LncRNA IKBKBAS Promotes Malignant Progression Of Lung Adenocarcinoma By Inhibiting Ubiquitination Of Prohibitin

Lung cancer is the malignant tumor with the highest incidence and mortality in the world.Among them,non-small cell lung cancer(NSCLC)is the most common histological type of lung cancer,accounting for about 85%of lung cancer.In recent years,the incidence of NSCLC in China has increased greatly,which poses a great threat to people's health.Because there are no obvious symptoms in the initial stage of NSCLC,a large number of patients are already in the late stage at the time of diagnosis,but the effect of radiotherapy and chemotherapy on improving the survival time of patients with advanced NSCLC is relatively limited.With the advent of the era of precision medicine,molecular typing and targeted therapy under the guidance of driving genes has become a key medical means for the treatment of advanced NSCLC.However,there are still a large number of NSCLC patients have not detected known driving genes,so we further explore the molecular mechanism of the occurrence and development of NSCLC.It is of great significance to find the key clinical treatment targets.long non-coding RNAs(lncRNAs)are a class of functional RNA molecules that are longer than 200nt and have not obvious protein coding ability.lncRNA is transcribed by RNA polymerase ?(Pol ?)in most cases.It has many characteristics in common with mRNA,but also shows some unique properties.It has been found that lncRNA is abnormally expressed in many types of tumors,and a series of abnormal-expressed lncRNAs can act as upstream regulators or downstream effectors in oncogenesis by interacting with other molecules.It has been demonstrated that IncRNAs play an important regulatory role in the tumorigenesis.However,the underline mechanism of lncRNAs functions is still unknown and need further study.Human Prohibitin(PHB,also known as PHB1,UniProtKB-P35232)is abnormally expressed in many types of tumors.In the past decade,there has been controversy about the role of PHB1 protein in malignant tumors.Recent studies have shown that membrane-located PHB1 plays an important regulatory role in the MAPK signaling pathway,which is necessary for activation of C-Raf mediated by K-Ras.Its specific inhibitor Rocaglamides or siRNA can significantly slow down tumor growth,indicating that inhibition of PHB1 activity may be a promising cancer treatment strategyIt is known that lncRNAs can participate in the regulation of signaling pathways through interaction with proteins.Whether the lncRNAs take part in the activation of K-Ras:C-Raf:PHB1 interaction through RNA-protein interaction has not been reported.However,the exploration of these potential regulatory mechanisms will be of great significance to find effective prognostic indicators and therapeutic targets,to achieve" individualized treatment"and improve the quality of life of patients with NSCLC.Three pairs of lncRNA chips of lung adenocarcinoma and para-cancerous normal tissues were tested in the early stage of our research group.According to the chip data,a new antisense IncRNA LOC101929897 was screened and named IKBKBAS.The results of Gain-and-loss-of-function experiments and molecular mechanism studies showed that IKBKBAS functioned as competing endogenous RNA(ceRNA)by competitively binding to miR-4741 to upregulate the expression of IKK? at the post-transcriptional level,thereby activating the classic NF-?B signaling pathway,and ultimately promoting the proliferation,migration and invasion of lung adenocarcinoma cells.On the basis of confirming the positive regulatory effect of IKBKBAS on NF-?B signal pathway,and considering that lncRNA often has multiple regulatory functions at the same time,we further studied the other potential roles of IKBKBAS in lung adenocarcinoma.In this study,the lung adenocarcinoma cell lines was taken as the research object,and the functional and molecular mechanisms were studied at multiple levels.The results were as follows:We performed RNA pulldown combined with mass spectrometry and bioinformatics analysis to detect the interaction proteins of IKBKBAS and found that IKBKBAS may target to PHB1.The RNA pulldown and RIP experiments proved the interaction between IKBKBAS and PHB1 protein.Because the biological effect of PHB1 is tissue-specific,but there is no systematic study in lung adenocarcinoma.Thus we first tested the biological function of PHB1 in lung adenocarcinoma cells.The detection of basic expression showed that PHB1 was significantly up-regulated in lung adenocarcinoma tissues and cells.Survival analysis showed that the high expression of PHB1 was associated with poor prognosis of lung adenocarcinoma.We used a variety of experimental methods such as plate clone formation experiment,Transwell chamber,CCK8 to explore the effect of PHB1 on lung adenocarcinoma cells.Compared with control group,plate clone formation experiment showed that over-expression of PHB1 increased the size and number of cell clones of BEAS-2B cells.While the size and number of cell clones in the si-PHB1 transfection group were significantly reduced than those in the control group.CCK8 assay showed that compared with the control group,over-expression of PHB1 promoted the proliferation ability of BEAS-2B cells,and knock-down of PHB1 inhibited the proliferation ability of H1299 and HCC827 cells.The results of Transwell assay showed that,compared with control group,over-expression of PHB1 promoted the migration and invasion ability of BEAS-2B cells,and knock-down of PHB1 inhibited the migration and invasion ability of H1299 and HCC827 cells.These results suggest that PHB1 promotes the proliferation,invasion and migration ability of lung adenocarcinoma cells.Spearman correlation analysis showed that IKBKBAS and PHB1 protein expression levels were positively correlated in lung adenocarcinoma tissues.We found that over-expression of IKBKBAS effectively increased the PHB1 protein level in A549 cells,but was significantly inhibited in HCC827 cells with IKBKBAS knockdown.Subsequent immunofluorescence experiment suggested that there was positive correlation between IKBKBAS and PHB1 protein expression levels.However,there was no significant correlation between IKBKBAS and PHB1 mRNA expression level.It has been confirmed that IKBKBAS can promote the migration and invasion of lung adenocarcinoma cells.We performed rescue experiments to explore whether PHB1 is involved in the malignant progression which mediated by IKBKBAS of lung adenocarcinoma cells.Plate colony formation assay showed that the enhancement of clone formation ability induced by overexpression of IKBKBAS could be partially reversed by knockdown PHB1 and vice versa in lung adenocarcinoma cells.Transwell assay showed that the over-expression of IKBKBAS improved the migration and invasion ability of A549 cells,while the knock-down of PHB1 could be partially reversed it.The decrease of migration and invasion ability caused by knockdown IKBKBAS in H1299 and HCC827 cells could be partially reversed by overexpression of PHB1.CCK8 assay showed that the enhancement of proliferation ability induced by overexpression of IKBKBAS could be partially reversed by knockdown PHB1 and vice versa in lung adenocarcinoma cells.These data demonstrated that IKBKBAS promoted malignant progression of lung adenocarcinoma cells partly through PHB1.The above experiments have demonstrated that IKBKBAS is involved in the proliferation,invasion and migration of lung adenocarcinoma cells,which is partly mediated by PHB1.We further explore its inherent molecular mechanism.Over-expression of IKBKBAS can inhibit the nuclear translocation of PHB1,increase the plasma membrane PHB1 content,and enhance the activation of the phosphorylation cascade of C-Raf/MEK/ERK.This effect can be attenuated by knocking down PHB1.The Knock-down of IKBKBAS in HCC827 cells can inhibit C-Raf/MEK/ERK phosphorylation cascade activation,which can be partially reversed by over-expression of PHB1.The above results show that IKBKBAS promotes the excessive activation of MAPK signaling pathway by inducing PHB1 membrane translocation and inhibiting PHB1 nuclear translocation,which will finally promotes malignant behaviors,such as proliferation,migration and cloning formation ability,in lung adenocarcinoma cells.The previous results showed that IKBKBAS up-regulated PHB1 at the protein level rather than mRNA level and there was interaction between IKBKBAS and PHB1.We thus inferred that IKBKBAS increases PHB1 protein stability by binding PHB1 to inhibit its ubiquitination degradation.Cycloheximide(CHX),an inhibitor of protein synthesis,was used to treat A549 cells.Western Blot results showed that the half-life of PHB1 protein was significantly prolonged after overexpression of IKBKBAS.MG132 experiment showed that IKBKBAS inhibited the degradation of PHB1 protein via ubiquitin-proteasome pathway.In order to explore whether IKBKBAS is involved in the ubiquitin process of PHB1,the following two groups of plasmids were co-transfected into HEK293T cells:?:pCDNA3.1-IKBKBAS+p-ENTER-PHB1+pCDNA3.1-UB;?:pCDNA3.1+p-ENTER-PHB1+pCDNA3.1-UB;Then,Immunoprecipitation experiments results showed that IKBKBAS overexpression could stabilize PHB1 protein by inhibiting its ubiquitin degradation.We further explored the molecular mechanism of IKBKBAS regulating the ubiquitin level of PHB1.According to database prediction combined with previous reports,SKP2B is an E3 ubiquitin ligase targeting PHB1.Co-immunoprecipitation(Co-IP)assay showed that there was endogenous binding between the two proteins in A549 cells and overexpression of IKBKBAS can inhibit the binding of SKP2B to PHB1.The above results prove that IKBKBAS inhibits SKP2B-mediated ubiquitination degradation and thus stabilizes PHB1 protein.Conclusion:Generally,SKP2B binds to PHB1 and degrades it through the ubiquitination pathway.In lung adenocarcinoma cells,overexpressed IKBKBAS inhibits SKP2B-mediated ubiquitination by binding to PHB1.Meanwhile,IKBKBAS could increase the transport of PHB1 to the membrane,which will activate the MAPK signaling pathway and finally promote the malignant progression of lung adenocarcinoma.