| Background:According to the latest research results of the 2022 American Cancer Society Statistics,lung cancer has the highest morbidity of malignant tumors(22%),the highest mortality rate in male,and the second in female(17%),which is next to breast cancer(18%).Cause the onset of lung cancer is hidden and the clinical symptoms are not obvious,it is often found in the middle and late stage,and the opportunity of operation is often lost in the diagnosis.Although with the deepening of molecular typing research and the gradual application of targeted drugs for driver gene(EGFR/ALK/ROS1/BRAF/MET)mutations in clinical practice,the treatment of lung cancer has made a breakthrough.However,low mutation rate and early secondary drug resistance are still severe challenges in the treatment of lung cancer.Studies have shown that the biological characteristics of local infiltration and distant metastasis of lung tumors are the main causes of high incidence rate and mortality of lung cancer.Therefore,in-depth study of the biological process and molecular regulation mechanism of lung cancer invasion and migration has very important clinical significance and application prospect.RNA Binding Motif Protein 10(RBM10),is one of the most important members of RNA Binding Protein(RBP)family.The previous study achievements of our research demonstrated that the changes of its abundance and activity can affect the proliferation of lung tumor cells.However,as an RBP,its function of binding RNA,especially non-coding RNA,is still unclear.Using CLIP-seq high-throughput sequencing technology,we found that RBM10 can bind to lots of lnc RNA.Among them,Metastasis-Associated Lung Adenocarcinoma Transcript 1(MALAT1)is not only a biomarker for clinical diagnosis,but also a candidate drug target for tumor treatment.It can participate in the regulation of a variety of pathophysiological processes and has been proved to be involved in the invasion and metastasis of many tumors.As the most common(more than 60%)RNA epigenetic modification,N6methyladenosine(m6A)methylation has great significance for the pathogenesis of tumors.MALAT1 is a lnc RNA with a highly abundant m6 A methylation site,which has been confirmed in multiple cell lines.Other studies have shown that RBM15 and IGF2 BP families can participate in m6 A methylation related processes.Similarly,RBM10,which is also an RBP,may also be involved in regulating m6 A methylation of RNA and further affect the invasion and metastasis of lung cancer.Objective:The intention of this study is to deeply explore and establish the mechanism of RBM10 regulating m6 A methylation of MALAT1 by recruiting m6 A methyltransferase METTL3,and then affecting the phosphorylation level of downstream PI3K/AKT/m TOR protein,and finally affecting the invasion and migration of NSCLC,so as to establish a solid scientific theory foundation for the development of new targets for NSCLC treatment.Methods:1.To explore the expression of RBM10 and MALAT1 in NSCLC at the histological level(1)Western Blot and immunohistochemical staining(IHC)were applied for the abundance of RBM10 protein in human NSCLC and adjacent tissues.(2)The correlation between RBM10 protein expression and clinical characteristics(histological type,age,gender,smoking history and TNM stage)was statistically analyzed.Using GEPIA database to connect the relevance between RBM10 protein with overall survival and disease-free survival.(3)QRT-PCR was applied for testing the level of MALAT1 in human NSCLC and adjacent tissues.2.Cytological level to explore the effect of RBM10 on the invasion and migration of NSCLC(1)A549 and H460 cell lines were stably transfected by lentivirus transfection to construct RBM10 overexpression and silencing cell lines.The transfection efficiency was evaluated Western Blot.(2)Apply Transwell experiment and cell scratch healing experiment to respectively calculate the changes of invasion and migration ability after overexpressing and silencing RBM10 in A549 and H460 cell lines.Utilize Western Blot and immunofluorescence(IF)to verify whether overexpressing or silencing RBM10 affect the expression of EMT related protein E-cadherin and vimentin.(3)Use Western Blot to clarify whether overexpressing or silencing RBM10 could affect the total and phosphorylation level of downstream PI3K/AKT/m TOR pathway protein.3.To study the mechanism of RBM10 directly binding and regulating MALAT1 affecting the invasion and migration of NSCLC(1)To deeply clarify the mechanism of RBM10 affecting the invasion and migration ability,CLIP-seq was carried on to screen the RNAs bound by RBM10 at the whole genome level.The binding peak(maxheight)and the number of binding tags(tags)were statistically analyzed to screen the lnc RNAs highly bound to RBM10.(2)RNA immunocoprecipitation-real time fluorescence quantitative PCR(RIP-q PCR)was used to verify the direct binding relationship between RBM10 and MALAT1 at the molecular level.(3)QRT-PCR was used to detect the effect of overexpressing and silencing RBM10 on MALAT1 RNA level.(4)To verify that RBM10 regulate the phosphorylation of downstream PI3K/AKT/m TOR pathway protein through MALAT1,and finally affect the invasion and migration of NSCLC: In RBM10 silencing cell line,MALAT1 was knocked down by si RNA,before and after restoring MALAT1,the level of total and phosphorylated protein of PI3 K,AKT,m TOR were detected by Western Blot.The changes of cell invasion and migration ability were detected by Transwell X assay and cell scratch healing assay.4.Clarify the mechanism of RBM10 recruiting METTL3 to regulate m6 A methylation of MALAT1(1)To further clarify the mechanism of RBM10 regulating MALAT1,the m6 A methylation site of MALAT1 was predicted by database SRAMP,and specific primers were designed for MALAT1 m6 A methylation site.The samples of each group were compared with input.The IP experimental samples of each group were precipitated with specific m6 A antibody.The total RNA of negtive control,RBM10 overexpression group and silencing group was extracted.The genome digestion and the degradation degree of samples was identified by RNA electrophoresis.(2)The m6 A methylation level of MALAT1 was quantitatively detected by Me RIPq PCR: The methylation level of exogenous negative,exogenous positive and endogenous positive m6 A modification sites was detected to ensure the effective enrichment of m6A;M6A methylation level(input%)of all methylated sites of MALAT1 was detected respectively;The effects of overexpression and silencing RBM10 on m6 A level of all methylated sites of MALAT1 were detected and analyzed.(3)The protein-protein interaction between METTL3 and RBM10 was analyzed by STRING database.Immunoprecipitation was used to clarify whether RBM10 recruiting with the m6 A key methyltransferase METTL3 of MALAT1.Results:1.Expression of RBM10 and MALAT1 in NSCLC tissues(1)Western Blot and IHC showed that the expression of RBM10 in NSCLC tissues was lower than that in adjacent tissues,including LUAD(P<0.0001)and LUSC(P<0.001).(2)The expression level of RBM10 protein was correlated with gender(P=0.0151).It was generally lower in male patients,and was not related to histological type,age,TNM stage and smoking history;Patients with high expression of RBM10 protein had longer overall survival(P=0.0027)and disease-free survival(P<0.0001).(3)QRT-PCR showed that the level of MALAT1 in adjacent tissues was lower than that in NSCLC tissues(P<0.0001).2.Overexpressing and silencing RBM10 affect the invasion and migration of NSCLC(1)The stable overexpression and silencing RBM10 cell lines were successfully constructed.Overexpression of RBM10 significantly reduced the ability of cell invasion and migration(P<0.05),while silencing RBM10 significantly enhanced cell invasion and migration(P<0.05).(2)Overexpression of RBM10 up-regulated the expression of E-cadherin and downregulated the expression of vimentin(P<0.05);When RBM10 was silent,it was the opposite(P<0.05).(3)RBM10 decreased the levels of p-PI3 K,p-AKT and p-m TOR(P<0.05),while when RBM10 was silenced,the phosphorylation level of PI3 K,AKT and m TOR increased(P<0.05).There was no significant change in the total protein levels of PI3 K,AKT and m TOR.3.RBM10 directly binds and regulates MALAT1,which affects the invasion and migration of NSCLC(1)To clarify the mechanism of RBM10 affecting the invasion and migration ability of NSCLC,CLIP-seq was used to show that RBM10 could bind 4040 RNAs,including 357 lnc RNAs,of which MALAT1 had a binding maxheight of 49,the third place at the whole genome level,and the number of binding tags was 680,which ranks first in the whole genome level.(2)RIP-q PCR was used to verify that RBM10 directly binds to MALAT1.Taking input group and Ig G antibody group as control,it was both proved that RBM10 directly binds to MALAT1(P<0.0001).(3)QRT-PCR results showed that the level of MALAT1 decreased in RBM10 overexpression cell(P<0.05),but increased in RBM10 silencing cell(P<0.05).(4)To further verify that RBM10 affects the phosphorylation of downstream PI3K/AKT/m TOR protein by regulating MALAT1,and finally affects the invasion and migration of NSCLC,in RBM10 silencing cells,MALAT1 was knocked down by XII si RNA: The level of MALAT1 and the phosphorylation level of PI3K/AKT/m TOR pathway increased after RBM10 silencing.After MALAT1 was rescued in this cell line,the phosphorylation level of the pathway decreased again(P<0.05),and the total protein was not affected.After RBM10 silencing,the level of MALAT1 and the cell invasion and migration ability increased.After MALAT1 was rescued in RBM10 silencing cell,the invasion and migration ability decreased again(P< 0.05).4.RBM10 regulates m6 A methylation of MALAT1 by recruiting methyltransferase METTL3(1)To clarify the mechanism of RBM10 regulating MALAT1,the database SRAMP was used to predict the m6 A methylation site of MALAT1.The results showed that there were 6 m6 A methylation high reliability sites in MALAT1(2539,2601,2635,2649,2656,2744).Specific primers were designed based on the principle of full coverage,and these sites were divided into three groups(cluster1,site12 and cluster3).(2)Me RIP-q PCR was used to quantitatively detect MALAT1 m6 A level.The results showed that when RBM10 was overexpressed,the m6 A levels of all methylated sites of MALAT1(cluster1,site12 and cluster3)decreased,and when RBM10 was silent,the m6 A levels of all methylated sites increased,indicating that RBM10 could inhibit the m6 A methylation levels of all methylated sites in MALAT1(P< 0.05).(3)STRING PPI network analysis indicated that METTL3 interacted with RBM10.Protein immunoprecipitation(IP)results showed that there was a binding relationship between RBM10 and m6 A methyltransferase METTL3,suggesting that RBM10 may regulate the m6 A methylation process of MALAT1 by recruiting METTL3.Conclusion:1.The expression of RBM10 in NSCLC tissues was lower than that in adjacent tissues,and the expression of MALAT1 in NSCLC tissues was higher than that in adjacent tissues.Patients with high RBM10 expression had longer overall survival and disease-free survival.2.RBM10 inhibit the EMT process and invasion and migration ability of NSCLC.3.RBM10 down regulates the phosphorylation level of PI3K/AKT/m TOR pathway protein by regulating the level of MALAT1,and finally inhibits the invasion and migration of NSCLC.4.RBM10 could directly bind MALAT1 by recruiting m6 A methyltransferase METTL3 to inhibit the m6 A methylation level of MALAT1 and realize the epigenetic regulation of MALAT1. |
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