| Background:RHBDD1,a new member of the rhomboid fam ily,is overexpressed in a variety of malignancies,including non-small cell lung cancer(NSCLC),which is associated with the overall low survival rate of NSCLC patients.RHBDD1 highly regulates apoptosis by cleaving the pro-apoptotic Bcl-2 family protein BIK.Therefore,the high expression of RHBDD1 in tumor tissues may directly affect tumor progression by preventing apoptosis.The fragment produced by catalytic cleavage of RHBDD1 can be degraded by proteasome through ER-associated degradation(ERAD).Ubiquitination has been shown to be involved in a variety of physiological processes.The ubiquitination process involves kinds of ERAD components,including E2 binding enzymes,E3 ligases and E4 factors.K48 linked ubiquitin chains were found as degradation signals.The fragment produced by the catalytic cleavage of ubiquitination related protein RHBDD1 can be degraded by proteasome through ERAD mechanism.RHBDD1 plays an important role in the development of non-small cell lung cancer.Objective:To clarify the expression of RHBDD1 in NSCLC and explore its clinical significance;to clarify the relationship between RHBDD1 and NSCLC cell proliferation.To explore the effect of RHBDD1 on apoptosis and its possible molecular mechanism,in order to provide a theoretical basis for the potential possibility of RHBDD1 as a treatment for non-small cell lung cancer.Methods:1.The online tool gepia2(http://gepia2.cancer-pku.cn/)was used to verify the expression level of RHBDD1 in non-small cell lung cancer from clinical samples of lung adenocarcinoma(LUAD)and lung squamous cell carcinoma(LUSC)in TCGA(The Cancer Genome Atlas,https://portal.gdc.cancer.gov/).At the same time,the correlation between the expression level of RHBDD1 and the pathological stage of non-small cell lung cancer was analyzed by online data.The expression level and location of RHBDD1 in NSCLC tissues were detected by RT-qPCR and immunohistochemistry.In addition,we used the Kaplan Meier plotter public database(http://kmplot.COM/analy sis/)to analyze the prognostic value of RHBDD1 in patients with non-small cell lung cancer.At the same time,the relationship between the expression level of RHBDD1 and clinicopathological factors in patients with non-small cell lung cancer was also counted.2.Western blot was proceeded to detect the protein expression level of RHBDD1.Human RHBDD1 gene sequence was found in Genebank database,siRNA and pcDNA-RHBDD1 targeting RHBDD1 gene were designed,and then the knockdown and overexpression vectors targeting RHBDD1 gene were constructed.After verification and transfection,RHBDD1 gene was knocked down or overexpressed in H1299 and A549 cell lines.CCK-8 assay was used to detect cell activity and cell clone formation assay was used to study the role of RHBDD1 in cell proliferation in vitro.The regulatory effect of RHBDD1 on cell cycle was detected by flow cytometry.The xenograft tumor experiment was carried out to verify the regulatory effect of RHBDD1 on tumor growth in mice,and the expression level of tumor growth related molecules was detected by immunohistochemistry.The secretion level of TGF-α was detected by ELISA,the protein expression level of main molecules of EGFR/Raf/MEK/ERK signaling pathway was determined by Western blot,the cell viability of H1299 or A549 cells treated with EGFR or MEK inhibitor was evaluated by CCK-8 assay,and the proliferation of H1299 or A549 cells treated with EGFR or MEK inhibitors was detected by colony formation assay.3.The proportion of apoptotic cells was detected by flow cytometry,and the protein expression levels of cleaved caspase-3 and cleaved caspase-7,full-length BIK and cleaved BIK were measured by western blot.The expression of K48 ubiquitin chain(K48-Ub)was detected by immunofluorescence assay.Results:1.RHBDD1 was up-regulated in clinical samples of lung adenocarcinoma and lung squamous cell carcinoma in TCGA(cancer genome map)database,as well as in non-small cell lung cancer tissues we collected.At the same time,we found that the expression level of RHBDD1 was up-regulated with the increase of pathological stage.In 45 pairs of non-small cell lung cancer tissues and their corresponding adjacent tissues,RHBDD1 was significantly up-regulated in NSCLC tissues,which was related to pathological tumor stage,and the expression level increased with the increase of the pathological stage.The protein expression levels of RHBDD1 in H1299 and A549 cell lines were significantly higher than that in the other three cell lines.In addition,Kaplan survival analysis showed that the overall survival rate of NSCLC patients with higher RHBDD1 expression levels in NSCLC was lower than that of NSCLC patients with lower RHBDD1 expression levels.At the same time,it was found that the expression of RHBDD1 was significantly correlated with lymph node metastasis,tumor size,pT stage and Ki-67 index in collected clinical samples.These results suggest that RHBDD1 may promote the occurrence and development of non-small cell lung cancer.2.The expression level of RHBDD1 in the selected non-small cell lung cancer cell lines was higher than that in the control cell lines.The expression levels of RHBDD1 protein in H1299 and A549 cells were significantly higher than that of the other three cell lines,which were then selected for further experiments.RHBDD1 knockdown inhibited the proliferation of H1299 and A549 cells in vitro,resulting in cell cycle arrest in G0/G1 phase.On the contrary,RHBDD1 overexpression significantly increased the proliferation of cells in vitro and the number of S-phase cells.In vivo experiments showed that RHBDD1 gene knockdown significantly reduced the size of tumor and inhibited the expression level of PCNA and Ki-67.On the contrary,RHBDD1 overexpression significantly increased the size of tumor and increased the expression level of PCNA and Ki-67.RHBDD1 knockdown inhibited TGF-αsecretion in H1299 or A549 cells,and then inhibited EGFR/Raf/MEK/ERK signaling pathway.In contrary,overexpression of RHBDD1 significantly stimulated TGF-αsecretion and activated the EGFR/MEK/ERK signaling pathway.H1299 or A549 cells were treated with EGFR or MEK inhibitors,and the cell growth activity and proliferation ability were significantly inhibited,especially after treatment with EGFR inhibitors,the cells almost did not grow.3.Knockdown and overexpression of RHBDD1 gene resulted in the decrease and increase of BIK cleavage,respectively,but this effect could be blocked by treating cells with proteasome inhibitors.At the same time,we also detected the expression of ubiquitinated protein K48 and found that the overexpression of RHBDD1 in H1299 cells promoted the accumulation of endogenous K48 ubiquitinated protein.In contrast,knockdown of RHBDD1 further inhibited the expression of K48 polyubiquitinated protein.Conclusion:1.In conclusion,through online tool analysis and clinical sample detection,it was found that RHBDD1 was significantly up-regulated in NSCLC tissues and cells,and survival analysis showed that the overall survival rate of NSCLC patients with higher RHBDD1 expression was significantly lower than that of NSCLC patients with lower RHBDD1 expression,indicating that RHBDD1 is very important in the development of NSCLC.2.RHBDD1 promoted the growth activity and cell proliferation of non-small cell lung cancer cells in vitro,and enhances the growth level and speed of tumors in vivo.At the same time,at the cellular level,our research showed that RHBDD1 could enhance cell viability and promote cell proliferation by regulating EGFR/Raf/MEK/ERK signaling pathway.3.RHBDD1 promoted the progress of NSCLC by promoting BIK cleavage,inhibiting apoptosis,and promoting the degradation of proteins unfavorable to cancer cells(unfolded/misfolded proteins,etc.). |
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