CN109100514B - Research method for analyzing Klotho interaction protein in non-small cell lung cancer - Google Patents
Research method for analyzing Klotho interaction protein in non-small cell lung cancer Download PDFInfo
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Abstract
The invention discloses a research method for analyzing Klotho interacting protein in non-small cell lung cancer, which comprises three steps of screening the Klotho interacting protein, verifying the mutual combination of Rab8 and Klotho gene and analyzing the mechanism of Rab8 and Klotho interaction. The invention discovers for the first time that Rab8 and Klotho are mutually combined in non-small cell lung cancer cells and are co-localized in subcellular, and Rab8 is combined with Klotho through a protein synthesis pathway and promotes the transportation of Klotho to the surface of a membrane in a vesicle form, so that the distribution level of Klotho on the surface of the membrane is improved.
Description
Technical Field
The invention relates to a protein, in particular to a research method for analyzing Klotho interaction protein in non-small cell lung cancer, belonging to the field of biological application.
Background
The incidence and mortality of lung cancer are the first malignant tumors worldwide. Among lung cancers, non-small cell lung cancer (NSCLC) accounts for over 80%, and the 5-year survival rate is about 10%, and the incidence rate is still increasing. Therefore, it is of great significance to explore the potential mechanism of NSCLC and develop safer and more effective treatment methods. The lung cancer is an aging-related disease, the median age of lung cancer is diagnosed to be 70 years old, and the age range of 75-79 years old is the peak stage of lung cancer. With the aging process, genome defects, epigenetic gene silencing, oxidative stress, immune surveillance defects, telomere abnormalities, chronic inflammation, and changes in tissue microenvironment, the risk of tumorigenesis is significantly increased. Thus, aging is an important risk factor for carcinogenesis. In addition, functional defects or deletions of cancer suppressor genes are also one of the important factors in cancer development, and the role of cancer suppressor genes in the development of cancer development has been a hot spot for studying the pathogenesis of cancer.
The subject group is continuously dedicated to the research on the function and the related mechanism of the anti-aging gene Klotho in lung cancer in recent years, and the result shows that the Klotho has the cancer inhibition effect in the lung cancer besides the anti-normal cell aging, the expression of the Klotho is obviously reduced in the lung cancer tissue compared with the normal tissue, and the abnormal expression of the Klotho participates in the occurrence and development of the lung cancer.
In addition to having a recognized anti-aging effect, the Klotho gene has been discovered in recent years to play a role in a cancer suppressor gene in various tumors. Klotho plays a potential cancer suppressor gene role in various tumors such as breast cancer, gastric cancer, prostatic cancer, colon cancer, cervical cancer, renal cancer and the like. In recent years, serial researches on the role of Klotho in lung cancer have been carried out by applicants and teams, and the results prove that: in the non-small cell lung cancer, Klotho can inhibit the growth and migration of lung cancer cells, promote the apoptosis of the lung cancer cells and improve the sensitivity of the lung cancer cells to chemotherapeutic drugs by regulating signal pathways such as insulin/IGF-1, Bax/Bcl-2, Wnt/beta-catenin, PI3K/AKT and the like. Thus, its cancer suppressing effect is evident from multiple studies of the role of Klotho in tumors. Although there are many studies around the cancer-suppressing effect of Klotho, the studies are mainly focused on its regulation of downstream signals, and the recognition of the regulation of Klotho itself, the interaction between Klotho-binding proteins, and the like is still very limited.
Disclosure of Invention
The present invention has been made to solve the above problems, and an object of the present invention is to provide a method for analyzing Klotho interacting protein in non-small cell lung cancer.
The invention achieves the aim through the following technical scheme, and the research method for analyzing the Klotho interaction protein in the non-small cell lung cancer comprises the following steps:
a. screening for Klotho interacting proteins
Co-transfecting 293T cells by using a vector plasmid pUltra-Klotho, a packaging plasmid psPAX2 and an envelope plasmid pMD2G, analyzing virus titer by using a flow cytometer to obtain a Klotho lentivirus expression vector Lenti-FLAG-Klotho, infecting a lung adenocarcinoma cell line A549 by using the Lenti-FLAG-Klotho, performing an immune co-precipitation experiment on extracted proteins by using the Lenti-FLAG-GFP as a control, screening proteins combined with the Klotho at a high probability, and finally selecting 20 Rab family members with protein scores exceeding 60, wherein the Rab8 protein score is the highest;
b. verification of mutual binding of Rab8 and Klotho protein comprises exogenous and endogenous co-immunoprecipitation experiments
Exogenous co-immunoprecipitation HEK293 cells were co-transfected by FLAG-Klotho and HA-Rab8, in which FLAG-Klotho and HA-Rab8 are Klotho and Rab8 overexpression plasmids, and then the co-immunoprecipitation experiments using labeled antibodies were performed to analyze the correlation between the two, showing that both Klotho and Rab8 bind to each other; endogenous co-immunoprecipitation experiments were performed by lysing a549 cells and performing Rab8/Klotho co-immunoprecipitation, which showed that endogenous Rab8 and Klotho proteins also bound to each other under physiological conditions;
c. mechanistic analysis of Rab8 interaction with Klotho
(1) Observation of Klotho and Rab8 subcellular localization
The subcellular co-localization of Klotho and Rab8 in A549 cells is observed by an immunofluorescence cytochemistry method, and the result shows that the Klotho and Rab8 have co-localization in the subcellular;
(2) analysis of the Effect of Rab8 expression changes on Klotho expression
Constructing Rab8 interfering sequence siRNAs, wherein the specific sequence is as follows:
Rab8 siRNA1#siRab8-1:5'-CGA GAA GTC CTT CGA CAA CAT-3'；
Rab8 siRNA2#siRab8-2:5'-CGG AAC TGG ATT CGC AAC ATT-3'；
Rab8 siRNA3#siRab8-3:5'-TCG CCA GAG ATA TCA AAG CAA -3'；
rab8 siRNA negative control siNC 5'-ATG TTG TCG AAG GAC TTC TCG-3';
transfecting A549 cells with the siRab8 and the siNC respectively, evaluating the interference efficiency, and indicating that the interference efficiency of the siRab8-3 is the highest and is 91 percent, transfecting the A549 cells with the siRab8-3 or the Rab8 overexpression plasmid Myc-Rab8, and detecting the changes of the protein and mRNA levels of Klotho by Western blot and qRT-PCR respectively, and indicating that the expression changes of the Rab8 do not influence the expression levels of the Klotho total protein or mRNA;
(3) observation of the Effect of Rab8 on Klotho Membrane surface expression
By utilizing a biotin surface labeling technology, whether Rab8 participates in membrane surface transport of Klotho in A549 cell lines and H1299 cell lines is researched, Rab8T22N, Rab8Q67L and SiRab8 are respectively transfected into lung cancer cells, and the density of the cancer cells is 1 x 106The cells are planted in a/ml mode and are carried out according to the previous experimental steps, and the experimental result shows that the activated mutant Rab8Q67L can obviously improve the lung cancer cellsThe expression level of Klotho on the surface of the membrane is obviously reduced by the inactivated mutants of Rab8T22N and SiRab 8;
(4) analysis of the relationship between Klotho localization and its protein metabolism
Different activated forms of Rab8, namely Rab8Q67L, Rab8WT and Rab8T22N are transfected into A549 cells respectively, cycloheximide CHX which is a protein translation inhibitor is used for blocking Klotho biosynthesis, and western blot is used for detecting the expression level of Klotho protein, so that the Klotho protein level in an activated form mutant Rab8Q67L group is obviously higher than that in a Rab8 wild group Rab8WT and an inactivated form mutant Rab8T22N group.
Preferably, the method comprises analyzing the difference of the different activity states of the Rab8 and the binding capacity of the Klotho, constructing Rab8GDP binding inactivation state mutant Rab8T22N and Rab8GTP binding activation state mutant Rab8Q67L through PCR-mediated site-directed mutagenesis technology, co-transfecting Rab8T22N and Rab8Q67L with FLAG-Klotho to A549 cells or H1299 cells respectively, and indicating that the inactivation state mutant Rab8T22N is more tightly bound with the Klotho protein and the Klotho protein is not easily released compared with the activation state mutant Rab8Q67L through co-immunoprecipitation.
Preferably, in the step c, Rab8 and Klotho qRT-PCR are both performed by using a Bio-rad 480PCR instrument, and the primer sequences are as follows:
Rab8-189bp：
and (3) sense: 5'-AAAGCTGGCCCTCGACTATG-3', respectively;
antisense: 5'-CTGCTCCTCTTCTGCTGGTC-3', respectively;
Klotho-374bp：
and (3) sense: 5'-CGACCACTTCAGGGATTACGC-3', respectively;
antisense: 5'-GGATAGTCACCATCAATAAATACGG-3', respectively;
preferably, the density of the cancer cell culture in the step C (3) is measured, and the immunoreaction zone is scanned and quantified by using NIH Image J.
Preferably, both the FLAG-Klotho and HA-Rab8 are pCDNA3.1(+) expression vectors.
The invention has the beneficial effects that: the invention utilizes a plurality of technologies of co-immunoprecipitation combined with mass spectrometry, immunofluorescence cytochemistry, biotin surface labeling and the like, finds that Rab8 and Klotho are mutually combined in non-small cell lung cancer cells for the first time, and co-localization exists in subcells, and Rab8 is combined with Klotho through a protein synthesis way and promotes the transportation of the Klotho to the surface of a membrane in a vesicle form, so that the distribution level of the Klotho on the surface of the membrane is improved.
Drawings
FIG. 1 is a diagram showing the result analysis of co-immunoprecipitation experiment using Flag antibody according to the present invention;
FIG. 2 is a diagram of co-immunoprecipitation mass spectrometry analysis according to the present invention;
FIG. 3 is an analysis chart of the results of the exogenous co-immunoprecipitation experiment of the present invention;
FIG. 4 is an analysis chart of the results of the endogenous co-immunoprecipitation experiment of the present invention;
FIG. 5 is the maps of Rab8GDP binding inactivation mutant Rab8T22N and Rab8GTP binding activation mutant Rab8Q 67L;
FIG. 6 is the result analysis chart of the co-immunoprecipitation of Rab8 GDP-binding inactivated mutant Rab8T22N and Rab8 GTP-binding activated mutant Rab8Q67L of the invention;
FIG. 7 is an analysis chart of the results of the Klotho and Rab8 immunofluorescence of the present invention;
FIG. 8 is a diagram of the interference efficiency of the interfering sequence siRNAs of Rab8 evaluated by the result of western blot;
FIG. 9 is a graph of the change in Rab8 expression versus the level of Klotho total protein expression of the invention;
FIG. 10 is a graph of the present invention analyzing the effect of the recombinant Klotho membrane surface protein on the cells transfected with Rab8T22N, Rab8Q67L and SiRab8 by using biotin surface labeling technique;
FIG. 11 is a graph showing the effect of Rab8 in different active forms on the expression level of Klotho total protein in A549 cells observed using western blot after translational inhibition of Klotho protein according to the present invention;
FIG. 12 is a graph of the rate of Klotho internalization observed using the cleaved surface biotin labeling technique following transfection of Rab8 with A549 cells in accordance with the present invention;
FIG. 13 is a graph of a recycling assay for the detection of Klotho protein using the fluorescent living cell ratio cycling assay of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
A study method for analyzing Klotho-interacting proteins in non-small cell lung cancer, comprising the steps of:
1. screening for Klotho interacting proteins
1.1 construction and infection of Lung cancer cells by the Klotho lentivirus expression vector Lenti-FLAG-Klotho
pUltra lentiviral vectors were purchased from Addgene. The human Klotho cDNA was cloned into the XbaI-BamHI site downstream of the 2a peptide coding sequence in frame with the enhanced GFP sequence, allowing for enhanced bicistronic expression of GFP. To obtain the target viral vector, the vector plasmids, i.e., pUultra-Klotho, packaging plasmid psPAX2, and envelope plasmid pMD2G were co-transfected into 293T cells and viral titers were analyzed by flow cytometry to obtain the Klotho lentiviral expression vector, Lenti-FLAG-Klotho.
Addgene model number is plasmid 24129, and enveloped plasmid pMD2G model number is adddge plasmids 12259, 12260.
Infecting a lung adenocarcinoma cell line A549 by utilizing Lenti-FLAG-Klotho, taking Lenti-FLAG-GFP as a control to eliminate irrelevant protein, and performing a co-immunoprecipitation experiment after extracting the protein, wherein the method specifically comprises the following steps: 1mg of protein was mixed with 2ug of rabbit IgG or Flag M2 antibody, 40ul of protein G + agarose was added, and overnight at 4 ℃. These beads were then eluted five times with lysis buffer. After the final centrifugation, 4 xSDS-PAGE sample buffer suspends the particles and boils for 5 minutes, and then immunoblotting is carried out to obtain co-immunoprecipitated products, and silver staining results show that compared with a control group, the experimental group has more different bands which are suggested to be Klotho interacting proteins, and the results are shown in figure 1.
In order to ensure the reliability of results, three groups of independent co-immunoprecipitates of the experimental group and the control group are respectively subjected to mass spectrometry. As a result, 77 kinds of proteins binding to Klotho were found. We selected 37 proteins that bind Klotho with high probability, including 20 Rab family members with a score over 60, with Rab8 protein scoring the highest, as shown in figure 2.
1.2 exogenous and endogenous co-immunoprecipitation experiments, respectively, demonstrated that Rab8 binds to Klotho and assessed the differences in binding ability between Rab8 and Klotho in different activity states
First, Klotho and Rab8 overexpression plasmids FLAG-Klotho and HA-Rab8 were constructed, and C-terminal FLAG, Myc and/or HA epitope tags were added before the stop codon by PCR, and all of the constructed plasmids were sequenced for identification. Then, to further confirm that Klotho binds Rab8, we performed exogenous and endogenous co-immunoprecipitation experiments, respectively. Exogenous co-immunoprecipitation assay HEK293 cells were co-transfected with FLAG-Klotho and HA-Rab8 and then analyzed for correlation using tag antibodies in co-immunoprecipitation assays, which showed that both Klotho and Rab8 bound to each other, as shown in FIG. 3.
Both FLAG-Klotho and HA-Rab8 are pCDNA3.1(+) expression vectors.
In addition, we observed endogenous binding of Rab8 and Klotho to each other under physiological conditions, lysed a549 cells, and performed Rab8/Klotho co-immunoprecipitation experiments, which showed that endogenous Rab8 and Klotho proteins also bound to each other under normal conditions, as shown in fig. 4.
Rab proteins cycle between a soluble state and a membrane-bound state, where the membrane-bound state is the predominant form in which they participate in trafficking. While the nucleotide binding state of Rab molecules affects its state and activity, where GDP binding state is inactive and GTP binding state is active, we further analyzed the difference in the different activity states of Rab8 from Klotho's binding ability.
First, Rab8 GDP-binding inactivation state mutant Rab8T22N and Rab8 GTP-binding activation state mutant Rab8Q67L were constructed, respectively, by PCR-mediated site-directed mutagenesis technology, as shown in fig. 5. Then, Rab8T22N, Rab8Q67L were co-transfected with FLAG-Klotho a549 cells or H1299 cells, respectively, and the co-immunoprecipitation results suggest that the inactive state mutant Rab8T22N binds more tightly to Klotho protein and the Klotho protein is not easily released compared to the active state mutant Rab8Q67L, as shown in fig. 6.
2. Mechanism research of interaction between Rab8 and Klotho
2.1 Observation of Klotho and Rab8 subcellular localization by immunocytochemistry
The subcellular co-localization of Klotho and Rab8 in a549 cells was observed by immunofluorescence cytochemistry methods, the brief steps are as follows:
a549 cells are planted on a slide, transfection is carried out after the cells grow to a proper density, the cells are fixed by 4% PFA for 10 minutes after 48 hours of transfection, then 0.4% Triton X-100 is used for permeabilization for 10 minutes, after 10% of normal goat serum is sealed for 60 minutes, the slide is placed into a primary antibody diluted in TBS for 1 hour of incubation, after PBS is washed, the slide is incubated for 1 hour by Alexa Fluor 488-or 594-fluorescent secondary antibody 1:1000, and finally DAPI counterstaining, mounting and observing are carried out under a fluorescence microscope. Immunofluorescence results showed that Klotho co-localized with Rab8 in the vesicles, as shown in fig. 7.
2.2 Effect of Rab8 expression changes on Klotho expression
Next, we observed whether Rab8 expression levels affected Klotho expression. First, we constructed several Rab8 interfering sequence siRNAs, the specific sequences are as follows:
Rab8 siRNA1#siRab8-1:5'-CGA GAA GTC CTT CGA CAA CAT-3'；
Rab8 siRNA2#siRab8-2:5'-CGG AAC TGG ATT CGC AAC ATT-3'；
Rab8 siRNA3#siRab8-3:5'-TCG CCA GAG ATA TCA AAG CAA-3'；
rab8 siRNA negative control siNC:5'-ATG TTG TCG AAG GAC TTC TCG-3'.
The above siRab8 and siNC were transfected into a549 cells, respectively, and the interference efficiency was evaluated, which suggests that the siRab8-3 interference efficiency was the highest at 91%, as shown in fig. 8.
A549 cells were then transfected with either siRab8-3 or Rab8 overexpression plasmid Myc-Rab8 and changes in Klotho protein and mRNA levels were detected by Western blot and qRT-PCR, respectively, using Bio-rad 480PCR instrument with the following primer sequences:
Rab8-189bp：
and (3) sense: 5'-AAAGCTGGCCCTCGACTATG-3', respectively;
antisense: 5'-CTGCTCCTCTTCTGCTGGTC-3' are provided.
Klotho-374bp：
And (3) sense: 5'-CGACCACTTCAGGGATTACGC-3', respectively;
antisense: 5'-GGATAGTCACCATCAATAAATACGG-3' are provided.
The above results show that changes in Rab8 expression do not affect Klotho total protein or mRNA expression levels, as shown in figure 9.
3. Biotin surface labeling technology for observing influence of Rab8 on Klotho membrane surface expression
Using the biotin surface labeling technique, we investigated whether Rab8 was involved in membrane surface transport in Klotho in the A549 and H1299 cell lines. First, Rab8T22N, Rab8Q67L and siRab8 were transfected into lung cancer cells, respectively, and the procedure was briefly as follows: at a density of 1 × 106Cells/ml were plated, washed twice with ice PBS, cells were incubated for 45 minutes in PBS containing sulfonated N-hydroxysuccinimide-biotin at 4 ℃ to biotinylate cell surface proteins, Tris buffer removed unreacted biotin, the bioacylated cells were lysed in extraction buffer, the extract was centrifuged at 12000g × 15 minutes, and then the cell biotinylated protein agarose beads were separated from the cell extract by streptavidin-bound agarose bead precipitation, the washed beads were eluted with SDS sample buffer, and the proteins were separated by SDS-PAGE. The experimental result shows that the activated form mutant Rab8Q67L can obviously improve the expression level of Klotho on the surface of the cell membrane of the lung cancer, and the inactivated form mutants Rab8T22N and siRab8 obviously reduce the expression level of the Klotho on the surface of the cell membrane, as shown in figure 10.
Sulfonated N-hydroxysuccinimide-biotin was purchased from Pierce corporation, and was available in a model size of 300. mu.g/ml.
The extraction buffer included 1.0% Triton X-100, 10mM Tris-HCl, pH 7.5, 120mM sodium chloride, 25mM potassium chloride, 1. mu.g/ml leupeptin, 1. mu.g/ml aprotinin, 2. mu.g/ml aprotinin, 0.5mM phenylmethylsulfonyl fluoride by volume.
For densitometry analysis, immunoreactive bands were scanned and quantified using NIH Image J.
4. Rab8 promotes the distribution of Klotho to membrane surfaces through protein posttranslational regulatory pathways
Next, we examined whether the localization of Klotho is related to its protein metabolism. First, different activated forms of Rab8, namely Rab8Q67L, Rab8WT and Rab8T22N, were transfected into a549 cells, respectively, and Klotho biosynthesis was blocked using the protein translation inhibitor cycloheximide CHX, and then the expression level of Klotho total protein was measured using western blot. The results show that the Klotho protein level in the activated form mutant Rab8Q67L group is significantly higher than that in the wild group of Rab8 and the inactivated form mutant Rab8T22N group, as shown in fig. 11. Thus, Rab8 indirectly inhibits degradation of Klotho by promoting its translocation to the membrane surface.
The membrane surface level of proteins depends on the balance between insertion and endocytosis, which are two opposite receptor transport processes. To determine which link Rab8 specifically influences membrane surface transport of Klotho, we observed the Klotho internalization process using a cleaved surface biotin labeling technique. The results showed that 30 minutes after biotin labeling, 40% ± 5% of topical Klotho internalized, and Rab8 overexpression group was not significantly different from the control group, as shown in fig. 12. Thus, these results indicate that Rab8 is not involved in the Klotho internalization process. After endocytosis, cells have two fates and are degraded or recycled to the plasma membrane, and the recycling of proteins to the plasma membrane can cause functional re-sensitization. Thus, we observed whether Rab8 could adjust the recycle loop of Klotho. We used a fluorescence-based live cell ratio cycling assay to measure Klotho recycling, the brief steps are as follows:
FLAG-Klotho-expressing A549 cells were incubated with Alex-488 fluorescent-binding anti-FLAG antibody at 4 ℃ for 30 minutes, then in DMEM at 37 ℃ for 30 minutes, and a first rewarming was performed, with green A488-M1 used to label internalized Klotho protein, and non-internalized anti-FLAG antibody removed by EDTA, followed by addition of 10ug/ml anti-Alexa-594 murine antibody and continued incubation for 45 minutes, and a second rewarming was performed to observe recycled Klotho protein, which was now red. The experimental results demonstrate that Rab8 does not affect the Klotho recycle loop, as shown in fig. 13.
The model specification of the anti-FLAG antibody which binds Alex-488 fluorescence is A488-M1, 2 ug/ml.
The results show that Rab8 and Klotho are mutually combined in the non-small cell lung cancer cell and are co-localized in the subcellular, and Rab8 is combined with Klotho and promotes the transportation to the membrane surface in a vesicle form through a protein synthesis pathway, so that the distribution level of the Klotho on the membrane surface is improved.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (5)
1. A study method for analyzing Klotho interacting proteins in non-small cell lung cancer, comprising: the method comprises the following steps:
a. screening for Klotho interacting proteins
Co-transfecting 293T cells by using a vector plasmid pUltra-Klotho, a packaging plasmid psPAX2 and an envelope plasmid pMD2G, analyzing virus titer by using a flow cytometer to obtain a Klotho lentivirus expression vector Lenti-FLAG-Klotho, infecting a lung adenocarcinoma cell line A549 by using the Lenti-FLAG-Klotho, performing an immune co-precipitation experiment on extracted proteins by using the Lenti-FLAG-GFP as a control, screening proteins combined with the Klotho at a high probability, and finally selecting 20 Rab family members with protein scores exceeding 60, wherein the Rab8 protein score is the highest;
b. verification of mutual binding of Rab8 and Klotho protein comprises exogenous and endogenous co-immunoprecipitation experiments
Exogenous co-immunoprecipitation HEK293 cells were co-transfected by FLAG-Klotho and HA-Rab8, in which FLAG-Klotho and HA-Rab8 are Klotho and Rab8 overexpression plasmids, and then the co-immunoprecipitation experiments using labeled antibodies were performed to analyze the correlation between the two, showing that both Klotho and Rab8 bind to each other; endogenous co-immunoprecipitation experiments were performed by lysing a549 cells and performing Rab8/Klotho co-immunoprecipitation, which showed that endogenous Rab8 and Klotho proteins also bound to each other under physiological conditions;
c. mechanistic analysis of Rab8 interaction with Klotho
(1) Observation of Klotho and Rab8 subcellular localization
The subcellular co-localization of Klotho and Rab8 in A549 cells is observed by an immunofluorescence cytochemistry method, and the result shows that the Klotho and Rab8 have co-localization in the subcellular;
(2) analysis of the Effect of Rab8 expression changes on Klotho expression
Constructing Rab8 interfering sequence siRNAs, wherein the specific sequence is as follows:
Rab8 siRNA1#siRab8-1:5'-CGA GAA GTC CTT CGA CAA CAT-3'；
Rab8 siRNA2#siRab8-2:5'-CGG AAC TGG ATT CGC AAC ATT-3'；
Rab8 siRNA3#siRab8-3:5'-TCG CCA GAG ATA TCA AAG CAA-3'；
rab8 siRNA negative control siNC 5'-ATG TTG TCG AAG GAC TTC TCG-3';
a549 cells are transfected by the siRab8-1, the siRab8-2, the siRab8-3 and the siNC respectively, interference efficiency is evaluated, and the result indicates that the interference efficiency of the siRab8-3 is the highest and is 91 percent, the A549 cells are transfected by the siRab8-3 or an Rab8 overexpression plasmid Myc-Rab8, and the change of the protein level and the mRNA level of Klotho is detected by Western blot and qRT-PCR respectively, and the result indicates that the change of the expression of the Rab8 does not influence the expression level of the Klotho total protein or the mRNA;
(3) observation of the Effect of Rab8 on Klotho Membrane surface expression
By utilizing a biotin surface labeling technology, whether Rab8 participates in membrane surface transport of Klotho in A549 cell lines and H1299 cell lines is researched, Rab8T22N, Rab8Q67L and SiRab8 are respectively transfected into lung cancer cells, and the density of the cancer cells is 1 x 106The cells are planted in a/ml mode and are carried out according to the previous experimental steps, and the experimental result shows that the activated form mutant Rab8Q67L can obviously improve the expression level of Klotho on the surface of the cell membrane of the lung cancer, and the inactivated form mutant Rab8T22N and siRab8 can obviously reduce the expression level of the Klotho on the surface of the cell membrane;
(4) analysis of the relationship between Klotho localization and its protein metabolism
Different activated forms of Rab8, namely Rab8Q67L, Rab8WT and Rab8T22N are transfected into A549 cells respectively, cycloheximide CHX which is a protein translation inhibitor is used for blocking Klotho biosynthesis, and western blot is used for detecting the expression level of Klotho protein, so that the Klotho protein level in an activated form mutant Rab8Q67L group is obviously higher than that in a Rab8 wild group Rab8WT and an inactivated form mutant Rab8T22N group.
2. The assay for the presence of a Klotho-interacting protein in non-small cell lung cancer according to claim 1, wherein: the method comprises the steps of analyzing the difference of different activity states of Rab8 and the binding capacity of Klotho, constructing Rab8 GDP-binding inactivation state mutant Rab8T22N and Rab8 GTP-binding activation state mutant Rab8Q67L through a PCR-mediated site-directed mutagenesis technology, co-transfecting Rab8T22N and Rab8Q67L with FLAG-Klotho or H1299 cells, and indicating that the inactivation state mutant Rab8T22N is more tightly bound with the Klotho protein and the Klotho protein is not easily released compared with the activation state mutant Rab8Q67L through the co-immunoprecipitation result.
3. The assay for the presence of a Klotho-interacting protein in non-small cell lung cancer according to claim 1, wherein: in the step c, Rab8 and Klotho qRT-PCR are both carried out by using a Bio-rad 480PCR instrument, and the primer sequences are as follows:
Rab8-189bp：
and (3) sense: 5'-AAAGCTGGCCCTCGACTATG-3', respectively;
antisense: 5'-CTGCTCCTCTTCTGCTGGTC-3', respectively;
Klotho-374bp：
and (3) sense: 5'-CGACCACTTCAGGGATTACGC-3', respectively;
antisense: 5'-GGATAGTCACCATCAATAAATACGG-3' are provided.
4. The assay for the presence of a Klotho-interacting protein in non-small cell lung cancer according to claim 1, wherein: and c (3) measuring the density of the cancer cell culture in the step c, and scanning and quantifying the immunoreaction zone by using NIH Image J.
5. The assay for the presence of a Klotho-interacting protein in non-small cell lung cancer according to claim 1, wherein: both the FLAG-Klotho and HA-Rab8 were pCDNA3.1(+) expression vectors.
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| Title |
|---|
| Klotho and (tumor+ or cancer+);陈波;《中国优秀硕士学位论文数据库 医药卫生科技辑》;20110615;E065-152 * |
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| CN109100514A (en) | 2018-12-28 |
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