Application of ARL14 as tumor marker in preparation of medicine for predicting lung adenocarcinoma prognosis and target spot
阅读说明：本技术 Arl14作为肿瘤标志物在制备预测肺腺癌预后及靶点药物中的用途 (Application of ARL14 as tumor marker in preparation of medicine for predicting lung adenocarcinoma prognosis and target spot ) 是由 邵春林 郭飞 潘燕 张江虹 朱琳 张俊伶 于 2018-07-13 设计创作，主要内容包括：本发明属于生物医学技术领域,涉及ARL14作为肿瘤标志物在制药中的新用途,本申请经实验显示,ARL14通过KRAS/CIDEC/ERK/p38信号通路调控肺腺癌细胞增殖、迁移和侵袭,影响细胞周期分布,并与细胞休眠密切相关以及显示γ射线照射对休眠细胞损伤作用微弱；结果表明,ARL14可作为临床肺腺癌治疗靶点和预后评估的指标,或成为临床肺腺癌患者放疗指针,所述的ARL14作为肿瘤标志物可用于制备预测肺腺癌尤其是针对肺腺癌细胞预后的试剂及靶点药物。(The invention belongs to the technical field of biomedicine, and relates to a new application of ARL14 in pharmacy as a tumor marker, and experiments show that ARL14 regulates and controls the proliferation, migration and invasion of lung adenocarcinoma cells through a KRAS/CIDEC/ERK/p38 signal channel, influences the cell cycle distribution, is closely related to cell dormancy and shows that gamma-ray irradiation has weak damage effect on dormant cells; the result shows that ARL14 can be used as an index for clinical lung adenocarcinoma treatment target and prognosis evaluation or a clinical lung adenocarcinoma patient radiotherapy pointer, and ARL14 can be used as a tumor marker for preparing a reagent and a target medicine for predicting lung adenocarcinoma, particularly lung adenocarcinoma cell prognosis.)
Use of ARL14 as a tumour marker in the manufacture of a product for determining the prognosis of lung adenocarcinoma.
Use of ARL14 as a tumor marker in the preparation of a diagnostic reagent for screening and judging the prognosis of lung adenocarcinoma.
Use of ARL14 as a tumor marker in the preparation of a lung adenocarcinoma treatment target drug.
The application of ARL14 as a tumor marker in screening and judging lung adenocarcinoma radiotherapy pointer preparations.
Technical Field
The invention belongs to the technical field of biomedicine, relates to a new application of ARL14 as a tumor marker in pharmacy, and particularly relates to an application of ARL14 in preparing a medicine for predicting lung adenocarcinoma prognosis and target spots; the invention relates to a relation between ARL14 expression and proliferation, migration, invasion, radiation sensitivity and prognosis of lung adenocarcinoma and a mechanism thereof, in particular to ARL14 which can be used for preparing a reagent and a target medicine for predicting the prognosis of lung adenocarcinoma.
Background
Disclosure of Invention
The invention aims to provide a new application of ARL14 in pharmacy as a tumor marker, in particular to an application of ARL14 in preparing a medicine for predicting lung adenocarcinoma prognosis and target spots; the invention relates to a relation and a mechanism of proliferation, migration, invasion, radiation sensitivity and prognosis of ARL14 expression and lung adenocarcinoma, and the ARL14 as a tumor marker can be used for preparing a reagent and a target medicine for predicting the prognosis of lung adenocarcinoma (especially for lung adenocarcinoma cells).
According to the invention, 10 pairs of mRNA differentially expressed in lung adenocarcinoma tissues and corresponding paracancer normal tissues are screened out through a TCGA database and are related to lung adenocarcinoma prognosis, and experimental detection shows that the expression of ARL14 in lung adenocarcinoma cells is obviously higher than that in lung normal cells;
in the invention, during detection, the TCGA database is firstly used for analyzing the expression difference of mRNA in lung adenocarcinoma tissues and paracancer normal tissues, and the expression of ARL14 in the lung adenocarcinoma tissues is found to be higher than that in the paracancer normal tissues; secondly, analyzing the influence of ARL14 expression level on lung adenocarcinoma prognosis by using a TCGA database, and finding that ARL14 is highly expressed and the prognosis is poor; cytological experimental detection proves that the expression level of ARL14 in lung adenocarcinoma cells is higher than that of lung normal cells, and the ARL14 is related to activities such as lung adenocarcinoma cell proliferation, migration, invasion, radiation resistance and the like; the result shows that ARL14 can be used as a screening marker for judging the prognosis of lung adenocarcinoma and can be used for preparing a diagnostic preparation for lung adenocarcinoma radiotherapy indications.
In the invention, the cell proliferation and survival capacity is detected by adopting a CCK-8 method and a clonogenic experiment, and the knocking-down of ARL14 gene expression is found to obviously inhibit the proliferation and clonogenic capacity of lung adenocarcinoma cells, but has no influence on the proliferation and clonogenic capacity of lung normal cells; meanwhile, the irradiation resistance of lung adenocarcinoma cells can be enhanced by knocking down the expression of ARL14 gene; the migration and invasion capabilities are detected by a Transwell method, and the migration and invasion capabilities of lung adenocarcinoma cells are weakened after the expression of ARL14 gene is knocked down, but the migration and invasion capabilities of irradiated cells can be slightly enhanced, which shows that the radiation resistance of ARL14 low-expression lung adenocarcinoma cells is increased from another point of view.
In the invention, cell cycle detection and immunofluorescence technology detection Ki67 are adopted to detect Ki67, and the study of ARL14 low expression inhibiting lung adenocarcinoma cell proliferation, migration, invasion and radiation sensitivity is carried out, and the results show that after ARL14 expression is knocked down, the proportion of G0/G1 cells in lung adenocarcinoma cells is increased, after irradiation for 24h, the G2/M retardation degree is weakened, and the proportion of Ki67 positive cells is reduced; and after the CIDEC gene expression is knocked down, the proportion of cells in the G0/G1 phase and the proportion of cells in the S phase and the G2/M phase in lung adenocarcinoma cells are increased, which shows that the CIDEC and ARL14 are possibly in negative correlation, and the fact that the ARL14 generates dormancy by inducing the lung adenocarcinoma cells to enter the G0 phase is clear, so that activities such as proliferation, migration and invasion are reduced, radiotherapy is avoided, and the experimental result provides a new idea for related basic and clinical researches.
According to the invention, a Western blot technology is adopted to research an ERK/p38 signal channel when the expression of ARL14/CIDEC is changed, and the result shows that the protein and phosphorylation levels of ERK1/2, p38, p16, p21, p27, p53 and CyclinD1 in cells are obviously increased after the expression of ARL14 is knocked down, and the mRNA and protein water level of the CIDEC is averagely increased; and the ERK1/2, p38, p16, p21, p27, p53, Cyclin D1 protein and phosphorylation level in the cell with reduced CIDEC expression are obviously reduced, which indicates that ARL14 and CIDEC can both activate ERK/p38 signal channel, CIDEC is a downstream negative related gene of ARL14, and the experimental result indicates that ARL14 can be used as a drug target for screening drugs for treating lung adenocarcinoma.
According to the invention, qRT-PCR and Western blot technologies are adopted to detect the influence of KRAS gene expression change on ARL14 expression, and the result shows that the KRAS gene expression level is knocked down to enable the mRNA and protein level of ARL14 in cells to be obviously increased on average, and the KRAS is the most common driving gene of lung cancer of small cells, so that the result shows that ARL14 can be used as a judgment index of lung adenocarcinoma prognosis and a drug target for screening drugs for treating lung adenocarcinoma.
The invention provides a new application of ARL14 in pharmacy as a tumor marker, and ARL14 as the tumor marker can be used for preparing a reagent for predicting the prognosis of lung adenocarcinoma (especially lung adenocarcinoma cells) and a target medicine.
Drawings
FIG. 1, ARL14 expression in lung adenocarcinoma tissue and paracancerous normal tissue.
Figure 2, prognostic significance of ARL14 in lung adenocarcinoma.
FIG. 3, ARL14 expression in lung gland cells and lung normal cells.
FIG. 4 shows the effect of inhibiting ARL14 expression on the proliferative capacity of lung adenocarcinoma cells and lung normal cells,
panel A and B shows the interference efficiency of the interference fragment ARL14 detected by qRT-PCR and western blot method in A549 and PC9 cells, respectively; FIGS. C and D are the effect of inhibiting ARL14 expression on the proliferation capacity of A549 and PC9 cells, respectively, detected by the CCK8 method; FIG. E is a graph of the effect of inhibiting ARL14 expression on clonality of A549 and PC9 cells; FIGS. F and G are the interference efficiencies of the interference fragment ARL14 in MRC-5 and BEAS-2B cells, respectively, as measured by qRT-PCR and western blot methods; FIGS. H and I are the effect of the CCK8 method to detect inhibition of ARL14 expression on the proliferative capacity of MRC-5 and BEAS-2B cells, respectively; panel J is the effect of inhibition of ARL14 expression on the clonality of MRC-5 and BEAS-2B cells; FIGS. K and L show the effect of inhibiting ARL14 expression on
FIG. 5, inhibition of the effect of ARL14 expression on the ability of lung adenocarcinoma cells to migrate and invade, wherein,
panels a and B are the Transwell assay to examine the effect of inhibiting ARL14 expression on the ability of a549 and PC9 cells to migrate and invade, respectively.
FIG. 6 shows the effect of inhibiting ARL14 expression on lung adenocarcinoma cell proliferation marker Ki67 and related signal protein, wherein, panel A shows the effect of inhibiting ARL14 expression on Ki67 expression detected by immunofluorescence; panel B is a quantitative analysis of Ki67 immunofluorescence assay; FIGS. C and D are graphs of the effect of inhibition of ARL14 expression on the levels of protein and phosphorylation in A549 and PC9 cells in ERK and p38 and downstream p16, p21, p27, p53 and Cyclin D1, respectively, as measured by the western blot method.
FIG. 7, CIDEC is a downstream gene of
FIG. 8 shows the effect of inhibiting CIDEC expression on the proliferative capacity of lung adenocarcinoma cells and lung normal cells,
graphs A and B are the interference efficiency of the qRT-PCR and western blot methods for detecting CIDEC interference fragments in A549 and PC9 cells, respectively; panels C and D are the effect of CCK8 method on the proliferation potency of a549 and PC9 cells, respectively, to detect inhibition of CIDEC expression; e is the influence of inhibiting CIDEC expression on the clonogenic capacity of A549 and PC9 cells; FIGS. F and G are the interference efficiencies of the CIDEC interference fragment in MRC-5 and BEAS-2B cells, respectively, as measured by qRT-PCR and western blot methods; FIGS. H and I are the effect of CCK8 method on the ability of MRC-5 and BEAS-2B cells to inhibit CIDEC expression; panel J is the effect of inhibition of CIDEC expression on the clonality of MRC-5 and BEAS-2B cells; panels K and L are the effect of CCK8 method to detect inhibition of CIDEC expression on
FIG. 9, inhibition of the effect of CIDEC expression on the migration and invasiveness of lung adenocarcinoma cells, wherein,
panels a and B are the Transwell assay to examine the effect of inhibition of CIDEC expression on the ability of a549 and PC9 cells to migrate and invade, respectively.
FIG. 10, inhibition of the effect of CIDEC expression on dormancy associated proteins in cells, wherein,
pan-blot method to examine the effect of inhibition of CIDEC expression on the protein and phosphorylation levels of ERK and p38 and downstream p16, p21, p27, p53 and Cyclin D1 in A549 and PC9 cells, respectively.
FIG. 11, KRAS is the upstream gene of ARL14, wherein,
graphs A and B are the interference efficiency of the KRAS interference fragment detected by qRT-PCR and western blot methods in A549 and PC9 cells, respectively; panel C and D are the qRT-PCR and western blot methods, respectively, to detect the effect of inhibiting KRAS expression in A549 and PC9 cells on the mRNA and protein expression levels of
Detailed Description
Specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Unless otherwise specified, all technical means involved in the present invention are known to those skilled in the art. Furthermore, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the reagents involved in the present invention can be made without departing from the spirit and scope of the invention. The cells and reagents involved in the present invention are commercially available.
The invention relates to:
1. method of producing a composite material
1.1 cell lines
Human bronchial epithelial cell line BEAS-2B cells were donated by Nanjing medical university; human embryonic fibroblast cell line MRC-5 cells and human lung adenocarcinoma cell line A549 cells are purchased from Shanghai cell bank of Chinese academy of sciences; the human lung adenocarcinoma cell line PC9 cells are all donated by the Life sciences college of the university of Compound Dan.
1.2 Primary reagents
Fetal bovine serum was purchased from Gibco, usa; DMEM and alpha-MEM medium were purchased from Gibco, USA, SYBRGreen PCR kit was purchased from Bao bioengineering (Dalian) Co., Ltd; transwell cells and matched 24-well plates were purchased from corning life science ltd; the qRT-PCR primer is synthesized by Shanghai Saibaosheng Gene technology Limited, and the purification mode is PAGE; siRNAs for ARL14, CIDEC, and KRAS were purchased from Ruibo Biotech, Inc., Guangzhou; beta-actin, p-p21, p-p27, p38 and ARL14 antibodies were purchased from abcam, UK; ERK, p-ERK, p27, p53, p-p53, p-p38, and Ki67 are available from CST corporation, USA; p16, p21, and Cyclin D1 antibodies were purchased from Santa Cruz, USA; p-p16 and p-CyclinD1 antibodies were purchased from SAB, USA; protein-tech corporation, CIDEC and KRAS, USA; alexa594 Goatanti-mouse IgG (H + L) (fluorescent secondary antibody) was purchased from Life Technology, USA; cell cycle detection kit purchased from American BDA driver; CCK-8 detection kit, DAPI Fluorocount-GTM, and Pre-dyed Trichrome protein Marker were purchased from Shanghai assist in san Francisco Biotechnology, Inc.
1.3 cell culture
The culture conditions of human lung adenocarcinoma cell strains A549 and PC9 and human lung normal cells BEAS-2B and MRC-5 are DMEM or alpha-MEM medium containing 10% fetal calf serum at 37 deg.C in 5% C02Cultured in an incubator.
1.4 transfection of cells
BEAS-2B, MRC-5, A549 and PC9 cells were transiently transfected with siRNA to ARL14/CIDEC/KRAS, whose interference sequences are shown in Table 1.
TABLE 1 siRNA interference sequences
1.5 qRT-PCR Gene detection
qRT-PCR detects the expression of ARL14, CIDEC and KRAS in transfected cells, each group of cells is collected 48h after transfection, total RNA in the cells is extracted by a column centrifugation method, and quantitative detection is carried out by a Nano Vue nucleic acid protein detector. After synthesizing cDNA from an RNA sample by reverse transcription reaction, detecting the contents of ARL14, CIDEC and KRAS in the sample by using a SYBRGreen PCR kit, wherein the primer sequences are shown in Table 2, and the reaction conditions are pre-denaturation at 95 ℃ for 30s, pre-denaturation at 95 ℃ for 5s, and pre-denaturation at 55 ℃ for 30 s; and (3) carrying out 40 cycles at 72 ℃ for 1min, and obtaining the basic cycle numbers (Ct values) of ARL14, CIDEC, KRAS and internal reference beta-actin of all samples after the reaction is finished. And calculating the relative content of the target gene according to a formula, namely the relative content of ARL14, CIDEC and KRAS, and repeating the experiment for three times.
TABLE 2 Gene primer sequences
1.6 CCK8 method for detecting cell proliferation and radiosensitivity
A control group and an experimental group were set for each cell. The cell lines of the control group and the experimental group in the logarithmic growth phase were trypsinized and the cells were diluted to 5 in103Each cell/ml culture solution, respectively taking 200ul to 96-well culture plates, inoculating 6 identical wells of each cell plate as multiple wells, 1 × 103For each cell/well, 200. mu.l of culture medium was used as a blank and 5% CO was placed2And continuously culturing in a constant-temperature cell culture box at 37 ℃. On
1.7 clone formation experiments
A control group and an experimental group were set for each cell. Cell lines of the control and experimental groups in the logarithmic growth phase were trypsinized and the cells were diluted to a concentration of 2.5X 102Taking 1ml of each cell/ml culture solution, adding into 6-well culture plate containing 2ml culture solution, setting 3 parallel samples for each treatment, and placing 5% CO2And continuously culturing for 10-14 days in a constant-temperature cell culture box at 37 ℃.
1.8 Transwell method for detecting invasion and migration capacity of cells
The cells of different groups are changed into serum-free DMEM medium 24 hours before the experiment, and the 24-hole plate, the Transwell chamber, the used EP tube and the used gun head are placed in a refrigerator at the temperature of-20 ℃, and meanwhile, the BD matrigel is placed in the refrigerator at the temperature of 4 ℃ overnight; diluting matrigel matrix with precooled serum-free DMEM medium according to the ratio of 1: 40 and mixing uniformly; add 110. mu.l of diluted matrigel (3 replicates) vertically in the center of the upper chamber of the transwell chamber to avoid bubbling, then place 5% CO2Incubating the cells in a cell incubator at 37 ℃ for at least 2h to allow the cells to coagulate; digesting the cells, washing the cells by serum-free DMEM medium, resuspending the cells, and diluting the cells to 1-1.4 multiplied by 105Per ml of cells/ml, 0.5ml of cell suspension (5-7X 10) was added to each chamber4Chamber), simultaneously adding 0.9ml of DMEM medium containing 10% fetal calf serum into the lower chamber, setting 3 parallel samples for each group treatment, and placing the samples in an incubator at 37 ℃ for culturing for 24 hours; adding 0.8ml of methanol solution into each hole, and fixing for 30min at room temperature; absorbing the fixing solution, washing with 1 × PBS for 3 times, adding 0.8ml crystal violet staining solution into each well, staining for 30min at room temperature, and washing with clear water for 3 times; cells that did not invade the lower surface of the transwell chamber were carefully wiped off with a 1 × PBS pre-wetted cotton swab, dried, placed under a microscope for observation and photographed.
The cell migration ability detection method is the same as the cell invasion ability, and only does not need to spread glue.
1.9 immunofluorescence assay
Cells grown to log phase were digested with pancreatin containing 0.25% EDTA and counted, seeded on 8-well cell culture plates, and cell concentration was adjusted to 2-4 × 10 per well depending on the cell line4A plurality of; washing with 1 × PBS (precooling at 4 deg.C) for three times during transfection for 48h, adding 0.2ml of immunostaining fixative into each well, fixing at room temperature for 15min, and washing with 1 × immunostaining fixative for three times; adding 0.2ml of immune staining permeation liquid into each hole, standing for 15min at room temperature, and cleaning with 1 Ximmune staining cleaning solution for three times; adding 0.2ml of the immunostaining sealing liquid into each hole, placing on a shaking table, and slowly shaking for 1-2 hours at room temperature; preparing primary antibody of Ki67 with immunostaining primary antibody diluent at a ratio of 1: 500, adding 0.2ml diluted Ki67 primary antibody into each well, and then placing in a refrigerator at 4 ℃ overnight; washing with 1 Ximmunostaining washing solution three times, and preparing Alexa with immunofluorescence staining secondary antibody diluent according to the ratio of 1: 100594 goal anti-mouse IgG (H + L) secondary antibody, 0.2ml of diluted secondary antibody is added into each hole, the mixture is placed on a shaking table, and the shaking table is kept in the dark at room temperature and slowly shaken for 1-2 hours; discarding the secondary antibody, and washing with 1 Ximmunostaining washing solution for three times; carefully prying the glass slide by using a tool matched with the glass slide, keeping the bottom, adding a proper amount of DAPI Fluromount-GTM, covering a cover glass, lightly pressing by using non-woven gauze, and dyeing for 5min at room temperature in a dark place; in Zeiss AxioplaObserving under an n-type upright fluorescence microscope and a 40-time microscope, randomly selecting at least 10 visual fields for photographing, and processing and synthesizing images by adopting ZEN 2.
1.10 cell cycle experiments
Digesting and counting the cells growing to logarithmic phase with pancreatin containing 0.25% EDTA, inoculating into six-well cell plate, and adjusting cell concentration to 2.5-4 × 10 per well according to different cell lines5A plurality of; carrying out gamma ray irradiation 48h after transfection, starting cell collection 24h after irradiation, firstly collecting the culture solution in a 15ml centrifuge tube, washing with 1 XPBS for three times, and adding the culture solution into the 15ml centrifuge tube for collecting the culture solution; then, digesting the cells by pancreatin containing 0.25% of EDTA until the cells can be lightly blown down by a sample adding gun, adding a new culture solution to stop digestion, lightly blowing down all adherent cells and blowing up the adherent cells, and then collecting the adherent cells into the same corresponding centrifuge tube; centrifuging at 4 ℃, washing for three times by 1000r/min multiplied by 10min with PBS, and transferring to a 1.5ml EP tube; carefully sucking off the supernatant with a sample adding gun, slowly adding 1ml of precooled 70% ethanol along the tube wall, lightly flicking the bottom of a 1.5ml EP tube to uniformly disperse cells to avoid agglomeration, and placing the mixture in a refrigerator at-20 ℃ for overnight; centrifuging at 4 ℃, 12000 r/min multiplied by 10min, washing with PBS for three times, carefully sucking off supernatant by using a sample adding gun, then adding 0.5ml of PI dye solution, uniformly mixing, and dyeing for 30min at room temperature in a dark place; flow detection was performed according to the instrument protocol using an excitation wavelength of 488 nh.
1.11 Western blot experiment
Western blot detection of expression of related proteins in transfected cells, collecting each group of cells 48h after transfection, extracting total protein in the cells by RIPA lysate (strong) and carrying out BCA protein quantification, then adding 5 × loading buffer with 0.25 times volume, mixing uniformly, and carrying out protein denaturation by a boiling method. SDS-PAGE conditions: preparing separation gel with proper concentration and 5% concentrated gel according to the relative molecular weight of the protein, adding the same amount of deformed protein sample into each hole, and adding a pre-dyed trichrome protein Marker into the holes on the two sides of the sample; and (3) turning on a power supply, setting the voltage to a constant voltage of 60V, changing the voltage to 100V when the protein of the sample to be detected runs to the separation gel, and stopping electrophoresis when the distance between the blue strip at the lowest surface of the Marker and the gel bottom is about 1 cm. Film transferring conditions: ice water bath, 100V, 1-2.5 h (the specific time is adjusted according to the relative molecular weight of the target protein), and the closed condition is as follows: put into a confining liquid (5% skimmed milk powder) prepared by 1 XTSST, put on a horizontal shaker, and slowly shake for 2h at room temperature. Primary anti-incubation conditions: the primary antibody dilutions were used to prepare the corresponding primary antibody incubators according to the instructions (1: 1000) and incubated overnight at 4 ℃. And (3) secondary antibody incubation: recovering redundant primary antibody, and washing with 1 × TBST for three times; prepare the desired volume of secondary antibody incubation with 1 XTSST according to the instructions (1: 4000), immerse the strip, place on a vertical shaker, and slowly shake up and down for 2h at room temperature. And (3) developing: washing with 1 XTBST for three times, preparing a chemiluminescence substrate (solution A: solution B is 1: 1) according to the instruction, uniformly covering the surface of a target strip film, starting a chemiluminescence imaging system, carrying out development imaging, and finally analyzing the gray values of all detection target strips by using Quantity One software.
2. Statistical treatment
The experimental results are average values of 3-5 repeated experiments, SPSS 20.0 statistical software is adopted for analysis, Student's-t test is adopted for statistical analysis, and if P is less than 0.05, the difference is considered to have statistical significance.