CN111323590B - Application of anti-TIF 1 gamma-IgA and anti-TIF 1 gamma-IgG serving as combined diagnostic markers in lung cancer diagnosis - Google Patents
Application of anti-TIF 1 gamma-IgA and anti-TIF 1 gamma-IgG serving as combined diagnostic markers in lung cancer diagnosis Download PDFInfo
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Abstract
The invention provides application of anti-TIF 1 gamma-IgA and anti-TIF 1 gamma-IgG serving as combined diagnosis markers in early diagnosis of lung cancer, and belongs to the technical field of medical diagnosis. The invention discovers that the TIF1gamma protein has abnormal high expression in the cancer tissues of patients with early lung cancer for the first time; meanwhile, strong humoral immune response aiming at TIF1gamma is found in a patient with early lung cancer, wherein serum anti-TIF 1 gamma-IgA and TIF1 gamma-IgG autoantibodies can be a marker for early diagnosis of lung cancer, and the combined detection of the anti-TIF 1 gamma-IgA and the anti-TIF 1 gamma-IgG can obviously improve the sensitivity of early diagnosis of the patient with the lung cancer on the basis of ensuring the specificity, and is suitable for being developed into a combined detection kit with clinical application value. Therefore, the combined detection of the anti-TIF 1 gamma-IgA and the anti-TIF 1 gamma-IgG is helpful for further improving the early serological screening quality of the lung cancer patients, so that the cure rate and the 5-year survival rate of the lung cancer are improved.
Description
Technical Field
The invention belongs to the technical field of medical diagnosis, and particularly relates to application of anti-TIF 1 gamma-IgA and anti-TIF 1 gamma-IgG serving as combined diagnosis markers in early diagnosis of lung cancer.
Background
Lung cancer is the leading malignant tumor with high morbidity and mortality in the world today, and has great harm to human health. Early diagnosis and timely radical surgical resection have great significance for prognosis of Lung Cancer patients, and research shows that 5-year survival rate of Lung Cancer patients receiving surgery in Stage I can reach 92% [ Goldstraw P, Chansky K, Crowley J, et al.the IASLC Lung Cancer Stage Project: Propusals for review of the TNM Stage group in the Forthcoming (Eighth) Edition of the TNM Classification for Lung cancer.JThorac Oncol.2016; 11(1) 39-51. ]; however, lung cancer often has no specific clinical symptoms at the early stage and is difficult to detect, and most patients are in the late stage of the disease at the visit, so that the optimal treatment period of the surgery is lost, and the 5-year survival rate is only 5% -15% [ Jantus-lewitre E, Uso M, Sanmartin E, cams c.update on biomarkers for the detection of lung cancer. lung cancer (auckl) 2012; 3:21-29.]. Therefore, increasing the efficiency of early diagnosis of lung cancer plays an important role in improving the prognosis of patients.
The serological indexes have the advantages of convenience in detection, safety and the like, but the currently common tumor antigen markers have the problems of detection sensitivity or specificity in different degrees, the clinical practical application effect is not ideal, and particularly the early diagnosis value is limited. Recent studies have found that a small amount of abnormally expressed Tumor Associated Antigens (TAAs) can stimulate B cells to produce a large amount of autoantibodies during the development of cancer, which makes the detection of serum autoantibody markers useful for early diagnosis of cancer. The literature reports that autoantibodies to TAAs can be detected in serum in lung cancer patients 5 years before imaging changes occur [ Zhong L, Coe SP, StrombergAJ, Khattar NH, Jett JR, hirschlowz ea. profiling tumor-associated antibodies for early detection of non-small cell cancer. jthorac one.2006; 1(6):513-519.].
More and more studies have demonstrated that TAAs abnormally expressed in early cancer stages induce humoral immune responses in the body, generate autoantibodies against TAAs and release them into peripheral blood, and normal humans and cancer patients can be distinguished by detecting autoantibodies against TAAs in peripheral blood. Even if the TAA level is very low, the immune system can still detect the existence of the specific protein, and a large amount of generated antibodies play a certain amplification role on antigen signals, so that the autoantibodies have higher sensitivity in the early stage of cancer compared with antigen markers; secondly, the generated tumor-associated autoantibodies have long half-life, can stably exist in serum for a long time, and are easy to detect clinically. These features of TAAs-related autoantibodies make it promising as an ideal serological marker for early diagnosis of lung cancer. Therefore, it is crucial to find new early lung cancer biomarkers as non-invasive screening tests to improve patient survival.
Transcription mediator 1gamma (transcription intermediary factor-1 gamma, TIF1 gamma), a regulator of TGF-beta/Smad pathway, has E3 ubiquitin protein ligase activity. Research shows that TIF1gamma can be involved in regulation and control of cell proliferation, differentiation and migration invasion by inhibiting Smad4 from regulating TGF-beta signaling pathway. In recent years, the relevance of TIF1gamma to tumors has attracted attention. The studies of Jain S et al showed that TIF1 γ is abnormally highly expressed in intestinal cancer tissues, as expressed by TIF1 γ protein in stage i and ii intestinal cancer tissues significantly upregulated compared to normal intestinal mucosal tissues, and correlated with the progression of the disease course in patients [ Jain S, Singhal S, Francis F, et al.association of overexpression of TIF1gamma with clinical cancer and advanced clinical anticancer. 17(35):3994-4000.]. Abnormal high expression of TIF1gamma is also found in breast cancer, Kassem L and the like find that positive staining rate of nuclear TIF1gamma and cytoplasmic TGF-beta 1 is as high as 35.9% and 30.4% through immunohistochemical detection of 248 breast cancer tissues, and high expression of TIF1gamma and TGF-beta 1 is closely related to poor prognosis of patient far-end metastasis, relapse and the like [ Kassem L, Deygas M, Fattet L, et al. TIF1gamma interferons with TGFbeta1/SMAD4 signaling to protein pore acoustic in optimal breast cancer. BMC cancer. 2015; 15:453.]. The above studies indicate that abnormally high expression of TIF1 γ may be involved in the development and progression of intestinal cancer and breast cancer, but the literature reports that TIF1 γ is significantly down-regulated in the expression of monocytes and pancreatic cancer [ Aucagne R, Droin N, paggeti J, et al. transcription interface factor 1gamma is a tumor tissue in mouse and human serum myelogenous leukemia. J. toxin invest.2011; 2361-; 180(6) < 2214 > -2221 >, which suggests that the action modes of the TIF1gamma involved in the development of different tumors may be different, and the manifestations are complicated.
In addition, Wang L et al tested the expression of TIF1 γ mRNA in cancer tissues and matched paracancerous tissues of 13 patients with non-small cell lung cancer (NSCLC) using Real-time PCR, and showed that TIF1 γ was attenuated in cancer tissues [ Wang L, Lei Z, Liu X, Liu R, Zhang H. [ Association of stimulation and methylation in the promoter region of TIF1gamma with non-small cell lung cancer ]. Zhongguo Fei Ai Za Zhi 2013; 16(5):227-232.].
Disclosure of Invention
The inventor shows that the positive expression rate of TIF1gamma in cancer tissues is 83.33% (50/60) and is obviously higher than 25.00% (15/60) of matched paracancerous tissues (P is less than 0.01) through immunohistochemical detection of cancer tissues and matched paracancerous tissues of 60 cases of early-stage lung cancer patients, and the result indicates that high-level autoantibodies aiming at TIF1gamma possibly exist in serum of the early-stage lung cancer patients, so that the abnormal high expression of TIF1gamma in the cancer tissues of the lung cancer patients is proved on a protein layer, and the prejudice of the prior art is overcome.
In view of the abnormally high expression of protein TIF1gamma in the cancer tissues of patients in early stage of lung cancer, the inventors speculate that TIF1gamma may stimulate the body to produce autoantibodies against TIF1 gamma. Furthermore, the inventor thinks that the same antigen can stimulate the organism to produce autoantibodies of different Ig types, so that an ELISA method aiming at four Ig type anti-TIF 1gamma antibodies is constructed, and the detection result shows that the levels and the positive rates of serum anti-TIF 1 gamma-IgM and TIF1 gamma-IgE of a patient group in the early stage of lung cancer are not obviously different compared with those of LBL and HC groups (P is more than 0.05); however, it is exciting that the level and positive rate of anti-TIF 1 gamma-IgG and TIF1 gamma-IgA are both significantly higher than LBL and HC group (P <0.05), especially the sensitivity of anti-TIF 1 gamma-IgA to LC early diagnosis reaches 28.2% (specificity 95.93%), AUC 0.704, and is higher than the sensitivity of anti-TIF 1 gamma-IgG at 18.54% (specificity 94.25%) and AUC 0.622. In addition, through the identification of a western blot method, the serum of early-stage lung cancer patients of anti-TIF 1 gamma-IgA and TIF1 gamma-IgG antibodies (+) detected by ELISA is only combined with target protein and is not combined with tag protein GST; LBL patients who detect autoantibodies (-) by ELISA, and serum of HC are not combined with target protein and label protein. The above results further demonstrate that there are a number of IgA-and IgG-type autoantibodies to TIF1 γ in sera from patients with early stage lung cancer.
Because the sensitivity of the single detection of IgA and IgG anti-TIF 1gamma antibodies to the early diagnosis of lung cancer is limited, the inventor further carries out the analysis of the combined detection of anti-TIF 1 gamma-IgA and TIF1 gamma-IgG, and the result shows that the combination of the two can improve the diagnosis sensitivity of a patient with early lung cancer to 38.31 percent and improve the AUC to 0.734 on the basis of ensuring the specificity (92.34 percent), which shows that the combined detection of anti-TIF 1 gamma-IgA and TIF1 gamma-IgG autoantibody is very helpful to further improve the early diagnosis performance of lung cancer.
Compared with the prior art, the invention has the advantages and the progressiveness that: the TIF1gamma protein is found to be abnormally and highly expressed in cancer tissues of patients in early lung cancer for the first time; meanwhile, strong humoral immune response aiming at TIF1gamma is found in a patient with early lung cancer, wherein serum anti-TIF 1 gamma-IgA and TIF1 gamma-IgG autoantibodies can be a marker for early diagnosis of lung cancer, and the combined detection of the anti-TIF 1 gamma-IgA and the anti-TIF 1 gamma-IgG can obviously improve the sensitivity of early diagnosis of the patient with the lung cancer on the basis of ensuring the specificity, and is suitable for being developed into a combined detection kit with clinical application value. Therefore, the combined detection of the anti-TIF 1 gamma-IgA and the anti-TIF 1 gamma-IgG is helpful for further improving the early serological screening quality of the lung cancer patients, so that the cure rate and the 5-year survival rate of the lung cancer are improved.
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FIG. 1: immunohistochemical detection of TIF1gamma protein in cancer tissues and paired paracarcinoma tissues of patients with early stage lung cancer (x 400); (a, c) TIF1gamma is strongly expressed in cancer tissues, and is respectively 3+ and 2 +; (b, d) TIF1gamma negative or weak expression in paraneoplastic tissues.
FIG. 2: comparison of the levels of autoantibodies against different subtypes of TIF1 γ from each group of sera; (a) TIF1 γ -IgA; (b) TIF1 gamma-IgG; (c) TIF1 γ -IgM; (d) TIF1 γ -IgE (. about.. about.indicates P <0.01, Early LC: Early lung cancer, LBL: benign lung lesion, HC: healthy control).
FIG. 3: comparing ROC curves of early lung cancer patients by single and combined detection of anti-TIF 1 gamma-IgA and IgG serum autoantibody; (a) ROC curves for TIF1 γ -IgA and TIF1 γ -IgG alone; (b) ROC curve for TIF1 γ -IgA combined with TIF1 γ -IgG.
FIG. 4: western blot identification of serum TIF1 γ -IgA and TIF1 γ -IgG; (a) TIF1 γ -IgA; (b) TIF1 gamma-IgG.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The present invention will be described in further detail with reference to the following embodiments. It will be understood by those skilled in the art that the following examples are illustrative of the present invention only and should not be taken as limiting the scope of the invention. In addition, the specific technical operation steps or conditions not indicated in the examples are performed according to the technical or conditions described in the literature in the field or according to the product specification. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1
1 materials and methods
1.1 study object
248 patients with early lung cancer, 200 patients with benign lung disease (LBL), and 218 Healthy Controls (HC), all from the hospital in fujian province during 2016, 9 months to 2018, 10 months. All patients strictly meet the international or professional society recognized diagnostic criteria and have not previously received any form of anti-tumor therapy, and the clinical and pathological data of the patients are shown in table 1. HC is a health examiner from the hospital examination center of Fujian province, and eliminates malignant tumors of the lung and other parts and benign lung lesions with non-malignant tumor properties, such as pneumonia, lung abscess, and the like. Collecting 5ml of fasting peripheral venous blood of all the study objects before operation, centrifuging at 4 ℃ and 3000rpm for 10min, collecting serum, and freezing and storing at-80 ℃ for later use; cancer tissues and paired paracarcinoma tissues were randomly selected from 60 of 248 early-stage lung cancer patients for IHC detection. All of the above subjects were informed and given informed consent.
TABLE 1 clinical pathological data of early lung cancer, LBL and HC groups
LBL: benign lesions of the lung; HC: healthy controls
1.2 immunohistochemical detection (IHC)
The IHC detection Kit of TIF1gamma is UltraSensitive SAP (Mouse/Rabbit) IHC Kit (New Biotechnology development company, Mich, Fuzhou), and the operation is strictly performed according to the instruction. The preparation method comprises the following steps of flattening cancer tissues of a lung cancer patient and matched paracancerous tissue section specimens in a water bath, baking for 24h at 37 ℃, dewaxing, hydrating and antigen retrieval, sealing serum, dropwise adding a 1:1000 diluted anti-TIF 1gamma antibody (Cell Signaling Technology, USA), a biotin-labeled secondary antibody and a streptavidin-peroxidase solution, finally performing DAB color development, hematoxylin counterstaining, dehydrating to be transparent, and sealing. Each section was randomly viewed through 5 high power fields and read by two experienced pathologists. The staining intensity was graded as: negative 0, weak positive 1+, medium positive 2+, strong positive 3 +.
1.3 enzyme-linked immunosorbent assay (ELISA)
The GST-labeled recombinant TIF1gamma protein 50 ng/hole is coated on a 96 enzyme-labeled pore plate, 100 mu l/hole, and the temperature is kept at 4 ℃ overnight; PBST washing 3 times, 3% BSA in 37 degrees C water bath blocking for 60 min; washing PBST for 1 time, adding diluted serum samples (anti-TIF 1 gamma-IgG, anti-TIF 1 gamma-IgM antibody are diluted at a ratio of 1:500, anti-TIF 1 gamma-IgA, anti-TIF 1 gamma-IgE antibody are diluted at a ratio of 1: 100), and incubating for 1h at 37 ℃; PBST was washed 4 times, diluted HRP-labeled goat anti-human IgG, IgM antibody (1:40000) (Jackson Immuno, USA), anti-IgA antibody (1:20000) (Jackson Immuno, USA) and anti-IgE antibody (1:20000) (Thermofish scientific, USA) were added, and PBST was washed 4 times after incubation at 37 ℃ for 30 minutes; adding Tetramethylbenzidine (TMB) and 3% H2O250 mul each, and developing for 10 minutes in dark; 2mol/LH2SO4The color development was stopped at 50. mu.l and the absorbance (OD) at a wavelength of 450nm was measured per well by a microplate reader.
1.4 immunoblot assay (Western blot)
After boiling denaturation of GST-labeled recombinant proteins TIF1gamma (150kD)200ng and GST (26kD)50ng, performing vertical electrophoresis at 100V on 8% SDS-PAGE; transferring the separated proteins to a PVDF membrane; after blocking the non-specific binding sites with 5% BSA, the cells were incubated with relevant sera (anti-TIF 1. gamma. -IgA 1:100 dilution, anti-TIF 1. gamma. -IgG 1:500 dilution) or GST antibody (1:2000 dilution, Beijing kang, century Co., Ltd.) overnight at 4 ℃; TBST washing, adding corresponding HRP-labeled goat anti-human IgA (Jackson Immuno, USA) and IgG secondary antibody (Beijing kang, century Co., Ltd.) diluted at 1:5000 respectively, shaking table incubating at room temperature in dark place for 2h, washing with TBST, developing with ECL developing kit, and taking picture.
1.5 statistical analysis
Experimental data were statistically analyzed using SPSS 22.0 statistical software. The Cut-off value using "OD mean value detected in HC group + 2-fold standard deviation" as an index. The comparison of the qualitative data among the groups was performed by chi-square test, and the comparison of the quantitative data among the groups was performed by Mann-Whitney U-test. Graph Prism5 was plotted, and ROC curves were generated for Medcalc v18.11, and AUC values were calculated, with P <0.05 being statistically significant.
2 results
2.1 expression of protein TIF1Gamma in Lung cancer tissues
Immunohistochemistry results showed that TIF1 γ protein localized to the nucleus and cytoplasm, and that patient's cancer tissues presented focally or diffusely distributed tan or brownish particles in early lung cancer (fig. 1a, c), while it was not or weakly expressed in paired paracarcinoma cells (fig. 1b, d). The positive expression rate of TIF1gamma in cancer tissue is 83.33% (50/60), which is obviously higher than that of paired para-cancer tissue by 25.00% (15/60) (P < 0.01); in addition, there was no significant difference in the positive expression rate of adenocarcinoma, squamous carcinoma and small cell lung carcinoma TIF1 γ (P >0.05) (table 2).
TABLE 260 comparison of Positive rates of TIF1 γ expression in cancer tissues of patients with early stage lung cancer and paired paracarcinoma tissues
2.2 expression of anti-TIF 1Gamma antibody in serum of patients with early Lung cancer
The ELISA detection result shows that the anti-TIF 1 gamma-IgA and anti-TIF 1 gamma-IgG levels of the early lung cancer group are obviously higher than those of the LBL group and the HC group (P <0.01, and figures 2a and b), and the expression of the anti-TIF 1 gamma-IgM and anti-TIF 1 gamma-IgE among the groups is not obviously different (P >0.05, and figures 2c and d). 28.2% of sensitivity (specificity 95.93%) of anti-TIF 1 gamma-IgA for early diagnosis of lung cancer, 0.704 of AUC, 18.54% of sensitivity (specificity 94.25%) of anti-TIF 1 gamma-IgG for early diagnosis of lung cancer, 0.622 of AUC, 38.31% of sensitivity (specificity 92.34%) of combined detection of the two for early diagnosis of lung cancer and 0.734 of AUC (Table 3, FIG. 3).
TABLE 3 comparison of early diagnosis of Lung cancer by serum TIF1 γ -IgA and TIF1 γ -IgG
**Represents P<0.01。
2.3 Western blot verification of ELISA results
We performed a western blot assay using the GST-tagged recombinant protein TIF1 γ expressed in yeast to verify the serum reactivity observed in the ELISA assay. As shown in FIGS. 4a and b, the early lung cancer patient sera tested by ELISA for anti-TIF 1 γ -IgA and anti-TIF 1 γ -IgG (+) all bound to the target protein only and not to the GST tag protein; LBL patients tested by ELISA for anti-TIF 1 γ -IgA, anti-TIF 1 γ -IgG (-), and HC sera were not bound to the target protein and GST tag protein.
Claims (2)
1. The application of a combined diagnosis marker consisting of a serum anti-TIF 1 gamma-IgA autoantibody and an anti-TIF 1 gamma-IgG autoantibody in preparing a kit for early diagnosis of lung cancer.
2. The use according to claim 1, wherein the kit is a multiplex assay kit based on ELISA technology.
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| CN110275026A (en) * | 2019-07-03 | 2019-09-24 | 中日友好医院 | A kind of molecular marker and its application for diagnosing idiopathic inflammatory myopathies |
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