CN117487913A - Application of STOX1-A in diagnosis and treatment of liver cancer - Google Patents

Abstract

The invention proves for the first time that the expression level of STOP-1-A in HCC hepatocellular carcinoma tissue is obviously increased, the higher the SI score of an HCC sample with high STOP-1-A expression is, the higher the severity of HCC is, the poor survival prognosis of HCC patients is obviously positively correlated, and the silencing STOP-1-A can effectively inhibit the in vivo tumor formation of HCC hepatocellular carcinoma cells, so that the expression level of STOP-1-A can be used for diagnosing hepatocellular carcinoma patients and can be used for prognosis analysis of hepatocellular carcinoma patients by detecting the expression level of STOP-1-A, and the preparation for silencing STOP-1-A can be used for preparing hepatocellular carcinoma drugs, thereby providing a new way for preventing and treating hepatocellular carcinoma.

Description

Application of STOX1-A in diagnosis and treatment of liver cancer

Technical Field

The invention belongs to the field of biological pharmacy, and in particular relates to application of STOX1-A in diagnosis and treatment of liver cancer.

Background

Hepatocellular carcinoma (Hepatocellular carcinoma-HCC) is one of the common tumors with high morbidity and mortality; meanwhile, the liver cancer is one of the most common subtypes of primary liver cancer, and accounts for 80% of all liver cancers. Common causative factors for HCC include hepatitis b and c, alcohol consumption, diabetes, food contaminated with aflatoxin, and nonalcoholic liver disease, as well as abnormal activation of oncogenes such as K-RAS or inactivation of the oncogenes p53 and PTEN.

Common treatments for primary liver cancer include surgical excision, liver transplantation, chemoradiotherapy, molecular targeted therapy, and the like. However, since the liver cancer is hidden, no obvious symptoms exist in the early stage, most patients are in the middle and late stages when they are diagnosed, and the optimal treatment time is often lost. Because, searching for a potential therapeutic target and an early diagnosis and prediction prognosis molecular marker of the hepatocellular carcinoma provides a new way for improving the life quality of hepatocellular carcinoma patients.

The transcription factor Storkhead box 1 (STOX 1) gene belongs to the large family of FOX transcription factors. STOX1 is reported to have 6 subtypes, with STOX1-A being one of six subtypes. Abnormal expression of STOX1-A was shown to be associated with Alzheimer's disease, preeclampsia, and trophoblast dysfunction. However, the clinical significance and biological function of STOX1-A in the tumor area other than neuroblastoma is not known, and there is no report on whether STOX1-A can be used as a target for diagnosis and treatment of hepatocellular carcinoma.

For this purpose, the present invention is proposed.

Disclosure of Invention

The invention aims to provide a new application of STOX1-A in preparing liver cancer diagnosis products and medicines.

To this end, the invention provides one or more of the following embodiments:

the application of the preparation for detecting STOX1-A expression level in preparing liver cancer diagnosis products or liver cancer prognosis evaluation products.

Further, the STOX1-A expression level includes an mRNA level or a protein level.

Further, the formulation includes primers or probes that specifically detect STOX1-AmRNA or cDNA.

Further, the formulation includes an antibody or aptamer that specifically detects STOX1-A protein.

Further, the liver cancer is hepatocellular carcinoma.

Further, STOX1-A expression levels were up-regulated in liver cancer patients.

Further, prognosis evaluation of liver cancer includes prediction of survival time.

The preparation for silencing STOX1-A is used for preparing liver cancer drugs.

Further, the formulation includes interfering RNA, antisense oligonucleotides or CRISPRi designed for STOX 1-A.

Further, the liver cancer is hepatocellular carcinoma.

Further, the medicament inhibits the in vivo tumor formation of liver cancer cells.

The beneficial effects of the invention include:

the invention proves for the first time that the expression level of STOP-1-A in HCC hepatocellular carcinoma tissue is obviously increased, the higher the SI score of an HCC sample with high STOP-1-A expression is, the higher the severity of HCC is, the poor survival prognosis of HCC patients is obviously positively correlated, and the silencing STOP-1-A can effectively inhibit the in vivo tumor formation of HCC hepatocellular carcinoma cells, so that the expression level of STOP-1-A can be used for diagnosing hepatocellular carcinoma patients and can be used for prognosis analysis of hepatocellular carcinoma patients by detecting the expression level of STOP-1-A, and the preparation for silencing STOP-1-A can be used for preparing hepatocellular carcinoma drugs, thereby providing a new way for preventing and treating hepatocellular carcinoma.

Drawings

Fig. 1: mRNA levels of STOX1 in 50 paracancestor normal tissues and 351 HCC hepatocellular carcinoma in the tumor genetic map (TCGA) database;

fig. 2: analysis of STOX1 different subtype expression levels in the TCGA hepatocellular carcinoma database;

fig. 3: mRNA levels of STOX1-a in paracancerous normal tissue and HCC hepatocellular carcinoma tissue in 10 clinical specimens;

fig. 4: protein levels of STOX1-A in paracancerous normal tissue and HCC hepatocellular carcinoma tissue in 10 clinical specimens;

in the above figures, ANT represents a paracancerous tissue, tumor represents a hepatocellular carcinoma tissue, and P1-P10 are paired sample numbers;

fig. 5: immunohistochemical (IHC) staining analyzes expression of STOX1 in benign liver disease tissue, benign proliferative liver disease tissue, and different levels of hepatocellular carcinoma tissue; information in the figure shows benign liver disease tissue, hyperplasia shows benign proliferative liver disease tissue, HCC (G1-G4) shows different grades of hepatocellular carcinoma tissue;

fig. 6: SI score of STOX1-a in benign liver disease tissue and HCC hepatocellular carcinoma tissue;

fig. 7: number of cases under SI score based on STOX1-a in benign liver disease tissue and HCC hepatocellular carcinoma tissue;

fig. 8: SI score of STOX1-a in benign liver disease tissue and HCC hepatocellular carcinoma tissue of different grade;

fig. 9: SI scores of STOX1-a in benign liver disease tissue and HCC hepatocellular carcinoma tissue of different T stages;

fig. 10: SI score of STOX1-a in benign liver disease tissue and HCC hepatocellular carcinoma tissue of different clinical stages;

fig. 11: kaplan-Meier survival analysis of hepatocellular carcinoma patients divided at high and low levels of STOX 1-A;

benign in the above figures represents Benign liver disease tissue and magnancy represents hepatocellular carcinoma tissue;

fig. 12: protein levels of STOX1-A in STOX1-A high-expression and low-expression stable cell lines are established in HepG2 and HuH7, wherein STOX1-A represents a STOX1-A high-expression cell line, and Vec-OE, vec-sh, sh#1 and sh#2 represent a STOX1-A low-expression cell line;

fig. 13: schematic of in vivo model of subcutaneous injection of designated hepatocellular carcinoma cells;

fig. 14: tumor weights of different mouse experimental groups;

fig. 15: tumor volumes of different mouse experimental groups;

fig. 16: analysis of Ki-67 expression in tumor tissues of different mouse experimental groups;

fig. 17: analysis of STOX1-A expression in tumor tissues of different mouse experimental groups;

in the above figures, STOX1-A represents a STOX1-A high expression experimental group, and Con-OE, con-sh, sh#1 represents a STOX1-A low expression experimental group.

Specific embodiments:

through extensive and intensive research, the invention detects the mRNA level and protein level of STOX1-A in HCC liver cell cancer tissues and paracancer normal tissues through real-time quantitative PCR and Western blot, and combines the analysis of a tumor gene map database, the invention discovers that the expression level of STOX1-A in the liver cell cancer tissues is obviously increased for the first time, the high expression of STOX1-A means that the severity of HCC is higher, and the poor survival prognosis of HCC patients is obviously positively correlated. Further experiments demonstrated that high expression of STOX1-A promoted silencing of STOX1-A inhibited HCC cell tumor formation in vivo. Therefore, STOX1-A can be used as a target for diagnosis and treatment of liver cancer and can be used for prognosis analysis of hepatocellular carcinoma patients.

Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still further embodiments.

Embodiments of the present invention include: the application of the preparation for detecting STOX1-A expression level in preparing liver cancer diagnosis products or liver cancer prognosis evaluation products. In some embodiments, the expression level of STOX1-A in an ex vivo sample from a subject can be assessed qualitatively or quantitatively to determine whether the subject has liver cancer, or to determine the severity of liver cancer, and/or to predict survival of a liver cancer patient, e.g., to predict progression free survival of a liver cancer patient. In some aspects, detecting STOX1-A expression levels can be used to distinguish between a patient with a defined liver cancer and a healthy person, in some aspects, detecting STOX1-A expression levels can be used to determine a grade, T-grade, or clinical total grade of a liver cancer, in some aspects, e.g., a grade of G2-G4 of a liver cancer, a grade of T1-T4, and a clinical total grade of I-IV. The skilled artisan can employ various means of detection to quantify or characterize STOX1-A expression levels. In some embodiments, the STOX1-A expression level comprises mRNA level or protein level. In some embodiments, the formulation includes a primer or probe that specifically detects STOX 1-AmRNA. For example, one skilled in the art can use fluorescent quantitative PCR, northern blot hybridization, in situ hybridization, and the like to obtain STOX1-AmRNA levels in an isolated sample of a subject. In some embodiments, the formulation includes an antibody or aptamer that specifically detects STOX1-A protein. For example, one skilled in the art can use immunohistochemistry, enzyme-linked immunosorbent, immunochromatography, electrochemical methods, and the like to obtain STOX1-A protein levels in an ex vivo sample of a subject. In some embodiments, the liver cancer is hepatocellular carcinoma. In some embodiments, STOX1-A expression levels are upregulated in liver cancer patients. In some embodiments, the ex vivo sample comprises tissue, cells, body fluids, secretions, excretions, and the like, such as whole blood, serum, plasma, and the like.

Embodiments of the present invention also include: the preparation for silencing STOX1-A is used for preparing liver cancer drugs. Drugs are understood to have an intervention effect on the patient, such as but not limited to preventing the occurrence of a disease, slowing the progression of a disease, ameliorating a disease state, curing a disease, etc. In some embodiments, the liver cancer is hepatocellular carcinoma. In some embodiments, the agent inhibits tumor formation in a liver cancer cell. In some embodiments, the formulation comprises an interfering RNA, an antisense oligonucleotide, or CRISPRi designed for STOX 1-A. Interfering RNAs (RNAi) refer to agents capable of specifically knocking out or shutting down expression of a particular gene, including, for example, siRNA, shRNA. Antisense oligonucleotides are molecular drugs that inhibit the expression of a target gene by binding the sequence specifically to the DNA or mRNA of that gene, and regulate at the gene level. CRISPRi is an agent that alters gene expression from the transcriptional level using CRISPR technology.

The experimental procedures, which are not specified in the following examples, are generally carried out according to conventional procedures, such as those reported in the literature book or those recommended by the reagent manufacturers.

Cell lines and cell cultures

Primary normal liver epithelial cells (PHs) were purchased from procall (marsupium procall life sciences technologies limited in china). Liver cancer cell lines, including HepG2 and Huh-7, are derived from the cell bank of Shanghai cell biology institute of China academy of sciences. PHs the HCC cell line was cultured in CM-H023 medium (Procell, china) and in rpm-1640 medium (Life Technologies, carlsbad, calif., US) to which penicillin G (100U/ml), streptomycin (100 mg/ml) and 10% fetal bovine serum (FBS, life Technologies) were added. All cell lines were incubated at 37℃with 5% CO ₂ Is cultured in a moist environment.

Patient and tumor tissue

10 pairs of paired hepatocellular carcinoma tissue, paracancerous normal tissue, 47 benign liver disease tissues, and 211 hepatocellular carcinoma tissues were all derived from thyroid surgery in the United states of the university of Jilin, 2016, 1 month to 12 months of the United states of the hospitals. The patient diagnoses according to clinical and pathological evidence, and the specimen is stored in a liquid nitrogen tank by adopting a quick freezing method. For scientific use of clinical specimens, approval by the institutional research ethics committee of patients was obtained in advance.

Plasmid and transfection

Human STOX1-AcDNA (Vigene Biosciences, shandong, china) was cloned into pcDNA3.1 (+) plasmid. Silencing of STOX1-A was achieved by cloning two short hairpin RNA (shRNA) oligonucleotides into the GV493 vector (GenChem, shanghai, china). The primers used for cloning PCR are shown in the following table, and reference is made to the plasmid transfection method (N.Wu, D.Ren, S.Li, W.Ma, S.Hu, Y.Jin, S.Xiao, RCC over-expression in tumor cells alters apoptosis and drug sensitivity by regulating Rac1 activation, BMC cancer,18 (2018) 67.).

RNA extraction, reverse transcription and real-time PCR

RNA was extracted from tissues and cells according to the instructions of the reagent manufacturer (TRIzol, life Technologies)). Messenger RNA (mRNA) was reverse transcribed from total RNA using the RevertAid First Strand cDNA kit (Thermo, USA) according to the manufacturer's instructions. Complementary DNA (cDNA) was amplified and quantified on the ABI 7500HT system (Applied Biosystems, foster City, calif., USA) using SYBR Green I (Applied Biosystems). The primers used in the RT-qPCR reaction are shown in the following table. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control for mRNA. The relative level is represented by a comparison threshold cycle calculation.

Western blot analysis

Western blot Standard methods reference (Y.Li, L.Deng, X.Zhao, B.Li, D.Ren, L.Yu, H.Pan, Q.Gong, L.Song, X.Zhou, T.Dai, tripartite motion-rotation 37 (TRIM 37) promotes the aggressiveness of non-small-cell lung cancer cells by activating the NF-kappa B path, the Journal ofpathology, (2018)). STOX1-A antibody, CDK1, CDK2, CDK4, CDK6, cyclin D1, cyclin E1, cyclin A1, cyclin B1, oxygenated PTEN, phosphorylated AKT1 (Ser 473), total PTEN and total AKT1 were purchased from Cell Signaling Technology. Cytosolic load control was performed by removal and protection of the cell membrane with anti-alpha-tubulin antibodies (Cell Signaling Technology).

Kit-8 cell count analysis

2×10 ³ Cells were seeded in 96-well plates, staining methods reference (X.Zhang, D.Ren, L.Guo, L.Wang, S.Wu, C.Lin, L.Ye, J.Zhu, J.Li, L.Song, H.Lin, Z.He, thymosin beta 10is a key regulator of tumorigenesis and metastasis and a novel serum marker inbreast cancer,Breast cancer research:BCR,19(2017)15.)。

Colony formation assay

Cells were broken down into single cells using trypsin and suspended in medium containing 10% fetal bovine serum. Indicator cells (300 cells per well) were seeded in 6-well plates and cultured for 10-14 days. Colonies were fixed with 10% formaldehyde for 5 min and stained with 1% crystal violet for 10 min. The method for calculating the dyeing rate is described in (M.Wang, D.Ren, W.Guo, S.Huang, Z.Wang, Q.Li, H.Du, L.Song, X.Peng, N-cadherin promotes epithelial-mesenchymal transition and cancer stemcell-like traits via ErbB signaling in prostate cancer cells, internationaljournal ofoncology,48 (2016) 595-606.). The different colony morphologies were observed by optical microscopy (Olympus).

Immunohistochemistry

Immunohistochemical procedure and STOX1-A expression evaluation methods are described in reference (X.Wu, X.Zhang, L.Yu, C.Zhang, L.Ye, D.Ren, Y.Li, X.Sun, L.Yu, Y.Ouyang, X.Chen, L.Song, P.Liu, X.Lin, zinc finger protein 367promotes metastasis by inhibiting the Hippo pathway in breast cancer,Oncogene, (2020)). The following is a brief description: frozen sections were fixed with 4% formalin solution for 10min, added with TE (pH 9.0) buffer, microwaved for 10min for antigen retrieval, and antibody diluted with antibody dilution (Abcam, USA) at a ratio of 1:100 followed by sequential addition of hydrogen peroxide and goat serum blocking solution and incubation overnight in a wet chamber at 4 ℃. After the incubation, the sections were washed with TBS/0.05% Tween 20, added with biotin-conjugated secondary antibodies (Proteintech, china) and peroxidase-conjugated streptavidin (Proteintech, china), incubated at 37℃for 30 min, and stained with 3,3' -Diaminobenzidine (DAB) enhanced liquid substrate system (Sigma-Aldrich, USA). The expression of STOX1-A was further assessed by taking the average of the Staining Index (SI) given by two independent investigators. Tumor cell proportion scoring: 0 (no positive tumor cells); 1 (positive tumor cells < 10%); 2 (10-35% positive tumor cells); 3 (35-70% positive tumor cells) and 4 (> 70% positive tumor cells). The staining intensity was graded according to the following criteria: 0 (no staining); 1 (weak staining, pale yellow); 2 (medium dyeing, yellow-brown) and 3 (intense dyeing, brown). SI is calculated as the product of staining intensity score and positive tumor cell proportion. Based on this evaluation method, SI was used to evaluate STOX1-A expression in hepatocellular carcinoma samples, scoring 0, 1, 2, 3, 4, 6, 8, 9, or 12 points. SI score 4 is the median of SI for all sample organization. High and low the expression of STOX1-A was stratified according to the following criteria: SI.ltoreq.4 is used to define tumors with STOX1-A low expression and SI score >4 as high expression tumors.

Animal study

8 week old BALB/c-nu mice were purchased from the university of Guangzhou traditional Chinese medicine laboratory animal center and bred according to institutional guidelines. Mice were randomly divided into 3 groups (6 per group) and indicator cells HepG2 (1X 10) ⁶ ) Is injected into the subcutaneous tissues of the inguinal folds of mice, forms any primary tumor, euthanizes at the end point, resects tumor tissue and is embedded in paraffin. H for tumor tissue section&e staining and histological examination of STOX1-A and Ki-67 staining, methods for counting tumor cell numbers are described in (J.Chen, A.Liu, Z.Lin, B.Wang, X.Chai, S.Chen, W.Lu, M.Zheng, T.Cao, M.Zhong, R.Li, M.Wu, Z.Lu, W.Pang, W.Huang, L.Xiao, D.Lin, Z.Wang, F.Lei, X.Chen, W.Long, Y.Zheng, Q.Chen, J.Zeng, D.Ren, J.Li, X.Zhang, Y.Huang, downregulation of the circadian rhythm regulator HLF promotes multiple-organ distant metastases in non-small cell lung Cancer through PPAR/NF-kappa signaling, cancer letters,482 (2020) 56-71.).

Statistical analysis

All values are expressed as mean ± Standard Deviation (SD). Significant differences were determined using GraphPad 5.0 software (united states). Statistical differences between the two groups were determined using student t-test. Statistical differences between multiple tests were determined using one-way anova. P < 0.05 is statistically significant for the differences. All experiments were repeated three times.

Example 1

We first analyzed the expression of STOX1 in HCC in the Tumor genomic profile (the Cancer Genome Atlas (TCGA)) dataset, and the results showed high expression of STOX1 in HCC hepatocellular carcinoma tissue (Tumor) relative to paracancerous normal tissue (ANT), as shown in fig. 1.

To further clarify the expression levels of the STOX1 isoforms in HCC hepatocellular carcinoma tissues, the expression of STOX1-A (NM-152709) and STOX1-B (NM-001130159) were tested, and the difference in expression of STOX1-A isoforms was statistically more significant than that of STOX1-B isoforms in paracancerous normal tissues, as shown in FIG. 2.

Example 2

We further analyzed STOX1-A expression in clinical specimens. Using real-time quantitative PCR and Westernblot analysis, STOX1-A mRNA and protein levels were expressed in HCC hepatocellular carcinoma tissue at a higher level than in paracancerous normal tissue, and the results are shown in FIGS. 3 and 4.

STOX1-A expression was further tested by Immunohistochemistry (IHC) in 47 benign liver disease tissues and 221 HCC hepatocellular carcinoma tissues, and staining was performed as shown in FIG. 5. The results of significantly elevated levels of STOX1-A expression in HCC hepatocellular carcinoma tissue compared to benign liver tissue are shown in FIG. 6. Further stratification of HCC samples according to SI score, the higher the SI score of the STOX1-A highly expressed HCC samples, the higher the severity of HCC, and the results are shown in FIG. 7.

Further analysis of STOX1-A expression levels was able to determine HCC severity, and the results showed that the expression levels were further increased as HCC was fractionated, T-staging and clinical total staging were increased, and the results are shown in FIGS. 8-10. The survival curve analysis results showed that STOX1-A high expression correlated significantly positively with poor progression free survival prognosis for HCC patients, as shown in FIG. 11.

Thus, the present invention has demonstrated that STOX1-A can be a diagnostic marker for HCC; STOX1-A has also been shown to be a prognostic marker for HCC.

Example 3

Stable cell lines for high and low expression of STOX1-A were established in HCC cell lines HepG2 and HuH7, as shown in FIG. 12. The effect of intervention of STOX1-A expression on tumorigenesis in HCC was examined by in vivo animal experiments in mice. As shown in FIGS. 13-16, silencing STOX1-A reduces tumor weight and volume, and staining index of Ki-67; silencing STOX1-A reduced SI score of mouse tumors, as shown in FIG. 17. In conclusion, silencing STOX1-A inhibits HCC hepatoma cells from forming tumors in vivo.

Thus, the present invention has demonstrated that silencing STOX1-A can inhibit tumor growth and improve tumor progression, and thus STOX1-A can be a drug target for HCC treatment.

Claims (10)

1. The application of the preparation for detecting STOX1-A expression level in preparing liver cancer diagnosis products or liver cancer prognosis evaluation products.

2. The use according to claim 1, wherein said STOX1-a expression level comprises mRNA level or protein level.

3. The use according to claim 1, wherein the preparation comprises a primer or probe for specifically detecting STOX1-AmRNA or cDNA.

4. The use according to claim 1, wherein the formulation comprises an antibody or aptamer that specifically detects the STOX1-a protein.

5. The use according to claim 1, wherein the liver cancer is hepatocellular carcinoma.

6. The use according to claim 1, wherein the prognosis evaluation of liver cancer comprises prediction of survival time.

7. The preparation for silencing STOX1-A is used for preparing liver cancer drugs.

8. The use according to claim 7, wherein the preparation comprises interfering RNA, antisense oligonucleotides or CRISPRi designed for STOX 1-a.

9. The use according to claim 7, wherein the liver cancer is hepatocellular carcinoma.

10. The use according to claim 7, wherein the medicament inhibits in vivo tumor formation in liver cancer cells.