| Objective: The expression pattern of TMEM120 B in humanity tissues,particularly in malignant tumors,remains unclear.The results of bioinformatics and immunohistochemical staining indicated that the expression of TMEM120 B in lung cancer,breast cancer,gastric cancer,colon cancer and ovarian cancer was significantly higher than that in corresponding normal tissues.High cytoplasmic expression of TMEM120 B in breast cancer was positively correlated with high TNM staging,lymph node metastasis and poor prognosis.TMEM120 B promotes proliferation,invasion and stemness of breast cancer cells through TAZ-mTOR signaling.Further,TMEM120 B activates β1-integrin by binding its coil-coiled domain to the coil-coiled domain of MYH9,and accelerates the assembly of local adhesion plaques(FAs)to activate TAZ-mTOR signaling.Overexpression of TMEM120 B inhibits DNA damage and increases the IC50 of docetaxel and doxorubicin.After overexpression of TMEM120B-CCD,proliferation,invasion,stemness and inhibition of DNA damage mediated by TMEM120 B were cancelled.In human breast cancer tissues,TMEM120 B is significantly correlated with the expression of TAZ,p-mTOR and SOX2.The expression of TMEM120 B in breast cancer patients receiving neoadjuvant chemotherapy is significantly higher than that before chemotherapy.While its expression in treatment-resistant patients(MP1-2)was significantly higher than that in treatment-sensitive patients(MP3-5).In summary,we found that TMEM120 B combined with MYH9 enhanced breast cancer cell stemness and chemotherapy resistance through the β1-integrin/FAK-TAZ-mTOR signaling axis.Methods: 1.The expression of TMEM120 B in malignant tumors was universal was detected by TCGA database pancarcinoma analysis and immunohistochemical pancarcinoma.2.The m RNA expression of TMEM120 B was detected by the breast cancer in the TCGA database in paired or unpaired samples,and the histopathologic sections of breast cancer patients were collected for immunohistochemical staining.Meanwhile,the relationship between the expression level and the clinicopathological factors of the disease was also analyzed.3.The relationship between TMEM120 B expression and prognosis of breast cancer patients was evaluated by Kaplan-Meier analysis and statistical analysis after immunohistochemical staining,and the relationship between TMEM120 B m RNA expression and SBR grading and distant metastasis was evaluated by bioinformatics.4.16 pairs of fresh breast cancer tissues and paired adjacent tissues were collected and the TMEM120 B level was detected by western blotting.5.The expression levels in seven types of breast cancer cells and normal breast cells were detected by western blotting,and the subcellular localization was verified by immunofluorescence.6.Transfection plasmid overexpression and Crispr-Cas9 knockout of TMEM120 B were used,and the effect of overexpression or knockout of TMEM120 B on the migration and invasion ability of breast cancer cells was detected by MTT,EDU,Colony formation,Wound healing,and Transwell.7.The effect of overexpression of TMEM120 B on the proliferation and invasion of breast cancer was verified by Tumor formation and Tail injection in nude mice.8.The genes were analysised by high-throughput RNA-sequencing analysis,GO and KEGG analysis.9.The effect of TMEM120 B on the stemness of breast cancer was confirmed by western blotting,Aldeflour analysis,Sphere forming and Secondary sphere forming,and Tumor formation,and Diluted injection of nude mice in vivo.10.The mechanism of TMEM120 B regulating stemness was explored by array analysis of phosphorylated antibody and GSEA analysis.11.The mechanism of the TMEM120 B regulatory pathway was verified by q PCR,nucleoplasm separation,immunofluorescence and CHX protein stability,CO-IP.The experiment was also restored by the pathway inhibitor.12.The potential binding protein of TMEM120 B was searched by Mass Spectrometry(MS),and the binding relationship was verified by endogenous CO-IP,exogenous CO-IP and GST-pull down test.13.The specific domain of their combination was detected by a series of truncated mutants of TMEM120 B and MYH9.14.To verify the effect of TMEM120 B on 3D invasion was verified by 3D collegen gel experiment.15.The effect of TMEM120 B on Integrin/FAK signals was verified by the Nocontazole model.16.The effect of TMEM120 B on doxorubicin and docetaxel chemoresistance was detected by bioinformatics and IC50 assays.Miller Grade I and Ⅳ pathological sections were collected before and after chemotherapy and immunohistochemical staining was performed on TMEM120 B.18.Immunohistochemical experiments confirmed that the expression of TMEM120 B was positively correlated with the expression of TAZ and p-mTOR and the expression of SOX2,a marker of breast cancer stem cells,respectively.Results: 1.The expression of TMEM120 B protein in lung cancer(P=0.029),breast cancer(P=0.009),gastric cancer(P=0.031),colon cancer(P=0.013)and ovarian cancer(P=0.029)was higher than that in adjacent normal tissues.The results of immunohistochemical staining in 139 cases of breast cancer and 42 cases of adjacent breast tissue indicated that TMEM120 B was mainly located in cytoplasm and a small amount in nuclear.The positive expression rate of TMEM120 B was 50.3%(70139),which was significantly higher than that of adjacent normal breast tissue(23.8%,10/ 42,P=0.001).Further analysis of the relationship between the expression of TMEM120 B and clinicopathological features,the positive expression of TMEM120 B was significantly positively correlated with high TNM stage(P= 0.017)and lymph node metastasis(P=0.005),but had no significant correlation with age and triple-negative breast cancer(P0.05).The survival time of patients with high TMEM120 B expression was higher than negative expression.2.Overexpression or knockout TMEM120 B can promote or inhibit the proliferation of breast cancer cells.Furthermore,in vivo tumor growth indicated that the volume of transplanted tumors was significantly higher than the control group after overexpression of TMEM120 B,and the number of lung metastases was significantly increased.3.The differential genes that overexpress TMEM120 B are mainly concentrated in the biological processes of stem cells,cytoskeleton and focal adhesion plaques,etc.TMEM120 B can enhance the stemness of breast cancer cells by western blotting assay,aldeflour flow analysis,pellet formation and secondary pellet formation assay,and in vivo tumor dilution injection in nude mice.4.The TMEM120 B differential gene was mainly involved in Hippo,Wnt,and AKTmTOR signaling through the analysis of RNA-seq,and our experiment found that overexpression TMEM120 B was involved in the regulation of AKT-mTOR and Hippo pathways.Subsequently,we further explored the up-and downstream relationship between TAZ and mTOR signals,and found that TMEM120 B may promote the stemness of breast cancer through TAZ-mTOR signals.Further through q PCR,nucleoplasma separation,immunofluorescence,CHX stability and CO-IP tests,we found that TMEM120 B may maintain the stemness of breast cancer by affecting the stability of TAZ.5.Next,we designed a series of truncated mutants of TMEM120B and MYH9 to detect their binding domains.The CO-IP assays suggested that TMEM120 B might bind to the coil-coiled domain of MYH9 through its coil-coiled domain.6.GO analysis of the potential binding protein of TMEM120 B obtained by mass spectrometry showed that the potential binding protein of TMEM120 B was mainly enriched in the regulatioin of actin cytoskeleton and focal adhesion.Western blotting showed that overexpression of TMEM120 B could up-regulate p-FAK(Tyr397)and active-β1-integrin,while overexpression of TMEM120B-CCD could not.Subsequently,Nocontazole model system was used to study FAs turnover,and TMEM120 B promoted breast cancer progression by accelerating the β1-Integrin cycle to regulate FAs assembly.7.Bioinformatics analysis,IC50 test and drug-resistant pathological sections showed that the IC50 of doxorubicin and docetaxel increased significantly after overexpression of TMEM120 B,and western blotting and immunofluorescence showed that γ-H2AX(marker of DNA damage)decreased significantly.The expression of TMEM120 B was significantly higher in breast cancer patients after neoadjuvant chemotherapy than before chemotherapy,and was significantly higher in treatment-resistant patients(MP 1-2)than in treatment-sensitive patients(MP 3-5).8.Correlation analysis showed that the expression of TMEM120 B was positively correlated with the expression of TAZ,p-mTOR and SOX2.Conclusion: 1.TMEM120 B is highly expressed in breast cancer tissues,and is associated with high TNM staging,lymph node metastasis and poor prognosis of breast cancer patients.2.TMEM120 B promotes the proliferation and invasion of breast cancer in vivo and vitro.3.Overexpression TMEM120 B enhanced the stemness of breast cancer cells.4.TMEM120 B enhances stemness of breast cancer through TAZ-mTOR signal.5.TMEM120 B combines with MYH9’s coil-coiled domain through its coil-coiled domain.6.TMEM120 B combines with MYH9 to activate the TAZ-mTOR signal by accelerating theβ1-integrin cycle to facilitate FAs assembly.7.TMEM120 B promotes chemoresistance to docetaxel and doxorubicin. |
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