CN117192127A - Method for discovering HCC therapeutic target - Google Patents
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Abstract
The invention discloses a method for finding HCC therapeutic targets, and relates to the technical field of liver cancer. The invention clarifies a molecular fine regulation mechanism of FBXW8 on AZGP1, determines the influence of FBXW8 on PPT1 ubiquitination, searches a phosphorylation site for regulating AZGP1 ubiquitination, constructs an HCC cell line for stable expression and knockout of FBXW8/AZGP41, determines the influence of FBXW8-PPT1 axis and HCC, determines the influence of FBXW8-PPT1 axis on HCC malignant phenotype, clarifies the molecular mechanism of the influence of FBXW8-PPT1 axis on HCC development, and verifies the molecular mechanism of the FBXW8-PPT1 axis in animal level liver cancer development. The invention discovers a potential substrate PPT1 of FBXW8 through screening by an immunoprecipitation-mass spectrometry technology, verifies that FBXW8 and PPT1 interact and degrade, shortens the half-life of PPT1, and PPT1 can promote the occurrence and development of HCC, thus being a prognosis marker and a treatment target of HCC.
Description
Technical Field
The invention belongs to the field of liver cancer, and particularly relates to a method for discovering an HCC treatment target.
Background
About 46 cases of new liver cancer occur in China every year, and about 38 tens of thousands of people die, which accounts for half of the total number of liver cancer deaths, and generally, cancers can be classified into primary liver cancer and metastatic liver cancer according to their tissue origins, wherein primary cancers include hepatocellular carcinoma (Hepatocellular carcinoma, HCC), cholangiocarcinoma and other rare types of cancers, and HCC is the most common primary cancer type, accounting for more than 90% of all primary liver cancers.
HCC occurs due to the underlying disease of the patient, but the general order is liver injury, chronic inflammation, fibrosis, cirrhosis and liver cancer, HCC has few obvious symptoms in early stages of the disease, once it is found that almost all patients are in middle and late stages, treatment is difficult, and at present, the survival rate of HCC patients in the world is only 20% in 5 years, early symptom concealment, recurrence and metastasis after treatment are major causes of poor cancer treatment effect and prognosis, so research on HCC occurrence and development, search for new prognostic indexes and treatment targets, and have important significance for improving clinical treatment effect of HCC.
The present invention has been made in view of this.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a method for finding an HCC therapeutic target spot, and solves the technical problems in the prior art.
In order to solve the technical problems, the invention adopts the basic conception of the technical scheme that:
a method of discovering HCC therapeutic targets, comprising:
step one: elucidating a molecular fine regulation mechanism of FBXW8 on AZGP1, determining the influence of FBXW8 on PPT1 ubiquitination, and searching a phosphorylation site for regulating AZGP1 ubiquitination; the 293T cells are transfected with GFP-tagged PPT1, F-box proteins with Flag tags are simultaneously co-transferred respectively, MG132 is added 6 hours before sample collection, flag antibodies are immunoprecipitated, whether PPT1 is contained in the precipitate is detected by western blot, and whether the PPT1 only interacts with FBXW8 is determined; the 293T cells are transfected with GFP-tagged PPT1, F-box proteins of Flag tags are simultaneously co-transferred respectively, the content of the PPT1 is detected by western blot, and whether the PPT1 is only degraded by FBXW8 is determined; 293T cells were co-transformed with pEGFP-PPT1, pBABE-Flag-FBXW8, HA-UB (plasmid expressing HA-tagged ubiquitin) and MG132 was added 6 hours before collection, GFP antibody immunoprecipitated, and HA antibody detected UB in the pellet to determine if FBXW8 promoted ubiquitination of PPT 1.
Step two: elucidating a molecular fine regulation mechanism of FBXW8 on AZGP1, determining the influence of FBXW8 on PPT1 ubiquitination, and searching a phosphorylation site for regulating AZGP1 ubiquitination; performing a truncated body experiment (Mapping), cutting the PPT1 protein into a plurality of small fragments with different sizes, respectively co-transferring the small fragments with FBXW8 into 293t cells, performing immunoprecipitation on the FBXW8, and detecting whether different truncated bodies exist in the precipitate by using a western blot; because the PPT1 protein is smaller, serine and threonine sites in the minimum necessary structural domain are not too many, the sites can be mutated into alanine respectively, the mutants are respectively co-transformed with FBXW8, and whether interaction with the FBXW8 disappears or not is detected; and (3) co-transferring the mutant and the FBXW8 into 293t cells, detecting whether the degradation of the FBXW8 is disappeared, detecting whether the ubiquitination of the PPT1 is disappeared, constructing a glutamic acid mutant, and detecting whether the interaction of the glutamic acid mutant and the FBXW8 is enhanced and the half-life is shortened.
Step three: determining the effect of the FBXW8-PPT1 axis on HCC; six well plates were aliquoted with common HCC cell lines Bel7402, hepG2, huh7, HCCLM3 and normal hepatocytes LO2, BCA quantification after RIPA lysis, western blot detection of FBXW8, PPT1 protein expression levels, comparison of whether HCC was reduced relative to normal hepatocytes, whether FBXW8 was elevated, FBXW8 antibodies were purchased from Invitrogen, cat No. PA 5-58555, PPT1 from three hawk organisms, cat No. 67699-1-Ig; extracting total RNA of cell lines such as Bel7402, hepG2, huh7, HCCLM3, LO2 and the like, reversing and performing RT-PCR, wherein beta-actin is used as an internal reference.
Ordering a liver cancer tissue sample chip, HE staining, performing immunohistochemical detection on the expression difference of FBXW8 and PPT1 in cancer and paracancer tissues, analyzing the correlation of the expression level of the FBXW8 and the PPT1 with tumor TNM stage, performing semi-quantitative immunohistochemical detection on the protein level of the FBXW8 and the PPT1, evaluating the cell staining intensity and the percentage of stained cells under a low-power mirror, randomly selecting 5 high-power visual fields for observation, performing double-blind reading, and determining the final immune response score after multiplying the intensity score by the positive score of the stained cells for comprehensive analysis, wherein the liver cancer tissue sample chip is purchased from Shanghai Weiao biotechnology Co, paracancer and autologous cancer, detailed biochemical indexes, TNM stage, with recurrence data, radiotherapy and chemotherapy treatment and 5-year survival period.
Step four: determining the effect of the FBXW8-PPT1 axis on HCC malignancy phenotype; the stable expression and knocking-out cell line of FBXW8/PPT1 of HCC cells such as Bel7402, hepG2, huh7, HCCLM3 and the like is constructed, the stable transfection cell line is constructed by adopting a slow virus infection mode, pBABE-Flag-FBXW8/PPT1 and auxiliary plasmids are co-transferred into 293t cells, HCC cells are infected by virus, puromycin screening is carried out, and after the expression of FBXW8 is verified by western blot, the over-expression cell line of FBXW8/PPT1 of HCC can be obtained, wherein the degron phosphorylation site of PPT1 is an alanine mutant. The gene knockout cell line is constructed by adopting a CRISPR-cas9 technology, the HCC cells are transfected with FBXW8/PPT1PX459 plasmid, the split single clone is adopted, sequencing screening is carried out on the FBXW8/PPT1 frame shift mutation single clone, the FBXW8/PPT1 knockout cell line can be obtained after western blot verification on the FBXW8/PPT1 knockout effect, gDNA sequence design in the FBXW8/PPT1PX459 plasmid is completed in a CCtop website, and 4 gDN genes are designed each; after each HCC cell line was constructed, CCK8 and EdU experiments detected HCC cell proliferation, cell scratch and transwell experiments detected HCC cell migration, matrigel matrix gel model detected HCC cell invasion, CCK8 experiments detected HCC cell line resistance to sorafenib, and the effects of stable FBXW8/PPT1 expression and knockdown on HCC cell proliferation, migration, invasion and resistance were compared.
Step five: elucidating the molecular mechanism of the influence of the FBXW8-PPT1 axis on HCC development; determining the influence of the FBXW8-PPT1 axis on HCC autophagy and the molecular mechanism thereof; treatment of each cell line with 3-MA, an activator of autophagy, rapamycin (RAP), CCK8 and EdU experiments to detect proliferation, cell scratch and transwell experiments to detect migration, matrigel matrix gel model to detect invasion, CCK8 experiments to detect resistance of HCC cell lines to sorafenib to determine whether the effect of the FBXW8-PPT1 axis on the malignant phenotype of FBXW8-PPT1 axis on HCC was dependent on autophagy, to determine the effect of the FBXW8-PPT1 axis on HCC autophagy, changes in autophagy levels in FBXW8/PPT1 over-expression and knocked out HCC cell lines were observed, single-pellet (sulfoglutamine) staining to detect the number of autophagies, LC3 protein Blot to detect initiation of autophagy, and p62 protein Western Blot to detect autophagy level.
Determining the effect of the FBXW8-PPT1 axis on HCC-associated oncoproteins; interference or overexpression of AEG-1 it was verified whether the effect of the FBXW8-PPT1 axis on HCC malignancy phenotype was dependent on AEG-1, CCK8 and EdU experiments to detect proliferation, cell scratch and transwell experiments to detect migration, matrigel matrix gel model to detect invasion, CCK8 experiments to detect HCC cell line resistance to sorafenib, western blot to detect AEG-1 protein in FBXW8/PPT1 overexpression and knockdown HCC cell lines, RT-PCR to detect downstream genes PTEN, CDKN1A (p 21), SPRY2, TGFBR2 expression.
Step six: the molecular mechanism of the FBXW8-PPT1 axis in the occurrence and development of animal-level liver cancer is verified. An in situ liver planting model; nude mice were divided into 6 groups of 5 nude mice each, huh7-FBXW8 KO, huh7-PPT1 KO, huh7-Flag-FBXW8, huh7-Flag-PPT1 mut, 2X 106 cells were suspended in 40. Mu.L of a serum-free DMEM/Matrigel (1:1 volume) mixture, each nude mouse was inoculated in situ under anesthesia to the left lobe by a microinjector through a lateral incision in the upper abdomen, after about 4 weeks, the mice were sacrificed, tumors were dissected subcutaneously and weighed, the tumors were aliquoted into two parts, one part was stored in liquid nitrogen for western blot and RT-PCR, the other part was immersed in paraformaldehyde for immunohistochemical analysis, and the level of autophagy-related marker expression, and AEG-1 and its downstream related gene expression changes were detected.
Tail vein injection lung transfer model; nude mice were divided into 6 groups of 5 nude mice each, huh7-FBXW8 KO, huh7-PPT1 KO, huh7-Flag-FBXW8, huh7-Flag-PPT1 mut, 3X 106 cells were injected into the lateral tail vein of nude mice, after 9 weeks, mice were sacrificed, tumors and individual liver and lung tissues were excised, tumors were aliquoted into two parts, one part was stored in liquid nitrogen for western blot and RT-PCR, the other part was immersed in paraformaldehyde for immunohistochemical analysis, and the level of autophagy-related marker expression, and AEG-1 and downstream-related gene expression changes were detected.
After the technical scheme is adopted, compared with the prior art, the invention has the following beneficial effects, and of course, any product for implementing the invention does not necessarily need to achieve all the following advantages at the same time:
according to the invention, a potential substrate PPT1 of the FBXW8 is discovered through screening by an immunoprecipitation-mass spectrometry technology, the FBXW8 is verified to be interacted with the PPT1 and degraded, the half life of the PPT1 is shortened, the PPT1 can promote the development of HCC, the PPT1 is a prognosis marker and a treatment target of HCC, an experimental result that the PPT1 is negatively regulated by the FBXW8 indicates that the FBXW8 can possibly inhibit the development of the liver cancer, a liver cancer tissue chip is used for verifying the expression of the FBXW8 in the liver cancer, the expression quantity of the FBXW8 in the liver cancer tissue is obviously reduced, the FBXW8 mediates the inhibition of HCC autophagy and the reduction of AEG-1 stability by promoting the degradation of the PPT1, and finally the inhibition of the development of HCC is caused.
The following describes the embodiments of the present invention in further detail with reference to the accompanying drawings.
Drawings
The drawings in the following description are only examples of embodiments from which other drawings may be derived by those skilled in the art without the exercise of inventive faculty. In the drawings:
FIG. 1 is a schematic diagram of a technical route for finding HCC therapeutic targets according to an embodiment of the present invention;
FIG. 2 is a schematic illustration of a potential substrate PPT1 according to one embodiment of the present invention;
FIG. 3 is a schematic diagram of a potential substrate for FBXW8 in accordance with an embodiment of the invention;
FIG. 4 is a schematic diagram of an embodiment of the present invention in which FBXW8 can be combined with PPT1;
FIG. 5 is a schematic diagram of PPT1 recovery according to an embodiment of the present invention;
FIG. 6 is a schematic illustration of PPT1 half-life reduction according to one embodiment of the present invention;
FIG. 7 is a schematic illustration of PPT1 half-life extension according to one embodiment of the present invention;
FIG. 8 is a graph showing the down-regulation of the FBXW8 expression level according to an embodiment of the invention.
It should be noted that these drawings and the written description are not intended to limit the scope of the inventive concept in any way, but to illustrate the inventive concept to those skilled in the art by referring to the specific embodiments.
Detailed Description
The invention will now be described in further detail with reference to the accompanying drawings.
Referring to fig. 1-8, in this embodiment, a method for discovering HCC therapeutic targets is provided, including:
step one: elucidating a molecular fine regulation mechanism of FBXW8 on AZGP1, determining the influence of FBXW8 on PPT1 ubiquitination, and searching a phosphorylation site for regulating AZGP1 ubiquitination; the 293T cells are transfected with GFP-tagged PPT1, F-box proteins with Flag tags are simultaneously co-transferred respectively, MG132 is added 6 hours before sample collection, flag antibodies are immunoprecipitated, whether PPT1 is contained in the precipitate is detected by western blot, and whether the PPT1 only interacts with FBXW8 is determined; the 293T cells are transfected with GFP-tagged PPT1, F-box proteins of Flag tags are simultaneously co-transferred respectively, the content of the PPT1 is detected by western blot, and whether the PPT1 is only degraded by FBXW8 is determined; 293T cells were co-transformed with pEGFP-PPT1, pBABE-Flag-FBXW8, HA-UB (plasmid expressing HA-tagged ubiquitin) and MG132 was added 6 hours before collection, GFP antibody immunoprecipitated, and HA antibody detected UB in the pellet to determine if FBXW8 promoted ubiquitination of PPT 1.
Step two: constructing an FBXW8/AZGP41 stable expression and knockout HCC cell line; performing a truncated body experiment (Mapping), cutting the PPT1 protein into a plurality of small fragments with different sizes, respectively co-transferring the small fragments with FBXW8 into 293t cells, performing immunoprecipitation on the FBXW8, and detecting whether different truncated bodies exist in the precipitate by using a western blot; because the PPT1 protein is smaller, serine and threonine sites in the minimum necessary structural domain are not too many, the sites can be mutated into alanine respectively, the mutants are respectively co-transformed with FBXW8, and whether interaction with the FBXW8 disappears or not is detected; and (3) co-transferring the mutant and the FBXW8 into 293t cells, detecting whether the degradation of the FBXW8 is disappeared, detecting whether the ubiquitination of the PPT1 is disappeared, constructing a glutamic acid mutant, and detecting whether the interaction of the glutamic acid mutant and the FBXW8 is enhanced and the half-life is shortened.
Step three: determining the effect of the FBXW8-PPT1 axis on HCC; six well plates were aliquoted with common HCC cell lines Bel7402, hepG2, huh7, HCCLM3 and normal hepatocytes LO2, BCA quantification after RIPA lysis, western blot detection of FBXW8, PPT1 protein expression levels, comparison of whether HCC was reduced relative to normal hepatocytes, whether FBXW8 was elevated, FBXW8 antibodies were purchased from Invitrogen, cat No. PA 5-58555, PPT1 from three hawk organisms, cat No. 67699-1-Ig; extracting total RNA of cell lines such as Bel7402, hepG2, huh7, HCCLM3, LO2 and the like, reversing, RT-PCR, taking beta-actin as an internal reference, and leading the information to be shown in table 1.
TABLE 1 RT-PCR primer information
Ordering a liver cancer tissue sample chip, HE staining, performing immunohistochemical detection on the expression difference of FBXW8 and PPT1 in cancer and paracancer tissues, analyzing the correlation of the expression level of the FBXW8 and the PPT1 with tumor TNM stage, performing semi-quantitative immunohistochemical detection on the protein level of the FBXW8 and the PPT1, evaluating the cell staining intensity and the percentage of stained cells under a low-power mirror, randomly selecting 5 high-power visual fields for observation, performing double-blind reading, and determining the final immune response score after multiplying the intensity score by the positive score of the stained cells for comprehensive analysis, wherein the liver cancer tissue sample chip is purchased from Shanghai Weiao biotechnology Co, paracancer and autologous cancer, detailed biochemical indexes, TNM stage, with recurrence data, radiotherapy and chemotherapy treatment and 5-year survival period.
Step four: determining the effect of the FBXW8-PPT1 axis on HCC malignancy phenotype; the stable expression and knocking-out cell line of FBXW8/PPT1 of HCC cells such as Bel7402, hepG2, huh7, HCCLM3 and the like is constructed, the stable transfection cell line is constructed by adopting a slow virus infection mode, pBABE-Flag-FBXW8/PPT1 and auxiliary plasmids are co-transferred into 293t cells, HCC cells are infected by virus, puromycin screening is carried out, and after the expression of FBXW8 is verified by western blot, the over-expression cell line of FBXW8/PPT1 of HCC can be obtained, wherein the degron phosphorylation site of PPT1 is an alanine mutant. The gene knockout cell line is constructed by adopting a CRISPR-cas9 technology, the HCC cells are transfected with FBXW8/PPT1PX459 plasmid, the split single clone is adopted, sequencing screening is carried out on the FBXW8/PPT1 frame shift mutation single clone, the FBXW8/PPT1 knockout cell line can be obtained after western blot verification on the FBXW8/PPT1 knockout effect, gDNA sequence design in the FBXW8/PPT1PX459 plasmid is completed in a CCtop website, and 4 gDN genes are designed each; after each HCC cell line was constructed, CCK8 and EdU experiments detected HCC cell proliferation, cell scratch and transwell experiments detected HCC cell migration, matrigel matrix gel model detected HCC cell invasion, CCK8 experiments detected HCC cell line resistance to sorafenib, and the effects of stable FBXW8/PPT1 expression and knockdown on HCC cell proliferation, migration, invasion and resistance were compared.
Step five: elucidating the molecular mechanism of the influence of the FBXW8-PPT1 axis on HCC development; determining the influence of the FBXW8-PPT1 axis on HCC autophagy and the molecular mechanism thereof; treatment of each cell line with 3-MA, an activator of autophagy, rapamycin (RAP), CCK8 and EdU experiments to detect proliferation, cell scratch and transwell experiments to detect migration, matrigel matrix gel model to detect invasion, CCK8 experiments to detect resistance of HCC cell lines to sorafenib to determine whether the effect of the FBXW8-PPT1 axis on the malignant phenotype of FBXW8-PPT1 axis on HCC was dependent on autophagy, to determine the effect of the FBXW8-PPT1 axis on HCC autophagy, changes in autophagy levels in FBXW8/PPT1 over-expression and knocked out HCC cell lines were observed, single-pellet (sulfoglutamine) staining to detect the number of autophagies, LC3 protein Blot to detect initiation of autophagy, and p62 protein Western Blot to detect autophagy level.
Determining the effect of the FBXW8-PPT1 axis on HCC-associated oncoproteins; interference or overexpression of AEG-1 it was verified whether the effect of the FBXW8-PPT1 axis on HCC malignancy was dependent on AEG-1, CCK8 and EdU experiments to detect proliferation, cell scratch and transwell experiments to detect migration, matrigel matrix gel model to detect invasion, CCK8 experiments to detect HCC cell line resistance to sorafenib, western blot to detect AEG-1 protein in FBXW8/PPT1 overexpression and knockdown HCC cell lines, RT-PCR to detect downstream gene PTEN, CDKN1A (p 21), SPRY2, TGFBR2 expression, AEG-1 antibodies were purchased from abcam Corp., cat No. (ab 205646), each downstream gene PCR primer is shown in Table 2.
TABLE 2 RT-PCR primer information
Step six: the molecular mechanism of the FBXW8-PPT1 axis in the occurrence and development of animal-level liver cancer is verified. An in situ liver planting model; nude mice were divided into 6 groups of 5 nude mice each, huh7-FBXW8 KO, huh7-PPT1 KO, huh7-Flag-FBXW8, huh7-Flag-PPT1 mut, 2X 106 cells were suspended in 40. Mu.L of a serum-free DMEM/Matrigel (1:1 volume) mixture, each nude mouse was inoculated in situ under anesthesia to the left lobe by a microinjector through a lateral incision in the upper abdomen, after about 4 weeks, the mice were sacrificed, tumors were dissected subcutaneously and weighed, the tumors were aliquoted into two parts, one part was stored in liquid nitrogen for western blot and RT-PCR, the other part was immersed in paraformaldehyde for immunohistochemical analysis, and the level of autophagy-related marker expression, and AEG-1 and its downstream related gene expression changes were detected.
Tail vein injection lung transfer model; nude mice were divided into 6 groups of 5 nude mice each, huh7-FBXW8 KO, huh7-PPT1 KO, huh7-Flag-FBXW8, huh7-Flag-PPT1 mut, 3X 106 cells were injected into the lateral tail vein of nude mice, after 9 weeks, mice were sacrificed, tumors and individual liver and lung tissues were excised, tumors were aliquoted into two parts, one part was stored in liquid nitrogen for western blot and RT-PCR, the other part was immersed in paraformaldehyde for immunohistochemical analysis, and the level of autophagy-related marker expression, and AEG-1 and downstream-related gene expression changes were detected.
The potential substrate PPT1 of the FBXW8 is discovered through screening by an immune coprecipitation-mass spectrometry technology, the FBXW8 is verified to interact with the PPT1 and degrade the PPT1, the half life of the PPT1 is shortened, the PPT1 can promote the development of HCC, the PPT1 is a prognosis marker and a treatment target of HCC, the experimental result that the PPT1 is negatively regulated by the FBXW8 indicates that the FBXW8 can inhibit the development of liver cancer, a liver cancer tissue chip is used for verifying the expression of the FBXW8 in the liver cancer, the expression level of the FBXW8 in the liver cancer tissue is obviously reduced, the FBXW8 mediates the inhibition of HCC autophagy and the reduction of AEG-1 stability by promoting the degradation of the PPT1, and finally the inhibition of the development of HCC is caused.
In one aspect of this example, as shown in fig. 1, 293T cell lines stably expressing 3 x Flag-FBXW8 and 3 x Flag empty were constructed, FBXW8 was enriched by Flag immunoprecipitation experiments, and it was observed that FBXW8 was specifically enriched into Flag-FBXW8 cells, and that the cells samples were acetone precipitated and then sent to large genes for mass spectrometry; after 6 hours of MG132 (10 μm) treatment, lysates from 293T cells stably expressing either FLAG or FLAG-FBXW8 were immunoprecipitated with anti-FLAG M2 agarose beads, bound proteins eluted with FLAG peptide, all separated cell lysate fractions were separated on 10% sds-PAGE gel, stained with coomassie brilliant blue, arrow indicated FBXW8, and it was seen that most exogenously expressed FBXW8 accumulated in the elution group.
In one aspect of this example, as shown in FIG. 2, mass spectrometry detection in FBXW 8-enriched pellet found large amounts of potential substrates for FBXW8, plasmid construction of cDNA from these substrates, cotransformation of 293t cells with FBXW8, and detection found that FBXW8 could down-regulate PPT1; GFP-labeled substrate plasmid was co-transfected with Flag-FBXW8 or control into 293T cells, and after 36 hours, whole cell lysates were Western blotted.
In one aspect of this example, as shown in FIG. 3, mass spectrometry detection in FBXW 8-enriched pellet found large amounts of FBXW8 potential substrates, cDNA construction of these substrates plasmid, co-transfer of 293t cells with FBXW8, flag immunoprecipitation detection found that FBXW8 can bind specifically to PPT1; GFP-labeled vector was co-transfected with Flag-FBXW8 into 293T cells, and after 36 hours, whole cell lysates were immunoprecipitated with Flag antibody and Western blot to detect interactions between FBXW8 and indicated proteins.
In one aspect of this example, as shown in fig. 4, FBXW8 and PPT1 are transfected in 293T cells, PPT1 decreasing with increasing FBXW8 dose. PPT1 resumes expression after addition of the proteasome inhibitor; 293T cells were transfected with GFP-PPT1 and Flag-FBXW8, the plasmid dose was increased with Flag-FBXW8, 10. Mu.M MG132 was added 36 hours later, and after 6 hours, the whole cell lysates were subjected to Western blotting.
In one aspect of this example, as shown in fig. 5, PPT1 was transfected in 293T cells while co-transferring FBXW8, protein synthesis inhibitor was added, and PPT1 half-life was detected, it was seen that FBXW8 could accelerate PPT1 disappearance, shortening PPT1 half-life; PPT1 was transfected in 293T cells while co-transferring FBXW8 or control plasmid, after 36 hours, 10 μm CHX was added and incubated with cells for the indicated time, and then western blotting was performed on the whole cell lysates.
In one aspect of this example, as shown in fig. 6, PPT1 was transfected in 293T and 293T FBXW8 KO cells, protein synthesis inhibitor was added, and PPT1 half-life was detected, it was seen that knocking out FBXW8 delayed PPT1 from disappearing, prolonging PPT1 half-life; 293T and 293T FBXW8-/-cells were transfected with GFP-PPT 1. After 36 hours 10 μm CHX was added and incubated with cells to the indicated time and the whole cell lysates were western blotted.
In one aspect of this example, as shown in FIG. 7, the cancer and paracancestral tissue chip FBXW8 of a liver cancer patient is subjected to immunohistochemical staining, IHC scoring is performed, and the difference of the expression level of FBXW8 in the cancer tissue and paracancestral tissue of the patient is compared, so that the expression level of FBXW8 in the cancer tissue of the patient is found to be significantly reduced; 81 liver cancer patients were stained for cancer and paracancerous tissue by immunohistochemical staining, FBXW8, IHC-SCORE = Σ (pi×i), where pi represents positive signal pixel area/cell number ratio, i represents staining intensity, and IHC-SCORE is data between 0 and 300, the larger the data indicates stronger overall positive intensity.
The present invention is not limited to the above embodiments, and any person who can learn the structural changes made under the teaching of the present invention can fall within the scope of the present invention if the present invention has the same or similar technical solutions. The technology, shape, and construction parts of the present invention, which are not described in detail, are known in the art.
Claims (10)
1. A method of discovering a target for HCC treatment, comprising:
step one: elucidating a molecular fine regulation mechanism of the FBXW8 on the AZGP1, determining the influence of the FBXW8 on the ubiquitination of the PPT1, searching a phosphorylation site for regulating the ubiquitination of the AZGP1, and entering a step two;
step two: constructing an FBXW8/AZGP41 stable expression and knockout HCC cell line, and entering a step three;
step three: determining the influence of the FBXW8-PPT1 axis and the HCC, and entering a step four;
step four: determining the effect of the FBXW8-PPT1 axis on HCC malignancy phenotype and proceeding to step five;
step five: elucidating the molecular mechanism of the influence of the FBXW8-PPT1 axis on HCC development, and proceeding to the step six;
step six: the molecular mechanism of the FBXW8-PPT1 axis in the occurrence and development of animal-level liver cancer is verified.
2. The method according to claim 1, wherein in step one, 293T cells are transfected with GFP-tagged PPT1 and simultaneously co-transduce Flag-tagged F-box proteins, MG132 is added 6 hours before sample collection, flag antibodies immunoprecipitate, western blot detects whether the precipitate contains PPT1, and determines whether PPT1 only interacts with FBXW 8; the 293T cells are transfected with GFP-tagged PPT1, F-box proteins of Flag tags are simultaneously co-transferred respectively, the content of the PPT1 is detected by western blot, and whether the PPT1 is only degraded by FBXW8 is determined; 293T cells were co-transformed with pEGFP-PPT1, pBABE-Flag-FBXW8, HA-UB, MG132 was added 6 hours before sampling, GFP antibody was immunoprecipitated, and the HA antibody detected UB in the pellet to determine if FBXW8 promoted ubiquitination of PPT 1.
3. The method according to claim 2, wherein in the second step, a truncated experiment is performed, PPT1 protein is truncated into several small fragments of different sizes, and 293t cells are co-transformed with FBXW8, and after FBXW8 is immunoprecipitated, western blot is used to detect the presence of different truncations in the precipitate; because the PPT1 protein is smaller, serine and threonine sites in the minimum necessary structural domain are not too many, the sites can be mutated into alanine respectively, the mutants are respectively co-transformed with FBXW8, and whether interaction with the FBXW8 disappears or not is detected; and (3) co-transferring the mutant and the FBXW8 into 293t cells, detecting whether the degradation of the FBXW8 is disappeared, detecting whether the ubiquitination of the PPT1 is disappeared, constructing a glutamic acid mutant, and detecting whether the interaction of the glutamic acid mutant and the FBXW8 is enhanced and the half-life is shortened.
4. The method of claim 1, wherein in step three, the common HCC cell lines Bel7402, hepG2, huh7, HCCLM3 and normal hepatocytes LO2 are aliquoted into six well plates, BCA is quantified after RIPA lysis, western blot detects FBXW8, PPT1 protein expression levels, compares HCC relative to normal hepatocytes, PPT1 is decreased, FBXW8 is increased, FBXW8 antibodies are purchased from Invitrogen, product No. PA 5-58555, PPT1 is purchased from three hawk organisms, product No. 67699-1-Ig; extracting total RNA of cell lines such as Bel7402, hepG2, huh7, HCCLM3, LO2 and the like, reversing and performing RT-PCR, wherein beta-actin is used as an internal reference.
5. The method according to claim 4, wherein in step three, a liver cancer tissue sample chip is ordered, HE staining is performed, the expression difference of FBXW8 and PPT1 in cancer and paracancerous tissues is detected by immunohistochemical analysis, correlation between the two expression levels and tumor TNM stage is analyzed, semi-quantitative immunohistochemical detection is used for determining the protein level of FBXW8 and PPT1, the cell staining intensity and the percentage of stained cells are evaluated under a low-power microscope, 5 high-power fields are randomly selected for observation, a double-blind method is adopted for reading, the final immune response score is determined after comprehensive analysis by multiplying the intensity score and the degree of the positive score of the stained cells, and the liver cancer tissue sample chip is purchased from shanghai wei austocha, liver cancer and paraautologous cancers, detailed biochemical indexes, TNM stage, with recurrent data, radiotherapy and chemotherapy treatment, and 5-year survival.
6. The method for finding HCC therapeutic targets according to claim 1, wherein in step four, a stable FBXW8/PPT1 expression and knockout cell line of Bel7402, hepG2, huh7 and HCCLM3d HCC cells is constructed, the stably transfected cell line is constructed by adopting a slow virus infection mode, pBABE-Flag-FBXW8/PPT1 and helper plasmids are co-transferred to 293t cells, HCC cells are infected by viruses, puromycin screening is carried out, and a FBXW8/PPT1 over-expression cell line of HCC can be obtained after verification of FBXW8 expression by western blot, wherein the degron phosphorylation site of PPT1 is an alanine mutant; the gene knockout cell line is constructed by adopting a CRISPR-cas9 technology, the HCC cells are transfected with FBXW8/PPT1PX459 plasmid, the split single clone is adopted, sequencing screening is carried out on the FBXW8/PPT1 frame shift mutation single clone, the FBXW8/PPT1 knockout cell line can be obtained after western blot verification on the FBXW8/PPT1 knockout effect, gDNA sequence design in the FBXW8/PPT1PX459 plasmid is completed in a CCtop website, and 4 gDN genes are designed each; after each HCC cell line was constructed, CCK8 and EdU experiments detected HCC cell proliferation, cell scratch and transwell experiments detected HCC cell migration, matrigel matrix gel model detected HCC cell invasion, CCK8 experiments detected HCC cell line resistance to sorafenib, and the effects of stable FBXW8/PPT1 expression and knockdown on HCC cell proliferation, migration, invasion and resistance were compared.
7. The method of claim 1, wherein in step five, the effect of the FBXW8-PPT1 axis on HCC autophagy and its molecular mechanism are determined; treatment of each cell line with 3-MA, an activator of autophagy, rapamycin, CCK8 and EdU experiments to detect proliferation, cell scratch and transwell experiments to detect migration, matrigel matrix gel model assay invasion, CCK8 experiments to detect resistance of HCC cell lines to sorafenib to determine whether the effect of the FBXW8-PPT1 axis on the malignant phenotype of HCC was dependent on autophagy, to determine the effect of the FBXW8-PPT1 axis on HCC autophagy, changes in autophagy levels in FBXW8/PPT1 overexpression and knockdown HCC cell lines were observed, single dansyl pentanediamine staining to detect autophagosome numbers, LC3 protein Western Blot to detect initiation of autophagy, and p62 protein Blot to detect autophagosome clearance levels.
8. The method of claim 7, wherein in step five, the effect of the FBXW8-PPT1 axis on HCC-associated oncoproteins is determined; interference or overexpression of AEG-1 it was verified whether the effect of the FBXW8-PPT1 axis on HCC malignant phenotype was dependent on AEG-1, CCK8 and EdU experiments to detect proliferation, cell scratch and transwell experiments to detect migration, matrigel matrix gel model to detect invasion, CCK8 experiments to detect HCC cell line resistance to sorafenib, western blot to detect AEG-1 protein in FBXW8/PPT1 overexpression and knockdown HCC cell lines, RT-PCR to detect downstream genes PTEN, CDKN1A, SPRY, TGFBR2 expression.
9. The method of claim 1, wherein in step six, the model is used for in situ liver implantation; nude mice were divided into 6 groups of 5 nude mice each, each group being Huh7, huh7-FBXW8 KO, huh7-PPT1 KO, huh7-Flag-FBXW8, huh7-Flag-PPT1 mut, and 2X 10 were mixed 6 Individual cells were suspended in 40 μl freeIn serum DMEM/Matrigel mixtures, each nude mouse was inoculated under anesthesia to the left lobe of the liver by a micro-injector through a lateral incision in the upper abdomen, after about 4 weeks, the mice were sacrificed, tumors were dissected subcutaneously and weighed, the tumors were aliquoted into two parts, one part was stored in liquid nitrogen for western blot and RT-PCR, the other part was immersed in paraformaldehyde for immunohistochemical analysis, and the autophagy-related marker expression levels, and AEG-1 and its downstream related gene expression changes were detected.
10. The method of claim 9, wherein in step six, the pulmonary metastasis model is injected tail vein; nude mice were divided into 6 groups of 5 nude mice each, respectively Huh7, huh7-FBXW8 KO, huh7-PPT1 KO, huh7-Flag-FBXW8, huh7-Flag-PPT1 mut, 3X 106 cells were injected into the lateral tail vein of nude mice, after 9 weeks, the mice were sacrificed, tumors and individual liver and lung tissues were excised, tumors were aliquoted into two parts, one part was stored in liquid nitrogen for western blot and RT-PCR, the other part was immersed in paraformaldehyde for immunohistochemical analysis, the level of autophagy-related marker expression was detected, and
AEG-1 and related gene expression changes downstream thereof.
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| US20100130527A1 (en) * | 2008-11-18 | 2010-05-27 | Lehrer Raphael | Individualized cancer treatment |
| US20200010557A1 (en) * | 2017-01-17 | 2020-01-09 | The University Of Chicago | Dysfunctional antigen-specific cd8+ t cells in the tumor microenvironment |
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