Application of PLCB1 gene as a biomarker in invasive pituitary adenomas

Abstract

The present invention belongs to the technical field of medical molecular biology, and specifically relates to the application of PLCB1 gene as a biomarker in invasive pituitary adenoma. The present invention provides the application of PLCB1 gene as a biomarker in the preparation of a product for diagnosing invasive pituitary adenoma and / or evaluating the invasiveness of pituitary adenoma. The present invention finds that compared with non-invasive pituitary adenoma samples, the expression of PLCB1 gene in invasive pituitary adenoma samples is significantly increased, which has an indicative effect on the diagnosis of invasive pituitary adenoma. Furthermore, by detecting the expression of PLCB1 gene in the sample to be tested, invasive pituitary adenoma and non-invasive pituitary adenoma can be accurately distinguished, providing a new idea for the research and development of invasive pituitary adenoma treatment drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical molecular biology, and in particular relates to the application of PLCB1 gene as a biomarker in invasive pituitary adenoma. Background Art

[0002] Pituitary adenoma is a common neuroendocrine tumor, accounting for 10% to 15% of central nervous system tumors, with a prevalence of 80 to 10 cases per 100,000 cases. The vast majority of pituitary adenomas are benign tumors that are easily treated with surgery or drugs. However, about 35% of pituitary adenomas are classified as invasive, which is defined and graded by the extent of invasion of the adjacent sphenoid sinus and cavernous sinus. Compared with non-invasive pituitary adenomas, invasive pituitary adenomas are not only more difficult to achieve complete resection, but also show drug resistance and multiple local recurrence characteristics. Ki-67 ≥ 3%, increased mitosis and p53 gene expression are generally common signs for identifying invasive pituitary adenomas, but they are not completely applicable to all invasive pituitary adenomas.

[0003] Early identification of invasive pituitary adenomas is challenging but of great clinical significance, as they are associated with increased morbidity and mortality even in the absence of metastasis. Although a large amount of basic research has been conducted on invasive pituitary adenomas and many advances have been made in clinical prognostic classification, there is no clear biomarker that can reliably predict the aggressiveness of pituitary adenomas. Summary of the invention

[0004] The purpose of the present invention is to provide an application of the PLCB1 gene as a biomarker in invasive pituitary adenomas. The PLCB1 gene as a biomarker can accurately identify whether a pituitary adenoma is invasive and effectively distinguish between non-invasive pituitary adenomas and invasive pituitary adenomas.

[0005] The present invention provides the use of PLCB1 gene as a biomarker in preparing a product for diagnosing invasive pituitary adenoma and / or evaluating the invasiveness of pituitary adenoma.

[0006] Preferably, the product comprises one or more of a reagent, a kit and a chip.

[0007] Preferably, the product includes components for detecting the expression level of the PLCB1 gene, a positive control and a negative control.

[0008] The present invention also provides a kit for diagnosing invasive pituitary adenoma or evaluating the invasiveness of pituitary adenoma, wherein the kit comprises a component for detecting the expression amount of PLCB1 gene.

[0009] Preferably, the reagent comprises a gene detection reagent or an immunohistochemistry reagent.

[0010] Preferably, the immunohistochemical reagent includes an immunoblotting reagent or an immunohistochemical staining reagent; and the gene detection reagent includes a sequencing-related reagent.

[0011] The present invention also provides the use of the expression inhibitor of the PLCB1 gene in preparing a drug for treating invasive pituitary adenoma and / or reducing the invasiveness of pituitary adenoma.

[0012] Preferably, the expression inhibitor includes a biochemical agent that inhibits or reduces the expression level of the PLCB1 gene.

[0013] The present invention also provides a drug for treating invasive pituitary adenoma and / or reducing the invasiveness of pituitary adenoma, wherein the drug comprises a biochemical preparation and an auxiliary material for inhibiting or reducing the expression amount of PLCB1 gene.

[0014] Beneficial effects:

[0015] The present invention provides the use of the PLCB1 gene as a biomarker in the preparation of a product for diagnosing invasive pituitary adenomas and / or evaluating the invasiveness of pituitary adenomas. The present invention finds that, compared with non-invasive pituitary adenoma samples, the expression level of the PLCB1 gene in invasive pituitary adenoma samples is significantly increased, which has an indicative effect on the diagnosis of invasive pituitary adenomas. Furthermore, by detecting the expression level of the PLCB1 gene in the sample to be tested, invasive pituitary adenomas and non-invasive pituitary adenomas can be accurately distinguished, providing a new idea for the research and development of invasive pituitary adenoma treatment drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0017] Figure 1 The volcano plot of differentially expressed genes in the non-invasive pituitary adenoma sample group and the invasive pituitary adenoma sample group in Example 1;

[0018] Figure 2 is a correlation heat map of the top 50 differentially expressed genes in the non-invasive pituitary adenoma sample group and the invasive pituitary adenoma sample group in Example 1;

[0019] Figure 3 GO functional enrichment analysis diagram of differentially expressed genes in the non-invasive pituitary adenoma sample group and the invasive pituitary adenoma sample group in Example 1;

[0020] Figure 4 This is a KEGG pathway enrichment analysis diagram of differentially expressed genes in the non-invasive pituitary adenoma sample group and the invasive pituitary adenoma sample group in Example 1;

[0021] Figure 5This is a disease pathway enrichment analysis diagram of differentially expressed genes in the non-invasive pituitary adenoma sample group and the invasive pituitary adenoma sample group in Example 1;

[0022] Figure 6 This is an analysis diagram of 11 invasion characteristic genes obtained by the lasso regression model in Example 2;

[0023] Figure 7 This is the analysis graph of 118 invasion characteristic genes obtained by the SVM-RFE regression model in Example 2;

[0024] Figure 8 It is a Venn diagram of the intersection genes of the invasion characteristic genes in the lasso regression model and the SVM-RFE regression model in Example 2;

[0025] Figures 9 to 16 The correlation analysis diagrams of the expression levels of BAMBI, CDH12, PLCB1, GALNT13, TOX, PAPPA, ESRRG and MPL genes and clinical samples in Example 3 are shown in sequence;

[0026] Fig.17 This is a receiver operating characteristic (ROC) curve analysis of the PLCB1 gene using the GSE26966 and GSE51618 gene expression chips in Example 4;

[0027] Fig.18 This is a receiver operating characteristic (ROC) curve analysis of the PLCB1 gene using the GSE169498 gene expression chip in Example 4;

[0028] Fig.19 This is the expression diagram of PLCB1 gene detected by immunohistochemical staining in invasive and non-invasive pituitary adenoma samples in Example 5;

[0029] Fig. 20 This is the expression diagram of PLCB1 gene in invasive and non-invasive pituitary adenoma group samples detected by Western Blot in Example 6;

[0030] Fig.21 The figure is the result of the Western blot (WB) method in Example 7 to verify the knockdown of PLCB1 in the cell line;

[0031] Fig. 22 This is a diagram showing the results of the CCK8 proliferation experiment in Example 7 to verify the knockdown of PLCB1 in the cell line;

[0032] Fig.23 This is a diagram showing the results of the cell scratch experiment in Example 7 to verify the knockdown of PLCB1 in the cell line;

[0033] Fig.24This is a diagram showing the results of the Transwell cell invasion experiment in Example 7 to verify the knockdown of PLCB1 in the cell line. DETAILED DESCRIPTION

[0034] The present invention provides the use of the PLCB1 gene as a biomarker in the preparation of a product for diagnosing invasive pituitary adenoma and / or evaluating the invasiveness of pituitary adenoma. The product of the present invention preferably includes one or more of a reagent, a kit and a chip, further preferably includes a kit or a chip, and more preferably a kit. The product of the present invention preferably includes a component for detecting the expression amount of the PLCB1 gene.

[0035] The present invention uses pituitary adenoma chips GSE26966 and GSE51618 in the Gene Expression Omnibus (GEO) as research samples, analyzes differentially expressed genes in invasive pituitary adenoma samples and non-invasive pituitary adenoma samples in the two chips GSE26966 and GSE51618; verifies the obtained differentially expressed genes by using a validation group chip GSE169498 and an immunohistochemical test, screens genes with significant expression differences in the invasive pituitary adenoma sample group and the non-invasive pituitary adenoma sample group (P<0.05), and obtains the PLCB1 gene, the accession number of the PLCB1 gene in the NCBI database is Gene ID: 23236, and the website is https: / / www.ncbi.nlm.nih.gov / gene / 23236.

[0036] In the above application, by detecting the expression level of the PLCB1 gene in the sample to be tested, it is judged whether the sample to be tested is invasive. When the expression level of the PLCB1 gene in the sample to be tested is significantly different from the negative control and not significantly different from the positive control, it can be judged that the sample to be tested is an invasive pituitary adenoma or the pituitary adenoma in the sample to be tested is invasive or the risk of invasiveness is higher. In the present invention, the negative control is a non-invasive pituitary adenoma sample, and the positive control is an invasive pituitary adenoma sample. The present invention preferably uses an immunohistochemical reagent or a gene detection reagent to detect the expression level of the PLCB1 gene in the sample to be tested. The immunohistochemical reagent in the present invention preferably includes but is not limited to an immunoblotting reagent or an immunohistochemical staining reagent; the reference antibody used in the immunohistochemical reagent is preferably GAPDH. The present invention does not specifically limit the source of the immunohistochemical reagent, and commercially available kits or reagents can be used. The gene detection reagents of the present invention preferably include sequencing-related reagents, more preferably but not limited to transcriptome sequencing reagents or genome sequencing reagents. For example, the transcriptome sequencing data in GSE26966, GSE51618 and GSE169498 used in the embodiments of the present invention are obtained by transcriptome sequencing of invasive pituitary adenoma samples and non-invasive pituitary adenoma samples.

[0037] The present invention also provides a kit for diagnosing invasive pituitary adenoma or evaluating the invasiveness of pituitary adenoma, the kit comprising a component for detecting the expression amount of PLCB1 gene, a positive control and a negative control. The components of the present invention preferably include gene detection reagents or immunohistochemical reagents. The gene detection and immunohistochemical reagents of the present invention are as described above and will not be repeated. The present invention does not specifically limit the source of the reagents, and they can be purchased through conventional commercial channels in the field. The positive control of the present invention is preferably a normal brain tissue sample; the negative control is preferably a normal liver tissue sample.

[0038] Based on the influence of the expression level of the PLCB1 gene on the properties of pituitary adenomas, the present invention also provides the use of an expression inhibitor of the PLCB1 gene in the preparation of a drug for treating invasive pituitary adenomas and / or reducing the invasiveness of pituitary adenomas. The expression inhibitor of the present invention preferably includes a biochemical agent that inhibits or reduces the expression of the PLCB1 gene; the biochemical agent preferably includes but is not limited to RNAi-related reagents, transcription inhibitors, post-transcriptional processing inhibitors, functional inhibitors after transcriptional processing maturation, or DNA fragments, sgRNA or shRNA that inhibit or reduce the expression of the PLCB1 gene in cells.

[0039] The present invention also provides a drug for treating invasive pituitary adenoma and / or reducing the invasiveness of pituitary adenoma, the drug comprising a biochemical agent and an excipient that inhibits or reduces the expression of the PLCB1 gene. The biochemical agent of the present invention preferably includes, but is not limited to, RNAi-related reagents, transcription inhibitors, post-transcriptional processing inhibitors, functional inhibitors after transcriptional processing maturation, or DNA fragments and sgRNA that inhibit or reduce the expression of the PLCB1 gene in cells. The excipient of the present invention is preferably compatible with the biochemical agent or pharmaceutical dosage form contained in the drug.

[0040] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0041] Example 1

[0042] The steps for screening the PLCB1 gene are as follows:

[0043] 1. The GSE26966 and GSE51618 gene expression chips in the GEO database were selected as research samples and divided into invasive pituitary adenoma sample group and non-invasive pituitary adenoma sample group. The specific conditions of the samples are shown in Table 1. The invasive pituitary adenoma samples were used as the experimental group and the non-invasive pituitary adenoma samples were used as the control group. The difference analysis was performed by t test. When the absolute value of log2FC was ≥1 and p <0.05, the difference was considered statistically significant.

[0044] Table 1 Samples in GSE26966 and GSE51618 gene expression chips

[0045]

[0046] The results showed that compared with the non-invasive pituitary adenoma sample group, there were 118 differentially expressed genes in the invasive pituitary adenoma sample group, of which 27 genes were upregulated and 91 genes were downregulated. Figure 1 As shown, in Figure 1 The red dots indicate genes with upregulated differential expression, the green dots indicate genes with downregulated differential expression, and the black dots indicate genes with no significant difference.

[0047] The top 50 genes with differential expression are Figure 2 As shown, in Figure 2 In the Type color block, green indicates non-invasive pituitary adenoma samples, and pink indicates invasive pituitary adenoma samples. The color of the right color bar gradually increases from 0 to 2, indicating that the correlation between differentially expressed genes gradually increases, and from 0 to -2, the color gradually increases, indicating that the correlation between differentially expressed genes gradually decreases.

[0048] 2. Functional enrichment analysis of differentially expressed genes

[0049] 1) Perform GO functional enrichment analysis on the 118 differentially expressed genes obtained in step 1 to analyze the possible functions of the 118 differentially expressed genes at the three levels of biological process (BP), cell composition (CC) and molecular function (MF). The results are as follows: Figure 3 As shown, the horizontal axis represents the gene ratio, which indicates the ratio of the number of differentially expressed genes enriched in the pathway to the total number of differentially expressed genes; the vertical axis represents the pathway name; the color bar represents the q-value, which indicates the significance of the enriched genes; the bubble size indicates the number of enriched genes; the larger the bubble, the more genes there are.

[0050] Through GO functional enrichment analysis, it can be concluded that the 118 differentially expressed genes in the present invention mainly function through the following pathways:

[0051] Biological processes (BP) include: type I interferon signaling pathway, cellular response to type I interferon, response to type I interferon, positive regulation of T cell-mediated immunity, antigen processing and presentation of endogenous peptide antigens, antigen processing and presentation of endogenous peptide antigens by MHC class I, antigen processing and presentation by MHC class Ib molecules, antigen processing and presentation of endogenous antigens, positive regulation of T cell-mediated cytotoxicity, and regulation of T cell-mediated cytotoxicity;

[0052] The cell components (CC) include: ion channel complexes, transmembrane transporter complexes, transporter complexes, transport vesicle membranes, cation channel complexes, potassium channel complexes, MHC protein complexes, components of the luminal side of the endoplasmic reticulum membrane, luminal side of the endoplasmic reticulum membrane, and transport vesicle membranes from the endoplasmic reticulum to the Golgi apparatus;

[0053] Molecular functions (MF) include: metal ion transmembrane transporter activity, monovalent inorganic cation transmembrane transporter activity, organic acid transmembrane transporter activity, carboxylic acid transmembrane transporter activity, organic anion transmembrane transporter activity, symporter activity, sodium ion transmembrane transporter activity, peptide antigen binding amino acid transmembrane transporter activity, alditol: NADP + 1-oxidoreductase activity.

[0054] 2) KEGG pathway enrichment analysis was performed on the 118 differentially expressed genes obtained in step 1. The results are as follows: Figure 4 As shown, the horizontal axis represents the gene ratio, which indicates the ratio of the number of differentially enriched genes to pathway genes to the total number of differentially enriched genes; the vertical axis represents the pathway name; the color bar represents the q-value, which indicates the significance of the enriched genes; the bubble size represents the number of enriched genes; the larger the bubble, the more genes there are.

[0055] Through KEGG pathway enrichment analysis, it can be concluded that the 118 differentially expressed genes in the present invention mainly function through the following pathways:

[0056] Human papillomavirus infection, cell adhesion molecules, focal adhesions, phagosomes, cellular senescence, allograft rejection, graft-versus-host disease, type I diabetes, autoimmune thyroid disease, viral myocarditis, antigen processing and presentation, ECM-receptor interactions, and glutamatergic synapses.

[0057] 3) Disease enrichment analysis was performed on the 118 differentially expressed genes obtained in step 1. The disease enrichment analysis was completed using R software, DO enrichment (Disease Ontology), and the up-regulated and down-regulated genes were linked to the Enrichr website (https: / / maayanlab.cloud / Enrichr / ) to complete the disease enrichment analysis. The results are as follows: Figure 5 As shown, the horizontal axis represents the gene ratio, which indicates the ratio of the number of differentially enriched genes to pathway genes to the total number of differentially enriched genes; the vertical axis represents the pathway name; the color bar represents the q-value, which indicates the significance of the enriched genes; the bubble size represents the number of enriched genes; the larger the bubble, the more genes there are.

[0058] Disease enrichment analysis showed that 3 of the 118 differentially expressed genes in the present invention may be associated with neuroendocrine cancer.

[0059] Example 2

[0060] This example uses the "glmnet" package of R software to carry out LASSO regression analysis. SVM is a linear classifier that uses the maximum margin principle based on SVM to train samples, iterates continuously, and finally selects the required number of features. The importance of genes significantly associated with the invasion group and the control group is obtained by constructing random forest selection features.

[0061] 1) The machine learning algorithm lasso regression algorithm was used to screen the invasive characteristic genes from the 118 differentially expressed genes in the invasive pituitary adenoma sample group, and the lasso regression model was used to cross-validate the differentially expressed genes. The results are as follows Figure 6 As shown, in Figure 6 In the figure, the dotted line is the lowest point of error, and the lowest point corresponds to the characteristic genes of the disease, that is, 11 invasion characteristic genes related to invasive pituitary adenoma, of which 8 genes are BAMBI, CDH12, PLCB1, GALNT13, TOX, PAPPA, ESRRG and MPL genes.

[0062] 2) The machine learning algorithm SVM-RFE algorithm was used to screen the invasive characteristic genes from the 118 differentially expressed genes in the invasive pituitary adenoma sample group, and the SVM-RFE regression model was used to cross-validate the differentially expressed genes. The results are as follows: Figure 7 As shown, in Figure 7 In the figure, the blue dot is the point with the lowest error, and the lowest point corresponds to the characteristic gene of the disease, which is 118 invasion characteristic genes.

[0063] 3) The Venn program (http: / / bioinformatics.psb.ugent.be / webtools / Venn / ) was used to identify the intersection of the 11 invasion characteristic genes obtained by lasso regression and the 118 invasion characteristic genes obtained by SVM-RFE algorithm. The results are as follows: Figure 8 As shown. Figure 8 It can be concluded that there are 11 invasion characteristic genes with intersection obtained by the lasso regression algorithm and the SVM-RFE algorithm in step 2), of which 8 genes are BAMBI, CDH12, PLCB1, GALNT13, TOX, PAPPA, ESRRG and MPL genes.

[0064] Example 3

[0065] The gene expression levels and clinical data of the samples in the gene expression chip GSE169498 were used to perform correlation analysis on the 8 invasion characteristic genes obtained in Example 2, wherein the sample conditions in the gene expression chip GSE169498 are shown in Table 2. The correlation between the expression levels of the 8 invasion characteristic genes and the invasive clinical characteristics was analyzed, and the results were as follows: Figures 9 to 16 As shown, the ordinate represents the gene expression level, the blue Con represents the non-invasive pituitary adenoma group samples, and the red Treat represents the invasive pituitary adenoma group samples.

[0066] Table 2 Samples in GSE169498 gene expression chip

[0067]

[0068]

[0069] Depend on Figures 9 to 16It can be concluded that the p values ​​of the expression differences of BAMBI, CDH12, PLCB1, GALNT13, TOX, PAPPA, ESRRG and MPL genes in the non-invasive pituitary adenoma group samples and the invasive pituitary adenoma group samples were 0.097, 0.32, 0.0042, 0.9, 0.83, 0.67, 0.28 and 0.95, respectively. It can be seen that the expression levels of the PLCB1 gene in the non-invasive pituitary adenoma group samples and the invasive pituitary adenoma group samples in the validation group were significantly different, and the PLCB1 expression level was significantly correlated with the sample invasiveness (P=0.0042<0.05), and the PLCB1 expression level in the invasive pituitary adenoma group samples was significantly higher than that in the non-invasive pituitary adenoma group.

[0070] Example 4

[0071] 1) The receiver operating characteristic (ROC) curve analysis of the PLCB1 gene in the gene expression chips GSE26966 and GSE51618 was performed to analyze the accuracy and sensitivity of the PLCB1 gene in predicting the invasiveness of pituitary adenoma samples. The area under the ROC curve (AUC value) of 0.5-0.7 was moderate, 0.7-0.9 was good, and >0.9 was optimal. The results are as follows Fig.17 shown.

[0072] 2) The receiver operating characteristic (ROC) curve analysis of the PLCB1 gene was performed using the gene expression chip GSE169498. The results are as follows Fig.18 shown.

[0073] Depend on Fig.17 and 18 It can be concluded that the AUC value of the PLCB1 gene in the gene expression chip GSE26966 and GSE51618 samples was 0.991, and the AUC value in the gene expression chip GSE169498 sample was 0.705, both of which were > 0.7, indicating that the accuracy and sensitivity of using the PLCB1 gene to predict the aggressiveness of pituitary adenomas are high.

[0074] Example 5

[0075] Immunohistochemical assay to detect the expression level of PLCB1 gene in invasive and non-invasive pituitary adenomas

[0076] Immunohistochemical staining was used to detect the expression of PLCB1 gene in invasive and non-invasive pituitary adenoma samples collected clinically. Four biological replicates were set for invasive pituitary adenoma samples and non-invasive pituitary adenoma samples respectively. The positive control samples were normal brain samples, which showed strong positive expression. The specific steps are as follows:

[0077] Fresh tissues of invasive pituitary adenoma group samples, non-invasive group samples (samples were obtained from postoperative tissues of patients undergoing pituitary adenoma surgery in Beijing Tiantan Hospital, with ethical approval and written consent from patients) and positive control samples were collected respectively, and the preparation process was carried out according to the instructions in the Absin immunohistochemistry kit (abs957).

[0078] Observe the sections of invasive pituitary adenoma samples, non-invasive pituitary adenoma samples and positive control samples, and the results are as follows Fig.19 As shown by Fig.19 It can be concluded that compared with the non-invasive pituitary adenoma sample sections, the yellow-brown staining in the invasive pituitary adenoma sample sections is darker and the expression intensity is higher, indicating that the expression level of the PLCB1 gene in the invasive pituitary adenoma samples is significantly higher than that in the non-invasive pituitary adenoma samples.

[0079] Example 6

[0080] Western Blot assay was used to detect the expression level of PLCB1 gene in invasive and non-invasive pituitary adenomas

[0081] Immunohistochemical staining was used to detect the expression of PLCB1 gene in invasive and non-invasive pituitary adenoma samples collected clinically. Five biological replicates were set for invasive pituitary adenoma samples and non-invasive pituitary adenoma samples, respectively. GAPDH was used as the internal reference. The antibody information used in the process is shown in Table 3. The results are shown in Fig. 20 As shown in the figure, invasive represents invasive pituitary adenoma samples, and Non-invasive represents non-invasive pituitary adenoma samples.

[0082] Table 3 Antibody information used in Western Blot assay

[0083]

[0084] Image J software was used to analyze the grayscale value of PLCB1 gene in invasive and non-invasive pituitary adenomas in Western Blot experiments. Statistical analysis of the results showed that the P value between the two groups was 0.022 < 0.05, and the difference was statistically significant. The expression of PLCB1 gene in invasive pituitary adenoma samples was significantly higher than that in non-invasive pituitary adenoma.

[0085] Example 7

[0086] The role of PLCB1 in invasive pituitary adenomas was verified by knockdown as follows:

[0087] The specific method of packaging of lentivirus was entrusted to Shanghai GeneCare Gene Medical Technology Co., Ltd., and was specifically carried out in accordance with GeneCare's RNAi Lentivirus Vector Construction and Packaging Manual Version 4.0:

[0088] According to the RNA sequence design principles, multiple RNA target sequences are designed for the target gene sequence, and the target with the best kinetic parameters is selected to enter the subsequent experimental process.

[0089] Specifically include shRNA-1, shRNA-2 and shRNA-3, the specific sequences are as follows:

[0090] shRNA-1: 5'-ccggGCTGTCTTTGTCTACATAGAActcgagTTCTATGTAGACAAAGACAGCtttttg-3' (SEQ ID NO. 1);

[0091] shRNA-2: 5'-ccggGCAGATAAACATGGGCATGTActcgagTACATGCCCATGTTTATCTGCtttttg-3' (SEQ ID NO. 2);

[0092] shRNA-3: 5'-ccggCGCATGATAACTGTGGTGTATctcgagATACACCACAGTTATCATGCGtttttg-3' (SEQ ID NO. 3).

[0093] The vector numbered GV115 was selected for subsequent experiments. The element sequence was: hU6-MCS-CMV-EGFP. The plasmid map can be found on the official website of Genechem (http: / / www.genechem.com.cn / service / index.php?ac=gene&at=vector_search&keyword=GV).

[0094] Lentiviruses packaged with shRNA-1, shRNA-2 and shRNA-3 were obtained respectively.

[0095] Lentiviral transfection

[0096] Three cell lines were prepared for subsequent experiments, including ATT20, GT1-1 and GH3 pituitary adenoma cells. The MOI of lentiviral transfection was explored in the preliminary experiment. 4Inoculate 96-well plates at a concentration of 10 cells / mL, observe the cell density regularly, and start transfection when the cell density is about 30%, setting 3 different gradients of MOI (10, 50, 100). According to the MOI obtained in the pre-experiment, add appropriate amount of virus and complete medium for transfection, observe the cell morphology for 6-8 hours, and do not change the medium if the cell morphology is normal, and continue transfection for 24 hours. Replace with new complete medium, and detect cell fluorescence at 72 hours of transfection to verify the cell transfection efficiency. Screen monoclonal cell lines, and refer to the lentivirus operation manual for puromycin concentration. After screening stable strains, remove the old medium, wash 3 times with sterile PBS, and set aside.

[0097] Western blot (WB) was used to verify the knockdown of PLCB1 in cell lines.

[0098] 1. Protein Collection

[0099] 1) Take a RIPA aliquot (1 mL), add protease inhibitor (25×, 40 μL) and mix well. Add an appropriate volume to each sample according to the cell volume. The experimental group was transfected with PLCB1 knockdown lentivirus, and the control group was transfected with empty vector lentivirus;

[0100] 2) Lyse on ice for 30 minutes, shake after 15 minutes, and turn on the centrifuge to precool (4°C);

[0101] 3) Centrifugation at 12000 rpm / min for 15 min;

[0102] 4) After centrifugation, aspirate the supernatant into a new EP tube and shake to mix evenly;

[0103] 5) Measure protein concentration using the Solebol protein quantification kit (BCA method);

[0104] 6) Detect the sample absorbance at 562 nm using an ELISA reader.

[0105] 2. Electrophoresis:

[0106] 1) Sample preparation: add loading buffer according to the dosage in the instruction manual, cook the protein in a metal bath for 5 minutes, and centrifuge the protein sample;

[0107] 2) Electrophoresis: Place the gel into the electrophoresis tank, add the running buffer, and after ensuring that there is no water leakage, load 10ul of sample into each well, and add equal amounts of markers at both ends of the sample. The voltage of the stacking gel is 80V, and when the sample runs to the separation gel, the voltage is adjusted to 200V. The electrophoresis time is determined according to the molecular weight of the protein.

[0108] 3. Transfer (wet transfer)

[0109] 1) Prepare quick transfer buffer* (1000mL), 50mL 20× transfer buffer, 100mL anhydrous ethanol, 850mL deionized water. It needs to be prepared in advance when running electrophoresis, and the filter paper and PVDF membrane required for transfer are soaked in the transfer buffer and pre-cooled in a refrigerator at 4 degrees. The PVDF membrane needs to be thoroughly wetted in methanol before being placed in the transfer buffer;

[0110] 2) Place thick filter paper, thin filter paper, glue, PVDF membrane, thin filter paper, and thick filter paper (from black to white) in the transfer tank in sequence, and remove bubbles after each layer.

[0111] 3) Constant current 400mA for 25min for small protein and 300min for large protein.

[0112] 4. Blocking and incubation with antibodies:

[0113] 1) Take out the PVDF membrane after transfer, fix it with methanol, wash it 3 times with PBS-T, dry it, cut the required protein band according to the marker, and mark it;

[0114] 2) Place the cut membrane in 5% milk-PBS blocking solution, block on a shaker for 1 hour, wash 3 times with PBS-T, and blot dry;

[0115] Primary antibody: dilute abcam plcb1 (ab182359) with antibody diluent at 1:1000, add it into the incubation box together with the transferred membrane, shake on a rocker for 1 hrs, and then put it into a 4°C refrigerator;

[0116] 4) On the next day, remove the membrane and wash it in PBS-T for 3 min each time, for a total of 3 times;

[0117] 5) Secondary antibody: Prepare blocking solution at 1:1000, incubate for 1 hour, and then wash the membrane;

[0118] 6) Exposure: Prepare the color developing solution in a 1:1 ratio, add it onto the membrane, incubate for 3 to 5 minutes, and then expose it.

[0119] WB results:

[0120] By performing WB tests on the three pituitary adenoma cell lines in the experimental group and the control group, we found that among the three-protection-one virus, shRNA-3 had the best knockdown effect on PLCB1, so the subsequent experiments in this example used shRNA-3 virus to knock down PLCB1 ( Fig.21 ).

[0121] CCK8 proliferation assay

[0122] 1) Take pituitary tumor cells in logarithmic growth phase and in good cell state and stable cells after transfection, inoculate them in 96-well plates, add 100 μL of cell suspension containing (ATT20: 3000, GT1-1: 5000, GH3: 2000) cells to each well, and divide them into 3 groups: knockdown group, vector control group and blank control group. Set up 3 replicate wells in each group, add 200 μL of PBS around to reduce evaporation, and culture in an incubator;

[0123] 2) Add 10 μL (10% volume) of CCK8 solution to each well (add slowly to prevent bubbles from forming while cells adhere to the wall) and incubate in an incubator for 4 h until cells adhere to the wall;

[0124] 3) Use a microplate reader to measure the OD value of each well at 450 nm at 24 h, 48 h, and 72 h;

[0125] CCK8 proliferation assay results:

[0126] The CCK8 cell proliferation experiment was performed on the PLCB1 knockdown group and the empty group of three cell lines. The results showed that compared with the empty group, the growth rate of cells after PLCB1 knockdown was significantly reduced at 24h, 48h and 72h ( Fig. 22 ), which indicates that knocking down the expression of PLCB1 in pituitary adenoma cells can reduce the proliferation ability of cells.

[0127] Cell scratch assay

[0128] Experimental equipment: Marker pen, ruler, 6-well plate

[0129] Experimental methods:

[0130] 1) Rulers and other experimental equipment should be placed in a biological safety cabinet for irradiation and disinfection in advance;

[0131] 2) Use a marker pen to draw even lines along the ruler on the back of the 6-well plate, about 0.5-1 cm across the holes, 3 lines per hole, to mark them;

[0132] 3) Add 5×10 5 Cells were cultured overnight in a 5% CO2 37°C incubator;

[0133] 4) The next day, take out the 6-well plate and observe the cell density under a microscope. When the cells are fully grown, prepare for scratching.

[0134] 5) Use the tip of the gun to scratch along the ruler, try to make a mark perpendicular to the horizontal line on the back, and keep the tip of the gun vertical and not tilted when scratching;

[0135] 6) Wash three times with sterile PBS to remove the scratched cells and add serum-free medium;

[0136] 7) Place in a 37°C 5% CO2 incubator for culture. Take samples at 0, 24, and 48 hours and take photos.

[0137] Cell scratch assay results:

[0138] The results of the cell scratch test on the PLCB1 knockdown group and the empty group of the three cell lines showed that compared with the empty group, the 24h and 48h scratch healing ability of the cells after PLCB1 knockdown was significantly reduced ( Fig.23 ), which indicates that knocking down the expression of PLCB1 in pituitary adenoma cells can reduce the migration ability of cells.

[0139] Transwell cell invasion assay

[0140] 1) The day before the experiment, take out the matrix gel from the -20℃ refrigerator and put it in a 4℃ refrigerator, and pre-cool the pipette tips and centrifuge tubes required for the experiment at 4℃;

[0141] 2) On the day of the experiment, take out the matrix gel and place it on an ice box. Dilute the matrix gel at a ratio of 1:8: matrix gel: serum-free medium, add 60 μL of the diluent to the upper chamber, and place it in the incubator for 8 hours to allow the matrix gel to solidify.

[0142] 3) digestion, centrifugation, resuspending cells in serum-free medium and counting;

[0143] 4) Add 600 μL of 20% FBS culture medium to the lower chamber, and add 160 μL of cell suspension containing 50,000 cells to the upper chamber. Avoid generating bubbles during the operation, and then place in an incubator for 48 hours;

[0144] 5) Gently wash the chamber 2-3 times with PBS, then add 1 mL of anhydrous ethanol to each well to fix the cells for 20 minutes, pour out the ethanol in the chamber, add 1 mL of crystal violet to stain for 15-20 minutes, gently wash off the residual crystal violet with PBS buffer, and wipe off the cells on the inner wall of the chamber that did not pass through the chamber with a cotton swab;

[0145] 6) Observe under a microscope and take photos and count.

[0146] Transwell cell invasion assay results:

[0147] The Transwell cell invasion assay was performed on the PLCB1 knockdown group and the empty group of the three cell lines. The results showed that compared with the empty group, the number of cells passing through the Transwell chamber was significantly reduced after PLCB1 knockdown ( Fig.24 ), which indicates that knocking down the expression of PLCB1 in pituitary adenoma cells can weaken the invasive ability of cells.

[0148] It can be concluded from the above embodiments that the PLCB1 gene can be used as a biomarker for diagnosing invasive pituitary adenomas or evaluating the invasiveness of pituitary adenomas, and its expression level in non-invasive pituitary adenoma samples is significantly higher than that in invasive pituitary adenoma samples; and the knockdown experiment can prove that the reagent for inhibiting the expression of the PLCB1 gene can be used to prepare a drug for treating invasive pituitary adenomas and / or reducing the invasiveness of pituitary adenomas.

[0149] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Detection PLCB1 Use of a gene reagent in the preparation of a product for diagnosing invasive pituitary adenoma and / or evaluating the invasiveness of pituitary adenoma.

2. The use according to claim 1, characterized in that: The product includes one or more of a reagent, a kit and a chip.

3. PLCB1 The invention relates to the use of a gene expression inhibitor in the preparation of a drug for treating invasive pituitary adenoma and / or reducing the invasiveness of pituitary adenoma; the expression inhibitor comprises shRNA-1, shRNA-2 or shRNA-3; the nucleotide sequences of the shRNA-1, shRNA-2 and shRNA-3 are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively.

4. A drug for treating invasive pituitary adenoma and / or reducing the invasiveness of pituitary adenoma, characterized in that: The medicine includes biochemical preparations and auxiliary materials that inhibit or reduce the expression of PLCB1 gene; PLCB1 The biochemical agent for regulating the expression amount of the gene includes shRNA-1, shRNA-2 or shRNA-3; the nucleotide sequences of the shRNA-1, shRNA-2 and shRNA-3 are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively.

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