CN117327797B - Application of RRP1 gene in treating non-small cell lung cancer - Google Patents
Application of RRP1 gene in treating non-small cell lung cancer Download PDFInfo
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Abstract
The invention discloses application of an RRP1 gene in treating non-small cell lung cancer, wherein the nucleotide sequence of the RRP1 gene is shown as SEQ ID NO. 1. The application also discloses the application of the polypeptide of the targeted RRP1 gene in preparing a non-small cell lung cancer therapeutic drug and the application of the interfering siRNAs of the targeted RRP1 gene in preparing the non-small cell lung cancer therapeutic drug. The application knows how RRP1 plays a role in non-small cell lung cancer, helps us to understand the underlying molecular mechanisms of non-small cell lung cancer occurrence and development, and facilitates the development of new targeted therapeutic drugs.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to application of an RRP1 gene in treating non-small cell lung cancer.
Background
Non-small cell lung cancer is one of the highly malignant tumors, with high incidence and mortality at position 1 in the world. Despite the positive combination of treatments, most patients are still unavoidably relapsed from tumor recurrence and metastasis and have a poor prognosis. The molecular mechanism of the occurrence and development of non-small cell lung cancer is not completely clear at present, however, a great deal of research has shown that the occurrence and development of the non-small cell lung cancer are closely related to various cancer related gene changes (such as gene mutation, gene overexpression, cancer suppressing gene inactivation and the like). Various targeted drugs have been developed for these genetic alterations, resulting in significant improvements in survival of lung cancer patients. Therefore, the further exploration of unknown lung cancer related gene changes is of great significance in revealing the precise molecular mechanism of occurrence and development of non-small cell lung cancer, designing reasonable therapeutic drugs, judging prognosis and further improving diagnosis and therapeutic level.
RRP1, called ribosomal RNA processing protein 1 (Ribosomal RNA Processing, RRP 1), has its coding gene which was found in yeast by Fabian et al in the earliest 1987, and then Jansen et al found a similar gene in humans (named NNP-1 at that time). At present, the human is very little aware of the gene, the research result is limited, and preliminary evidence shows that the gene has the functions of regulating the processing of rRNA, regulating the generation of mature ribosome subunits, participating in the formation of mitotic late nucleolus, repairing DNA damage and the like, and more functions of the gene are still to be further researched.
Disclosure of Invention
The invention aims to provide an application of RRP1 gene in treating non-small cell lung cancer, which researches how RRP1 plays a role in the non-small cell lung cancer, helps us to know potential molecular mechanisms of occurrence and development of the non-small cell lung cancer and promotes research and development of new targeted therapeutic drugs.
The technical problems to be solved by the invention are realized by the following technical scheme:
the first aim of the application is to provide an application of RRP1 gene in preparing medicaments for treating non-small cell lung cancer, wherein the nucleotide sequence of the RRP1 gene is shown as SEQ ID NO. 1.
Preferably, the agent is capable of inhibiting or knockdown RRP1 gene expression.
Preferably, the agent is capable of inhibiting proliferation, invasiveness or migration of non-small cell lung cancer cells.
The second aim of the application is to provide an application of RRP1 gene serving as a target point in preparing medicines for treating non-small cell lung cancer, wherein the nucleotide sequence of the RRP1 gene is shown as SEQ ID NO. 1.
Preferably, the pharmaceutical product is capable of inhibiting or knockdown RRP1 gene expression.
The third object of the present application is to provide an application of a polypeptide targeting an RRP1 gene in preparing a drug for treating non-small cell lung cancer, wherein the polypeptide sequence of the RRP1 gene is shown as SEQ ID NO. 2.
The fourth object of the present application is to provide an application of interference siRNAs targeting RRP1 genes in preparing a medicament for treating non-small cell lung cancer, wherein the nucleotide sequence of the interference siRNAs is shown as SEQ ID No. 3.
The fifth aim of the application is to provide an application of a recombinant vector and a recombinant cell for over-expressing RRP1 genes or knocking down RRP1 genes in preparation of medicines for treating non-small cell lung cancer, wherein the nucleotide sequence of the RRP1 genes is shown as SEQ ID NO. 1.
A sixth object of the present application is to provide an application of a biomarker or a reagent for detecting the biomarker in preparing a product for treating or diagnosing non-small cell lung cancer, wherein the biomarker is an RRP1 gene.
The technical scheme of the invention has the following beneficial effects:
according to the application, RRP1 is used as a non-small cell lung cancer treatment target, pathogenic effects of RRP1 on non-small cell lung cancer are confirmed from multiple angles, proliferation, invasiveness or migration capacity of RRP1 gene non-small cell lung cancer cells are reduced, and the inhibition or the knocking down of RRP1 can be used as an effective strategy for treating the non-small cell lung cancer, so that research and development of new targeted therapeutic drugs are promoted.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description, serve to explain the principles of the invention.
FIG. 1 is a graph showing that RRP1 is significantly highly expressed in non-small cell lung cancer and is associated with poor prognosis for non-small cell lung cancer; wherein, the A diagram is the analysis of the expression difference of RRP1 gene in the TCGA database and the tissue sample beside the cancer, the B diagram is the analysis of the expression of RRP1 gene in the tissue beside the cancer, the C diagram is the analysis of the accumulated survival rate of the patient.
FIG. 2 shows RT-qPCR detection of non-small cell lung cancer cells with knockdown RRP1 gene; wherein, the A diagram is qPCR detection of mRNA level RRP1 gene knockdown efficiency in A549 cells, the B diagram is WB detection of protein level RRP1 gene knockdown efficiency in A549 cells, the C diagram is qPCR detection of mRNA level RRP1 gene knockdown efficiency in H1299 cells, and the D diagram is WB detection of protein level RRP1 gene knockdown efficiency in H1299 cells.
FIG. 3 is a graph demonstrating the effect of RRP1 loss on A549 cell proliferation capacity; wherein, the A diagram shows the effect of MTT detection RRP1 gene knockdown on A549 cell proliferation (490 nm absorbance value), and the B diagram shows the fold change of MTT detection RRP1 gene knockdown on A549 cell proliferation.
FIG. 4 is a graph demonstrating the effect of RRP1 deletion on the proliferation capacity of H1299 cells; wherein, the A diagram shows the effect of MTT detection RRP1 gene knockdown on H1299 cell proliferation (490 nm absorbance value), and the B diagram shows the fold change of MTT detection RRP1 gene knockdown on H1299 cell proliferation.
FIG. 5 is a graph demonstrating the effect of RRP1 deletion on A549 cell clone formation efficiency; wherein, the A diagram is a fluorescent diagram of the influence of Celigo detection RRP1 gene knockdown on A549 cell proliferation, and the B diagram is a quantitative analysis diagram of the influence of Celigo detection RRP1 gene knockdown on A549 cell proliferation.
FIG. 6 is a graph demonstrating the effect of RRP1 deletion on H1299 cell clone formation efficiency; wherein, the A diagram is a fluorescent diagram of the influence of Celigo to detect RRP1 gene knockdown on H1299 cell proliferation, and the B diagram is a quantitative analysis diagram of the influence of Celigo to detect RRP1 gene knockdown on H1299 cell proliferation.
FIG. 7 is a graph demonstrating the effect of RRP1 depletion on A549 cell invasiveness; wherein, the A diagram is a representative diagram of the influence of Celigo scratch detection RRP1 gene knockdown on A549 cell migration, and the B diagram is a quantitative analysis diagram of the influence of Celigo scratch detection RRP1 gene knockdown on A549 cell migration.
FIG. 8 is a graph demonstrating the effect of RRP1 depletion on the invasive capacity of H1299 cells; wherein, the A diagram is a representative diagram of the influence of Celigo scratch detection RRP1 gene knockdown on H1299 cell migration, and the B diagram is a quantitative analysis diagram of the influence of Celigo scratch detection RRP1 gene knockdown on H1299 cell migration.
FIG. 9 is a graph demonstrating the effect of RRP1 depletion on A549 cell transfer ability; wherein, the A diagram is a representative diagram of the influence of the Mitigation (ECM-free Transwell) detection on the transfer of the A549 cells by the RRP1 gene knockout, and the B diagram is a statistical analysis result of the Mitigation (ECM-free Transwell) detection on the transfer number of the A549 cells by the RRP1 gene knockout.
FIG. 10 is a graph demonstrating the effect of RRP1 deletion on H1299 cell transfer capacity; wherein, the A diagram is a representative diagram of the effect of the Mitigation (ECM-free Transwell) detection of RRP1 gene knockdown on H1299 cell transfer, and the B diagram is a statistical analysis result of the Mitigation (ECM-free Transwell) detection of RRP1 gene knockdown on the H1299 cell transfer number.
Fig. 11 is a comparison of tumor weights in mouse experiments.
FIG. 12 is a comparison of tumor volume size in a mouse experiment; wherein, the A diagram is the change of the nodulation capacity of A549 cells after the RRP1 gene expression is knocked down detected by a nude mouse nodulation experiment, and the B diagram is the picture of the mice and the tumor after 44 days of subcutaneous injection.
Detailed Description
Various exemplary embodiments of the present invention will now be described in detail. It should be noted that: the relative arrangement of the components and steps, numerical expressions and numerical values set forth in these embodiments do not limit the scope of the present invention unless it is specifically stated otherwise.
Example 1
Correlation study of RRP1 with non-small cell lung cancer:
the clinical relevance of RRP1 gene expression and non-small cell lung cancer is analyzed through a TCGA database, and the expression of RRP1 in tumor tissues is found to be higher than that of normal tissues, and the high expression is related to prognosis of patients. Furthermore, we selected 43 pairs of non-small cell lung cancer/paracancestor specimens; the IHC method is adopted to detect the expression of RRP 1.
Constructing a cell model of RRP1 knockdown, and verifying knockdown efficiency:
and (3) carrying out the synthesis of RRP1 specific interference sequences (shRNA sequences) and the packaging of lentiviruses, carrying out lentivirus infection, and verifying the knockdown efficiency by utilizing RT-PCR and western blot. And constructing a cell model of RRP1 knockdown, and constructing a finished cell model for subsequent experiments.
RT-qPCR detects the knockdown efficiency of RRP1 gene:
1) Non-small cell lung cancer cells were plated in 12-well plates, and non-small cell lung cancer cell line a549 was infected with RRP1 shRNA lentivirus (SANTA CRUZ corporation) and control shRNA lentivirus (SANTA CRUZ corporation);
2) Screening shRNA stably expressed non-small cell lung cancer cells by using puromycin;
3) And detecting the expression level of RRP1 in the non-small cell lung cancer cells A549-RRP1-shRNA and A549-RRP1-mock by RT-qPCR.
The clinical relevance analysis and immunohistochemical results of fig. 1 show: RRP1 is significantly more expressed in non-small cell lung cancer tissues than in paracancestor normal tissues. In FIG. 2, it can be seen that the RRP1 gene is expressed in both A549 and H1299, and the expression of the RRP1 gene in A549 and H1299 is obviously reduced after knocking down.
Example 2
MTT experiment research RRP1 regulates the effect of non-small cell lung cancer cell proliferation:
1) Taking non-small cell lung cancer cells (RRP 1-shRNA) in logarithmic growth phase and control non-small cell lung cancer cells (RRP 1-mock), 1×10 4 Individual cells/wells were seeded into 96-well plates;
2) 5% CO at 37 DEG C 2 Cells from 2 groups of dishes were counted daily for 5 consecutive days while the proliferation capacity of 2 groups of cells was examined using the CCK-8 method.
As shown in fig. 3 and 4, the experimental results indicate that: after RRP1 knockdown in a549 and H1299, proliferation capacity of two non-small cell lung cancer cells was significantly reduced.
Example 3
Celigo cell count detects cell growth: the virus-infected cells usually have green or red fluorescence, and the Celigo instrument can read white light and the cells with fluorescence and take pictures, and then calculate the number of cells contained in different groups of the well plate through software analysis and treatment. After 5 days of continuous detection, a cell growth curve was drawn, thereby exhibiting a cell growth condition. Celigo cell count experiments study the role of RRP1 in regulating proliferation of non-small cell lung cancer cells:
1) Counting non-small cell lung cancer cells (RRP 1-shRNA) in logarithmic growth phase and control non-small cell lung cancer cells (RRP 1-mock);
2) Cell density was plated (2000 cells/well). 3 multiple wells per group, the culture system is 100 mu L/well, the cell number added into each well is ensured to be consistent in the plating process, the temperature is 37 ℃, and the CO content is 5% 2 Culturing in an incubator;
3) Starting from the second day after the plate is paved, celigo is detected and read once every day, and the plate is continuously detected and read for 5 days;
4) Accurately calculating the number of white light or cells with green fluorescence in each scanning pore plate by adjusting the input parameters of analysis settings; statistical plots were made on the data to plot 5 day cell proliferation curves.
The results are shown in fig. 5 and 6, and the experimental results show that: after RRP1 knockdown in a549 and H1299, proliferation capacity of two non-small cell lung cancer cells was significantly reduced.
Example 4
Cell scratch test (Cell scratch assay) is an experimental technique that investigates cell migration movement, repair ability and cell-cell interactions by scratching on a cell monolayer and periodically capturing images by a time lapse microscope.
Scratch experiment research on the effect of RRP1 on regulating the migration capacity of non-small cell lung cancer cells:
1) The cell density is 5-10×10 5 The non-small cell lung cancer cells A549-RRP1-shRNA and the control non-small cell lung cancer cells A549-RRP1-mock are paved on a 24-hole plate, and DMEM culture solution containing 10% FBS is added for culturing for 16-24h;
2) A 10 mu L pipette tip is used for forming a line-shaped scratch on the monolayer cells;
3) Washing 3 times by PBS, incubating for 24 hours, changing into DMEM culture solution containing 10% FBS, and incubating for 48 hours;
4) Observation, photographing and measurement were performed immediately after the scratch was completed, 24 hours after the scratch was completed and 48 hours after the scratch was completed, respectively.
The results are shown in fig. 7 and 8, and the experimental results show that: after RRP1 knockdown in a549 and H1299, the invasive capacity of both non-small cell lung cancer cells was significantly reduced.
Example 5
Transwell invasion assay is the most commonly used assay for detecting tumor cell invasion capacity, and the principle is that a layer of polycarbonate membrane is used for separating a high-nutrition culture solution from a low-nutrition culture solution, cells are inoculated into the low-nutrition culture solution, membrane holes are covered by Matrigel in general and imitate extracellular matrix, tumor cells must secrete hydrolytic enzymes and pass through a filter membrane paved with Matrigel through deformation movement, and the cell quantity entering a lower chamber can reflect the invasion capacity of the tumor cells.
Transwell invasion experiments study the effect of RRP1 on regulating the invasion ability of non-small cell lung cancer cells:
1) Coating the upper chamber surface of the bottom film of the Transwell chamber with Matrigel gel diluent;
2) Sucking out the residual liquid in the culture plate, adding 50ul of serum-free culture solution containing 10g/L BSA into each hole, and maintaining the temperature at 37 ℃ for 30min;
3) Serum starved cells were withdrawn for 12-24h, cells were resuspended in serum-free medium containing BSA (density: 1-5X 105/uL); 200ul of the cell suspension is taken and added into a Transwell chamber;
4) 500ul of 10% FBS culture medium is added into the lower chamber of the 24-well plate, and the culture is carried out for 12-48 hours in a conventional way;
5) Wiping out the matrigel and cells in the upper chamber with a cotton swab, and staining with 0.1% crystal violet;
6) Inverted Transwell cells, 10 fields were randomly selected for counting and photographing by microscope.
The results are shown in fig. 9 and 10, and the experimental results show that: after RRP1 knockdown in a549 and H1299, the invasive capacity of both non-small cell lung cancer cells was significantly reduced.
Example 6
Nude mouse tumorigenesis experiments study the effect of RRP1 gene on tumor growth:
since most tumor studies use human cells, we need immunodeficient mice as carriers for the model of transplanted tumors due to the presence of xenogenic rejection, which are judged for biological changes by injecting tumor cells into the mice to form tumors and observing their growth.
Animal experiment:
1) Establishing a nude mouse subcutaneous transplantation tumor model: culturing non-small cell lung cancer cell A549-RRP1-shRNA and control non-small cell lung cancer cell A549-RRP1-mock to logarithmic phase; non-small cell lung cancer cells were suspended in serum-free medium (1X 10) 7 Individual/100 uL); male Balb/c nude mice 6-8 weeks old were used, randomly divided into 2 groups (10/group); the syringe draws 100 mu L of non-small cell lung cancer cell suspension and inoculates the suspension under the right dorsal side near axillary part of the nude mice;
2) Drawing a graft tumor growth curve: the mice were weighed every 4 days from day 28 of inoculation and the long diameter (a) and short diameter (b) of the transplanted tumor body were measured with a vernier caliper, and 6 times of continuous observation were performed; calculating the average volume of the transplanted tumor according to a volume formula V= (a multiplied by b 2)/2, and drawing a tumor growth change curve;
3) Weighing the transplanted tumor: after the last 1 observation, the cervical dislocation of the mice is killed, and the tumors are taken out, weighed and photographed.
As shown in fig. 11 and 12, the volume of subcutaneous tumor of tumor-bearing mice with a549 cells knocked down by RRP1 was significantly lower than that of tumor-bearing mice with a549 cells normally expressed by RRP1 in fig. 11. It can be seen in fig. 12 that the weight of tumor-bearing mice with a549 cells knocked down with RRP1 was significantly lower than the weight of tumor-bearing mice with normal RRP1 expressing a549 cells (at the time of mice sacrifice).
Although the present invention has been described with reference to the above embodiments, it should be understood that the present invention is not limited thereto, and that various changes and modifications may be made by those skilled in the art without departing from the spirit and scope of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.
Claims (1)
1. The application of a reagent for detecting a biomarker in preparing a product for diagnosing non-small cell lung cancer is characterized in that the biomarker is an RRP1 gene, and the nucleotide sequence of the RRP1 gene is shown as SEQ ID NO. 1.
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| Ángela Marrugal et al..Inhibition of HSP90 in Driver Oncogene-Defined Lung Adenocarcinoma Cell Lines: Key Proteins Underpinning Therapeutic Efficacy.《International Journal o f Molecular Sciences》.2023,第第24卷卷第1-22页. * |
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