Use of a substance that inhibits the activity and / or expression of fam111b protein in the prevention of non-small cell lung cancer

Abstract

The application discloses application of a substance for inhibiting FAM111B protein activity and / or expression quantity in prevention of non-small cell lung cancer. The substance for inhibiting FAM111B protein activity and / or expression quantity is z1) or z2): z1) an RNAi molecule shown in SEQ ID NO:1; and z2) an shRNA synthesized by an shRNA expression system and taking the RNAi molecule as a target. Experiments prove that FAM111B is significantly highly expressed in non-small cell lung cancer tissues, and is obviously related to gender, histological type, tumor size, stage, and lymph node metastasis; FAM111B enhances the anti-apoptosis ability of non-small cell lung cancer cells by participating in a p53-regulated mitochondrial apoptosis pathway; and inhibition of the activity and / or expression quantity of FAM111B in non-small cell lung cancer cells can promote apoptosis of the non-small cell lung cancer cells, thereby preventing and / or treating non-small cell lung cancer. The application has important application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of substances that inhibit the activity and / or expression of FAM111B protein in the prevention of non-small cell lung cancer. Background Technology

[0002] Xuanwei lung cancer is a type of lung cancer, primarily adenocarcinoma, unique to the Xuanwei region of Yunnan Province. The clinical treatment outcomes of lung cancer exhibit significant individual variability; even patients with the same stage and subtype of lung cancer show varying sensitivities to standardized treatment regimens. Tumor heterogeneity is one of the unavoidable challenges in current clinical treatment. Tumor cells undergo multiple differentiations, resulting in genomic and behavioral changes due to external environmental factors or their own adaptations. This leads to adverse traits such as accelerated cell growth, clinical drug resistance, and poor prognosis, resulting in multiple genotypes and differentiation subtypes. This is the fundamental reason for the differences in clinical treatment outcomes and prognoses in lung cancer. The continuous development of modern biological technologies, such as proteomics and genomics, has greatly aided in the molecular subtyping, pathogenesis, and diagnosis of lung cancer. According to the WHO classification of lung cancer, histologically, lung cancer can be broadly divided into two categories: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), accounting for approximately 85% and 15% of all lung cancer cases worldwide, respectively. Lung adenocarcinoma (LUAD) is a type of non-small cell lung cancer (NSCLC) that primarily develops in the mucous glands of the bronchi in the lungs.

[0003] Effective diagnostic techniques are the most crucial link and method for reducing the mortality rate of lung cancer. Early diagnosis and treatment of lung cancer mainly include X-rays and sputum cytology. With the development of tumor diagnosis and treatment technologies, the application of various techniques such as automated fluorescence bronchoscopy (AFB), positron emission tomography (PET), X-rays, ultrasound, MRI, endoscopy, longitudinal fluorescence examination, and thoracoscopy has reduced the mortality rate of lung cancer patients to varying degrees in various trials. Molecular targeted screening technology, which emerged in the late 20th century, has become a precision technology for the early prevention and diagnosis of lung cancer by screening tumor markers through antigen-antibody binding. In terms of treatment, cisplatin and carboplatin combined with paclitaxel and gemcitabine have achieved good clinical efficacy in the treatment of lung adenocarcinoma. However, along with considerable efficacy and remission rates, serious problems such as unavoidable chemotherapy toxicity and tumor drug resistance remain. The most representative example is the clinical resistance of non-small cell lung cancer patients to ECFR-TKIs, clinically manifested as gefitinib resistance in NSCLC patients leading to insensitivity to similar tyrosine kinase inhibitors (TKIs). Driven by precision medicine, a product of modern medical development, new specific treatments such as targeted therapy and immunotherapy have emerged. While these specifically kill tumor cells, they also significantly reduce the scope of application. Nevertheless, targeted drugs still offer the best treatment efficacy for intermediate and advanced malignant tumors, as they can specifically kill tumor cells and minimize damage to the body.

[0004] Molecular targeted therapy has shown good efficacy in treating tumors, especially lung cancer with specific genetic mutations. Studies have shown that nearly 70% of non-small cell lung cancer patients carry potential therapeutic molecular targets. Molecular targeted therapy is most effective for young patients with adenocarcinoma and those with specific genetic abnormalities, as well as for advanced lung cancer patients with specific gene mutations who are not suitable for surgery. Next-generation sequencing can identify abnormal gene loci, and corresponding molecular targeted therapies can be used in combination to achieve good therapeutic outcomes. Currently, the most studied target genes include PD-1 / PD-L1, EGFR, ALK / ROS1, KRAS, MET, and RET. Abnormal changes in these genes are also used clinically as tumor markers for lung cancer diagnosis, greatly aiding in the diagnosis and treatment of lung adenocarcinoma.

[0005] Tumor immunotherapy is a novel molecularly targeted treatment method that has shown great potential in lung cancer treatment. Compared with traditional chemotherapy and radiotherapy, molecularly targeted techniques have demonstrated good efficacy and low toxicity. Although the scope of application has been correspondingly reduced, the development of molecularly targeted technology remains a powerful means to improve the survival of cancer patients. With the rapid development of modern medical technology, a series of new technologies under the background of precision medicine are gradually being applied to the life sciences. Technologies such as next-generation sequencing are shifting traditional medicine from hypothesis-driven to data-driven approaches. Combining multi-omics detection technologies such as reverse transcriptomics and proteomics, and utilizing large-scale biological database analysis, to identify susceptibility genes for tumor development and progression and to perform functional verification, represents a new approach and direction for breakthroughs in the treatment of lung adenocarcinoma. Summary of the Invention

[0006] The purpose of this invention is to prevent and / or treat non-small cell lung cancer.

[0007] This invention first protects the use of substances that inhibit the activity and / or expression level of FAM111B protein in the preparation of products; the function of said product may be at least one of the following C1) to C3):

[0008] C1) Prevention of non-small cell lung cancer;

[0009] C2) Treatment for non-small cell lung cancer;

[0010] C3) promotes apoptosis in non-small cell lung cancer cells.

[0011] In the above applications, the non-small cell lung cancer may be squamous cell carcinoma or adenocarcinoma of the lung.

[0012] In the above applications, the substance that inhibits the activity and / or expression level of FAM111B protein can be z1) or z2):

[0013] z1) The RNAi molecule shown in SEQ ID NO: 1;

[0014] z2) shRNA synthesized by the shRNA expression system with the RNAi molecule as the target.

[0015] The z2) can specifically be sh-FAM111B-2 in step four of embodiment 2.

[0016] This invention also protects the use of substances that use FAM111B as a drug target in the preparation of products; the function of said products may be at least one of the following C1) to C3):

[0017] C1) Prevention of non-small cell lung cancer;

[0018] C2) Treatment for non-small cell lung cancer;

[0019] C3) promotes apoptosis in non-small cell lung cancer cells.

[0020] In the above applications, the non-small cell lung cancer may be squamous cell carcinoma or adenocarcinoma of the lung.

[0021] This invention also protects a product that may contain a substance that inhibits the activity and / or expression level of the FAM111B protein; the function of said product may be at least one of the following C1) to C3):

[0022] C1) Prevention of non-small cell lung cancer;

[0023] C2) Treatment for non-small cell lung cancer;

[0024] C3) promotes apoptosis in non-small cell lung cancer cells.

[0025] The product may specifically consist of substances that inhibit the activity and / or expression level of the FAM111B protein.

[0026] In the above products, the non-small cell lung cancer may be squamous cell carcinoma or adenocarcinoma of the lung.

[0027] This invention also protects the use of substances that inhibit the activity and / or expression level of FAM111B protein in the development or screening of reagents; the use of said reagents may be at least one of the following C1) to C3):

[0028] C1) Prevention of non-small cell lung cancer;

[0029] C2) Treatment for non-small cell lung cancer;

[0030] C3) promotes apoptosis in non-small cell lung cancer cells.

[0031] In any of the above-described applications, the substance that inhibits the activity and / or expression level of FAM111B protein may be z1) or z2):

[0032] z1) The RNAi molecule shown in SEQ ID NO: 1;

[0033] z2) shRNA synthesized by the shRNA expression system with the RNAi molecule as the target.

[0034] The z2) can specifically be sh-FAM111B-2 in step four of embodiment 2.

[0035] This invention also protects the use of FAM111B protein as a biomarker in the diagnosis or auxiliary diagnosis of non-small cell lung cancer.

[0036] In the above applications, the activity and / or expression level of FAM111B protein in non-small cell lung cancer patients were higher than those in healthy individuals.

[0037] In any of the above-described applications, the non-small cell lung cancer may be squamous cell carcinoma or adenocarcinoma of the lung.

[0038] Experiments have demonstrated that FAM111B is significantly highly expressed in non-small cell lung cancer (NSCLC) tissues, and its expression is significantly correlated with gender, histological type, tumor size, stage, and lymph node metastasis. FAM111B enhances the anti-apoptotic ability of NSCLC cells by participating in the p53-regulated mitochondrial apoptosis pathway. Inhibiting the activity and / or expression level of FAM111B in NSCLC cells can promote apoptosis in NSCLC cells, thereby preventing and / or treating NSCLC. This invention has significant application value. Attached Figure Description

[0039] Figure 1 The melting curve and amplification curve are for the real-time fluorescence PCR process.

[0040] Figure 2 The gene expression of five candidate susceptibility genes in lung adenocarcinoma cells was compared with that of the negative control group. * indicates P<0.5, ** indicates P<0.01, and *** indicates P<0.001.

[0041] Figure 3 The expression differences of five candidate susceptibility genes in lung adenocarcinoma tissue microarrays are shown. * indicates P<0.5, ** indicates P<0.01, and *** indicates P<0.001.

[0042] Figure 4 The melting curve and amplification curve are for the real-time fluorescence PCR process.

[0043] Figure 5 The reverse transcription expression of FAM111B in clinical lung cancer tissues. * indicates P<0.5, ** indicates P<0.01, *** indicates P<0.001.

[0044] Figure 6 The expression of FAM111B protein in lung adenocarcinoma cells.

[0045] Figure 7 Immunohistochemical detection of FAM111B expression in lung adenocarcinoma tissue (×200, N=25). * indicates P<0.5, ** indicates P<0.01, *** indicates P<0.001.

[0046] Figure 8The expression of FAM111B protein in lung squamous cell carcinoma tissue and adjacent normal tissue is shown. A indicates negative expression of FAM111B protein in lung squamous cell carcinoma tissue; B indicates weak positive expression of FAM111B protein in lung squamous cell carcinoma tissue; C indicates positive expression of FAM111B protein in lung squamous cell carcinoma tissue; D indicates strong positive expression of FAM111B protein in lung squamous cell carcinoma tissue (AD: 200×); E is a bar chart of the positive expression rate of FAM111B protein in each group of lung squamous cell carcinoma; F is a bar chart of the expression score of FAM111B protein in each group of lung squamous cell carcinoma (***P<0.001).

[0047] Figure 9 The expression of FAM111B protein in lung adenocarcinoma tissue and adjacent normal tissue is shown. A indicates negative expression of FAM111B protein in lung adenocarcinoma tissue; B indicates weak positive expression of FAM111B protein in lung adenocarcinoma tissue; C indicates positive expression of FAM111B protein in lung adenocarcinoma tissue; D indicates strong positive expression of FAM111B protein in lung adenocarcinoma tissue (AD: 200×); E is a bar chart of the positive expression rate of FAM111B protein in each lung adenocarcinoma group; F is a bar chart of the expression score of FAM111B protein in each lung adenocarcinoma group (***P<0.001).

[0048] Figure 10 The expression of LAD1 protein in lung squamous cell carcinoma tissue and adjacent tissue is shown. A indicates negative expression of LAD1 protein in lung squamous cell carcinoma tissue; B indicates weak positive expression of LAD1 protein in lung squamous cell carcinoma tissue; C indicates positive expression of LAD1 protein in lung squamous cell carcinoma tissue; D indicates strong positive expression of LAD1 protein in lung squamous cell carcinoma tissue (AD: 200×); E is a bar chart of the positive expression rate of LAD1 protein in each group of lung squamous cell carcinoma; F is a bar chart of the expression score of LAD1 protein in each group of lung squamous cell carcinoma (***P < 0.001).

[0049] Figure 11 The expression of LAD1 protein in lung adenocarcinoma tissue and adjacent tissue is shown. A indicates negative expression of LAD1 protein in adjacent tissue of lung adenocarcinoma; B indicates weak positive expression of LAD1 protein in lung adenocarcinoma tissue; C indicates positive expression of LAD1 protein in lung adenocarcinoma tissue; D indicates strong positive expression of LAD1 protein in lung adenocarcinoma tissue (AD: 200×); E is a bar chart of the positive expression rate of LAD1 protein in each group of lung adenocarcinoma; F is a bar chart of the expression score of LAD1 protein in each group of lung adenocarcinoma (***P < 0.001).

[0050] Figure 12The expression of AHNAK2 protein in lung squamous cell carcinoma tissue and adjacent tissue is shown. A indicates negative expression of AHNAK2 protein in lung squamous cell carcinoma tissue; B indicates weak positive expression of AHNAK2 protein in lung squamous cell carcinoma tissue; C indicates positive expression of AHNAK2 protein in lung squamous cell carcinoma tissue; D indicates strong positive expression of AHNAK2 protein in lung squamous cell carcinoma tissue (AD: 200×); E is a bar chart of the positive expression rate of AHNAK2 protein in each group of lung squamous cell carcinoma; F is a bar chart of the expression score of AHNAK2 protein in each group of lung squamous cell carcinoma (***P<0.001).

[0051] Figure 13 The expression of AHNAK2 protein in lung adenocarcinoma tissue and adjacent normal tissue is shown. A indicates negative expression of AHNAK2 protein in lung adenocarcinoma tissue; B indicates weak positive expression of AHNAK2 protein in lung adenocarcinoma tissue; C indicates positive expression of AHNAK2 protein in lung adenocarcinoma tissue; D indicates strong positive expression of AHNAK2 protein in lung adenocarcinoma tissue (AD: 200×); E is a bar chart showing the positive expression rate of AHNAK2 protein in each lung adenocarcinoma group; F is a bar chart showing the expression score of AHNAK2 protein in each lung adenocarcinoma group (***P < 0.001).

[0052] Figure 14 To determine the expression of FAM111B in different non-small cell lung cancer cell lines. A: qRT-PCR was used to detect the mRNA expression level of FAM111B in non-small cell lung cancer cells; B: Western blot was used to detect the protein expression of FAM111B in non-small cell lung cancer cells.

[0053] Figure 15 This is a schematic diagram of the shRNA lentiviral vector GV248.

[0054] Figure 16 To validate the knockdown of FAM111B in the H1299 cell line. A: qRT-PCR detection of FAM111B mRNA expression in each transfected cell line, empty vector control, and blank control (*P<0.05, **P<0.01, #P: no statistical significance); B: Western blot detection of FAM111B protein expression in each transfected cell line, empty vector control, and blank control (***P<0.001, #P: no statistical significance).

[0055] Figure 17 The effect of FAM111B on apoptosis of non-small cell lung cancer cells H1299. A shows the apoptosis of sh-NC (empty vector control) and sh-FAM111B cells detected by flow cytometry; B shows the comparison of apoptosis rates between sh-NC and sh-FAM111B cells (***P<0.001).

[0056] Figure 18 The effect of FAM111B on apoptosis-related proteins in non-small cell lung cancer cells H1299. A shows the expression of apoptosis-related proteins of FAM111B in sh-NC and sh-FAM111B cells detected by Western blot; B shows the grayscale analysis of the expression of apoptosis-related proteins in sh-NC and sh-FAM111B cells (*P<0.05, **P<0.01).

[0057] Figure 19 The expression of FAM111B-cfDNA in the serum of non-small cell lung cancer patients and healthy volunteers was detected by qRT-PCR (**P<0.01). Detailed Implementation

[0058] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0059] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0060] The human lung adenocarcinoma cell line XWLC-05 in the following examples is a lung cancer cell line from Xuanwei.

[0061] Example 1: Discovery of five candidate susceptibility genes for lung adenocarcinoma and their verification at the reverse transcription level in lung adenocarcinoma cells.

[0062] I. Discovery of 5 candidate susceptibility genes for lung adenocarcinoma

[0063] Based on reverse transcriptome sequencing (RNA-seq) results from 4909 tumor tissues and 631 normal tissues involving 13 types of tumors, the inventors of this invention used Cross-Value Association Analysis (CVAA) scoring criteria to screen for the top 500 upregulated genes and the top 500 downregulated genes with significant differences in tumor tissues (fold change > 2, P < 0.05). Subsequently, five genes with significant differences in lung adenocarcinoma and relatively few related studies were selected as candidate susceptibility genes. These five candidate susceptibility genes are COL10A1, LAD1, IGSF9, AHNAK2, and FAM111B.

[0064] II. Detection of reverse transcription expression of five candidate susceptibility genes in lung adenocarcinoma cells

[0065] Using human bronchial epithelial cells Beas-2b as a negative control, the reverse transcription expression levels of candidate susceptibility genes (COL10A1, LAD1, IGSF9, AHNAK2, or FAM111B) in human lung adenocarcinoma cell lines (A549, NCI-H1299, SPC-A1, or XWLC-05) were detected by real-time fluorescence PCR. The specific steps are as follows:

[0066] 1. RNA was extracted from human lung adenocarcinoma cell lines (A549, NCI-H1299, SPC-A1 or XWLC-05) and human bronchial epithelial cells Beas-2b, respectively, and RNA integrity was detected by agarose gel electrophoresis.

[0067] The results showed that both the RNA from human lung adenocarcinoma cells and the RNA from human bronchial epithelial cells Beas-2b exhibited distinct three bands (5S, 18S, and 28S), with the 28S band being approximately twice that of the 18S band. This indicates that the extracted RNA from both human lung adenocarcinoma cells and human bronchial epithelial cells Beas-2b possessed integrity.

[0068] 2. Real-time quantitative PCR

[0069] Using RNA from human lung adenocarcinoma cell lines or human bronchial epithelial cells Beas-2b as templates, the expression levels of candidate susceptibility genes (COL10A1, LAD1, IGSF9, AHNAK2, or FAM111B) were detected by real-time fluorescence PCR.

[0070] The primers for detecting COL10A1 are 5'-ATGCTGCCACAAATACCCTTT-3' and 5'-GGTAGTGGGCCTTTTATGCCT-3'. The primers for detecting LAD1 are 5'-CTTGAGGCTCTCAGGGGTTG-3' and 5'-CTGAGTCCTCTCGGTTGCAG-3'. The primers for detecting IGSF9 are 5'-CCCCAAGAACAGCACAGTCA-3' and 5'-AGGTGAGGTTAGCAGGGTATG-3'. The primers for detecting AHNAK2 are 5'-GCAGAAACGGAAGATGACCAC-3' and 5'-CAGCCTCAGTCGTGTATTCGT-3'. The primers for detecting FAM111B are 5'-GCATATGGTAAACCCAGCGAG-3' and 5'-GAATCACTAGGCAGGCACTTG-3'.

[0071] Observe the melting curve and amplification curve. The detection results are shown below. Figure 1(A is the amplification curve, B is the melting curve): A clear melting single peak and amplification plateau phase are observed. This indicates that there was no non-specific amplification or primer dimer interference during the experiment.

[0072] Statistical results of expression levels of candidate susceptibility genes are shown in [the table]. Figure 2 The results showed that, compared with human bronchial epithelial cells Beas-2b, LAD1, COL10A1, and IGSF-9 exhibited certain differences in reverse transcription expression levels in A549, NCI-H1299, SPC-A1, or XWLC-05 cells. LAD1, FAM111B, and COL10A1 showed significant overexpression in Xuanwei lung cancer cells XWLC-05. LAD1, FAM111B, IGSF9, and COL10A1 all showed significant differences in gene overexpression in lung adenocarcinoma and adjacent tissue microarrays, with FAM11B showing approximately 11-fold overexpression.

[0073] 3. Reverse transcription expression of 5 candidate susceptibility genes in lung adenocarcinoma tissue

[0074] The reverse transcription levels of five candidate susceptibility genes were detected using a Roche lung adenocarcinoma cDNA microarray. The cDNA was derived from lung adenocarcinoma tissue and adjacent normal tissue, with 20 ng of cDNA template per well, and included complete clinical information.

[0075] The reverse transcription results are shown below. Figure 3 (Para-LUAD represents lung adenocarcinoma tissue, and LUAD represents lung adenocarcinoma tissue). The results showed that LAD1, FAM111B, IGSF9, and COL10A1 exhibited significant differences in gene expression between lung adenocarcinoma and lung adenocarcinoma tissue microarrays, and showed a clear overexpression trend.

[0076] III. Differences in gene expression in clinical lung cancer tissues

[0077] The inventors of this invention collected postoperative lung cancer tissue and paired adjacent normal tissue (LUAD, n=25; SCLC, n=26) from lung cancer patients at Yunnan Cancer Hospital from December 2018 to February 2020, and used real-time fluorescence PCR to detect the differences in reverse transcription expression of five candidate susceptibility genes in clinical lung cancer tissue.

[0078] Based on the melting curve and amplification curve during the instrumentation process (see...) Figure 4 The experiment showed a clear melting peak and an amplification plateau, indicating that there was no non-specific amplification or primer dimer effect during the experiment.

[0079] Real-time fluorescence PCR results are shown in Figure 5(A represents the differential expression of FAM1111B in 51 clinical lung cancer tissues; B represents the differential reverse transcription of FAM111B in clinical lung adenocarcinoma and lung squamous cell carcinoma tissues; Para-LUAD represents adjacent normal tissue of lung adenocarcinoma; LUAD represents lung adenocarcinoma tissue; Para-SCLC represents adjacent normal tissue of lung squamous cell carcinoma; Ca represents cancer tissue; Para-Ca represents adjacent normal tissue). The results showed that FAM111B exhibited an 8.5-fold differential in reverse transcription overexpression in lung cancer tissues (P<0.05); while the reverse transcription expression of FAM111B was higher in lung squamous cell carcinoma tissues compared to lung adenocarcinoma (P<0.05).

[0080] IV. Protein Expression of FAM111B in Lung Adenocarcinoma

[0081] 1. Western blotting was used to detect the protein expression of FAM111B in human bronchial epithelial cells (Beas-2b) and lung adenocarcinoma cells (primary antibody: Anti-FAM111B Antibody, a product of Novus Bioloicals). Results are shown below. Figure 6 See Table 1 (* indicates P<0.5, ** indicates P<0.01, *** indicates P<0.001). The results showed that FAM111B protein was almost not expressed in human bronchial epithelial cells Beas-2b, but was highly expressed in A549, NCI-H1299, SPC-A1 and XWLC-05 cells, and was more pronounced in Xuanwei lung cancer cells XWLC-05.

[0082] Table 1. Expression of FAM111B protein in lung adenocarcinoma cells

[0083] FAM111B / β-actin Beas-2b 0.0950±0.0235 A549 0.9986±0.1339*** NCI-H1299 1.5573±0.07275*** SPC-A1 0.5620±0.1531** XWLC-05 1.2106±0.1782***

[0084] 2. Immunohistochemical staining was used to detect the expression of the target protein in lung adenocarcinoma tissue.

[0085] Test results are shown Figure 7 (Ca represents cancerous tissue, Para-Ca represents adjacent normal tissue). The target protein FAM111B is located in the cell nucleus and cytoplasm. Under a microscope, blue (representing the cell nucleus) and brown (FAM111B protein) colors can be observed. The proportion and intensity of the brown color in lung adenocarcinoma tissue are significantly higher than those in adjacent normal tissue. Quantitative results show that the immune response score (IRS) of the lung adenocarcinoma group (Ca) is significantly higher than that of the paired negative control group (Para-Ca) (P<0.05).

[0086] The results showed that FAM111B was indeed highly expressed in reverse transcription and at the protein level in lung adenocarcinoma, and this expression was more pronounced in lung adenocarcinoma than in lung squamous cell carcinoma.

[0087] Example 2: Expression of FAM111B in non-small cell lung cancer and its effect on apoptosis

[0088] I. Expression of FAM111B, LAD1, and AHNAK2 in Non-Small Cell Lung Cancer Tissues

[0089] To determine the subjects for subsequent research, the inventors of this invention collected 146 non-small cell lung cancer tissue samples (including 74 squamous cell carcinoma tissues and 72 adenocarcinoma tissues) and used immunohistochemistry to detect the expression of FAM111B, LAD1, and AHNAK2 proteins in non-small cell lung cancer tissues. The results showed that all three proteins were expressed to some extent in both non-small cell lung cancer tissues and adjacent normal tissues, but the expression in non-small cell lung cancer tissues was significantly higher than that in adjacent normal tissues. Specifically, the expression level of FAM111B in squamous cell carcinoma and adenocarcinoma tissues was 4.36 times and 3.79 times higher than that in adjacent normal tissues, respectively; the expression level of LAD1 in squamous cell carcinoma and adenocarcinoma tissues was 3.20 times and 3.03 times higher than that in adjacent normal tissues, respectively; and the expression level of AHNAK2 in squamous cell carcinoma and adenocarcinoma tissues was 3.61 times and 3.95 times higher than that in adjacent normal tissues, respectively. Details are as follows:

[0090] 1. Expression of FAM111B in non-small cell lung cancer tissues

[0091] (1) Expression of FAM111B in lung squamous cell carcinoma tissue

[0092] The results showed that FAM111B was located in the nucleus and cytoplasm, and the brownish-yellow color indicated protein binding. In 74 cases of lung squamous cell carcinoma tissue, 63 cases (85.1%) were positive for FAM111B staining, of which 24 cases (32.4%) were strongly positive. FAM111B staining was negative in all adjacent tissues of lung squamous cell carcinoma, and the positive rate in lung squamous cell carcinoma tissue was significantly higher than that in adjacent tissues (P < 0.05). Figure 8 In the middle section (AE, Ca represents cancerous tissue, Para-Ca represents adjacent normal tissue),...

[0093] According to the expression score of FAM111B protein, the expression scores of FAM111B in lung squamous cell carcinoma tissue and adjacent normal tissue were 5.93±2.50 and 1.36±0.79, respectively. The expression of FAM111B protein in lung squamous cell carcinoma tissue was significantly higher than that in adjacent normal tissue (P<0.001). Figure 8 In the middle section, F and Ca represent cancerous tissue, while Para-Ca represents adjacent normal tissue.

[0094] (2) Expression of FAM111B in lung adenocarcinoma tissue

[0095] Of the 72 lung adenocarcinoma tissue samples, 42 (58.3%) were FAM111B positive, with 11 (15.3%) showing strong positivity. FAM111B staining was negative in all adjacent tissues of lung adenocarcinoma, and the positive rate in lung adenocarcinoma tissues was significantly higher than that in adjacent tissues (P < 0.05). Figure 9 In the middle section (AE, Ca represents cancerous tissue, Para-Ca represents adjacent normal tissue),...

[0096] According to the expression score of FAM111B protein, the expression scores of FAM111B in lung adenocarcinoma tissue and adjacent normal tissue were 4.47±2.56 and 1.18±0.42, respectively. The expression of FAM111B protein in lung adenocarcinoma tissue was significantly higher than that in adjacent normal tissue (P<0.001). Figure 9 In the middle section, F and Ca represent cancerous tissue, while Para-Ca represents adjacent normal tissue.

[0097] 2. Expression of LAD1 in non-small cell lung cancer tissues

[0098] (1) Expression of LAD1 in lung squamous cell carcinoma tissue

[0099] Immunohistochemical results showed that LAD1 was located in the extracellular matrix, and the brownish-yellow color indicated protein binding. In 74 cases of lung squamous cell carcinoma tissue, 70 cases (94.6%) were LAD1 positive, with 22 cases (29.7%) showing strong positivity. Four cases (5.4%) of adjacent tissues of lung squamous cell carcinoma were LAD1 positive, and the positive rate in lung squamous cell carcinoma tissue was significantly higher than that in adjacent tissues (P < 0.05). Figure 10 In the middle section (AE, Ca represents cancerous tissue, Para-Ca represents adjacent normal tissue),...

[0100] According to the LAD1 protein expression score, the LAD1 protein expression scores in lung squamous cell carcinoma tissue and adjacent normal tissue were 6.55±1.87 and 2.05±1.26, respectively. The LAD1 protein expression in lung squamous cell carcinoma tissue was significantly higher than that in adjacent normal tissue (P<0.001). Figure 10 In the middle section, F and Ca represent cancerous tissue, while Para-Ca represents adjacent normal tissue.

[0101] (2) Expression of LAD1 in lung adenocarcinoma tissue

[0102] Of the 72 lung adenocarcinoma tissue samples, 59 (81.9%) were LAD1 positive, with 8 (6.9%) showing strong positivity. Eight (11.1%) of the adjacent tissues of lung adenocarcinoma were also LAD1 positive, and the positivity rate in lung adenocarcinoma tissues was significantly higher than that in adjacent tissues (P < 0.05). Figure 11 In the middle section (AE, Ca represents cancerous tissue, Para-Ca represents adjacent normal tissue),...

[0103] According to the LAD1 protein expression score, the LAD1 protein expression scores in lung adenocarcinoma tissue and adjacent normal tissue were 5.61±1.80 and 1.85±1.07, respectively. The LAD1 protein expression in lung adenocarcinoma tissue was significantly higher than that in adjacent normal tissue (P<0.001). Figure 11 In the middle section, F and Ca represent cancerous tissue, while Para-Ca represents adjacent normal tissue.

[0104] 3. Expression of AHNAK2 in non-small cell lung cancer tissues

[0105] (1) Expression of AHNAK2 in lung squamous cell carcinoma tissue

[0106] Immunohistochemical results showed that AHNAK2 was located in the cell nucleus, and the brownish-yellow color indicated protein binding. In 74 cases of lung squamous cell carcinoma tissue, 38 cases (51.3%) were AHNAK2 positive, with 5 cases (6.8%) showing strong positivity. One case (1.4%) of adjacent tissue of lung squamous cell carcinoma was AHNAK2 positive, and the positive rate in lung squamous cell carcinoma tissue was significantly higher than that in adjacent tissue (P < 0.05). Figure 12 In the middle section (AE, Ca represents cancerous tissue, Para-Ca represents adjacent normal tissue),...

[0107] According to the AHNAK2 protein expression score, the AHNAK2 protein expression scores in lung squamous cell carcinoma tissue and adjacent normal tissue were 4.12±2.2 and 1.14±1.06, respectively. The AHNAK2 protein expression in lung squamous cell carcinoma tissue was significantly higher than that in adjacent normal tissue (P<0.001). Figure 12 In the middle section, F and Ca represent cancerous tissue, while Para-Ca represents adjacent normal tissue.

[0108] (2) Expression of AHNAK2 in lung adenocarcinoma tissue

[0109] Of the 72 lung adenocarcinoma tissue samples, 27 (37.5%) were AHNAK2 positive, with 5 (6.9%) showing strong positivity. AHNAK2 staining was negative in all adjacent tissues of lung adenocarcinoma, and the positive rate in lung adenocarcinoma tissues was significantly higher than that in adjacent tissues (P < 0.05). Figure 13 In the middle section (AE, Ca represents cancerous tissue, Para-Ca represents adjacent normal tissue),...

[0110] According to the AHNAK2 protein expression score, the AHNAK2 protein expression scores in lung adenocarcinoma tissue and adjacent normal tissue were 3.2±2.5 and 0.81±0.62, respectively. The AHNAK2 protein expression in lung adenocarcinoma tissue was significantly higher than that in adjacent normal tissue (P<0.001). Figure 13 In the middle section, F and Ca represent cancerous tissue, while Para-Ca represents adjacent normal tissue.

[0111] II. Relationship between the expression of FAM111B, LAD1, and AHNAK2 in non-small cell lung cancer tissues and clinicopathological markers

[0112] Immunohistochemistry was used to detect the expression of FAM111B, LAD1, and AHNAK2 in non-small cell lung cancer tissues. The results showed that the expression of the three proteins in non-small cell lung cancer tissues was higher than that in adjacent normal tissues. Based on this, the relationship between the expression of FAM111B, LAD1, and AHNAK2 proteins and the clinicopathological indicators of patients was further analyzed.

[0113] 1. Relationship between FAM111B expression and clinicopathological markers in non-small cell lung cancer tissues

[0114] The results are shown in Table 2. The results indicated that among FAM111B-positive patients, male patients showed significantly higher FAM111B protein expression than female patients (P < 0.001); the positive rate in lung squamous cell carcinoma tissue (85.14%) was higher than that in lung adenocarcinoma tissue (58.33%); FAM111B expression in advanced (stage III and IV) patients was higher than that in early (stage I and II) patients (P < 0.05); FAM111B expression in patients with tumors of T2-T4 size was higher than that in patients with tumors of T1 size (P < 0.05); and FAM111B protein expression in patients with lymph node metastasis was significantly higher than that in patients without lymph node metastasis (P < 0.05), but this was not related to the patient's age.

[0115] Table 2. Relationship between FAM111B protein expression in non-small cell lung cancer tissues and clinicopathological markers in patients.

[0116]

[0117] 2. Relationship between LAD1 expression and clinicopathological markers in non-small cell lung cancer tissues

[0118] The test results are shown in Table 3. The results showed that in LAD1-positive patients, LAD1 protein expression was higher in patients older than 55 years than in patients ≤55 years old (P<0.05); the positive rate in lung squamous cell carcinoma tissue (94.59%) was higher than that in lung adenocarcinoma tissue (81.94%); and LAD1 expression was higher in advanced (stage III and IV) patients than in early (stage I and II) patients (P<0.05). However, in LAD1-positive patients, LAD1 expression was not related to patient gender, tumor size, or lymph node metastasis.

[0119] Table 3. Relationship between LAD1 protein expression in non-small cell lung cancer tissues and clinicopathological markers in patients.

[0120]

[0121]

[0122] 3. Relationship between AHNAK2 expression and clinicopathological markers in non-small cell lung cancer tissues

[0123] The test results are shown in Table 4. The results showed that among AHNAK2-positive patients, only male patients showed significantly higher AHNAK2 protein expression than female patients (P < 0.01). AHNAK2 expression was not related to age, histological type, stage, tumor size, or lymph node metastasis.

[0124] Table 4. Relationship between AHNAK2 protein expression in non-small cell lung cancer tissues and clinicopathological markers in patients.

[0125]

[0126]

[0127] Further analysis was conducted to compare the positive expression rates of each gene in 146 non-small cell lung cancer tissues and their correlation with clinicopathological indicators (P < 0.05). The results are shown in Table 5. Among the three genes, the positive expression rate of FAM111B protein was 71.9%, and it was significantly correlated with gender, histological type, stage, tumor size, and lymph node metastasis (P < 0.05); the positive expression rate of LAD1 protein was 88.4%, and it was correlated with age, histological type, and stage (P < 0.05); while the positive expression rate of AHNAK2 protein was 51.4%, and it was only correlated with gender (P < 0.05).

[0128] Table 5. Comparison of protein expression positivity rate and pathological indicators of three genes in non-small cell lung cancer tissues.

[0129]

[0130] Therefore, among the 23 candidate genes FAM111B, LAD1, and AHNAK2, FAM111B is significantly associated with the malignant progression of non-small cell lung cancer, suggesting that FAM111B may have an impact on the malignant biological behavior of non-small cell lung cancer.

[0131] III. Expression levels of FAM111B in different non-small cell lung cancer cell lines

[0132] To investigate the biological function of FAM111B in non-small cell lung cancer cells, the mRNA and protein expression of FAM111B in different non-small cell lung cancer cells (HARA-B, H520, H1299 and XWLC-05) were detected by qRT-PCR and Western blot.

[0133] Test results are shown Figure 14 The results showed that FAM111B was universally expressed in four non-small cell lung cancer cell lines. Based on the combined results of qRT-PCR and Western blot experiments, the internationally recognized non-small cell lung cancer cell line H1299 was selected to construct a low-expression model of FAM111B.

[0134] IV. Construction and screening of FAM111B knockdown non-small cell lung cancer cell line H1299 stable cell line

[0135] 1. Obtaining shRNA lentiviral vectors

[0136] (1) Based on the FAM111B RNAi sequence, three RNAi were designed and synthesized by Shanghai Jikai Gene Medical Technology Co., Ltd. The names, accessions, sequences, corresponding CDS positions, and GC contents of the three RNAi are shown in Table 6. One RNAi was also synthesized as a control; the sequence of the control RNAi is shown in Table 6.

[0137] Table 6

[0138]

[0139] (2) Shanghai Jikai Gene Medical Technology Co., Ltd. separately coupled FAM111B-RNAi (96900-1), FAM111B-RNAi (96901-1), FAM111B-RNAi (96902-1) and control RNAi with shRNA lentiviral vector GV248 (see vector schematic diagram). Figure 15 The ligation was performed to obtain shRNA lentiviral vectors, which were named FAM111B-RNAi(96900-1), FAM111B-RNAi(96901-1), FAM111B-RNAi(96902-1) and a knockdown vector control, respectively.

[0140] 2. Lentiviral transfection of H1299 cells

[0141] The transfection experiment was conducted by Shanghai Jikai Gene Medical Technology Co., Ltd. The specific steps are as follows:

[0142] (1) When H1299 cells reach the logarithmic growth phase, digest the cells with trypsin, centrifuge, and adjust the cell concentration to 1×10⁻⁶ cells with an appropriate amount of complete culture medium. 5 Cells / ml were used to obtain a cell suspension.

[0143] (2) Take a 6-well plate, add the cell suspension and complete culture medium obtained in step (1) to each well to obtain a 2ml system; incubate at 37℃ for 24h.

[0144] (3) After completing step (2), replace with fresh complete culture medium.

[0145] (4) After completing step (3), add shRNA lentiviral vector (according to the preliminary experimental results, the optimal MOI value for shRNA lentiviral vector infection of H1299 cells is 10. Calculate the total volume of shRNA lentiviral vector required: (MOI × number of cells) / shRNA lentiviral vector titer) and HitransGP virus infection reagent, shake well, and continue to culture at 37℃ for 12h.

[0146] (5) After completing step (4), remove 500 μl of culture medium from the culture system and replace it with an equal volume of fresh complete culture medium; continue to culture at 37°C for 24 h.

[0147] (6) After completing step (5), remove all the culture medium from the culture system and replace it with an equal volume of fresh complete culture medium; continue to culture at 37°C for 48 hours.

[0148] 3. Screening of FAM111B knockdown non-small cell lung cancer cell line H1299 stable cell line

[0149] The screening experiments were conducted by Shanghai Jikai Gene Medical Technology Co., Ltd. The specific steps are as follows:

[0150] (1) When H1299 cells reach the logarithmic growth phase, digest the cells with trypsin, centrifuge, and adjust the cell concentration to 1×10⁻⁶ cells with an appropriate amount of complete culture medium. 5 Cells / ml were used to obtain a cell suspension.

[0151] (2) Take a 12-well plate, add the cell suspension and complete culture medium obtained in step (1) to each well to obtain a 1ml system; incubate at 37℃ for 24h and observe cell morphology.

[0152] (3) After completing step (2), remove the complete culture medium and wash twice with PBS. Replace with fresh complete culture medium containing different concentrations of puromycin and continue culturing at 37°C for 48 hours.

[0153] (4) After completing step (3), observe the cells and select the lowest concentration of puromycin that can kill all H1299 cells as the screening concentration.

[0154] The results showed that 2.5 μg / ml was the optimal concentration of puromycin.

[0155] (5) If the cells are in good condition after transfection, puromycin can be added to continue culturing, and the medium can be changed during the process.

[0156] (6) Observe the cells under a fluorescence microscope. If the fluorescence efficiency reaches 95% or more, the cells can be amplified. At the same time, the cells are collected for subsequent identification experiments.

[0157] When the shRNA lentiviral vectors were FAM111B-RNAi(96900-1), FAM111B-RNAi(96901-1), FAM111B-RNAi(96902-1) and control RNAi, sh-FAM111B-1, sh-FAM111B-2, sh-FAM111B-3 and sh-NC were obtained respectively.

[0158] 4. Using RNA from H1299 cells, sh-FAM111B-1, sh-FAM111B-2, sh-FAM111B-3, or sh-NC as templates, the expression level of FAM111B was detected by qRT-PCR.

[0159] The primers for detecting FAM111B are 5'-GCATATGGTAAACCCAGCGAG-3' and 5'-GAATCACTAGGCAGGCACTTG-3'.

[0160] qRT-PCR results showed that, compared with sh-NC, both sh-FAM111B-1 and sh-FAM111B-2 effectively reduced the expression of FAM111B at the mRNA level (P < 0.05, P < 0.01), but sh-FAM111B-3 did not show effective low expression. Figure 16 (A)

[0161] 5. Western blot was used to detect the expression of FAM111B protein in H1299 cells, sh-FAM111B-1, sh-FAM111B-2, sh-FAM111B-3 or sh-NC (primary antibody was Anti-FAM111B Antibody, a product of Novus Biologics).

[0162] Western blot analysis showed that, compared with sh-NC, only sh-FAM111B-2 significantly reduced the expression of FAM111B at the protein level (P < 0.001). Figure 16 In sh-NC, there was no statistically significant difference in the mRNA and protein expression of FAM111B compared to the blank control group (H1299). Figure 16 (AB).

[0163] sh-FAM111B-2 achieved low expression of both FAM111B mRNA and protein levels, and was referred to as sh-FAM111B or sh-FAM111B in subsequent experiments. Thus, a non-small cell lung cancer cell line stably inhibiting FAM111B expression was successfully established.

[0164] V. Effect of FAM111B on the apoptosis ability of non-small cell lung cancer cells H1299

[0165] (1) Flow cytometry detection of apoptosis in non-small cell lung cancer cells H1299

[0166] To investigate the effect of FAM111B on apoptosis in non-small cell lung cancer cells H1299, sh-NC and sh-FAM111B were stained with an apoptosis kit, and apoptosis was detected by flow cytometry.

[0167] The experimental results are shown in Figure 17 The results showed that the total apoptosis rate of sh-FAM111B was significantly increased compared with sh-NC. Therefore, knockdown of FAM111B can promote apoptosis in non-small cell lung cancer cells H1299.

[0168] (2) Western blot detection of changes in apoptosis-related proteins

[0169] To further investigate the effect of FAM111B on apoptosis in non-small cell lung cancer cells, the expression levels of apoptosis-related proteins in sh-NC and sh-FAM111B were detected by Western blot.

[0170] Test results are shown Figure 18 The results showed that, compared with sh-NC, sh-FAM111B significantly increased the expression of Bax and p53 proteins by 123.17% (1.44±0.09 vs 0.64±0.29, P<0.01) and 19.49% (0.70±0.06 vs 0.58±0.03, P<0.05), respectively, while decreasing Bcl-2 protein expression by 32.68% (0.08±0.007 vs 0.05±0.004, P<0.01). This indicates that FAM111B can enhance the anti-apoptotic ability of H1299 non-small cell lung cancer cells, and this ability may be achieved by regulating the expression of Bax, Bcl-2, p53, and Cleaved-caspase 9 proteins.

[0171] VI. FAM111B-cfDNA Levels in the Serum of Non-Small Cell Lung Cancer Patients

[0172] To detect the level of FAM111B in serum cfDNA of non-small cell lung cancer (NSCLC) patients, the inventors of this invention collected serum samples from 17 NSCLC patients and 17 healthy volunteers. The level of FAM111B in serum cfDNA was then detected using qRT-PCR. The primers for detecting FAM111B were 5'-GCATATGGTAAACCCAGCGAG-3' and 5'-GAATCACTAGGCAGGCACTTG-3'.

[0173] Test results are shown Figure 19 The results showed that the FAM111B-cfDNA level in the patient group composed of non-small cell lung cancer patients was significantly higher than that in the healthy control group composed of healthy volunteers (P < 0.01). Therefore, FAM111B may serve as a diagnostic biomarker for non-small cell lung cancer.

[0174] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Use of a substance that inhibits the activity and / or expression amount of FAM111B protein in the manufacture of a product; the function of the product is as follows C1) or C2): C1) treating non-small cell lung cancer; C2) promoting apoptosis of non-small cell lung cancer cells; the non-small cell lung cancer is lung squamous carcinoma; the substance that inhibits the activity and / or expression amount of FAM111B protein is z1) or z2): z1) an RNAi molecule represented by SEQ ID NO: 2; z2) connecting the RNAi molecule represented by SEQ ID NO: 2 with shRNA lentivirus vector GV248 to obtain shRNA. ​ ​ ​ ​ ​ ​

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