CN114767863A - Application of ENPP2 gene or protein in regulation and control of colorectal cancer cells - Google Patents
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Abstract
The invention provides an application of ENPP2 gene or protein in regulation and control of colorectal cancer cells, wherein the expression of the ENPP2 gene or protein promotes the proliferation and growth of the colorectal cancer cells HT 29 and HCT116 and the forming capability of cell colonies; therefore, the proliferation and growth of the colorectal cancer cells HT 29 and HCT116 and the formation capacity of cell colonies can be inhibited by inhibiting the expression of ENPP2 genes or proteins in the colorectal cancer cells HT 29 and HCT116, the purpose of inhibiting the colorectal cancer is further achieved, and the method has important significance for the research and development of small molecule targeted drugs for the colorectal cancer.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly discloses an application of ENPP2 gene or protein in colorectal cancer cell regulation.
Background
Colorectal cancer (cancer of colon and rectum) belongs to common malignant tumors in gastrointestinal tracts, has unobvious early symptoms, shows symptoms such as defecation habit change, hematochezia, diarrhea and constipation alternation, local abdominal pain and the like along with the increase of cancer, and shows general symptoms such as anemia, weight loss and the like at late stage. Its incidence and mortality is high in cancer.
Since the concept of tumor-targeted therapy comes out, the drug has become the core of precise medicine, and the development of small-molecule inhibitor drugs has become a research hotspot in clinical oncology. With the continuous development of new clinical oncology technologies, the small molecule inhibitors are more and more valued as powerful tools for tumor targeted therapy, and the development of new drugs has been shifted to the development of tumor targeted small molecule inhibitors. Small molecule targeted drugs are drugs that are chemically synthesized and target specific mutations of tumor cells, mainly focusing on protein tyrosine kinases, proteases and other species. With the rapid development of the major health industry in China and the changing demand of precise medical treatment, the small-molecule targeted drug is rapidly developed due to the expanding medical insurance coverage and the approval of more and more innovative small-molecule targeted drugs.
In China, small-molecule targeted antitumor drugs are developed in a blowout manner in recent years, but most of the research and development bases of innovative drugs come from action mechanisms and action targets adopted in European and American countries. In the development of targeted drugs, the procedures of confirmation of drug targets, manual design, clinical trials, drug screening and the like are mainly included, the targets serving as the origins of the whole technical chain are always key core technologies, however, no effective method or technology exists in the current Chinese research and development on drug targeting, and the domestic research and development of targeted drugs are still mainly me-to and me-beter. China has a large population and a high colorectal cancer prevalence rate, and is an important consumption market of small molecule targeted anticancer drugs in the world. However, at present, a targeted therapeutic drug capable of effectively targeting colorectal cancer patients is lacking in China, and the difficulty is mainly in target point confirmation and artificial design.
Disclosure of Invention
The invention aims to provide application of ENPP2 gene or protein in regulation and control of colorectal cancer cells, which can solve the technical problem that the target is not clear in the current research on colorectal cancer targeted drugs.
The invention provides an application of ENPP2 gene or protein in regulation and control of colorectal cancer cells, which is characterized in that the expression of the ENPP2 gene or protein promotes the proliferation and growth of the colorectal cancer cells and the forming capability of cell colonies.
Compared with the prior art, the invention at least has the following advantages and positive effects:
the invention provides an application of ENPP2 gene or protein in regulation and control of colorectal cancer cells, wherein the expression of the ENPP2 gene or protein can promote the proliferation and growth of colorectal cancer cells HT 29 and HCT116 and the formation capability of cell colonies, so that the proliferation and growth of the colorectal cancer cells HT 29 and HCT116 and the formation capability of the cell colonies can be inhibited by inhibiting the expression of the ENPP2 gene or protein in the colorectal cancer cells HT 29 and HCT116, the purpose of inhibiting colorectal cancer is further achieved, and the application has important significance in research and development of small molecule targeted drugs for colorectal cancer.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a graph of the discovery process of the colorectal cancer key metabolic protein ENPP2 provided in example 1 of the present invention, wherein A is an orthogonal partial least squares analysis (OPLS-DA) graph comparing metabolites in serum samples of colorectal cancer patients and healthy subjects; wherein B is a Venn diagram of the process of screening, comparing, identifying and confirming the different metabolites in the serum samples of the colorectal cancer patients and healthy subjects; wherein C is a heatmap of 61 differential metabolites in serum samples from colorectal cancer patients versus healthy subjects; wherein D is a related analysis chart of downstream related proteins of the related pathway of the differential metabolite and an ENPP2 screening process;
FIG. 2 is a graph showing the results of the effect of ENPP2 on colorectal cancer cell proliferation provided in example 2 of the present invention, wherein A is the expression level of ENPP2 detected in different colorectal cancer cell lines by qRT PCR; b is the mRNA protein expression level of ENPP2 in HCT116 cells and HT 29 cells after transfection by ENPP2 shRNA vector; c is the proliferation condition of HCT116 cells after the ENPP2 is knocked down; d is the proliferation condition of HT 29 cells after the ENPP2 is knocked down; e is the effect of ENPP2 knockdown on colony forming ability of HCT116 cells; f is the effect of ENPP2 knockdown on colony forming ability of HT 29 cells; g is the change in proliferation of HCT116 cells and HCT 29 cells following the use of ENPP2 small molecule inhibitor Ziritaxestat (GLPG 1690);
FIG. 3 is a graph showing the results of the effect of ENPP2 on colorectal cancer cell apoptosis provided in example 3 of the present invention, wherein A is HCT116 cell apoptosis after different concentrations of ENPP2 inhibitor Ziritaxestat (GLPG1690) were added; b is HT 29 apoptosis after adding different concentrations of ENPP2 inhibitor Ziritaxestat (GLPG 1690); c is the expression of apoptosis-related proteins Bax, Bcl2, Caspase-3 and cleared Caspase 3 after adding different concentrations of ENPP2 inhibitor Ziritaxestat (GLPG 1690);
FIG. 4 is a graph showing the results of knocking down ENPP2 to reduce the tumor burden in a xenograft model in vivo, wherein A is the expression level of ENPP2 mRNA protein in HCT116 cells after transfection with ENPP2 shRNA vector, according to example 4 of the present invention; b is the growth condition of the colorectal cancer cell subcutaneous xenograft tumor which is inhibited in vivo after the ENPP2 is knocked down; c is the growth condition of the tumor in the xenograft model after the knockdown of ENPP 2; tumor burden in xenograft models following knockdown of ENPP 2;
FIG. 5 is a graph showing the results of the ENPP2 inhibitor of the present invention inhibiting tumor growth in AOM/DSS (inflammatory-cancerous) model, wherein A is a flowchart of the experiment for evaluating the efficacy of ENPP2 inhibitor Ziritaxestat (GLPG1690) on AOM/DSS colorectal (inflammatory-cancerous) model; b is the DAI score evaluation curve for each group after use of ENPP2 inhibitor Ziritaxestat (GLPG 1690); c is the occurrence condition that ENPP2 inhibitor Ziritaxestat (GLPG1690) inhibits the intestinal colorectal tumor of the AOM/DSS model mouse; d is the condition that ENPP2 inhibitor Ziritaxestat (GLPG1690) reduces the number of intestinal tumors of AOM/DSS mice; e is the condition that ENPP2 inhibitor Ziritaxestat (GLPG1690) reduces the intestinal tumor burden of AOM/DSS mice; f is H & E staining analysis of colon tissue in mice in the colorectal (inflammatory-cancerous) model after the use of ENPP2 inhibitor Ziritaxestat (GLPG 1690).
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available.
It should be noted that, in the present application, the embodiments and features of the embodiments may be combined with each other without conflict. The present invention will be described in detail below with reference to specific examples.
The invention provides application of ENPP2 gene or protein in regulation and control of colorectal cancer cells, which is characterized in that the expression of ENPP2 gene or protein promotes the proliferation and growth of the colorectal cancer cells and the formation capability of cell colonies. Therefore, the proliferation and growth of the colorectal cancer cells HT 29 and HCT116 and the formation capability of cell colonies can be inhibited by inhibiting the expression of ENPP2 gene or protein in the colorectal cancer cells HT 29 and HCT116, so that the purpose of inhibiting colorectal cancer is achieved, and the method has important significance for the development of small molecule targeted drugs for colorectal cancer.
The invention also provides a medicine for treating or inhibiting colorectal cancer, which can inhibit the ENPP2 gene or protein expression inhibitor. The drug inhibits the activity of ENPP2 gene or protein by taking the ENPP2 gene or protein as a target spot, thereby realizing the regulation of the proliferation and growth of colorectal cancer cells and the formation capability of cell colonies, realizing the inhibition of the growth of colorectal cancer tumors, and finally achieving the purpose of inhibiting or treating colorectal cancer.
The inhibitor comprises an ENPP2-shRNA interference vector for inhibiting the expression of ENPP2 gene or protein, and the oligo sequence of the ENPP2-shRNA interference vector is shown as SEQ ID NO.1 and SEQ ID NO. 2. HCT116 cells and HT 29 cells can be effectively transfected by the interference vector, so that the expression of ENPP2 protein in the cells is inhibited, and the purpose of inhibiting the growth of colorectal cancer tumor is achieved.
The method for inhibiting the ENPP2 gene or protein comprises the following steps: ENPP2-shRNA interference vectors were transfected into colorectal cancer cells HT 29 or HCT 116. The transfection time is preferably about 48h, so as to ensure higher transfection efficiency.
Such inhibitors include Ziritaxestat. The CAS number of Ziritaxestat is 1628260-79-6, Ziritaxestat (GLPG1690) is an innovative Autotaxin (ATX) inhibitor, the IC50 and the Ki value are 131nM and 15nM respectively, and experimental research shows that the Ziritaxestat can effectively inhibit the activity of ENPP2 protein, and the main principle of the Ziritaxestat is that the expression of bcl2 protein in HCT116 and HT 29 colorectal cancer cells can be remarkably reduced, the expression of caspase 3 is not remarkably changed or slightly reduced, and the expression of bax and cleaved caspase 3 protein is remarkably increased, so that the apoptosis of the HCT116 and HT 29 colorectal cancer cells is promoted.
The concentration of the above Ziritaxestat is 5-40. mu.M. The concentration interval has the best effect of reducing the activity of HCT116 and HT 29 colorectal cancer cells, when the concentration is lower than the interval, the activity reducing effect is not obvious, and when the concentration is higher than the interval, the activity reducing effect is not obviously improved, and the use cost is increased and is not paid.
The method for inhibiting the ENPP2 gene or protein comprises the following steps: intravenous or intraperitoneal injection of Ziritaxestat. By intraperitoneal or intravenous injection to enable fast onset of the Ziritaxestat.
Example 1
Colorectal cancer key metabolic protein ENPP2 screening and finding process:
collecting samples: a total of 3482 serum samples (containing 1204 healthy subjects, 1183 patients with metabolic syndrome and 1095 patients with colorectal cancer) were collected from natural population cohort studies in the xiaoshan region, the taizhou hospital and the zhejiang tumor hospital. Each serum sample was collected and immediately dispensed, and then stored in a-80 ℃ freezer.
The inclusion criteria were selected as follows:
healthy subjects: the body weight index is 19-24 (body weight index: weight (kg)/height)2(m2) Without history of heart, liver, kidney, digestive tract, nervous system, metabolic abnormality and the like, general examination and laboratory examination are normal.
Colorectal cancer: the patient was judged to be colorectal cancer by colonoscopy and after finding the tumor, a portion of the sample was taken at the colonoscope for pathological biopsy and confirmed to be colorectal cancer by the pathologist.
Metabonomics detection methods: a detection instrument: agilent 1290 ultra high performance liquid chromatography-6545 type quadrupole-time of flight mass spectrometer.
Sample pretreatment: a50-microliter serum sample is taken, 3 times of methanol 150 microliter is added into a centrifuge tube of 1.5mL, the mixture is swirled for 30s and mixed evenly, and then the mixture is placed into a high-speed centrifuge to be centrifuged for 10min (4 ℃) at 13000 rpm. And sucking 75 mu L of centrifuged supernatant, respectively placing the supernatant into 2 centrifuge tubes with the volume of 1.5mL, and drying the supernatant by using a nitrogen blowing instrument. After drying, respectively adding 100 mu L of methanol complex solution containing internal standard solution (100 ng/mL of L-2-chlorophenylalanine under the condition of positive ion mode and 1 mu g/mL of ketoprofen under the condition of negative ion mode) for redissolution, then swirling for 30s for uniform mixing, and after uniform mixing, centrifuging for 10min (4 ℃) in a high-speed centrifuge at 13000 rpm. 80. mu.L of each centrifuged supernatant was aspirated and placed in a liquid vial for detection of positive and negative ions. And calculating the ratio of the peak area of each target metabolite beta-hydroxybutyric acid or alpha-hydroxybutyric acid to the peak area of the internal standard to serve as the relative content of the metabolite.
Chromatographic conditions are as follows: and (3) chromatographic column: waters BEH C8(100mm × 2.1mm I.D., 1.7 μm); acetonitrile (B) -water (a) solutions of the positive ion mobile phase (both containing 0.1% formic acid); negative ion mobile phase methanol (B) -water (A) solutions (both containing 10mmol/L ammonium acetate); flow rate: 0.4 mL/min; column temperature: 50 ℃; the temperature of the sample introduction chamber is 4 ℃, and the sample introduction amount is 1 mu L. Flow phase ratio: 0-1min, 5% B; 1-4min, 5% -30% of B; 4-9min, 30% -90% B; 9-10min, 90% -100% B; 10-12min, 100% B. The running time is 12 minutes, the later running time is 3 minutes, and the effluent after the column is directly introduced into a mass spectrometry system without being split for detection.
Mass spectrum conditions: the collision voltage is 100V, the atomizer pressure is 35psig, and the capillary voltage is 3500V; the temperature of the dryer is 300 ℃, the flow rate of the dryer is 10L/min, the drying gas and the taper hole gas are both high-purity nitrogen, and the temperature of the ion source is 100 ℃. Data acquisition is carried out for three times per second in a full scanning mode, and the scanning quality range is as follows: m/z is 100 to 1000. M/z 121.0508 and 922.0098 for the reference ions in positive ion mode, and m/z 112.9855 and 980.0163 for the reference ions in negative ion mode.
The data processing method comprises the following steps: performing multidimensional data analysis on the obtained spectrogram information by using SIMCA-P software (Version 14.1, Sweden), comparing the two groups by adopting PCA, simultaneously meeting VIP >1 and the characteristic peak of which P is less than 0.05 after correction, using a Human Metabolome Database and a METLIN Database as a differential metabolite to identify and compare primary and secondary spectrums of the differential metabolic peak, returning to the most original spectrogram after identification, comparing the peak-out time, and finally confirming by using a standard substance. Differential metabolite metabolic disorder pathways were analyzed by KEGG database.
In OPLS-DA of metabolites in serum samples of colorectal cancer patients and healthy subjects, R2X and R2Y is 0.324 and 0.953, Q2At 0.945, a significant difference in metabolites was seen between the two groups of serum samples (FIG. 1A). A total of 1793 signal peak ions were identified after 50% missing peak removal and peak alignment procedures for colorectal cancer and serum samples from healthy subjects. Colorectal cancer has 345 differential ions in total with serum samples from healthy subjects, with 305 differential ions being relatively highly expressed in healthy subjects and 45 differential ions being relatively highly expressed in colorectal cancer patients. In negative ion mode, there are 289 difference ions in the serum samples of colorectal cancer and healthy subjects, of which 249 difference ions are relatively highly expressed in healthy subjects and 40 differencesThe relative expression of the xeno ions is higher in colorectal cancer patients. And further comparing 639 different ion peaks with a Human Metabolome Database and a METLIN Database, finding 208 different ion peaks, comparing a secondary mass spectrogram with the Database, and finally comparing and identifying the secondary mass spectrogram with a standard library existing in a laboratory. Finally, a total of 61 differential metabolites were identified in serum samples from colorectal cancer patients and healthy subjects, and the differential metabolite screening procedure is shown in FIG. 1B. By performing a global analysis of 61 differential metabolites by heatmap we can see that the relative content of differential metabolites in serum samples of colorectal cancer patients is significantly lower than in healthy subjects, see fig. 1C. Further, by analyzing metabolic disorder pathway and related protein of the metabolites, the results show that through bioinformatics analysis of the same increased and decreased metabolites in the process from healthy subjects → metabolic syndrome → colorectal cancer, human ectonucleotide pyrophosphatase/phosphodiesterase family member 2 (ENPP 2) is found to be an independent and relatively important protein in poor metabolism foreign matter (see fig. 1D), indicating that the protein may play an important role in the occurrence and development of colorectal cancer.
Example 2
Effect of ENPP2 on colorectal cancer cell proliferation:
(1) construction and identification of ENPP2-shRNA interference vector
First, the expression level of ENPP2 in different colorectal cancer cell lines is detected by qRT PCR, and ENPP2 is found to be significantly highly expressed in colorectal cancer cell lines HT 29 and HCT116 (FIG. 2A), so that two colorectal cancer cell lines, namely HCT116 and HT 29, are selected as cell lines for subsequent proliferation research.
The ENPP2-shRNA interference vector adopted by the invention is respectively constructed by Nanjing Keruis biotechnology and Gilman biotechnology (Shanghai) and the designed oligo sequence is shown in Table 1:
TABLE 1 sequence Listing of oligos
The two designed ENPP2-shRNA interference vectors are verified by alignment, and the results show that the sequence of the inserted fragment in the recombinant clone is completely consistent with the designed oligo sequence, so that the vector construction is successful.
Then, HCT116 cells and HT 29 cells were transfected by two ENPP2 shRNA vectors and a control vector for 48h, and the results of detecting the mRNA protein expression level of ENPP2 in the HCT116 cells and the HT 29 cells by qRT PCR are shown in FIG. 2B: the transfection efficiencies of ENPP2 shRNA and a control vector are both higher, which shows that HCT116 ENPP2 knockdown cell line and HT 29-ENPP2 cell line are successfully constructed.
(2) Knocking down influence of ENPP2 on proliferation of colorectal cancer cells
The ENPP2 expression in HCT116 and HT 29 cells is knocked down by ENPP2-shRNA respectively, the CCK8 method is strictly according to the operation instruction of a CCK-8 kit, the time of adding CCK8 at 0h is taken as the standard, 10 mu l of CCK8 is added after 24h is separated, the machine is operated to detect for 3h till 96h, and the influence of the knocking-down ENPP2 on the proliferation of the colorectal cancer cells is examined. The results show that HCT116 and HT 29 cells significantly inhibited the proliferation of colorectal cancer cell lines at 72h and 96h after ENPP2 knockdown, as compared to the control group, see fig. 2C and 2D.
(3) Effect of knocking down ENPP2 on colony forming capability of colorectal cancer cells
The expression of HCT116 and ENPP2 in HT 29 cells is knocked down by an ENPP2-shRNA interference vector, after the culture is finished, the supernatant is discarded, and after cells are washed by PBS (phosphate buffer solution), 1mL of 95% ethanol is added into each hole for fixation for 15 min. After fixation, 1mL of 0.5% crystal violet dye solution was added to each well for 15 min. After the staining is finished, rinsing is carried out by PBS, image shooting is carried out after the rinsing is repeated three times to remove the redundant crystal violet, and the influence of the knock-down ENPP2 on the colorectal cancer cell colony forming capability is observed by using a plate clone test. The results show that ENPP2 can significantly inhibit colony formation ability of colorectal cancer cell lines after knockdown compared to the control group, see fig. 2E and 2F.
(4) Effect of ENPP2 inhibitors on proliferation of colorectal cancer cell sets
Further examining the effect of ENPP2 on proliferation of colorectal cancer cells, ENPP2 small molecule inhibitor Ziritaxostat (GLPG1690) was selected to treat HCT116 and HT 29 cells for 24h with concentrations of 2.5. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, and 40. mu.M, respectively, and the results showed that 5. mu.M, 10. mu.M, 20. mu.M, and 40. mu.M Ziritaxostat (GLPG1690) significantly inhibited proliferation of HCT116 and HT 29 cells compared to DMSO, and the results are shown in FIG. 2G.
Example 3
Effect of ENPP2 inhibitors on colorectal cancer cell apoptosis:
HT 29 or HCT116 cells were seeded in 24-well plates and treated with drug-containing media containing varying concentrations of the ENPP2 small molecule inhibitor Ziritaxestat (GLPG1690) when the cell confluence reached 70%. After 24h of administration, the cells were digested with EDTA-free trypsin, and 300g of the cells were harvested and centrifuged at 4 ℃ for 5 min. Cells were washed twice with pre-chilled PBS (300g, 4 ℃ C., centrifugation 5 min). Add 100. mu.L of 1 × Binding Buffer and mix the cells into a single cell suspension by gentle blowing with a pipette. Adding 5 μ L Annexin V-FITC or 5 μ L Annexin V-APC and 5 μ L PI stabilizing Solution, mixing well by blowing, shading, and incubating at room temperature for 20 min. After the staining was completed, 400. mu.L of 1 XBinding Buffer was added and mixed gently. The effect of the ENPP2 inhibitor Ziritaxestat (GLPG1690) on colorectal cancer apoptosis was examined by flow cytometry after filtration through a 200 mesh cell screen. The results show that the addition of Ziritaxestat (GLPG1690) at concentrations of 7.5. mu.M, 10. mu.M and 20. mu.M significantly promoted apoptosis of HT 29 as well as HCT116 cells, as compared to the control group, and the results are shown in FIG. 3A. The expression of apoptosis-related proteins, namely, bax, bcl2, caspase 3 and cleared caspase 3, is further detected by western blot, and the result shows that after the ENPP2 inhibitor Ziritaxestat (GLPG1690) is used, the expression of bcl2 protein for regulating and controlling the apoptosis of HCT116 and HT 29 colorectal cancer cells shows a remarkable descending trend, the protein expression of caspase 3 shows no obvious change or slight reduction, while the expression of bax and cleared caspase 3 protein shows a remarkable ascending trend, and the result is shown in a figure 3B.
Example 4
The ENPP2 gene knockout inhibited growth of colorectal cancer cells in the HCT116 xenograft model:
(1) construction and identification of HCT116 ENPP2 knockdown cell line
The result of detecting the mRNA protein expression level of ENPP2 in HCT116 cells by qRT PCR using HCT116 cells transfected 48h with ENPP2 shRNA vector and blank vector, which were constructed by gemma biotechnology (shanghai), respectively, is shown in a in fig. 4: the transfection efficiency of the ENPP2 shRNA interference vector and the control vector is over 90%, which shows that the HCT116 ENPP2 knockdown cell line is successfully constructed, and the figure is shown in figure 4A.
(2) Inhibiting the growth of the tumor of the xenograft model after knocking down ENPP2
BALB/c Nude mice (male, 5-6 weeks old) were randomly divided into two groups (n ═ 6), HCT116 and HCT116 ENPP2 in logarithmic growth phase were subjected to knockdown cell digestion, centrifuged, and the cells were washed twice with PBS, resuspended in PBS and counted in PBS, and adjusted to 4X 10 cell density with PBS6One per mL. The nude mice were injected subcutaneously on the ventral and dorsal sides with 100. mu.L of cell suspension. Tumor size was measured with a vernier caliper every 2 days after injection and tumor length and width were recorded. Tumor volume was calculated according to the following formula: length x width2X 0.5. The animals were euthanized 22 days after feeding, tumor tissues of the mice were taken, as shown in fig. 4B, and the tumor burden and the tumor weight were measured at the same time, as shown in fig. 4C and 4D, respectively, and it was revealed from the results of fig. 4C and 4D that the reduction of ENPP2 protein inhibited the growth of tumors in the xenograft model and reduced the tumor burden.
Example 5
Inhibitors of ENPP2 inhibit tumor growth in AOM/DSS (inflammatory-cancerous) models:
healthy 6-8 week old C57BL/6 male mice were randomized into 5 groups: a normal control group; ② colorectal cancer group; low dose group of colorectal cancer + ENPP2 inhibitor; and fourthly, in the colorectal cancer + ENPP2 inhibitor high-dose group, the ENPP2 inhibitor is Ziritaxestat (GLPG 1690). Normal food intake and water adaptation were given, AOM 10mg/kg was given 1 intraperitoneal injection 1 time after one week, then 2.5% DSS drinking water was given for 7 days, and normal water was changed for 14 days, and 3 cycles were repeated. Intraperitoneal injections of various concentrations of ENPP2 inhibitor were started after the end of cycle 2, with a total of 70 days for the low dose group of 30mg/kg Ziritaxostat (GLPG1690) and 60mg/kg Ziritaxostat (GLPG1690) for the high dose group, and the procedure is shown in FIG. 5A.
During the modeling process, the degree of damage to the colon was assessed daily for all mice by DAI scoring. The DAI score ranged from 0 to 4, and included weight change, degree of hematochezia, and degree of diarrhea, from the DAI assessment curve we can see that ENPP2 inhibitor Ziritaxestat (GLPG1690) significantly improved the health status of the mice, alleviating the clinical signs of colonic lesions, and the DAI results are shown in figure 5B. After 70 days of sacrifice, the colon of the mice was removed and washed in normal saline to remove residual stool, and then the colorectal status was recorded, as shown in fig. 5C. The results show that the ENPP2 inhibitor Ziritaxestat (GLPG1690) significantly inhibited the development of intestinal colorectal tumors in AOM/DSS model mice compared to the model group.
Intestinal tissue samples are taken and placed on a precooled carrying plate, the intestinal tissue samples are placed under a high-resolution stereomicroscope for photographing and observation, the splicing of full intestinal tissue micrographs is completed by adopting a 'large-field jigsaw puzzle' mode, tumor tissues are quantitatively analyzed by adopting professional graphic processing software, the number and the load of tumors are recorded, and the result shows that the number and the load of the tumors of an ENPP2 inhibitor Ziritaxestat (GLPG1690) treatment group are obviously lower than those of a model group, and the results are shown in figures 5D and 5E. Calculating tumor load, fixing a small part of colon in 4% paraformaldehyde, and storing the rest part of colon at-80 deg.C after quick freezing with liquid nitrogen. And (3) dehydrating the fixed colon tissues and other steps to prepare HE pathological sections, observing the relevant change of the pathological morphology of the colon by the prepared pathological sections, wherein HE staining shows that the degree of tumor dysplasia of the treatment group is obviously lower than that of the model group, and the result is shown in a figure 5F.
In conclusion:
the embodiment of the invention provides application of ENPP2 gene or protein in regulation and control of colorectal cancer cells, and the expression of the ENPP2 gene or protein can promote the proliferation and growth of colorectal cancer cells HT 29 and HCT116 and the formation capability of cell colonies, so that the proliferation and growth of colorectal cancer cells HT 29 and HCT116 and the formation capability of cell colonies can be inhibited by inhibiting the expression of ENPP2 gene or protein in the colorectal cancer cells HT 29 and HCT116, the purpose of inhibiting colorectal cancer is achieved, and the application has important significance in research and development of small molecule targeted drugs for colorectal cancer.
The embodiments described above are some, not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of protection of the present invention.
Claims (7)
1. Use of an ENPP2 gene or protein for regulating colorectal cancer cells, wherein the expression of the ENPP2 gene or protein promotes the proliferation, growth and colony formation of colorectal cancer cells.
2. A medicament for treating or inhibiting colorectal cancer, comprising an inhibitor capable of inhibiting the expression of ENPP2 gene or protein according to claim 1.
3. The medicament for treating or inhibiting colorectal cancer according to claim 2, wherein the inhibitor comprises an ENPP2-shRNA interference vector for inhibiting the expression of the ENPP2 gene or protein, and the oligo sequence of the ENPP2-shRNA interference vector is shown as SEQ ID No.1 and SEQ ID No. 2.
4. The medicament for treating or inhibiting colorectal cancer according to claim 3, wherein the method for inhibiting the ENPP2 gene or protein comprises the steps of: transfecting the ENPP2-shRNA interference vector into the colorectal cancer cell HT 29 or HCT 116.
5. The medicament for treating or inhibiting colorectal cancer according to claim 2, wherein the inhibitor comprises Ziritaxestat.
6. The medicament for treating or inhibiting colorectal cancer according to claim 5, wherein the concentration of Ziritaxestat is 5-40 μ M.
7. The medicament for treating or inhibiting colorectal cancer according to claim 5, wherein the method for inhibiting the ENPP2 gene or protein comprises the steps of: intravenously or intraperitoneally injecting the Ziritaxestat.
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