Method and application of TM9SF1 gene regulating lung cells

Abstract

The present invention provides a method and application of TM9SF1 gene regulation of lung cells, and regulates the growth of lung cells by regulating the expression of TM9SF1 gene. In the present invention, it is found that the growth of lung cells can be regulated by regulating the expression of TM9SF1 gene, and another targeted gene and method for regulating the growth of lung cells are provided, which is conducive to further improving the gene regulatory network of lung cell growth.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a method and application of TM9SF1 gene regulating lung cells. Background Art

[0002] Lung cancer cells are a type of lung cell, and their malignant proliferation leads to lung cancer, which is one of the most common tumors, rather than small cell carcinoma. At present, the treatment of lung cancer is still mainly based on traditional chemotherapy, but chemotherapy not only inhibits the growth of lung cancer cells, but also harms normal cells, so there will be great side effects.

[0003] In the field of molecular biology, targeted therapies for non-small cell carcinoma have emerged, making treatment plans more individualized and precise, and therefore with fewer side effects. Today, the genes found to be relevant for detection and targeted therapy are: epidermal growth factor receptor, anaplastic lymphoma kinase, c-ros proto-oncogene 1 tyrosine kinase, human epidermal growth factor receptor 2, mesenchymal epithelial transition factor, v-raf murine sarcoma viral oncogene homolog B1, murine sarcoma viral oncogene, transfection rearrangement gene and neurotrophic tyrosine receptor kinase.

[0004] However, according to research findings, lung cancer is the common result of complex regulation of multiple genes. Current research has not fully grasped the existing gene regulation mechanism of lung cancer, which has hindered the progress of targeted treatment of lung cancer. Summary of the invention

[0005] The purpose of the present invention is to provide a method and application of TM9SF1 gene regulating lung cells, aiming at another targeted gene for the growth of lung cells and improving the regulatory network of their growth.

[0006] To achieve the above object, the present invention provides a method for regulating the growth of lung cells, which regulates the growth of lung cells by regulating the expression of TM9SF1 gene.

[0007] Optionally, the lung lineage cells include human lung adenocarcinoma cells; and / or,

[0008] Controlling the growth of the lung cells by regulating the expression of the TM9SF1 gene in any subcell of the endoplasmic reticulum, lysosome and mitochondria of the lung cells; and / or,

[0009] The regulating the growth of lung cells comprises regulating the proliferation of the lung cells; and / or,

[0010] The regulating the growth of lung cells comprises reducing the ratio of G0 / G1 phase cells in the growth of lung cells; and / or,

[0011] The regulating the growth of lung lineage cells comprises increasing the ratio of cells in the S phase and G2 / M phase; and / or,

[0012] The growth of the lung cells is promoted by increasing the expression level of the TM9SF1 gene.

[0013] In addition, the present invention provides a method for regulating the growth of lung cells, which regulates the growth of lung cells by regulating the mTOR signaling pathway and / or protein synthesis through the TM9SF1 gene.

[0014] Optionally, the regulating the mTOR signaling pathway comprises:

[0015] At least one of regulating the phosphorylation of mTOR, regulating the phosphorylation of p70S6K, regulating the phosphorylation of S6 ribosomal protein, regulating the phosphorylation of 4EBP1, and regulating the activity of 4EBP1.

[0016] Optionally, the growth of the lung cells is promoted by increasing the expression level of the TM9SF1 gene to activate the phosphorylation of the mTOR signaling pathway molecules; and / or,

[0017] The growth of the lung cells is promoted by increasing the expression level of the TM9SF1 gene to promote protein synthesis.

[0018] In addition, the present invention provides a method for inhibiting the growth of lung-lineage cells, by silencing or inhibiting the TM9SF1 gene to control the growth of lung-lineage cells.

[0019] Optionally, RNA interference is used to silence or inhibit the TM9SF1 gene.

[0020] Optionally, the TM9SF1 gene is silenced or inhibited by using an RNA interference system, wherein the RNA interference system comprises:

[0021] The siRNA is shown in SEQ ID NO:1.

[0022] In addition, the present invention provides a TM9SF1 gene RNA interference system, the TM9SF1 gene RNA interference system comprising:

[0023] The siRNA is shown in SEQ ID NO:1.

[0024] In addition, the present invention provides a TM9SF1 gene or a protein encoded by it, any of the above methods for regulating the growth of lung cells, the above method for inhibiting the growth of lung cells, or the above TM9SF1 gene RNA interference system for use in preparing a preparation for treating lung cancer.

[0025] In the present invention, it is found that the growth of lung cells can be regulated by regulating the expression of TM9SF1 gene, which provides another targeted gene and method for regulating the growth of lung cells, and is conducive to further improving the gene regulatory network of lung cell growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 The expression level of TM9SF1 in lung cancer tissue and normal lung tissue is detected;

[0028] Figure 2 It is the identification diagram of TM9SF1 overexpression and subcellular localization in A549 cells;

[0029] Figure 3 This is an experimental diagram showing the effect of TM9SF1 on A549 cell proliferation and cell cycle;

[0030] Figure 4 A detection diagram showing that TM9SF1 enhances the tumorigenicity of A549 cells in vivo;

[0031] Figure 5 To perform microarray-based transcriptome analysis of A549 cells overexpressing TM9SF1;

[0032] Figure 6 This is a diagram showing the research on TM9SF1 and mTOR signaling pathway.

[0033] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0034] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them.

[0035] It should be noted that, in the embodiments, those without specifying specific conditions are carried out according to normal conditions or conditions recommended by the manufacturer. Those without specifying the manufacturer of reagents or instruments used are conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, and "A and / or B" is taken as an example, including schemes A, B, or A and B that meet the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of the technical schemes is contradictory or cannot be achieved, it should be considered that the combination of such technical schemes does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work, all belong to the scope of protection of the present invention.

[0036] Members of the TM9SF (Transmembrane 9 Superfamily member) family usually have a long N-terminal extracellular region and nine transmembrane domains. The human TM9SF1 gene is located on chromosome 14q12, with a DNA length of 6593 bp, 6 exons, a CDS length of 1821 nt, and encodes 606 amino acids. Its function in lung cancer is still unclear.

[0037] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0038] Example 1

[0039] To determine whether TM9SF1 is involved in regulating the occurrence of lung cancer, this example collects and analyzes RNA sequence data of cancer tissues and adjacent normal tissues of patients with LUAD (lung adenocarcinoma) and LUSC (lung squamous cell carcinoma) in the TCGA database (https: / / portal.gdc.cancer.gov / ). The paired t-test was used to evaluate the expression level of TM9SF1 between cancer tissues and adjacent normal tissues in the TCGA database.

[0040] The expression of TM9SF1 in patients with non-small cell lung cancer was analyzed based on the TCGA database. The results showed that the mRNA level of TM9SF1 in cancer tissues was significantly increased compared with the adjacent normal tissues of patients with LUAD (lung adenocarcinoma) and LUSC (lung squamous cell carcinoma). Figure 1A, 1B). Immunohistochemical detection of TM9SF1 expression in a commercial tissue microarray (produced by Biomax (USA)). The tissue microarray included 75 lung cancer tissues and 14 normal lung tissues. Representative images of immunohistochemical staining of TM9SF1 ( Figure 1 A) and its analysis showed that 69 lung cancer samples highly expressed TM9SF1, and its proportion (92.0%) was much higher than that of normal lung tissue (28.6%) ( Figure 1 C, 1D).

[0041] HE and immunohistochemical staining were performed on 22 clinical NSCLC specimens (obtained from 22 patients who underwent surgical resection in Xiangyang Central Hospital (Xiangyang, China). The samples were collected according to the random principle. The age of all patients ranged from 41 to 69 years. Informed consent was obtained from the donor patients. This study was approved by the Institutional Ethics Committee of Xiangyang Central Hospital (approval number: 2017-056) and was conducted in accordance with the principles of the Declaration of Helsinki). The results are shown in Table 1. Figure 1 E shows the image indicating the focus area of ​​the above image, and the scale bar is 200 μm.

[0042] Immunohistochemistry was used to detect the expression level of TM9SF1 protein in cancer tissues of 22 clinical non-small cell lung cancer patients, and it was found that the proportion of samples with high expression of TM9SF1 was 86.4% (n = 19), while the proportion of samples with low expression was 13.6% (n = 3). The above data show that TM9SF1 expression is positively correlated with the occurrence of lung cancer.

[0043] Example 2

[0044] To explore the effect of TM9SF1 on lung cancer cells, flag-tagged TM9SF1 was packaged with lentivirus and transfected into A549 cells (human non-small cell lung cancer cell line A549 was obtained from ATCC, USA and cultured in RPMI containing 10% fetal bovine serum (ScienCell, USA), 100 IU / ml penicillin and 100 μg / ml streptomycin (EallBio, Beijing, China) 1640 (Lifetech, USA), cultured at 37°C in a humidified atmosphere of 95% air and 5% CO2), the operation was as follows: the expression plasmid (Zhang Guoying, Yang Duo, Gao Nana, Tang Zihua, Li Na, Tong Yongjuan, Shang Ting, Xiao Juan*. Lentivirus-mediated recombinant TM9SF1 protein inhibits 293T cell growth by inducing autophagy and endoplasmic reticulum stress. Chinese Journal of Biochemistry and Molecular Biology 2017; (06): 600-6.) and packaging plasmid (purchased from Hanheng Biotechnology) were co-transfected into 293T cells to produce lentivirus, and the vector plasmid and packaging plasmid without the target fragment were used as empty vectors, and transfection was performed using Lipofectamine 3000 reagent (Invitrogen, USA) according to the manufacturer's instructions. The supernatant containing lentivirus and the vector supernatant were collected 48 hours after transfection. Some A549 cells were plated at 5×10 4 / well were seeded on a 6-well plate, and supernatant containing virus and 5 μg / ml polyvinyl alcohol were added. After 24 hours, the medium was replaced with fresh medium, and the cells were incubated for another 24 hours and studied. At the same time, some A549 cells were added with supernatant containing vector and 5 μg / ml polyvinyl alcohol. After 24 hours, the medium was replaced with fresh medium, and the cells were incubated for another 24 hours and studied.

[0045] To confirm whether the overexpression was successful, Western blotting was used to detect the TM9SF1 protein level. The operation was as follows:

[0046] 1. Identify the overexpression and temperature-based stability of TM9SF1 protein. The supernatant containing lentivirus was detected by Western blotting using a flag tag antibody fused to TM9SF1 protein at the N-terminus, and GAPDH was used as an internal reference. The results are shown in Figure 2 As shown in A, the results show that after denaturation treatment under different temperature conditions, the level of TM9SF1 protein gradually decreased with increasing temperature, indicating that the protein instability of TM9SF1 protein gradually increased when the temperature was higher than 37°C.

[0047] 2. Since the molecular function and mechanism depend largely on its intracellular localization, the distribution of TM9SF1 in cells was studied next. The expression and intracellular distribution of endogenous and overexpressed TM9SF1 were identified by immunofluorescence technique. The results are as follows: Figure 2As shown in B (scale = 25 μm), Anti-TM9SF1 staining represents the distribution of TM9SF1 protein, DAPI staining represents the cell nucleus, Organelle staining represents different organelles (ER represents endoplasmic reticulum, Lyso represents lysosome, Mito represents mitochondria, and Golgi represents Golgi apparatus), Merged represents the color development after two different colors of fluorescence are fused (showing co-localization), and the immunofluorescence results show that both endogenous and overexpressed TM9SF1 proteins are distributed in a dotted form.

[0048] 3. To determine the co-localization of TM9SF1 with important organelles. A549 cells were transfected with lentivirus expressing TM9SF1-ZsGreen fusion protein (purchased from Hanheng Biotechnology). After 48 hours, the organelles were stained with endoplasmic reticulum chromogen-blue, lysosome chromogen-red, mitochondrial chromogen-red, and Golgi apparatus chromogen-red, respectively, and then observed under a confocal laser scanning microscope. The arrows indicate co-localization. The results are shown in Figure 2. Figure 2 As shown in C, the scale bar in the figure = 10 μm, the results show that TM9SF1 co-localizes with multiple membrane systems, including the endoplasmic reticulum, lysosomes, and mitochondria, but does not show that TM9SF1 co-localizes with the Golgi apparatus ( Figure 2 C).

[0049] Example 3

[0050] 1) In order to study whether TM9SF1 affects cell proliferation, CCK8 (Cell Counting Kit-8 cell counting reagent) was used to measure the growth curve of A549 cells and vectors transfected with TM9SF1 lentivirus in Example 2. The results are as follows: Figure 3 As shown in A. The study found that overexpression of TM9SF1 significantly promoted the proliferation of A549 cells compared with the vector group.

[0051] 2) Colony formation assay to detect cell viability

[0052] The A549 cells transfected with TM9SF1 lentivirus and the cells containing the vector were cloned into three samples and their colony formation was observed. The results are as follows: Figure 3 As shown in B in Figure 1, and observing the micrograph in B, the results are as follows Figure 3 As shown in C, the results showed that after 9 days of cell culture, the number and size of colonies in the TM9SF1 group increased significantly. According to the gray value quantification by ImageJ, the number of colonies, total colony area and average colony area in the TM9SF1 group increased significantly compared with the vehicle group ( Figure 3 D).

[0053] Representative histograms of flow cytometric analysis showing cell cycle analysis of A549 cells transfected with lentivirus expressing TM9SF1. The relative DNA content of cells was determined by PI staining ( Figure 3 E), and the percentage of cells in each cycle stage was quantified simultaneously (n=4) ( Figure 3 F), The results showed that TM9SF1 decreased the ratio of cells in the G0 / G1 phase and significantly increased the ratio of cells in the S phase and G2 / M phase.

[0054] 3) Construct TM9SF1 inhibitor to further prove

[0055] 1. GenePharma (Suzhou, China) was commissioned to synthesize siRNA targeting TM9SF1. The siRNA sequence designed based on the human target gene was 5'-GGUUACGACCUGACGAGUUTT-3' (SEQ ID NO: 1). To verify its targeting, it was transfected into A549 cells and then transfected with siTM9SF1 into A549 cells using Lipofectamin 3000 (transfection reagent). The effectiveness and specificity of siRNA targeting TM9SF1 (si-TM9SF 1) were identified by qPCR. The results are as follows: Figure 3 As shown in G, the results showed that the expression levels of TM9SF3 and TM9SF4, which are homologous to TM9SF1, did not change, indicating that si-TM9SF1 has targeting specificity.

[0056] 2. CCK8 analysis showed that A549 cells transfected with TM9SF1 siRNA or interfering siRNA as negative control (si-NC) proliferated, P < 0.001. Figure 3 As shown in H, the results showed that compared with the control group, A549 cells transfected with TM9SF1 siRNA proliferated slower.

[0057] 3. A549 cells transfected with TM9SF1 siRNA and si-NC were cloned into three samples and their colony formation was observed. The results are as follows: Figure 3 As shown in (I), the number and area of ​​colonies from (I) were quantified using the ImageJ program, and the results are shown in Figure 3 As shown in J, **P<0.01; ***P<0.001, unpaired t-test was used between different groups, and the results showed that TM9SF1 successfully reduced the proliferation of A549 cells after expression inhibition.

[0058] Example 4

[0059] In order to reveal the role of TM9SF1 in promoting lung cancer in vivo, A549 cells transfected with TM9SF1 lentivirus were inoculated into the axilla of some nude mice, and A549 cells transfected with control lentivirus were inoculated into the axilla of another part of nude mice as a control group to establish a xenograft tumor model. The specific operation was as follows:

[0060] Male BALB / c nude mice (5-7 weeks old, 22 ± 3 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and randomly divided into two groups (10 mice in each group). The mice were placed in a laminar flow cabinet under specific pathogen-free conditions. A549 cells (2.5 × 106) transfected with lentivirus expressing TM9SF1 or control constructs were injected subcutaneously into the axilla. Tumor growth was monitored for 30 days and measured with a caliper. The formula V (mm 3 )=(AxB2) / 2 to calculate tumor volume. On day 30 of the experiment, mice were sacrificed and xenograft tumors were dissected for further analysis. Animal experiments were in accordance with ARCH guidelines and approved by the Institutional Ethics Committee of Xiangyang Central Hospital (approval number: 2017-056).

[0061] The images of nude mouse xenograft tumors were observed on day 30. Figure 4 The arrow in A indicates that the lesion was then dissected and observed. Figure 4 As shown in B, Figure 4 C is the tumor formation rate of nude mice injected with A549 cells. The results show that the tumor formation rate of nude mice transfected with TM9SF1 lentivirus A549 cells is 90%, while that of the control group is 30%.

[0062] From the observations at different times, we know that from the 17th day of the experiment, the tumor volume of the TM9SF1 group increased rapidly compared with the control group ( Figure 4 D), mean tumor volume of TM9SF1 group on day 30 ( Figure 4 E, 184.5±149.6cm 3 ) and weight ( Figure 4 F, 214.6±187.3mg) were much higher than those in the control group (3.8±6.4cm3 and 5.9±9.3mg, respectively).

[0063] Ki67 staining is an important marker of cell proliferation. Immunohistochemical staining was performed using Ki67 monoclonal antibody (#9449). Representative images of Ki67 immunohistochemical staining of xenograft tumors are shown in Figure 2. Figure 4 As shown in G, the scale bar in the figure is 20 μm, and the percentage of Ki67 positive cells in the figure is Figure 4As shown in H, n = 10. *P < 0.05; n = 10. *P < 0.05; ***P < 0.01. Unpaired t-test was used between the groups. TM9SF1 significantly increased the proportion of Ki67-positive cells in xenograft tumor tissues ( Figure 4 G, 4H), indicating that TM9SF1 has a significant effect in promoting lung cancer growth in vivo.

[0064] Example 5

[0065] In order to study the molecular mechanism by which TM9SF1 promotes the occurrence and development of lung cancer, this example performed a microarray-based transcriptome analysis to identify the gene function category and signal pathway by determining the differentially expressed genes. The operation is as follows:

[0066] Transcriptome analysis was performed by The expression profile of qualified RNA was determined by Clariom_D_Human Array (Affymetrix ) was determined according to the manufacturer's recommended protocol. Each group included three replicate samples. The gene chip command console scanned the gene chip. The data were analyzed using the Affymetrix default analysis settings and the robust multichip analysis (RMA) algorithm. Hierarchical clustering was performed using Cluster_Treeview software from Stanford University (Palo Alto, USA) to analyze differentially expressed mRNAs (RVM t test, P < 0.05). Gene ontology (GO) enrichment analysis was performed based on differentially expressed genes to study gene functions. Pathway enrichment analysis was performed based on the KEGG (Kyoto Encyclopedia of Genes and Genomes) database.

[0067] The results showed that there were significant differences in the transcriptional expression of thousands of genes in A549 cells between the TM9SF1 overexpression group and the vector group, of which 3948 were upregulated and 2513 were downregulated ( Figure 5 A, Volcano plot of differentially expressed genes under TM9SF1 overexpression conditions). In addition, the cluster analysis heat map showed that 884 mRNAs were upregulated and 1123 mRNAs were downregulated ( Figure 5 B, Cluster analysis heat map of significantly differentially expressed mRNAs in A549 cells between the TM9SF1 overexpression group and the control group. The differential expression of related genes is displayed by color gradient scale (log2). Each row represents a separate sample, and each column represents an mRNA / lncRNA probe).

[0068] In order to understand the biological functions of TM9SF1, functional enrichment analysis was performed based on significantly differentially expressed genes. The results showed that the enriched functions of upregulated genes were mainly related to anabolism and cell proliferation, such as mitotic cell cycle G1 / S transition, ribosomal large subunit generation, ribosomal large subunit nuclear export, RNA nuclear export, DNA replication, cell division, purine nucleotide biosynthesis and cell division ( Figure 5 C). Down-regulated genes were enriched in functions including type I interferon signaling pathway, PERK-mediated protein response and dealkenylation of nuclear transcribed mRNA ( Figure 5 D), upregulated (C) or downregulated (D) genes formed a total of 20 significant enrichment entries. Statistical analysis was performed using chi-square test and two-sided Fisher's exact test. P<0.01 was considered statistically significant. The vertical axis represents the functional enrichment category, and the horizontal axis represents the negative logarithm of the P value (-LgP). The numbers on the right side of the figure show the enrichment degree of the corresponding enrichment category.

[0069] To reveal the relevant signaling pathways involved in TM9SF1, this study used the KEGG database for signaling pathway analysis. The results showed that the top 10 upregulated gene enrichment pathways were mainly related to anabolism ( Figure 5 E), such as DNA replication, pyrimidine metabolism, cell cycle, pentose phosphate pathway, and ribosome biogenesis in eukaryotes. Meanwhile, the top 10 down-regulated gene enriched pathways included circadian rhythm, NOD-like receptor signaling pathway, TNF signaling pathway, and viral infection ( Figure 5 F). Transcriptome analysis results showed that TM9SF1 could promote biosynthesis and anabolism to regulate the growth of A549 cells.

[0070] Example 6

[0071] Based on the analysis results of Example 5, the inventor's research team selected multiple signal pathways for verification, including the MAPK signal pathway, the AKT signal pathway and the mTOR signal pathway. It was verified that the MAPK signal pathway and the AKT signal pathway were not related to TM9SF1 regulating the growth of A549 cells, while the mTOR signal pathway was related to the growth of A549 cells. mTOR (rapamycin target protein) is a serine / threonine kinase. The mTOR signal pathway has the functions of promoting material metabolism, participating in cell apoptosis and autophagy, and plays an important role in many diseases. For the verification of TM9SF1 regulation based on the mTOR signal pathway, the operation is as follows:

[0072] 1. Western blotting was used to analyze mTOR, phosphorylated mTOR (p-mTOR), p70S6K (P70 ribosomal protein S6 kinase, the main downstream target of mTOR), phosphorylated p70S6K (p-p70S7K), S6, phosphorylated p70S6 (p-S6), 4EBP1 (4E binding protein 1) and phosphorylated 4EBP1 (p-4EBP1) in A549 cells transfected with TM9SF1 lentivirus and A549 cells transfected with lentiviral vector. The results showed that TM9SF1 significantly enhanced the phosphorylation of mTOR, overexpression of TM9SF1 increased the phosphorylation of p70S6K and its downstream S6 ribosomal protein ( Figure 6 A), which means an increase in protein translation. In addition, phosphorylation of 4EBP1 was detected, indicating that TM9SF1 significantly increased the phosphorylation / inactivation of 4EBP1 ( Figure 6 A), demonstrating 5′ cap-dependent translation enhancement.

[0073] 2. To determine the effect of mTOR signaling on TM9SF1-mediated A549 cell proliferation, the mTOR inhibitor rapamycin was used. CCK8 analysis showed that rapamycin treatment successfully inhibited the proliferation of A549 cells transfected with TM9SF1 lentivirus and A549 cells transfected with lentiviral vectors. CCK8 analysis showed that the proliferation of A549 cells treated with 0.2 μM rapamycin (Rapa) was significantly inhibited. Figure 6 B1) and proliferation rate ( Figure 6 B2), the proliferation rate of the TM9SF1 group was not significantly reduced compared with the vector group ( Figure 6 B), where Figure 6 In B, the arrow indicates the administration time, and the cell proliferation rate is calculated using the absorbance value. The calculation formula is R=(EN) / N*100%, where R is the proliferation rate, E is the experimental group, and N is the negative control group.

[0074] 3. To investigate whether the proliferative effect of TM9SF1 on A549 cells is mediated by increased protein synthesis, cells were treated with a novel protein synthesis inhibitor, 0.2 μM cycloheximide (CHX), and 5 μg / ml puromycin. CCK8 analysis showed that treatment with CHX attenuated the proliferation of A549 cells and the increased proliferation rate induced by TM9SF1 ( Figure 6 C), indicating that protein synthesis plays a role in the proliferation effect of TM9SF1. Similar results were obtained for puromycin (Puro), which significantly reduced the increase in proliferation rate induced by TM9SF1 after treatment of cells ( Figure 6 D). Figure 6 C and Figure 6In D, the cell proliferation rate was calculated using the absorbance value according to the formula R = (EN) / N * 100% (R, proliferation rate; E, experimental group; N, negative control group). Figure 6 C and Figure 6 In D, the arrows indicate the time of drug administration.

[0075] Therefore, this study proposed that TM9SF1 promotes the proliferation of A549 cells by activating the mTOR signaling pathway and enhancing protein translation ( Figure 6 E).

[0076] In summary, TM9SF1 is highly expressed in non-small cell lung cancer, and its expression level is positively correlated with the incidence of non-small cell lung cancer. Confocal microscopy results showed that TM9SF1 was localized in the endoplasmic reticulum, lysosomes and mitochondria of A549 cells, but not in the Golgi apparatus. Overexpression of TM9SF1 significantly promoted the proliferation and cell cycle of A549 cells, while knocking out endogenous TM9SF 1 significantly inhibited the proliferation of A549 cells. Nude mouse tumor-bearing experiments confirmed that overexpression of TM9SF1 significantly increased the formation rate and tumor growth of nude mouse xenograft tumors. Transcriptome analysis results showed that overexpression of TM9SF1 widely promoted biosynthesis and anabolism. Molecular mechanism studies have shown that TM9SF1 promotes lung cancer growth by promoting protein synthesis and activating the mTOR signaling pathway.

[0077] Further data showed that high expression levels of TM9SF1 were positively correlated with the incidence of non-small cell lung cancer, and TM9SF1 promoted the proliferation of lung cancer A549 cells by activating the mTOR signaling pathway and protein synthesis, suggesting that TM9SF1 may be a new therapeutic target or prognostic indicator for lung cancer.

[0078] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. A method for regulating the growth of lung cells, characterized in that: The growth of the lung cells is regulated by regulating the expression of the TM9SF1 gene, wherein the lung cells are human non-small cell lung cancer cells, and the method is for purposes other than disease treatment or diagnosis.

2. The method for regulating the growth of lung cells according to claim 1, characterized in that: The growth of the lung cells is promoted by increasing the expression level of the TM9SF1 gene.

3. A method for promoting the growth of lung cells, characterized in that: The growth of lung cells is regulated by regulating the mTOR signaling pathway and / or protein synthesis through the TM9SF1 gene. The lung cells are human non-small cell lung cancer cells. The method is for non-disease treatment or diagnosis purposes.

4. The method for promoting the growth of lung cells according to claim 3, characterized in that: The regulation of the mTOR signaling pathway includes: At least one of regulating the phosphorylation of mTOR, regulating the phosphorylation of p70S6K, regulating the phosphorylation of S6 ribosomal protein, regulating the phosphorylation of 4EBP1, and regulating the activity of 4EBP1.

5. The method for promoting the growth of lung cells according to claim 3, characterized in that: By increasing the expression level of the TM9SF1 gene to activate the phosphorylation of the mTOR signaling pathway molecules to promote the growth of the lung cells; and / or, The growth of the lung cells is promoted by increasing the expression level of the TM9SF1 gene to promote protein synthesis.

6. A method for inhibiting the growth of lung cells, characterized in that: The growth of lung cells is controlled by silencing or inhibiting the TM9SF1 gene, wherein the lung cells are human non-small cell lung cancer cells. The method is for non-disease treatment or diagnosis purposes.

7. The method for inhibiting the growth of lung cells according to claim 6, characterized in that: RNA interference is used to silence or inhibit the TM9SF1 gene.

8. The method for inhibiting the growth of lung cells according to claim 6, characterized in that: The TM9SF1 gene is silenced or inhibited by using an RNA interference system, wherein the RNA interference system comprises: The siRNA has a nucleotide sequence as shown in SEQ ID NO:

1.

9. Use of siRNA having a nucleotide sequence as shown in SEQ ID NO: 1 in the preparation of a preparation for treating lung cancer, wherein the lung cancer is human non-small cell lung cancer.

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