1:100, and incubating at room temperature for 60 min or overnight at 4 deg.C;
(5) washing with PBS for 5min, and repeating for 3 times;
(6) the section was slightly blotted with absorbent paper, and 50. mu.l of secondary antibody: incubating goat anti-rabbit IgG working solution labeled by biotin for 20 minutes at room temperature;
(7) washing with PBS for 5min, and repeating for 3 times;
(8) slightly absorbing PBS on the slices by absorbent paper, dropwise adding 50 mu l of streptavidin-peroxidase solution to each slice, and incubating for 15 minutes at room temperature;
(9) washing with PBS for 3min, and repeating for 3 times;
(10) slightly absorbing PBS liquid on the slices by absorbent paper, dropwise adding 100 mul of freshly prepared DAB color developing agent (50 mul of each of 20 XDAB buffer solution, 20 XDAB substrate and 20 XDAB chromogen is dropwise added into 850 mul of double distilled water to prepare 1ml of fresh color developing solution, the effective use time is 30min) for color development, fully washing by tap water, re-dyeing, dehydrating and transparentizing, and sealing the slices;
(11) judging standard of immunohistochemical detection result: under a Leica fluorescence microscope, five high-power lens fields are randomly selected for each section to observe cells, positive cells are obtained when brown or brownish particles appear in cytoplasm and/or nucleus, and the experimental result is judged according to the staining intensity of the positive cells and the number proportion of the positive cells in the total cells. Scoring by staining intensity: colorless is 0 minutes, yellowish is 1 minute, tan is 2 minutes, and tan is 3 minutes; then scored by positive cell rate: the number of the patients without positive staining in the tumor cells is 0, the positive cell rate is less than or equal to 10%, 1, 11-50% 2, 51-75% 3 and more than 75% 4, and the product of the two integrals is used as the judgment standard of the staining result: 0 is determined as-negative; 1-4 are divided into plus and weak positive; 5-8 are divided into + +, positive; 9-12 are divided into +++, strong positive.
Example 3:
the reagent for detecting the prognosis of the prostate cancer by GLTSCR1 comprises the following components (20 parts):
the use method of the kit containing the formula comprises the following steps:
(1) placing the slices after deparaffinization, hydration and antigen restoration into the incubatorIn the incubator, the tissue is enclosed by a water-blocking pen to prevent the dripped reagent from diffusing, water on the section is slightly absorbed by absorbent paper without touching the tissue, and H is added2O250 μ l of the tissue was covered and incubated for 30min at room temperature to block the activity of endogenous peroxidase;
(2) washing with PBS for 3min, and repeating for 3 times;
(3) slightly absorbing PBS on the section by absorbent paper, dropwise adding 50 mu l of non-immune goat serum working solution, and incubating for 15 minutes at room temperature;
(4) gently removing serum without rinsing, adding 50 μ l of primary antibody dilution of rabbit-derived anti-GLTSCR 1 polyclonal antibody PBS 1:100, and incubating at room temperature for 60 min or overnight at 4 deg.C;
(5) washing with PBS for 5min, and repeating for 3 times;
(6) the section was slightly blotted with absorbent paper, and 50. mu.l of secondary antibody: incubating goat anti-rabbit IgG working solution labeled by biotin for 20 minutes at room temperature;
(7) washing with PBS for 5min, and repeating for 3 times;
(8) slightly absorbing PBS on the slices by absorbent paper, dropwise adding 50 mu l of streptavidin-peroxidase solution to each slice, and incubating for 15 minutes at room temperature;
(9) washing with PBS for 3min, and repeating for 3 times;
(10) slightly absorbing PBS liquid on the slices by absorbent paper, dropwise adding 100 mul of freshly prepared DAB color developing agent (50 mul of each of 20 XDAB buffer solution, 20 XDAB substrate and 20 XDAB chromogen is dropwise added into 850 mul of double distilled water to prepare 1ml of fresh color developing solution, the effective use time is 30min) for color development, fully washing by tap water, re-dyeing, dehydrating and transparentizing, and sealing the slices;
(11) judging standard of immunohistochemical detection result: under a Leica fluorescence microscope, five high-power lens fields are randomly selected for each section to observe cells, positive cells are obtained when brown or brownish particles appear in cytoplasm and/or nucleus, and the experimental result is judged according to the staining intensity of the positive cells and the number proportion of the positive cells in the total cells. Scoring by staining intensity: colorless is 0 minutes, yellowish is 1 minute, tan is 2 minutes, and tan is 3 minutes; then scored by positive cell rate: the number of the patients without positive staining in the tumor cells is 0, the positive cell rate is less than or equal to 10%, 1, 11-50% 2, 51-75% 3 and more than 75% 4, and the product of the two integrals is used as the judgment standard of the staining result: 0 is determined as-negative; 1-4 are divided into plus and weak positive; 5-8 are divided into + +, positive; 9-12 are divided into +++, strong positive.
Example 4:
the reagent for detecting the prognosis of the prostate cancer by GLTSCR1 comprises the following components (20 parts):
the use method of the kit containing the formula comprises the following steps:
(1) placing the slices subjected to dewaxing, hydration and antigen restoration in an incubation box, enclosing the tissues by a water-blocking pen to prevent the dropwise added reagent from diffusing, slightly absorbing water on the slices by absorbent paper without touching the tissues, and adding H2O250 μ l of the tissue was covered and incubated for 30min at room temperature to block the activity of endogenous peroxidase;
(2) washing with PBS for 3min, and repeating for 3 times;
(3) slightly absorbing PBS on the section by absorbent paper, dropwise adding 50 mu l of non-immune goat serum working solution, and incubating for 15 minutes at room temperature;
(4) gently removing serum without rinsing, adding 50 μ l of primary antibody dilution of rabbit-derived anti-GLTSCR 1 polyclonal antibody PBS 1:100, and incubating at room temperature for 60 min or overnight at 4 deg.C;
(5) washing with PBS for 5min, and repeating for 3 times;
(6) the section was slightly blotted with absorbent paper, and 50. mu.l of secondary antibody: incubating goat anti-rabbit IgG working solution labeled by biotin for 20 minutes at room temperature;
(7) washing with PBS for 5min, and repeating for 3 times;
(8) slightly absorbing PBS on the slices by absorbent paper, dropwise adding 50 mu l of streptavidin-peroxidase solution to each slice, and incubating for 15 minutes at room temperature;
(9) washing with PBS for 3min, and repeating for 3 times;
(10) slightly absorbing PBS liquid on the slices by absorbent paper, dropwise adding 100 mul of freshly prepared DAB color developing agent (50 mul of each of 20 XDAB buffer solution, 20 XDAB substrate and 20 XDAB chromogen is dropwise added into 850 mul of double distilled water to prepare 1ml of fresh color developing solution, the effective use time is 30min) for color development, fully washing by tap water, re-dyeing, dehydrating and transparentizing, and sealing the slices;
(11) judging standard of immunohistochemical detection result: under a Leica fluorescence microscope, five high-power lens fields are randomly selected for each section to observe cells, positive cells are obtained when brown or brownish particles appear in cytoplasm and/or nucleus, and the experimental result is judged according to the staining intensity of the positive cells and the number proportion of the positive cells in the total cells. Scoring by staining intensity: colorless is 0 minutes, yellowish is 1 minute, tan is 2 minutes, and tan is 3 minutes; then scored by positive cell rate: the number of the patients without positive staining in the tumor cells is 0, the positive cell rate is less than or equal to 10%, 1, 11-50% 2, 51-75% 3 and more than 75% 4, and the product of the two integrals is used as the judgment standard of the staining result: 0 is determined as-negative; 1-4 are divided into plus and weak positive; 5-8 are divided into + +, positive; 9-12 are divided into +++, strong positive.
Example 5:
the reagent for detecting the prognosis of the prostate cancer by GLTSCR1 comprises the following components (20 parts):
the use method of the kit containing the formula comprises the following steps:
(1) placing the slices subjected to dewaxing, hydration and antigen restoration in an incubation box, enclosing the tissues by a water-blocking pen to prevent the dropwise added reagent from diffusing, slightly absorbing water on the slices by absorbent paper without touching the tissues, and adding H2O250 μ l of the tissue was covered and incubated for 30min at room temperature to block the activity of endogenous peroxidase;
(2) washing with PBS for 3min, and repeating for 3 times;
(3) slightly absorbing PBS on the section by absorbent paper, dropwise adding 50 mu l of non-immune goat serum working solution, and incubating for 15 minutes at room temperature;
(4) gently removing serum without rinsing, adding 50 μ l of primary antibody dilution of rabbit-derived anti-GLTSCR 1 polyclonal antibody PBS 1:100, and incubating at room temperature for 60 min or overnight at 4 deg.C;
(5) washing with PBS for 5min, and repeating for 3 times;
(6) the section was slightly blotted with absorbent paper, and 50. mu.l of secondary antibody: incubating goat anti-rabbit IgG working solution labeled by biotin for 20 minutes at room temperature;
(7) washing with PBS for 5min, and repeating for 3 times;
(8) slightly absorbing PBS on the slices by absorbent paper, dropwise adding 50 mu l of streptavidin-peroxidase solution to each slice, and incubating for 15 minutes at room temperature;
(9) washing with PBS for 3min, and repeating for 3 times;
(10) slightly absorbing PBS liquid on the slices by absorbent paper, dropwise adding 100 mul of freshly prepared DAB color developing agent (50 mul of each of 20 XDAB buffer solution, 20 XDAB substrate and 20 XDAB chromogen is dropwise added into 850 mul of double distilled water to prepare 1ml of fresh color developing solution, the effective use time is 30min) for color development, fully washing by tap water, re-dyeing, dehydrating and transparentizing, and sealing the slices;
(11) judging standard of immunohistochemical detection result: under a Leica fluorescence microscope, five high-power lens fields are randomly selected for each section to observe cells, positive cells are obtained when brown or brownish particles appear in cytoplasm and/or nucleus, and the experimental result is judged according to the staining intensity of the positive cells and the number proportion of the positive cells in the total cells. Scoring by staining intensity: colorless is 0 minutes, yellowish is 1 minute, tan is 2 minutes, and tan is 3 minutes; then scored by positive cell rate: the number of the patients without positive staining in the tumor cells is 0, the positive cell rate is less than or equal to 10%, 1, 11-50% 2, 51-75% 3 and more than 75% 4, and the product of the two integrals is used as the judgment standard of the staining result: 0 is determined as-negative; 1-4 are divided into plus and weak positive; 5-8 are divided into + +, positive; 9-12 are divided into +++, strong positive.
The use method of the kit containing the formula comprises the following steps:
(1) placing slices subjected to deparaffinization, hydration and antigen retrieval in an incubation box, and using a stopperEnclosing the tissue with the water pen to prevent the dropwise added reagent from diffusing, slightly absorbing the water on the section with the absorbent paper without touching the tissue, and adding H2O250 μ l of the tissue was covered and incubated for 30min at room temperature to block the activity of endogenous peroxidase;
(2) washing with PBS for 3min, and repeating for 3 times;
(3) slightly absorbing PBS on the section by absorbent paper, dropwise adding 50 mu l of non-immune goat serum working solution, and incubating for 15 minutes at room temperature;
(4) gently removing serum without rinsing, adding 50 μ l of primary antibody dilution of rabbit-derived anti-GLTSCR 1 polyclonal antibody PBS 1:100, and incubating at room temperature for 60 min or overnight at 4 deg.C;
(5) washing with PBS for 5min, and repeating for 3 times;
(6) the section was slightly blotted with absorbent paper, and 50. mu.l of secondary antibody: incubating goat anti-rabbit IgG working solution labeled by biotin for 20 minutes at room temperature;
(7) washing with PBS for 5min, and repeating for 3 times;
(8) slightly absorbing PBS on the slices by absorbent paper, dropwise adding 50 mu l of streptavidin-peroxidase solution to each slice, and incubating for 15 minutes at room temperature;
(9) washing with PBS for 3min, and repeating for 3 times;
(10) slightly absorbing PBS liquid on the slices by absorbent paper, dropwise adding 100 mul of freshly prepared DAB color developing agent (50 mul of each of 20 XDAB buffer solution, 20 XDAB substrate and 20 XDAB chromogen is dropwise added into 850 mul of double distilled water to prepare 1ml of fresh color developing solution, the effective use time is 30min) for color development, fully washing by tap water, re-dyeing, dehydrating and transparentizing, and sealing the slices;
(11) judging standard of immunohistochemical detection result: under a Leica fluorescence microscope, five high-power lens fields are randomly selected for each section to observe cells, positive cells are obtained when brown or brownish particles appear in cytoplasm and/or nucleus, and the experimental result is judged according to the staining intensity of the positive cells and the number proportion of the positive cells in the total cells. Scoring by staining intensity: colorless is 0 minutes, yellowish is 1 minute, tan is 2 minutes, and tan is 3 minutes; then scored by positive cell rate: the number of the patients without positive staining in the tumor cells is 0, the positive cell rate is less than or equal to 10%, 1, 11-50% 2, 51-75% 3 and more than 75% 4, and the product of the two integrals is used as the judgment standard of the staining result: 0 is determined as-negative; 1-4 are divided into plus and weak positive; 5-8 are divided into + +, positive; 9-12 are divided into +++, strong positive.
Experimental example: selection assay for markers indicative of prostate cancer proliferation and metastasis
1. Expression of GLTSCR1 in prostate cancer and paraneoplastic tissues
The expression of GLTSCR1 was detected in 73 prostate cancers and 7 prostate normal tissues by immunohistochemical chips. As shown in fig. 1A-E, GLTSCR1 stained moderately in the cytoplasm of prostate cancer cells and slightly in the cytoplasm of normal prostate tissue. And of the 73 prostate cancer samples, 26 (35.6%) samples showed low expression of GLTSCR1, and 47 (64.4%) samples showed high expression of GLTSCR 1. The expression level of GLTSCR1 in prostate cancer tissue was significantly higher than in normal prostate tissue. (IRS: prostate cancer 4.82 ± 0.99vs. normal prostate tissue 3.86 ± 0.69, P0.015) (fig. 1B).
The samples were now fixed in 10% neutral formalin and then paraffin embedded. Paraffin sections were 4 μm thick, xylene deparaffinized, gradient ethanol hydrated, tissue blocked with peroxidase, and then incubated with GLTSCR1 primary antibody (rabbit polyclonal antibody, 10977-1-AP, Proteintech Group, inc. us) overnight at 4 ℃ at a dilution of 1: 500. DAB (China fir gold bridge) coloration after incubation of the secondary antibody. For each immunohistochemistry, no primary antibody was incubated as a negative control. The immunohistochemical staining intensity was assessed by double-blind methods and if the two were different, they were re-scored to be consistent.
2. GLTSCR1 related to prostate cancer clinicopathological parameters
The expression level of GLTSCR1 and the clinical and pathological parameters of the tissue chip were analyzed for correlation. Analysis indicated that high expression of GLTSCR1 was significantly associated with clinical staging of prostate cancer (P <0.001), tumor aggressiveness (P0.003), lymph node metastasis (P0.003) and distant metastasis (P0.001). However, we found that the expression level of GLTSCR1 had no significant correlation with patient age (P >0.05) (Table 1)
TABLE 1 relationship of expression of prostate cancer GLTSCR1 to the clinical pathology of patients
3. GLTSCR1 can be used as an independent prognostic factor for assessing survival of prostate cancer patients.
Kaplan-Meier analysis was first performed on the relationship between GLTSCR1 and clinical survival for prostate cancer patients in the TCGA database. The analysis indicated that the overall survival and the survival without BCR for the prostate cancer patients with high expression of GLTSCR1 were significantly lower than those with low expression of GLTSCR1 (P0.028 and P0.004) (FIG. 2). GLTSCR1 was then shown by one-way and multi-way Cox regression analysis to be an independent prognostic factor for prognosis of survival without biochemical recurrence in prostate cancer patients (Table 2), indicating that GLTSCR1 can be used as a tumor marker for prostate cancer recurrence.
TABLE 2 Single and Multi-factor analysis of prostate cancer survival time
4. GLTSCR1 enhances the proliferative capacity of prostate cancer cells.
The analysis through a CCK-8 experiment shows that: GLTSCR1 was knocked out in both a group of DU145 cell strains and LNCap cell strains, wherein GLTSCR1 knocked-out groups (DU145-sh-GLTSCR1-c and LNCap-sh-GLTSCR1-b) were experimental groups, and non-knocked-out groups (DU145-sh-NC and LNCap-sh-NC) were control groups. The CCK8 experiment results show that: compared with DU145-sh-NC and LNCap-sh-NC of a control group, the OD values of two groups of cells of two cell lines have no significant difference (all P is more than 0.05) when DU145-sh-GLTSCR1-c and LNCap-sh-GLTSCR1-b cells of the experimental group are 4 hours; there was a significant difference in the OD values of the cells in both groups for the remaining 24h, 48h and 72h cell lines (all P < 0.05). Indicating that the proliferative capacity of the prostate cancer cells is inhibited after knocking out GLTSCR1 (P < 0.05). The above experimental results suggest that GLTSCR1 enhances the proliferative capacity of prostate cancer cells. (see FIG. 3)
Fourthly, the method comprises the following steps: GLTSCR1 promotes the invasive capacity of prostate cancer cells.
The GLTSCR1 silent group (DU145-sh-GLTSCR1) was the experimental group and the non-silent GLTSCR1 group (DU145-sh-NC) was the control group. The Transwell experiment found that: the number of cells from the experimental group that DU145-sh-GLTSCR1 migrated to the lower chamber was 60.5% of the control group DU145-sh-NC (72. + -. 2.0 vs 119. + -. 2.8, P < 0.001). It is shown that the invasion capacity of DU145 cells is reduced after the expression of GLTSCR1 is reduced, and the promotion effect of GLTSCR1 on the invasion capacity of DU145 cells is shown (see FIGS. 4A and 4B). In the LNCap group cell lines performed simultaneously, the GLTSCR1 silencing group (LNCap-sh-GLTSCR1) was the experimental group and the non-silencing GLTSCR1 group (LNCap-sh-NC) was the control group. The Transwell experiment found that: the number of cells from the experimental group that LNCap-sh-GLTSCR1 migrated to the lower chamber was 85.3% of the control group LNCap-sh-NC (70. + -. 6.0 vs 82. + -.2.0, P < 0.05). Indicating that the invasion capacity of LNCap cells is reduced after the expression of GLTSCR1 is reduced, and indicating that GLTSCR1 has a promoting effect on the invasion capacity of LNCap cells (see FIGS. 4C and 4D). The two groups of cell lines were consistent, suggesting that GLTSCR1 has a promoting effect on prostate cancer cell invasion.
Fifthly: GLTSCR1 promotes the migratory capacity of prostate cancer cells.
The GLTSCR1 silent group (DU145-sh-GLTSCR1) was the experimental group and the non-silent GLTSCR1 group (DU145-sh-NC) was the control group. The result of the round Healing experiment shows that: compared with a control group (DU145-sh-NC), the mobility of the experimental group (DU145-sh-GLTSCR1) at 24h is higher than that of the experimental group (DU145-sh-GLTSCR1), and the experimental group (DU145-sh-GLTSCR1) has statistical significance (p is less than 0.05); due to the strong migration ability of DU145 cells, the experimental results of the experimental group and the control group have no obvious statistical significance at 48 h. (see FIGS. 5A and 5B) show that the migration capacity of DU145 cells is reduced after down-regulating the expression of GLTSCR1, indicating that GLTSCR1 has a promoting effect on the migration capacity of DU145 cells. In the LNCap group cell lines performed simultaneously, the GLTSCR1 silencing group (LNCap-sh-GLTSCR1) was the experimental group and the non-silencing GLTSCR1 group (LNCap-sh-NC) was the control group. The result of the round Healing experiment shows that: compared with the control group (LNCap-sh-NC), the mobility of the LNCap-sh-NC at 72h in the experimental group (LNCap-sh-GLTSCR1) is higher than that of the LNCap-sh-GLSCR1, and the statistical significance is achieved (p is less than 0.05). (see FIGS. 5C and 5D) show that upon downregulation of GLTSCR1 expression, the migratory capacity of LNCap cells is reduced, suggesting that GLTSCR1 has a promoting effect on the migratory capacity of LNCap cells. The two groups of cell lines were consistent, suggesting that GLTSCR1 indeed has a promoting effect on the migratory capacity of prostate cancer cells.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any simple modification, equivalent change and modification made to the above embodiment according to the technical spirit of the present invention are within the scope of the present invention without departing from the technical spirit of the present invention.
Claims (4)
1. The reagent for detecting the prognosis of the prostate cancer by GLTSCR1 comprises the following components (20 parts):
。
2. the GLTSCR1 prostate cancer prognostic test kit according to claim 1, comprising a detection reagent for GLTSCR 1.
3. The method of using GLTSCR1 prostate cancer prognostic test reagent according to claim 2, comprising the steps of:
(1) placing the slices subjected to dewaxing, hydration and antigen restoration in an incubation box, enclosing the tissues by a water-blocking pen to prevent the dropwise added reagent from diffusing, slightly absorbing water on the slices by absorbent paper without touching the tissues, and adding H2O250 μ l of the tissue was covered and incubated for 30min at room temperature to block the activity of endogenous peroxidase;
(2) washing with PBS for 3min, and repeating for 3 times;
(3) slightly absorbing PBS on the section by absorbent paper, dropwise adding 50 mu l of non-immune goat serum working solution, and incubating for 15 minutes at room temperature;
(4) gently removing serum without rinsing, adding 50 μ l of primary antibody dilution of rabbit-derived anti-GLTSCR 1 polyclonal antibody PBS =1:100, and incubating at room temperature for 60 min or overnight at 4 ℃;
(5) washing with PBS for 5min, and repeating for 3 times;
(6) the section was slightly blotted with absorbent paper, and 50. mu.l of secondary antibody: incubating goat anti-rabbit IgG working solution labeled by biotin for 20 minutes at room temperature;
(7) washing with PBS for 5min, and repeating for 3 times;
(8) slightly absorbing PBS on the slices by absorbent paper, dropwise adding 50 mu l of streptavidin-peroxidase solution to each slice, and incubating for 15 minutes at room temperature;
(9) washing with PBS for 3min, and repeating for 3 times;
(10) slightly absorbing PBS on the slices by absorbent paper, dropwise adding 100 mu l of freshly prepared DAB color developing agent for color development, fully washing by tap water, re-dyeing, dehydrating and sealing;
(11) judging standard of immunohistochemical detection result: under a Leica fluorescence microscope, randomly selecting five high-power lens fields for observing cells from each section, taking positive cells with brownish yellow or brownish brown particles appearing in cytoplasm and/or nucleus, and judging the experimental result according to the staining intensity of the positive cells and the number proportion of the positive cells in the total cells;
scoring by staining intensity: colorless is 0 minutes, yellowish is 1 minute, tan is 2 minutes, and tan is 3 minutes; then scored by positive cell rate: the number of the patients without positive staining in the tumor cells is 0, the positive cell rate is less than or equal to 10%, 1, 11-50% 2, 51-75% 3 and more than 75% 4, and the product of the two integrals is used as the judgment standard of the staining result: 0 is determined as-negative; 1-4 are divided into plus and weak positive; 5-8 are divided into + +, positive; 9-12 are divided into +++, strong positive.
4. The method of using GLTSCR1 prostate cancer prognostic test reagent according to claim 3, wherein: the DAB color developing agent preparation method in the step (10) is that 50 mul of 20 XDAB buffer solution, 20 XDAB substrate and 20 XDAB chromogen are respectively dripped into 850 mul of double distilled water to prepare 1ml of fresh color developing solution, and the effective service time is 30 min.
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