MKRN1, SNIP1 in the preparation / as a colorectal cancer drug, reagent

Abstract

The present application relates to the field of biotechnology, in particular, MKRN1 and SNIP1 are applied in preparation of drugs or reagents for colorectal cancer. It is proved in the present application that MKRN1 can promote the proliferation, migration, invasion ability and EMT process of CRC cells, and can be applied in preparation of reagents for promoting colorectal cancer. It is also proved that reducing the content of MKRN1 can inhibit the proliferation, migration and invasion ability of CRC cells, which indicates that MKRN1 inhibiting drugs can be prepared or directly used as anti-colorectal cancer drugs. It is indicated in the present application that SNIP1 can inhibit the EMT and migration ability of CRC cells, which indicates that SNIP1 protein can be used for preparation or directly used as anti-colorectal cancer drugs. It is also proved that reducing the content of SNIP1 can promote the EMT process and migration ability of CRC cells, which indicates that SNIP1 inhibiting drugs can be used for preparation or directly used as reagents for promoting colorectal cancer.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of MKRN1 and SNIP1 in the preparation / as drugs and reagents for colorectal cancer. Background Technology

[0002] Colorectal cancer (CRC) is the third most common malignant tumor worldwide. More than one million new cases are diagnosed globally each year, with a mortality rate as high as 33% in developed countries. Furthermore, the patient population is increasingly younger. CRC metastasis is the biggest factor affecting survival. Approximately one-quarter of patients already have metastases at diagnosis, directly contributing to the overall poor prognosis and high mortality rate of CRC patients. Therefore, understanding the underlying molecular mechanisms of CRC metastasis progression and finding new treatment methods for CRC is crucial.

[0003] Currently, there are no reports, either domestically or internationally, on the role of E3 ubiquitin ligase MKRN1 and Smad nuclear interfering protein 1 (SNIP1) in the development of CRC, especially the relationship between MKRN1, SNIP1 and CRC migration and invasion. Therefore, determining the expression levels of MKRN1 and SNIP1 in CRC tissues and their specific effects on CRC, particularly the relationship and molecular mechanisms between MKRN1 and SNIP1 and CRC invasion and metastasis, as well as focusing on the effects of MKRN1 and SNIP1 on CRC cell migration and invasion, is of great practical significance for the full application of MKRN1 and SNIP1 in CRC treatment, CRC model preparation, and CRC cell culture. Summary of the Invention

[0004] To address the aforementioned technical problems in the existing technology, this invention provides the application of MKRN1 and SNIP1 in the preparation / use as drugs and reagents for colorectal cancer. Specifically, this is achieved through the following technical solutions:

[0005] Application of MKRN1 protein in the preparation / use as a colorectal cancer promoting agent. Further, the colorectal cancer promoting agent is a reagent that promotes colorectal cancer metastasis or colorectal cancer proliferation.

[0006] A reagent that promotes the metastasis of colorectal cancer, containing the MKRN1 protein.

[0007] A reagent that promotes the proliferation of colorectal cancer, containing the MKRN1 protein.

[0008] The use of MKRN1 inhibitors in the preparation / as anti-colorectal cancer drugs. Further, the MKRN1 inhibitor is an MKRN1 antibody or an MKRN1 inhibitor.

[0009] An anti-colorectal cancer drug containing MKRN1 antibody or MKRN1 inhibitor.

[0010] Applications of SNIP1 protein in the preparation / as an anti-colorectal cancer drug.

[0011] An anti-colorectal cancer drug containing the SNIP1 protein.

[0012] The application of SNIP1 inhibitors in the preparation / use as colorectal cancer promoting agents. Further, the colorectal cancer promoting agent is an agent that promotes colorectal cancer metastasis or colorectal cancer proliferation.

[0013] A reagent that promotes the metastasis of colorectal cancer contains an SNIP1 inhibitor.

[0014] An agent that promotes the proliferation of colorectal cancer contains an SNIP1 inhibitor.

[0015] Compared with the prior art, the technical effects of this invention are reflected in:

[0016] (1) This application confirms that MKRN1 is highly expressed in CRC and is positively correlated with clinical TNM stage, indicating that an increase in MKRN1 content or concentration is more likely to cause colorectal cancer. CRC cells with high levels of MKRN1 exhibit a mesenchymal phenotype and can promote the proliferation, migration, invasion and EMT process of CRC cells. It is the first time that MKRN1 can be used as a tumor-promoting factor to promote the progression of CRC, indicating that MKRN1 protein can be used in the preparation / as a colorectal cancer promoting agent, and can be further used for colorectal cancer cell expansion, preparation of CRC tumor metastasis model, screening of colorectal cancer therapeutic drugs, etc.

[0017] (2) This application demonstrates that reducing MKRN1 content can inhibit the proliferation, migration and invasion of CRC cells, indicating that MKRN1 inhibitory drugs such as MKRN1 antibodies or MKRN1 inhibitors can be prepared or used directly as anti-colorectal cancer drugs.

[0018] (2) This application demonstrates that SNIP1 can inhibit the EMT and migration ability of CRC cells, confirms that silencing SNIP1 can promote the EMT and migration ability of CRC cells, and that overexpressing SNIP1 can inhibit the EMT and migration ability of CRC cells, indicating that SNIP1 protein can be used to prepare or directly as an anti-colorectal cancer drug.

[0019] (3) This application confirms that reducing SNIP1 content can promote the EMT process and migration ability of CRC cells, indicating that SNIP1 inhibitory drugs can be used to prepare or directly as colorectal cancer promoting agents, and can be used for colorectal cancer cell amplification, preparation of CRC tumor metastasis models, screening of colorectal cancer therapeutic drugs, etc. Attached Figure Description

[0020] Figure 1This is a comparison of MKRN1 expression in normal and cancerous colorectal tissues, demonstrating high MKRN1 expression in CRC and its association with poor prognosis in CRC patients. A. shows the expression distribution of the MKRN1 gene in different tumor cell lines, displaying the MKRN1 content in different tumor cell lines and normal colorectal epithelial cells, indicating increased MKRN1 content and expression in both tumors. B. shows the expression distribution of the MKRN1 gene in tumor and normal tissues, suggesting that patients with higher MKRN1 levels have a poorer prognosis and potentially shorter survival. C. Western blotting analysis of MKRN1 protein expression levels in CRC tissues and paired adjacent normal tissues further confirms the high MKRN1 content in colorectal cancer tissues, while the content is very low in normal tissues. D. IHC staining was used to detect MKRN1 expression in tissue sections from patients with colitis and CRC at different stages, and typical images are provided (Scale bar: 100 μm; Scale bar: 50 μm). E. MKRN1 involvement in the prognosis of CRC patients was obtained from the R2 genomic analysis platform. F. Western blotting was used to detect the MKRN1 protein expression level in CRC cell lines (HT29, HCT116, HCT15, RKO) and normal human colonic fibroblasts (CCD-18Co). n=3, compared with the control group, *P<0.05, **P<0.01, ***P<0.001. Increased MKRN1 may be involved in the prognosis of CRC patients; patients with high MKRN1 levels have poor prognosis and shorter survival time. F. Western blotting was used to detect the MKRN1 protein expression level in CRC cell lines (HT29, HCT116, HCT15, RKO) and normal human colonic fibroblasts (CCD-18Co). The results also revealed that MKRN1 levels were very low in normal colorectal cells, almost undetectable, while they were very high in colorectal cancer cells. Detectable MKRN1 levels indicate that increased MKRN1 levels or concentrations may suggest cancer, or tumor recurrence after surgery or chemotherapy. Undetectable MKRN1 levels suggest a lower likelihood of cancer, or better treatment outcomes after surgery or chemotherapy, potentially increasing or prolonging patient survival. In summary, MKRN1 can serve as a cancer biomarker or an indicator for treatment or prognosis in the diagnosis, treatment, or prognosis of colorectal cancer. Regular blood tests can provide predictive value.

[0021] Figure 2This study aimed to construct stable MKRN1 knockdown CRC cells HCT116 and HT29. A. Agarose gel electrophoresis images of PCR products after sh1, sh2, and sh3 amplification; B. Agarose gel electrophoresis images of sh1, sh2, sh3, and the vector pTripz after double enzyme digestion; D. Agarose gel electrophoresis images of colony PCR amplification after transformation; E. Sequencing results of positive clones; F. Fluorescent expression (Scale bar: 100 μm) in HEK-293T cells after lentiviral packaging with sh1, sh2, and sh3 for 72 h; G. Fluorescent expression (Scale bar: 100 μm) in HCT116 cells after infection with concentrated viral solutions of sh1, sh2, and sh3 for 48 h; MKRN1 transfection rate in HCT116 and HT29 cell lines was detected by HI-WB. n=3, compared with the control group, *P<0.05.

[0022] Figure 3 MKRN1 promotes the proliferation of CRC cells. A. Western blot analysis (WB) was used to detect the transfection rate of MKRN1 in the HCT15 cell line; BC. CCK-8 assay was used to determine CRC cell viability; and DE. Colony formation assay was used to detect CRC cell proliferation. n=3. Compared with the control group, *P<0.05; **P<0.01; ***P<0.001.

[0023] Figure 4 MKRN1 induces EMT in CRC cells. The assays included: AB. Scratch assay to detect the migration ability of CRC cells with different MKRN1 expression levels (Scale bar: 100 μm); CD. Transwell assay to detect the migration and invasion abilities of MKRN1 knockdown and overexpression cells (Scale bar: 50 μm); EF. Microscopic observation of cell morphology in HCT116 (Control, sh1-MKRN1) and HCT15 (Vector, OE-MKRN1) cell lines (Scale bar: 100 μm); GH. Expression of EMT-related markers after MKRN1 knockdown and overexpression. n=3. Compared with the control group, *P<0.05; **P<0.01; ***P<0.001.

[0024] Figure 5The interaction is between MKRN1 and SNIP1. Specifically: A. Predicting proteins interacting with MKRN1 using the STRING database; B. Predicting proteins interacting with MKRN1 using the IntAct database; C. The experimental process of quantitative proteomics and ubiquitination modification proteomics of MKRN1; D. Expression distribution of SNIP1 (top) and TRA2A (bottom) genes in tumor and normal tissues; E. Univariate and multivariate Cox regression analyses of p-values, hazard ratios (HR), and confidence intervals for gene expression and clinical characteristics; FG. Western blotting to detect the effect of MKRN1 expression on SNIP1 protein levels; H. Confocal microscopy to observe the localization of MKRN1 and SNIP1 (Scale bar: 20 μm); IJ. Validating the interaction between MKRN1 and SNIP1 in HCT116 and HCT15 cells using Co-IP and Western blotting (both forward and reverse); K. Validating the interaction between exogenous MKRN1 and SNIP1 in HCT15 cells using Co-IP and Western blotting. n=3, compared with the control group, ***P<0.001.

[0025] Figure 6 SNIP1 is a ubiquitination substrate of MKRN1. A. The degradation effect of MKRN1 on SNIP1. Expression vectors of Flag-MKRN1 (0, 1, 2, 4, and 6 μg) were introduced into HCT15 cells, and the expression levels of SNIP1 and MKRN1 were detected by Western blotting. B. Expression vectors of Flag-SNIP1 (1 μg) and Flag-MKRN1 (0, 1, 2, 4, and 6 μg) were introduced into HCT15 cells, and the expression levels of SNIP1 and MKRN1 were detected by Western blotting. C. MKRN1 affects the half-life of SNIP1. HCT116 cells (Control and sh1-MKRN1) and HCT15 cells (Vector and OE-MKRN1) were treated with 50 μg / mL CHX, and the results were analyzed by Western blotting. EF. RT-qPCR was used to detect the effect of MKRN1 on SNIP1 mRNA. G. MKRN1's proteasome-dependent degradation of SNIP1. In HCT15 cells overexpressing MKRN1, SNIP1 expression was enhanced after treatment with 10 μM MG132 for 6 h. MKRN1 mediates SNIP1 ubiquitination. The ubiquitination levels of SNIP1 after MKRN1 knockdown and overexpression were detected by Western blotting in HCT116 and HCT15 cells. The ubiquitination level of SNIP1 in HCT15 cells was detected by Western blotting after treatment with 10 μM MG132 for 6 h. n=3, compared with the control group, ***P<0.001.

[0026] Figure 7The E3 ligase-deficient mutant MKRN1 cannot mediate SNIP1 ubiquitination. A. Schematic diagram of the MKRN1 domain. B. Construction of the MKRN1 E3 ligase mutant H307E. C. Co-IP and Western blotting to verify the interaction between MKRN1(H307E) and SNIP1. D. Western blotting to detect the effect of the MKRN1(H307E) mutant on SNIP1 degradation. E. Expression vectors of MKRN1(H307E) (0, 1, 2, 4, and 6 μg) were introduced into HCT15 cells, and Western blotting was used to detect the expression levels of SNIP1 and MKRN1. F. Effect of MKRN1(H307E) on the half-life of SNIP1. HCT15 cells (Vector and MKRN1(H307E)) were treated with 50 μg / mL CHX, and Western blotting was performed. G. In HCT15 cells (Vector, OE-MKRN1, MKRN1(H307E)), the ubiquitination level of SNIP1 was detected by Western blotting after treatment with 10 μM MG132 for 6 h.

[0027] Figure 8 SNIP1 inhibits EMT in CRC cells. AB. Western blotting was used to detect the transfection rate of SNIP1 in HCT116 and HCT15 cells. CD. Western blotting was used to detect the expression of EMT-related markers after SNIP1 knockdown and overexpression. EF. Transwell assay was used to detect the migration ability of SNIP1 knockdown and overexpression cells (Scale bar: 50 μm). n=3, compared with the control group, **P<0.01, ***P<0.001.

[0028] Figure 9 MKRN1 promotes EMT in CRC cells by degrading SNIP1. The study included: A. Western blotting (WB) to detect the expression of EMT-related markers in HCT116 co-transfected Control, sh1-MKRN1, and sh-SNIP1. B. Western blotting to detect the expression of EMT-related markers in HCT15 co-transfected Vector, OE-MKRN1, and OE-SNIP1. C. Transwell assay to detect cell migration ability (Scale bar: 50 μm) after HCT116 co-transfected Control, sh1-MKRN1, and sh-SNIP1. D. Transwell assay to detect cell migration ability (Scale bar: 50 μm) after HCT15 co-transfected Vector, OE-MKRN1, and OE-SNIP1. n=3. Compared with the control group, *P<0.05; **P<0.01; ***P<0.001.

[0029] Figure 10SNIP1 inhibits the TGF-β pathway. The effect of SNIP1 on the TGF-β pathway was detected by AB and WB. n=3, and compared with the control group, ***P<0.001.

[0030] Figure 11 MKRN1 promotes EMT in CRC cells by activating the TGF-β signaling pathway. A. Spearman correlation analysis between MKRN1 and the TGF-β pathway. BC. Western blotting to detect the expression of TGF-β pathway-related markers after MKRN1 knockdown and overexpression. D. TGF-β1 treatment reversed the inhibition of EMT and TGF-β pathway-related markers induced by MKRN1 knockdown. E. LY2109761 treatment reversed the activation of EMT and TGF-β pathway-related markers induced by MKRN1 overexpression. F. TGF-β1 treatment reversed the decreased migration ability of HCT116 cells induced by MKRN1 knockdown (Scale bar: 50 μm). G. LY2109761 treatment reversed the increased migration ability of HCT15 cells induced by MKRN1 overexpression (Scale bar: 50 μm). n=3, compared with the control group, **P<0.01; ***P<0.001.

[0031] Figure 12 High levels of MKRN1 promote the TGF-β pathway by degrading SNIP1. The following data were presented: A. Expression levels of TGF-β pathway markers in HCT116 cells after co-transfection with Control, sh1-MKRN1, and sh-SNIP1. B. Expression levels of TGF-β pathway markers in HCT15 cells after co-transfection with Vector, OE-MKRN1, and OE-SNIP1. C. Representative images of MKRN1, SNIP1, and TGF-β1 expression in CRC cells detected by IHC (Scale bar: 100 μm; Scale bar: 50 μm). n=3. Compared with the control group, **P<0.01; ***P<0.001.

[0032] Figure 13 This diagram illustrates how MKRN1 promotes TGFβ signaling by ubiquitinizing and degrading SNIP1, thereby inducing EMT in CRC cells. Specifically, it shows a schematic diagram of the mechanism by which MKRN1 promotes CRC metastasis through ubiquitinization and SNIP1 degradation. Detailed Implementation

[0033] The technical solution of the present invention will be further defined below with reference to specific embodiments, but the scope of protection is not limited to the description.

[0034] 1. Materials and Methods

[0035] 1.1 Materials

[0036] 1.1.1 Clinical patient specimens

[0037] Pathological sections were collected from 4 patients diagnosed with colitis and 16 patients initially diagnosed with CRC who underwent radical colectomy at the Department of Colorectal Surgery, Affiliated Hospital of Guizhou Medical University, between September 2019 and January 2020. Tumor tissue and matched adjacent fresh tissue were collected from 3 patients with colorectal cancer. This study was approved by the Ethics Review Committee of XXX University, and informed consent was obtained from the patients before the study.

[0038] 1.1.2 Cells

[0039] Human colonic epithelial cells CCD-18Co and human embryonic kidney epithelial cells HEK-293T were purchased from the U.S. Standard Biological Collection Center. Human colorectal cancer cells HCT116, HT29, HCT15, and RKO were all purchased from the Cell Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.

[0040] 1.1.3 Main Reagents and Their Sources

[0041]

[0042]

[0043]

[0044]

[0045] 1.1.5 Preparation methods of main reagents

[0046] (1) 10× electrophoresis buffer:

[0047] Table 1. Preparation method of 10× electrophoresis buffer

[0048] name Dosage glycine 72.0g Tris 15.1g SDS 5.0g <![CDATA[ddH2O]]> 500mL

[0049] After dissolving, store at room temperature. Dilute the 10× electrophoresis buffer to 1× with ddH2O before use.

[0050] (2) 10× transfer buffer:

[0051] Table 2 Preparation method of 10× transfer buffer

[0052] name Dosage glycine 151.1g Tris 30.3g <![CDATA[ddH2O]]> 500mL

[0053] After dissolving, store at room temperature. When using, prepare according to the ratio of ddH2O:methanol:10×transfer buffer = 7:2:1, and pre-cool at 4℃ after preparation.

[0054] (3) 10×TBS:

[0055] Table 3 Preparation method of 10×TBS

[0056] name Dosage Tris 15.0g NaCl 40.0g KCl 1.0g <![CDATA[ddH2O]]> 500mL

[0057] After dissolving, store at room temperature. When using, dilute 10× electrophoresis buffer to 1× with ddH2O, add 0.5 mL of Tween-20, mix well, and store at room temperature.

[0058] (4) 5% blocking solution:

[0059] Table 4. Preparation method of 5% sealing solution

[0060] name Dosage Skim milk powder or bovine serum albumin (BSA) 5.0g 1×TBST 100mL

[0061] After accurate weighing and preparation, mix thoroughly on a horizontal shaker before use.

[0062] (5) Western Blot separating gel and stacking gel:

[0063] Table 5. SDS-PAGE gel preparation method

[0064]

[0065]

[0066] Prepare separating gels according to the molecular weight of the target protein as shown in the table above. Add the reagents sequentially, mix thoroughly, and then pour the separating gel mixture to the appropriate position on the glass plate. Next, add anhydrous ethanol on top of the separating gel to press it in place. After the lower layer of separating gel solidifies, pour off the anhydrous ethanol, absorb the residual ethanol with absorbent paper, and allow it to evaporate for 10 minutes. Then, add the stacking gel to the glass plate, quickly insert the comb teeth, and let it solidify at room temperature.

[0067] (6) Preparation of stock solution for cycloheximide (CHX)

[0068] Dissolve 50 mg CHX in 5 ml PBS to a final concentration of 10 mg / mL. After thorough mixing, filter through a 0.22 μm filter membrane and dispense into 500 μL tubes. Store at -80°C for later use.

[0069] (7) Preparation of MG132 storage solution

[0070] Dissolve 5 mg MG132 in 210.25 μL DMSO to prepare a 50 mM solution. After thorough mixing, dispense 20 μL per tube and store at -80°C for later use.

[0071] (8) Preparation of storage solution for TGF-β1 cytokine

[0072] Before opening, centrifuge the cytokine proteins at 2000 rpm for 30 seconds, add an appropriate amount of the specified buffer to dissolve them, without vortexing. Dilute the reconstituted cytokines with an appropriate amount of diluent to a final concentration of at least 10 μg / mL, gently pipette to mix, aliquot, and store at -80℃ for later use. Western blotting was performed using 20 ng / mL HCT116 cells treated for 4 hours, followed by protein extraction and detection. Transwell assays were performed using 10 ng / mL HCT116 cells treated for staining and observation.

[0073] (9) Preparation of storage solution for LY2109761

[0074] Dissolve 0.5 mg LY2109761 in 113.25 μL DMSO to prepare a 10 mM solution. After thorough mixing, aliquot the solution into 10 μL tubes and store at -80°C for later use. For Western blotting, treat HCT15 cells with 10 μM for 2 hours and then extract protein for detection. For Transwell assay, treat HCT15 cells with 10 μM and then stain for observation.

[0075] 1.2 Methods

[0076] 1.2.1 Cell Culture

[0077] HEK-293T, HCT116, HT29, HCT15, RKO, and CCD-18Co cell lines were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin / streptomycin in a 37°C, 5% CO2 incubator, with the complete medium changed every two days. Cells were passaged when the cell density reached 80%-90%. For passage, the original medium was discarded, the cells were washed once with PBS, and then digested with 0.25% trypsin. Microscopic observation showed that the cells became rounder and brighter, and a few adherent cells floated. Complete medium was then added to stop digestion. After centrifugation at 1200 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in fresh complete medium. Cells were passaged at a 1:3 ratio and then cultured in the incubator. All cells used in the experiments were in the logarithmic growth phase.

[0078] 1.2.2 Design of shRNA

[0079] Primers were designed as follows: Forword: 5′-CAG AAG GCT CGA GAA GGT ATA TTG CTG TTG ACA GTGAGC G-3′; Reverse: 5′-CTAAAG TAG CCC CTT GAA TTC CGAGGC AGTAGG CA-3′. Three shRNAs were selected, and their sequences are shown in Table 6.

[0080] Table 6 shows the three shRNA target sequences for the MKRN1 gene.

[0081]

[0082] 1.2.3 Vector Construction and Identification

[0083] (1) PCR amplification system: see Table 7. PCR conditions: 94℃ for 1 min, 25× (94℃ for 30 s, 54℃ for 30 s, 75℃ for 30 s), 75℃ for 10 min, 4℃ hold.

[0084] Table 7. PCR amplification system, enzyme digestion system, and colony PCR system for shRNA.

[0085]

[0086] (2) Enzyme digestion system: see Table 7.

[0087] (3) Ligation: Take 5 μL Solution I, 3 μL p-Tripz and 2 μL shRNA into a PCR tube (ligate overnight at 16℃).

[0088] (4) Transformation: Take 20 μL of competent DH-5α cells, add 10 μL of ligation product, incubate on ice for 30 min, heat shock at 42℃ for 90 s, quickly return to ice, add AMP-free medium, shake at 200 rpm / min at 37℃ for 1 h; take the shaken bacteria and concentrate the bacterial solution at 4000 rpm / min for 1 min, discard 400 μL of supernatant, resuspend the bacterial solution, plate it, and incubate at 37℃ overnight; the next day, pick a single colony for colony PCR.

[0089] (5) Colony PCR: See Table 7. PCR conditions: 95℃ for 10 min, 36× (95℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s), 72℃ for 7 min, 4℃ hold. Based on the colony PCR results, positive bacterial cultures were selected and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0090] (6) The bacterial culture with correct sequencing was cultured overnight by shaking (approximately 15-20 mL). The next day, plasmids were extracted using the endotoxin-free plasmid extraction kit from Tiangen Biotech Co., Ltd. (aseptic operation). The extracted plasmids were stored at -80℃.

[0091] 1.2.4 Lentiviral Packaging and Construction of CRC Cells with Stable MKRN1 Silencing

[0092] HEK-293T cells in logarithmic growth phase were digested into single cells with 0.25% trypsin, resuspended in fresh culture medium, and seeded into 100 mm culture dishes at a cell count of 1×10⁹. The next day, when the cell confluence reached 60%-70%, the virus was packaged using Lip2000. Add 30 μL of Lip2000 to 500 μL of serum-free and antibiotic-free DMEM. Take three other EP tubes and add shRNA (sh1, sh2, sh3), pSPAX2, and pMD2.G to 500 μL of serum-free and antibiotic-free DMEM at a ratio of 2:1:1. After standing for 5 min, add the plasmids to Lip2000 and stand for 15 min. Immediately after standing, add the plasmids to the culture medium of HEK-293T cells and incubate at 37℃ for 6-8 h. Replace with 15 mL of complete medium containing 1 mg / L doxycycline. Collect the viral supernatant the next day (48 h after virus packaging) and add another 15 mL of complete medium containing 1 mg / L doxycycline. Collect the viral supernatant again the following day (72 h after virus packaging). Finally, obtain 30 mL each of shRNA1, shRNA2, and shRNA3 viral solutions. Then, use 1500 rpm / min... Centrifuge the viral supernatant for 3 min, filter the supernatant through a 0.45 μm filter membrane, and transfer the filtrate to an ultrafiltration tube. Centrifuge at 3000 rpm for 20 min to a final volume of 500 μL, and divide into 5 portions for freezing at -80°C. Digest logarithmic growth phase CRC cells into single cells with 0.25% trypsin, resuspend in fresh culture medium, and seed 6 × 10⁸ cells per 60 mm culture dish. The next day, when the cell confluence reaches 60%-70%, add sh1, sh2, and sh3 viral solutions, a final concentration of 5 mg / L polybrene, and 3 mL of complete culture medium to the corresponding culture dishes. After 6-8 h of infection, replace with complete culture medium containing 1 mg / L doxycycline. Observe cell fluorescence expression after 48 h of infection. Select cells for stable MKRN1 gene silence by puromycin based on fluorescence expression and cell confluence.

[0093] 1.2.5 Western Blot

[0094] (1) Extraction of total protein

[0095] Cell samples: Discard the cell culture medium, wash twice with pre-cooled PBS, and remove residual PBS with a pipette. Add an appropriate amount of RIPA containing protease inhibitors and phosphatase inhibitors to each dish according to cell density to lyse cells. Scrape cells from each dish into 1.5 mL Eppendorf tubes and lyse on ice for 10 min. After complete cell lysis, use an ultrasonic homogenizer with a 3-second sonication followed by an 8-second pause at 25% amplitude, repeating this process 3 times per sample. Centrifuge at 12000 rpm / min for 20 min at 4°C. Transfer the protein supernatant to a new Eppendorf tube and store at -80°C. Determine protein concentration using the BCA method before loading.

[0096] Tissue samples: Weigh 10 mg of tissue and add 100 μL of RIPA containing protease inhibitors and phosphatase inhibitors. Lyse on ice for 15 min, then use an ultrasonic homogenizer placed on ice and sonicated for 6 seconds with a 10-second pause, at an amplitude of 25%. Subsequent procedures are the same as for cell protein extraction.

[0097] (2) BCA method for determining protein concentration

[0098] 1) Prepare BSA standards with gradient concentrations according to the table below.

[0099] Table 8. BCA Method Protein Quantification Reaction System

[0100] serial number Standard dilution solution (μL) BSA stock solution (μL) Final concentration (μg / mL) A 0.0 30.0BSA stock solution 2000 B 12.5 37.5BSA stock solution 1500 C 32.5 32.5BSA stock solution 1000 D 17.5 17.5ml B bottle solution 750 E 32.5 32.5 C bottle liquid 500 F 32.5 32.5ml E-bottle solution 250 G 32.5 32.5 F-bottle solution 125 H 40.0 10.0 G bottle liquid 25 I 40.0 0 0

[0101] 2) The protein sample to be tested was diluted 10 times and then subjected to BCA detection, i.e., 81 μL of IPA diluent + 9 μL of protein stock solution;

[0102] 3) Set up 3 replicates for each protein sample, 200 μL / well. Prepare BCA working solution according to the required number of wells. The working solution should be prepared at a ratio of BCA reagent:Cu reagent = 50:1 and should be used immediately after preparation.

[0103] 4) Add 200 μL of BCA working solution to each well of a 96-well plate, then add the serially diluted standard and the test sample at 25 μL per well, and incubate at 37°C for 30 min.

[0104] 5) After incubation, place the 96-well plate in a microplate reader and measure the optical density (OD) of each well at 570 nm.

[0105] 6) Plot a standard curve with the OD values ​​of serially diluted standards on the x-axis and protein concentration on the y-axis. Calculate the standard curve formula and R0 using Excel. 2 (Standard Curve R) 2 (Should be >0.99). Calculate the protein concentration of the sample to be tested based on the calculated standard curve formula.

[0106] (3) Protein denaturation

[0107] After mixing the 5× protein loading buffer with the extracted protein sample at a ratio of 1:4, the sample was placed in a 100℃ metal bath to denature the protein, and then aliquoted and stored in a -80℃ freezer.

[0108] (4) SDS-PAGE gel electrophoresis

[0109] 1) Gel preparation: Install a clean glass plate onto the gel preparation frame and prepare the appropriate separating gel according to the molecular weight of the target protein; after the separating gel solidifies, prepare a 5% stacking gel, quickly insert the comb teeth to prevent the gel from solidifying, and let it stand until the gel completely solidifies.

[0110] 2) After the gel solidifies, assemble the prepared gel into the electrophoresis clamp, add an appropriate amount of 1× electrophoresis solution to the inner tank, let it stand for 5 minutes, and observe whether there is any leakage. If there is no leakage, add an appropriate amount of electrophoresis solution to the outer tank and carefully pull out the comb teeth in the gel.

[0111] 3) Electrophoresis: Slowly add the protein sample and press the gel using a constant voltage of 80V. After the markers separate (about 30 minutes), adjust the voltage to 120V and continue electrophoresis. Stop electrophoresis when the bromophenol blue reaches the bottom of the separating gel.

[0112] 4) Transfer: Cut a PVDF membrane to the appropriate size according to the required protein molecular weight, and activate it with methanol. Then, with the positive electrode facing up, arrange the "transfer sandwich" from bottom to top in the following order: "sponge - two layers of thin filter paper - separating gel - PVDF membrane - two layers of thin filter paper - sponge". Place it in the electroporation cell (note that the positive electrode is to the positive electrode and the negative electrode is to the negative electrode). Pour in the pre-cooled transfer solution (placing ice packs around the transfer cell will improve the effect). According to the required molecular weight, maintain a constant current of 250mA and transfer the membrane at a rate of 1-1.5kD / min.

[0113] 5) Blocking: After the transfer is complete, remove the PVDF membrane and immerse the PVDF membrane with protein marker in 5% blocking solution (skim milk or BSA) and incubate it with gentle shaking on a horizontal shaker at room temperature for 2 hours.

[0114] 6) Primary antibody incubation: After blocking, discard the blocking solution and wash the PVDF membrane three times with TBST to remove any residual blocking solution. Dilute the antibody with the primary antibody dilution buffer according to the instructions, immerse the PVDF membrane in the corresponding antibody solution, and incubate overnight at 4°C with gentle shaking.

[0115] 7) Secondary antibody incubation: The next day, after the primary antibody incubation box has returned to room temperature, remove the PVDF membrane from the primary antibody solution and wash the PVDF membrane 4 times with TBST, 5 min each time. Then add HRP-labeled goat anti-rabbit or goat anti-mouse secondary antibody diluted with secondary antibody dilution buffer at a ratio of 1:10000, and incubate at room temperature for 1 h;

[0116] 8) Exposure: After the secondary antibody incubation, wash the PVDF membrane four times with TBST, 5 min each time. Place the PVDF membrane with the front side (protein side up) in an exposure apparatus, and evenly drop ECL luminescent solution onto the surface. Analyze the results.

[0117] 1.2.6 qRT-PCR

[0118] (1) Extraction of total RNA

[0119] 1) Chloroform, anhydrous ethanol, and isopropanol were pre-cooled to 4°C.

[0120] 2) Add 1 mL of Trizol to the cell culture dish and repeatedly pipette the cells to lyse them fully. Let it stand at room temperature for 5 min and then aspirate it into an enzyme-free Eppendorf tube.

[0121] 3) Add 200 μL of pre-cooled chloroform to the above lysis buffer, mix by inverting until the solution turns milky white, let stand at room temperature for 5 min, centrifuge at 4℃ and 12000 rpm / min for 5 min, at which point the solution will separate into three layers: a colorless supernatant (containing RNA), a middle protein layer, and a colored lower organic phase.

[0122] 4) Carefully pipette the colorless supernatant into an enzyme-free Ep tube, add 500 μL of pre-chilled isopropanol, invert the Ep tube to mix the solution thoroughly, let it stand at room temperature for 10 min, then centrifuge at 4℃, 12000 rpm / min for 10 min, discard the supernatant, and RNA precipitate will appear at the bottom of the Ep tube.

[0123] 5) Add 500 μL of anhydrous ethanol to wash the precipitate, centrifuge at 12000 rpm / min for 5 min at 4℃, discard the supernatant and collect the precipitate;

[0124] 6) Open the cap of the Eppendorf tube and let the precipitate dry at room temperature. After the precipitate is dry (do not let it dry too much, otherwise it will not dissolve easily), add an appropriate amount of DEPC water to dissolve the precipitate, which is the RNA solution.

[0125] 7) Detect RNA concentration and purity.

[0126] (2) Genomic DNA removal reaction

[0127] Prepare the reaction system on ice according to the table below:

[0128] Table 9. Genomic DNA Removal Reaction System

[0129]

[0130]

[0131] After mixing, incubate briefly. PCR conditions: 42℃, 2 min.

[0132] (3) Reverse transcription reaction

[0133] Prepare the reaction system on ice according to the table below:

[0134] Table 10 Reverse Transcription System

[0135] reagents Volume (μL) The reaction solution from the previous step 10.0 Prime Script RT Enzyme MixI 1.0 RT Primer Mix 1.0 5×Prime Script Buffer(for Real Time) 4.0 <![CDATA[RNase Free H2O]]> 4.0 Total Volume 20.0

[0136] After mixing, incubate briefly; PCR conditions: 37℃, 15 min; 85℃, 5 sec. After the reaction, aliquot the cDNA and store at -80℃.

[0137] (4) Real-time quantitative PCR reaction

[0138] 1) Primer sequence:

[0139] Table 11 Primer sequences

[0140]

[0141] 2) Prepare the reaction system according to the table below (protected from light):

[0142] Table 12. Real-time quantitative PCR system

[0143] reagents Volume (μL) TB Green Premix Ex TaqII (2×) 10.0 RCR Foward Primer (concentration 10 μM) 1.0 PCR Reverse Primer (10 μM) 1.0 RT reaction solution (cDNA solution) 2.0 <![CDATA[RNase-free H2O]]> 6.0 Total Volume 20.0

[0144] After mixing, incubate briefly; PCR conditions: 95℃ for 30s, 40×(95℃ for 3s, 60℃ for 30s), 4℃ hold.

[0145] 1.2.7 CCK-8 assay for cell viability

[0146] Cells were digested with 0.25% trypsin, centrifuged at 1200 rpm for 5 min, and the culture supernatant was discarded. The cell pellet was collected. 1 mL of complete culture medium was added to the cell pellet to resuspend the cells, and the cells were counted using a Newton-Bauer counting chamber. The cell suspension was diluted at a ratio of 1 × 10³ cells / well, and 200 μL of the total volume of cell suspension was added to each well of a 96-well plate. The plates were then incubated. After culturing the cells for 0, 1, 3, 5, and 7 days, the original culture medium was discarded, and 100 μL of serum-free culture medium containing 10% CCK-8 reagent was added to each well. The plates were incubated at 37°C for 2 h, and the OD value of each well was measured at 450 nm using a microplate reader.

[0147] 1.2.8 Colony formation assay for detecting cell proliferation

[0148] Cells were digested with 0.25% trypsin and seeded into 6-well plates at a rate of 500 cells per well. After 12 days of cell growth (with medium changed every 3-4 days), the original culture medium was discarded, the cells were washed 3 times with PBS, fixed with methanol for 30 min, washed twice with PBS, stained with 1% crystal violet for 15 min, and finally washed with PBS to remove excess stain. After drying, the cells were photographed and stored. Clusters with more than 50 cells were considered as one colony, and the number of colonies formed was counted using ImageJ.

[0149] 1.2.9 Scratch assay to detect cell migration ability

[0150] (1) Seed CRC cells into 6-well plates, ideally until the cell density reaches 90% or higher the next day;

[0151] (2) Use a sterile 200μL pipette tip to make a scratch on each well of cells. After washing the floating cells with PBS, add serum-free culture medium for culture. Take pictures of the cell scratches at 0h and 48h after scratching and use ImageJ to calculate the change in scratch area.

[0152] 1.2.10 Transwell assay to detect cell migration and invasion abilities

[0153] (1) Pre-culture cells in serum-free medium overnight to starve cells and remove the effect of serum;

[0154] (2) The next day, the cells were digested with 0.25% trypsin. After digestion was terminated, the cells were centrifuged at 1200 rpm / min for 5 min. The supernatant was discarded. The cell pellet was washed three times with PBS and resuspended in 1 mL of basal medium. The cells were counted using a Boehringer Institut. The concentration of HCT116 cells was adjusted to 5 × 10⁴ cells / well and the concentration of HCT15 cells was adjusted to 4 × 10⁴ cells / well. 600 μL of medium containing 10% FBS was added to the lower chamber of a 24-well plate. The Transwell chamber was placed in the 24-well plate containing medium (avoiding air bubbles). The cell suspension was added to the chamber at a rate of 200 μL / well (avoiding air bubbles). The cells were cultured for 48 h as usual.

[0155] (3) When detecting cell invasion, use basal medium to dilute the matrix gel at a ratio of 1:8, add 60 μL to the chamber, spread it evenly, avoid generating air bubbles, and place it in a 37℃ incubator for 2 h. At the same time, digest the starved cells, resuspend the cell pellet in 1 mL of basal medium, count the cells using a Newton-Bauer counting chamber, adjust the HCT116 cell concentration to 6 × 10⁴ cells / well and the HCT15 cell concentration to 5 × 10⁴ cells / well, add 600 μL of medium containing 20% ​​FBS to the lower chamber of a 24-well plate, place the Transwell chamber into the 24-well plate containing medium (avoid generating air bubbles), add the cell suspension to the chamber at a rate of 200 μL / well (avoid generating air bubbles), and culture for 48 h as usual.

[0156] (4) Fixation and staining: Discard the culture medium in the Transwell chamber and culture plate, wash twice with PBS, fix with 4% paraformaldehyde for 30 min, wash three times with PBS, stain with crystal violet for 15 min, and rinse thoroughly with PBS. Moisten a cotton swab and gently wipe away the cells on the upper layer of the chamber, wash thoroughly with PBS, and then take a picture.

[0157] 1.2.11 Immunohistochemistry (IHC) staining was used to observe the expression of MKRN1, SNIP1, TGF-β1, and E-cadherin in the tissue.

[0158] (1) Dewaxing paraffin sections to water: Place the sections in xylene I for 10 min, xylene II for 10 min, xylene III for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, and then wash with distilled water.

[0159] (2) Antigen retrieval: Place the slides in a slide rack and slowly immerse them in the antigen retrieval solution. Boil on high for 5 minutes, then on low for 20 minutes. During this process, prevent excessive evaporation of the buffer solution and avoid drying the slides. After natural cooling, place the slides in PBS and wash them three times on a shaker for 5 minutes each time.

[0160] (3) Blocking endogenous peroxidase: Place the slide in 3% hydrogen peroxide solution and incubate at room temperature in the dark for 25 min. Then place the slide in PBS and wash it three times on a shaker for 5 min each time.

[0161] (4) Serum blocking: Add 3% BSA evenly to the histochemistry zone and block for 30 min at room temperature. (Use rabbit serum for blocking if the primary antibody is of goat origin, and BSA for blocking if the primary antibody is of other origin.)

[0162] (5) Incubate with primary antibody: Gently shake off the blocking solution, add the prepared primary antibody in a certain proportion to the slide, and incubate the slide in a humidified box at 4°C overnight.

[0163] (6) Incubation with secondary antibody: Place the slide in PBS and wash it three times on a shaker for 5 minutes each time. After slightly drying the slide, add the secondary antibody (HRP-labeled) of the corresponding species to the primary antibody to cover the tissue and incubate at room temperature for 50 minutes.

[0164] (7) DAB staining: Place the slide in PBS and wash it three times on a shaker for 5 minutes each time. After slightly drying the slide, add freshly prepared DAB staining solution to the circle. Control the staining time under a microscope. The positive color is brownish-yellow. Rinse the slide with tap water to stop the staining.

[0165] (8) Counterstaining cell nuclei: Counterstain with hematoxylin for about 3 minutes, then rinse with tap water. Differentiate with hydrochloric acid alcohol for a few seconds, then rinse with tap water, then use hematoxylin blue solution to return to blue, and rinse with running water.

[0166] (9) Dehydration and mounting: Place the sections in 75% alcohol for 5 min, 85% alcohol for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, n-butanol for 5 min, and xylene I for 5 min to dehydrate and make them transparent. Remove the sections from the xylene and let them dry slightly. Then mount them with mounting glue.

[0167] (10) Microscopic examination: The results are interpreted under a white light microscope.

[0168] (11) Use Image-J software to calculate the optical density value (IOD). The result is expressed as the average optical density value (AOD). The calculation method is AOD = IOD / area.

[0169] 1.2.12 Immunofluorescence detection of co-localization of MKRN1 and SNIP1

[0170] (1) Place cell crawling sheets into 24-well plates and inoculate cells until the cell density reaches 50-60% the next day.

[0171] (2) The next day, discard the culture medium and wash once with PBS.

[0172] (3) Use pre-cooled 4% paraformaldehyde, fix at room temperature for 20 min, and wash with PBS 3 times, 5 min each time.

[0173] (4) Use 0.5% Triton X-100, allow to pass through at room temperature for 10 min, and wash with PBS 3 times for 5 min each time.

[0174] (5) Add 5% BSA blocking solution and block at room temperature for 1 hour.

[0175] (6) Discard the blocking solution, add MKRN1 and SNIP1 antibodies to cover the slides (approximately 200 μL / well, dilution ratio 1:200), and incubate overnight at 4°C. The next day, after returning to room temperature, wash three times with PBS for 5 min each time.

[0176] (7) Fluorescent secondary antibody (dilution ratio 1:500), incubated at room temperature in the dark for 1 hour, then washed 3 times with PBS for 5 minutes each time.

[0177] (8) Add DAPI staining solution, incubate at room temperature in the dark for 5 minutes, then wash with PBS 3 times, 5 minutes each time.

[0178] (9) Mount the slide with 90% glycerin and nail polish around the slide, then observe and photograph it under a confocal microscope.

[0179] 1.2.13 Quantitative proteomics and ubiquitination modification analysis of MKRN1

[0180] Cells from the HT29, HCT116 Control, and sh1-MKRN1 groups were collected for proteomics and ubiquitination modification studies. This research was conducted by Hangzhou Jingjie Biotechnology Co., Ltd.

[0181] 1.2.14 Immunoprecipitation assay for the interaction between MKRN1 and SNIP1

[0182] (1) Wash the cells in the logarithmic growth phase twice with pre-cooled PBS, scrape them off with a cell scraper, collect the cell pellet, add 600 μL of NP40 cell lysis buffer containing protease inhibitor to the cell pellet, place it on a shaker at 4°C and shake slowly for 1 h, centrifuge at 12000 rpm / min for 15 min, and aspirate the supernatant into an Ep tube.

[0183] (2) Take 100 μL of lysis supernatant, add 5× Loading Buffer, place in a metal bath at 100℃, and use as Input sample after 10 min;

[0184] (3) Add 20 μL of protein A / G agarose beads to the remaining 500 μL of supernatant, incubate with gentle shaking at 4°C for 0.5 h, then centrifuge at 3000 rpm for 10 min, and transfer the supernatant to a new Ep tube;

[0185] (4) The concentration of supernatant protein was detected by BCA method. 1 μg of antibody was added per mg of protein. The target antibody and IgG antibody with the same properties as the target antibody (IgG group and IP group) were added. The antibody and protein were incubated for more than 8 hours. 35 μL of protein A / G agarose beads were added and incubated overnight at 4°C with slow shaking.

[0186] (5) The next day, centrifuge at 3000 rpm / min for 10 min at 4℃ to get the agarose beads to the bottom of the tube. Carefully remove the supernatant and wash the agarose beads 3 times with 1 mL of pre-cooled PBS. Finally, add 70 μL of 1×Loading Buffer and incubate at 100℃ for 10 min in a metal bath as IgG and IP samples.

[0187] (6) Analyze the interactions between proteins using Western blotting.

[0188] 1.2.15 Ubiquitination Experiment

[0189] (1) Treat cells with 10 μM / L MG132 for 8 h in advance, harvest cells, add 150 μL of SDS lysis buffer containing protease inhibitor and 1 mM / L DTT to the precipitate, SDS lysis buffer: DTT = 100:1, boil in a metal bath at 100℃ for 40 min until the solution is clear and free of precipitate, and mix the Ep tube every 1.5 min during the process; (2) Add 500 μL of lysis buffer containing protease inhibitor NP40 to dilute the lysed protein solution, centrifuge at 12000 rpm / min at 4℃ for 15 min, and transfer the protein supernatant to a new Ep tube;

[0190] (3) Add 20 μL of protein A / G agarose beads to the protein supernatant, incubate with gentle shaking at 4°C for 0.5 h, centrifuge at 3000 rpm / min for 10 min at 4°C, and transfer the supernatant to a new Eppendorf tube;

[0191] (4) The concentration of supernatant protein was detected by BCA method. 1 μg of antibody was added per mg of protein. The target antibody was added and incubated with the protein for more than 6 hours. 35 μL of protein A / G agarose beads were added and incubated overnight at 4°C with slow shaking.

[0192] (5) The next day, centrifuge at 3000 rpm / min for 10 min at 4℃ to get the agarose beads to the bottom of the tube. Carefully remove the supernatant and wash the agarose beads 3-4 times with 1 mL of pre-cooled PBS. Finally, add 70 μL of 1×Loading Buffer and incubate at 100℃ for 10 min in a metal bath as an IP sample.

[0193] (6) The ubiquitination level of the target protein was analyzed by Western blotting.

[0194] 1.2.16 Statistical Analysis

[0195] ImageJ, IHC Profiler, SPSS 26.0, and GraphpadPrism 6 software were used for data analysis and statistical chart creation. All measurement data were expressed as mean ± standard deviation. The t-test was used for comparisons between two groups, with P < 0.05 considered statistically significant. Each experiment was repeated three times. *P < 0.05, **P < 0.01, ***P < 0.001.

[0196] 2 Results

[0197] 2.1 MKRN1 expression is upregulated in CRC and is associated with poor prognosis.

[0198] To investigate MKRN1 expression in tumors, we obtained the gene expression matrix of colorectal tumor cell lines from the Encyclopedia of Cancer Cell Lines dataset (https: / / portals.broadinstitute.org / ccle / about). The results showed that MKRN1 was highly expressed in CRC. Figure 1 A). Furthermore, we obtained RNA-seq data and corresponding clinical information for 620 colorectal tumors from The Cancer Genome Atlas (TCGA) dataset (https: / / portal.gdc.com), and analyzed the expression level of MKRN1 in CRC and normal tissues using TCGA combined with a genotype-tissue expression database. The results showed that MKRN1 was highly expressed in CRC compared to normal tissues. Figure 1 B). We examined the MKRN1 protein levels in three pairs of CRC and adjacent non-tumor tissues and found that MKRN1 protein expression was higher in CRC tissues than in adjacent non-tumor tissues. Figure 1 C).

[0199] IHC staining results showed that, compared with the histochemical results of colitis patients, MKRN1 expression was significantly increased in CRC tissues, and MKRN1 expression was correlated with the TNM stage of the tumor, with MKRN1 expression in stage III-IV patients being significantly higher than that in stage I-II patients. Figure 1 D). Furthermore, analysis using the R2 genomics platform showed that CRC patients with high MKRN1 expression had significantly shorter overall survival (OS) than CRC patients with low MKRN1 expression. Figure 1 E).

[0200] To investigate the role of MKRN1 in CRC development, we used Western blotting (WB) to detect MKRN1 protein expression in four CRC cell lines and normal human colonic fibroblasts (CCD-18Co). The results showed that compared with CCD-18Co cells, MKRN1 protein was highly expressed in CRC cells HT29, HCT116, and RKO, while low expression was observed in HCT15. Figure 1 F). In summary, these findings suggest that MKRN1 is significantly upregulated in CRC and is associated with poor prognosis in CRC patients.

[0201] 2.2 Construction of stably silenced MKRN1 CRC cells

[0202] We selected CRC cells HCT116 and HT29, which highly express MKRN1, for MKRN1 knockdown treatment. shRNAs targeting the MKRN1 gene were designed using the platform http: / / katahdin.csh1.org / siRNA / RNAi.cgi?type=shRNA, named sh1, sh2, and sh3. After agarose gel electrophoresis, the shRNA PCR products showed bands at approximately 100 bp, successfully amplifying three shRNA sequences. Figure 2 A); after gel recovery, the gel was double-digested with EcoRI and XhoI ( Figure 2 B); and double digestion of the vector pTripz ( Figure 2 C); After gel recovery of the enzyme-digested shRNA and vector pTripz, ligation and transformation were performed, followed by colony PCR. The PCR products were then subjected to agarose gel electrophoresis, and the results showed that sh1, sh2, and sh3 were all successfully ligated to the vector pTripz. Figure 2 D); The bacterial culture was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results of sh1-1~sh1-4, sh2-1, sh2-2, sh2-4, and sh3-1 were consistent with the designed shRNA sequences. Figure 2 E). The above results indicate that the sh1, sh2, and sh3 sequences were all successfully ligated into the pTripz vector.

[0203] Plasmids were extracted from correctly sequenced sh1, sh2, and sh3 bacterial cultures after shaking, and then lentiviruses were packaged. After 72 hours, the fluorescence expression in HEK-293T cells reached 50.0%, 40.0%, and 30.0%, respectively. Figure 2 F) indicates that the shRNA vector was successfully transfected into HEK-293T cells. Viral supernatants from doxycycline-induced expression at 48h and 72h were collected and used to infect HCT116 cells with the concentrated viral solution. After 48h of infection, the fluorescence expression of HCT116 cells was approximately 80.0% (F). Figure 2G). Western blotting verified the knockdown efficiency of MKRN1. The results showed that sequence 1 was effective in knocking down MKRN1, while sequences 2 and 3 had little effect. Figure 2 H). Subsequently, we successfully knocked down MKRN1 expression in HT29 cells using sequence number 1. Figure 2 I).

[0204] 2.3 MKRN1 promotes the proliferation of CRC cells

[0205] Based on the expression of MKRN1 in CRC cells, we selected HCT15 cells with low MKRN1 expression for overexpression. The overexpression effect of MKRN1 in HCT15 cells was then assessed. Figure 3 A). Cell viability was assessed using the CCK8 assay, and the results showed that knockdown of MKRN1 reduced the viability of HCT116 and HT29 cells. Figure 3 B); Overexpression of MKRN1 increased HCT15 cell viability ( Figure 3 C). Meanwhile, colony formation experiments showed that knocking down MKRN1 resulted in fewer and smaller colonies formed by HCT116 and HT29 cells. Figure 3 D), after overexpression of MKRN1, HCT15 cells formed more and larger colonies. Figure 3 E). The results suggest that MKRN1 promotes the proliferation of CRC cells.

[0206] 2.4MKRN1 promotes CRC cell migration, invasion, and EMT.

[0207] To investigate whether MKRN1 can promote CRC metastasis, we used highly invasive CRC cells HCT116 for experiments. The wound healing ability of the cells was assessed using a scratch assay. The results showed that knocking down MKRN1 in HCT116 cells inhibited the scratch healing ability of CRC cells. Figure 4 A); In HCT15 cells, overexpression of MKRN1 can promote the scratch healing ability of CRC cells (A); Figure 4 B). The migration and invasion abilities of HCT116 and HCT15 cells were assessed using Transwell assays. The results showed that knockdown of MKRN1 in HCT116 cells inhibited the migration and invasion abilities of CRC cells. Figure 4 C), conversely, in HCT15 cells, overexpression of MKRN1 can promote the migration and invasion of CRC cells (C). Figure 4 D).

[0208] EMT plays a crucial and complex role in tumor invasion and metastasis. Microscopic observation revealed that knocking down MKRN1 altered the morphology of HCT116 cells from fibroblasts (spindle-shaped) to epithelial cells (tightly bound); while overexpression of MKRN1 altered the morphology of HCT15 cells from epithelial cells to fibroblasts. Figure 4 Consistent with these morphological changes, knockdown of MKRN1 resulted in decreased N-cadherin expression, increased E-cadherin expression, and decreased Snail expression at the protein level; overexpression of MKRN1 resulted in increased N-cadherin expression, decreased E-cadherin expression, and increased Snail expression. Figure 4 GH). The results suggest that MKRN1 induces EMT in CRC cells.

[0209] 2.5 Interaction between MKRN1 and SNIP1

[0210] To elucidate the potential mechanisms by which MKRN1 functions, we searched for proteins that may interact with MKRN1 using the STRING and IntAct databases. Figure 5 AB) The intersection of the two databases yielded three proteins: SNIP1, TRA2A, and LUC7L. Subsequent proteomics and ubiquitination modification studies showed that MKRN1 interacts with SNIP1 and TRA2A. Figure 5 C). To explore the roles of SNIP1 and TRA2A in CRC, we analyzed the TCGA database and found that SNIP1 showed low expression in CRC compared to normal controls. Figure 5 (D), but TRA2A shows high expression in CRC ( Figure 5 (D below), and we obtained RNAseq data and corresponding clinical information for 620 CRCs from the TCGA dataset. Through univariate and multivariate Cox regression analysis, we found that SNIP1 can serve as an independent prognostic factor for CRC, but TRA2A cannot. Figure 5 E).

[0211] Studies have found that SNIP1 is significantly reduced in intestinal epithelial cells of patients with inflammatory bowel disease and mouse models of colitis. Meanwhile, TRA2A, an oncogene, is highly expressed in various tumors. Therefore, we further examined the effect of MKRN1 alterations on SNIP1. The results showed that knockdown of MKRN1 upregulated SNIP1 expression, while overexpression of MKRN1 downregulated SNIP1 expression. Figure 5FG) and SNIP1 showed a negative correlation. Since SNIP1 protein expression was significantly downregulated in cells with high MKRN1 expression, we sought to elucidate the relationship between MKRN1 and SNIP1 proteins in CRC cells. Therefore, we used confocal imaging to detect the spatial distribution of MKRN1 and SNIP1. The results showed that the expression of MKRN1 (red) and SNIP1 (green) partially overlapped, indicating that both are co-localized in the cytoplasm and nucleus (FG). Figure 5 H). Co-IP combined with Western blotting for both forward and reverse verification showed that endogenous MKRN1 and SNIP1 form a complex in HCT116 and HCT15 cells (H). Figure 5 IJ), exogenous MKRN1 and endogenous SNIP1 can still form a complex ( Figure 5 These results indicate that MKRN1 interacts with SNIP1.

[0212] 2.6MKRN1 induces SNIP1 proteasomal degradation and promotes SNIP1 ubiquitination.

[0213] Since MKRN1 overexpression leads to downregulation of SNIP1 protein expression, we then examined the stable levels of SNIP1 protein in HCT15 cells with increasing exogenous MKRN1 levels. The results showed that the stable levels of both endogenous and exogenous SNIP1 gradually decreased with increasing exogenous MKRN1 levels. Figure 6 Next, we treated CRC cells with CHX and determined the half-life of SNIP1. Kinetic analysis of SNIP1 degradation by MKRN1 in the presence of CHX showed that knockdown of MKRN1 increased the stability of SNIP1. Figure 6 C); Excessive use of MKRN1 can reduce the stability of SNIP1. Figure 6 D). Simultaneously, CHX treatment also disrupted the stability of MKRN1, supporting earlier research that MKRN1 is degraded through autoubiquitination. Although MKRN1 overexpression led to the degradation of SNIP1 protein, MKRN1 expression did not affect SNIP1 mRNA expression. Figure 6 This indicates that MKRN1 can affect the post-transcriptional level of SNIP1. Since MKRN1 is an E3 ubiquitin ligase, to further investigate the mechanism by which MKRN1 induces SNIP1 degradation, we added the proteasome inhibitor MG132 to examine whether SNIP1 degradation is dependent on the 26S proteasome. The results showed that MG132 could reverse the MKRN1-mediated SNIP1 degradation process, suggesting that MKRN1 degrades SNIP1 via the proteasome pathway. Figure 6 G). Further ubiquitination analysis showed that knocking down MKRN1 reduced the ubiquitination level of SNIP1. Figure 6H), after overexpression of MKRN1, the ubiquitination level of SNIP1 increased ( Figure 6 I). Treatment with MG132 further induced ubiquitination of SNIP1 ( Figure 6 In summary, these results indicate that SNIP1 is a ubiquitination substrate of MKRN1, and that MKRN1 promotes the degradation of SNIP1.

[0214] 2.7 The E3 ligase mutant H307E of MKRN1 cannot ubiquitinate SNIP1.

[0215] The RING domain is an important element of the RING domain family that functions as a ubiquitin ligase. MKRN1 is a member of the RING domain family, and its domain diagram is shown below. Figure 7 As shown in Figure A, amino acids 281-334 at the N-terminus constitute the RING domain. Studies have reported that a mutation of histidine to glutamate (H307E) at amino acid 307 in the RING domain of MKRN1 can cause MKRN1 to lose its E3 ligase activity. Therefore, we constructed the MKRN1 E3 ligase mutant H307E (… Figure 7 B). Although the MKRN1(H307E) mutant can bind to SNIP1 ( Figure 7 C), but MKRN1(H307E) does not induce SNIP1 degradation ( Figure 7 D), and as exogenous MKRN1 (H307E) gradually increases, the level of endogenous SNIP1 does not change significantly. Figure 7 E); meanwhile, CHX experiments showed that the ability of MKRN1 mutation to induce SNIP1 instability was blocked. Figure 7 F). Our ubiquitination analysis further confirmed that MKRN1(H307E) cannot induce SNIP1 ubiquitination. Figure 7 (G). In summary, MKRN1(H307E) cannot induce ubiquitination and degradation of SNIP1, indicating the importance of MKRN1's E3 ligase activity in mediating SNIP1 degradation. These data suggest that MKRN1 is a novel E3 ligase for SNIP1.

[0216] 2.8 SNIP1 inhibits CRC cell EMT and migration

[0217] To investigate whether SNIP1 plays a role in inhibiting CRC metastasis, we silenced and overexpressed SNIP1 in CRC cells. Figure 8 AB); Western blot results showed that knocking down SNIP1 increased N-cadherin expression, decreased E-cadherin expression, and increased Snail expression, promoting EMT in CRC cells. Figure 8C); Overexpression of SNIP1 decreased N-cadherin expression, increased E-cadherin expression, decreased Snail expression, and inhibited EMT in CRC cells. Figure 8 D). Transwell assay results showed that knocking down SNIP1 enhanced the migration ability of HCT116 cells. Figure 8 E); Overexpression of SNIP1 reduces the migration ability of HCT15 cells. Figure 8 F). These results demonstrate that SNIP1 can inhibit EMT and migration in CRC cells.

[0218] 2.9MKRN1 promotes CRC cell EMT and migration by inhibiting SNIP1.

[0219] To investigate whether SNIP1 is involved in MKRN1-promoted CRC cell migration, we conducted a reversal experiment, demonstrating that shSNIP1 can reverse the EMT inhibition induced by sh1-MKRN1 in HCT116 cells. Figure 9 A); Overexpression of SNIP1 can reverse OE-MKRN1-induced EMT activation in HCT15 cells ( Figure 9 B). Meanwhile, Transwell assays showed that silencing SNIP1 reversed the inhibition of HCT116 cell migration induced by sh1-MKRN1. Figure 9 C); Overexpression of SNIP1 can reverse the increased migration ability of HCT15 cells induced by OE-MKRN1. Figure 9 D). These findings suggest that in CRC cells, MKRN1 promotes EMT by downregulating SNIP1.

[0220] 2.10 In CRC cells, SNIP1 can inhibit TGF-β signaling.

[0221] SNIP1 is a nuclear protein cloned and identified based on its ability to inhibit the TGF-β signaling pathway. To investigate whether SNIP1 affects TGF-β signaling in CRC, Western blotting results showed that knockdown of SNIP1 increased the expression of p-Smad2 / 3 and TGF-β1, promoting TGF-β signaling; conversely, overexpression of SNIP1 decreased the expression of p-Smad2 / 3 and TGF-β1, inhibiting TGF-β signaling. Figure 10 AB).

[0222] 2.11 MKRN1 activates the TGF-β signaling pathway, thereby promoting EMT in CRC cells.

[0223] To investigate the relationship between MKRN1 and the TGF-β signaling pathway, we obtained RNAseq data and corresponding clinical information of CRC from the TCGA database. Spearman correlation analysis showed that MKRN1 was positively correlated with the TGF-β pathway. Figure 11 A). Western blotting results showed that knocking down MKRN1 reduced the expression of p-Smad2 / 3 and TGF-β1, inhibiting the TGF-β pathway; overexpression of MKRN1 increased the expression of p-Smad2 / 3 and TGF-β1, activating the TGF-β pathway. Figure 11 (BC). This suggests that high levels of MKRN1 may promote CRC progression through the TGF-β signaling pathway. Since the TGF-β pathway plays a crucial role in inducing EMT and is a key step in tumor invasion and metastasis, we further determined whether MKRN1 affects CRC cell EMT through the TGF-β pathway. Treatment with the TGF-β pathway inducer TGF-β1 reversed the inhibition of EMT and TGF-β pathway-related markers induced by MKRN1 knockdown. Figure 11 D); Treatment with the TGF-β pathway inhibitor LY2109761 reversed the increase in EMT and TGF-β pathway-related markers caused by MKRN1 overexpression. Figure 11 E). Furthermore, we performed corresponding phenotypic experiments using Transwell, and the results showed that TGF-β1 treatment significantly increased the migration ability of HCT116 sh1-MKRN1 cells. Figure 11 F); LY2109761 treatment significantly reduced the migration ability of HCT15 OE-MKRN1 cells (F). Figure 11 G). These results suggest that MKRN1 promotes EMT and migration of CRC cells through the TGF-β pathway.

[0224] 2.12MKRN1 promotes the TGF-β pathway by degrading SNIP1

[0225] To assess whether MKRN1 overexpression promoting the TGF-β pathway is related to MKRN1 degradation of SNIP1, we conducted a reversal experiment. Introducing sh-SNIP1 into HCT116 sh1-MKRN1 cells showed that SNIP1 interference could reverse the levels of p-Smad2 / 3 and TGF-β1 in MKRN1-deficient cells. Similarly, introducing OE-SNIP1 into HCT15 OE-MKRN1 cells showed that SNIP1 overexpression could reverse the levels of p-Smad2 / 3 and TGF-β1 in MKRN1-overexpressing cells. Figure 12(AB). To further confirm our study, we used IHC to verify the expression of MKRN1, SNIP1, and TGF-β1 in clinical CRC specimens. The results showed that in patients with high MKRN1 expression, SNIP1 was lowly expressed, while TGF-β1 expression was high (AB). Figure 12 C). These results indicate that MKRN1 can promote TGF-β signaling by degrading SNIP1.

[0226] In summary, our study demonstrates that MKRN1 promotes EMT and metastasis in CRC cells by ubiquitinizing and degrading SNIP1, thereby eliminating the inhibition of TGF-β signaling. Figure 13 ).

[0227] 3. Discussion:

[0228] This application provides sufficient evidence to demonstrate that MKRN1 promotes the progression of CRC. First, MKRN1 is highly expressed in CRC and is positively correlated with clinical TNM stage; CRC patients with high MKRN1 expression have a worse prognosis. Second, in vitro experiments demonstrate that CRC cells with high levels of MKRN1 exhibit a mesenchymal phenotype and can promote CRC cell proliferation, migration, invasion, and EMT processes. We are the first to discover that MKRN1 can act as a pro-oncogene to promote CRC progression.

[0229] Our research indicates that MKRN1 and SNIP1 co-localize. Co-IP results show that both endogenous and exogenous MKRN1 interact with SNIP1. MKRN1 can destabilize SNIP1, reduce its half-life, and mediate SNIP1 degradation via the ubiquitin-proteasome pathway, suggesting that SNIP1 is a ubiquitination substrate of MKRN1. However, although the E3 ligase-deficient mutant MKRN1 (H307E) can bind to SNIP1, it does not induce SNIP1 ubiquitination and degradation. This suggests that while this site may not be the key binding site for MKRN1 and SNIP1, it is essential for MKRN1 ubiquitination of SNIP1. Therefore, further research is warranted on the synthesis of targeted compounds against the H307E mutant site of MKRN1 for the inhibition of CRC.

[0230] SNIP1 can undergo SUMOylation at K5, K30, and K108 sites. After SNIP1 SUMOylation, its interference with Smad complex formation is weakened, leading to increased expression of TGF-β target genes and promoting TGF-β signaling-mediated cell migration and invasion. Therefore, we verified the role of SNIP1 in CRC cells. Western blotting and Transwell assays confirmed that silencing SNIP1 promotes EMT and migration in CRC cells, while overexpression of SNIP1 inhibits EMT and migration. Reversal experiments demonstrated that SNIP1 overexpression reversed the activation of EMT markers by overexpressed MKRN1, and SNIP1 silencing reversed the inhibition of EMT markers by sh1-MKRN1. This study is the first to report a negative correlation between MKRN1 and SNIP1 expression levels in CRC, confirming that SNIP1 can act as a tumor suppressor, being degraded at the protein level by MKRN1 ubiquitination. In this study, we demonstrated that MKRN1 promotes CRC cell metastasis and EMT processes through ubiquitination and degradation of SNIP1.

[0231] We confirmed a positive correlation between MKRN1 and the TGF-β pathway using bioinformatics analysis and Western blotting. Furthermore, using a reversion assay, by adding TGF-β signaling inducers and inhibitors, we demonstrated that high expression of MKRN1 can promote TGF-β signal transduction, thereby inducing EMT in CRC cells.

[0232] We validated the relationship between SNIP1 and the TGF-β signaling pathway in CRC cells. The results showed that overexpression of SNIP1 inhibited p-Smad2 / 3 expression, indicating that SNIP1 can suppress TGF-β signaling. However, it also inhibited TGF-β1 expression, suggesting a possible alternative regulatory mechanism between SNIP1 and TGF-β1, which requires further investigation. Furthermore, we demonstrated through reversal experiments that silencing SNIP1 could reverse the inhibition of TGF-β signaling pathway markers by sh1-MKRN1, and overexpression of SNIP1 could reverse the activation of TGF-β signaling pathway markers by OE-MKRN1.

[0233] We performed IHC staining on tissue sections from clinical patients, and the results showed that the MKRN1 low-expression group had higher levels of SNIP1, E-cadherin, and reduced TGF-β1 expression. This study, by combining clinical specimens with cellular level findings, demonstrates that MKRN1 overexpression promotes TGF-β signaling transduction through ubiquitination and degradation of SNIP1, thus playing an important role in the development of CRC.

[0234] In this study, we discovered that MKRN1 is a novel driver of CRC progression. First, we found that MKRN1 expression in CRC tissues was significantly higher than in non-tumor tissues and was positively correlated with CRC TNM stage. Second, high MKRN1 expression also promoted CRC cell invasion, migration, and EMT. Next, we identified SNIP1, the ubiquitination substrate of MKRN1, and revealed that MKRN1 can degrade SNIP1 protein via the ubiquitin-proteasome pathway, and that the E3 ubiquitin ligase activity of MKRN1 is crucial for MKRN1 ubiquitination and degradation of SNIP1. Furthermore, high levels of MKRN1 can activate the TGF-β signaling pathway. Finally, we demonstrated that MKRN1 promotes TGF-β signaling transduction through ubiquitination and degradation of SNIP1, thereby inducing CRC metastasis. These results suggest that MKRN1 may play a unique role in the molecular mechanisms of CRC development. In this study, we provided a reliable molecule that can serve as a potential biomarker and prognostic indicator for predicting CRC metastasis, contributing to CRC treatment. Furthermore, the MKRN1 / SNIP1 / TGF-β axis may provide new targets for the development of CRC anti-metastasis drugs.

[0235] 4. Conclusion

[0236] 1. MKRN1 is highly expressed in CRC cells and tissues, and it can promote the metastasis of CRC cells.

[0237] 2. SNIP1 is a novel ubiquitination substrate of MKRN1. MKRN1 degrades SNIP1 via the ubiquitin-proteasome pathway, thereby promoting EMT and metastasis in CRC cells.

[0238] 3. High expression of MKRN1 can promote TGF-β signal transduction, thereby inducing EMT in CRC cells.

[0239] 4. MKRN1 promotes EMT and metastasis of CRC cells by ubiquitinizing and degrading SNIP1, thereby promoting TGF-β signal transduction.

[0240] 5. MKRN1 may serve as a potential biomarker and prognostic indicator for predicting CRC metastasis, and developing therapeutic drugs targeting MKRN1 could contribute to the treatment of CRC.

[0241] Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.

Claims

1. The application of shRNA that inhibits the MKRN1 gene in the preparation of reagents that promote SNIP1 expression, characterized in that, The shRNA that inhibits the MKRN1 gene contains any one or more of the following three sequences: sh1: 5′-TGC TGT TGA CAG TGA GCG CCC TGG TTA TCC CAT ATA ATA ATA GTG AAGCCA CAG ATG TAT TAT TAT ATG GGA TAA CCA GGT TGC CTA CTG CCT CGG A- 3′ sh2: 5′-TGC TGT TGA CAG TGA GCG ACG GAA CTA TGT GAT CCC GGA ATA GTG AAGCCA CAG ATG TAT TCC GGG ATC ACA TAG TTC CGG TGC CTA CTG CCT CGG A- 3′ sh3: 5′-TGC TGT TGA CAG TGA GCG CTG CTG TGT GGG ATA AAC AGT ATA GTG AAGCCA CAG ATG TAT ACT GTT TAT CCC ACA CAG CAA TGC CTA CTG CCT CGG A- 3′.

2. The application of shRNA that inhibits the MKRN1 gene and increases SNIP1 expression in the preparation of anti-colorectal cancer drugs, characterized in that, The shRNA that inhibits the MKRN1 gene and increases SNIP1 expression contains any one or more of the following three sequences: sh1: 5′-TGC TGT TGA CAG TGA GCG CCC TGG TTA TCC CAT ATA ATA ATA GTG AAGCCA CAG ATG TAT TAT TAT ATG GGA TAA CCA GGT TGC CTA CTG CCT CGG A- 3′ sh2: 5′-TGC TGT TGA CAG TGA GCG ACG GAA CTA TGT GAT CCC GGA ATA GTG AAGCCA CAG ATG TAT TCC GGG ATC ACA TAG TTC CGG TGC CTA CTG CCT CGG A- 3′ sh3: 5′-TGC TGT TGA CAG TGA GCG CTG CTG TGT GGG ATA AAC AGT ATA GTG AAGCCA CAG ATG TAT ACT GTT TAT CCC ACA CAG CAA TGC CTA CTG CCT CGG A- 3′.

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