Function And Mechanism Study Of Mex3a In Lung Adenocarcinoma

Background:Lung cancer is currently the malignant tumor with the highest incidence and the highest mortality in the world.Lung cancer is classified into non-small cell lung cancer and small cell lung cancer according to pathological types.Among them,lung adenocarcinoma accounts for more than 80%of non-small cell lung cancer.Early screening,reform of surgical methods,and in-depth research on radiotherapy and chemotherapy have improved the survival rate of patients with lung adenocarcinoma to a certain extent,but it is still not effective for patients with metastasis and resistance to drug resistance during treatment.The survival rate is still very low.The pathogenesis of tumors is a complex process of multi-step and multi-factor regulation,which is currently unclear.Reports have shown that the incidence of lung adenocarcinoma is mainly the result of genetic,environmental and mutations.The activation of proto-oncogenes and the inactivation of tumor suppressor genes are important factors in the development of tumors.Molecular targeted therapy is to inhibit the key molecules in tumorigenesis and development to achieve the effect of inhibiting tumor growth and metastasis.For example,EGFR tyrosine kinase inhibitors were aimed at the most common mutant gene EGFR in lung adenocarcinoma.Studies have shown that the median survival time of lung adenocarcinoma patients receiving targeted therapy is 3 years,while the median survival time of lung adenocarcinoma patients who have not received targeted drug therapy is only about one year;The emergence of immune checkpoint inhibitor against PDL1 and PD1 has opened a new era of immunotherapy for lung adenocarcinoma.Molecular targeted therapy has brought great help to the treatment of patients with lung adenocarcinoma,but there are still some patients with lung adenocarcinoma that have not found existing molecular targeted drug targets,and most patients have secondary drug resistance.Therefore,exploring new molecular targets for the occurrence and development of tumor cells is essential to further increase the treatment of lung adenocarcinoma and improve the prognosis of patients.Existing studies have proved that RNA binding protein(RBP)plays a vital role in the occurrence and development of tumors.RBP participates in all the steps of post-transcriptional regulation and determines the fate and function of each transcript in the cell to ensure the homeostasis of the cell.It is now clear that some RBPs are dysregulated in different types of cancer,thereby affecting the expression and function of oncoproteins and tumor suppressor proteins.They form a complex with other proteins,can interact with coding or non-coding RNA,regulate RNA splicing,polyadenylation,stability,localization,translation and degradation.Although the research on RBP is progressing rapidly,most of the functions of RBP are still unknown,and the reports of RBP related to lung adenocarcinoma are even rarer.Mex3a belongs to a class of RBP family with conserved gene sequence.The Mex3 family was first discovered in C.elegans,including Mex3a,Mex3b,Mex3c,and Mex3d,which play an important role in the pathophysiological process of differentiation,proliferation,autophagy,apoptosis,inflammation,and tumors.The structure of Mex3a protein has a KH domain capable of binding RNA and a ring finger(RF)domain as an E3 ubiquitin ligase.The initial study found that Mex3a participates in maintaining the homeostasis of the intestinal mucosal epithelium through post-transcriptional modification of the CDX2 gene.The expression of Mex3a is increased in glioma,esophageal squamous cancer,liver cancer,gastric cancer,breast cancer and pancreatic cancer,and high expression of Mex3a is associated with poor prognosis.A large number of studies have shown that Mex3a promotes the proliferation,invasion and migration of tumor cells.But what role Mex3a plays in lung adenocarcinoma is still unknown.Therefore,discovering the function of Mex3a and deciphering the complex interaction network between Mex3a and its RNA targets related to lung adenocarcinoma will provide the basis for revealing the molecular mechanism of lung adenocarcinoma invasion and metastasis,and may reveal new lung adenocarcinoma treatments target.In this experiment,after clarifying the role of Mex3a in the progression of lung adenocarcinoma,transcriptome sequencing and RNA immunoprecipitation analysis were used to screen out the gene laminin alpha 2 chain(laminin subunit alpha 2,LAMA2)that interacts with Mex3a.LAMA2 belongs to the extracellular glycoprotein family and is an important part of the extracellular matrix(ECM).Current studies have found that LAMA2 mutation is an important cause of muscular dystrophy(myodystrophy,MDS).In colon cancer,ovarian cancer,and pituitary tumors,it was found that the expression of LAMA2 mRNA was reduced and there was a lot of methylation in DNA.Functional experiments found that knocking down LAMA2 can increase the metastatic characteristics of tumor cells and promote the activation of the PI3K/AKT pathway.However,the study of LAMA2 in lung adenocarcinoma has not been reported.In this study,the RNA-binding protein Mex3a was screened by the lung adenocarcinoma tissue mRNA expression profile chip,and then the expression of Mex3a was detected in the lung adenocarcinoma tissue samples,and its relationship with the clinicopathological parameters of lung adenocarcinoma was analyzed.In vivo and in vitro functional experiments are conducted to explore the effects of Mex3a on the proliferation,invasion and metastasis of lung adenocarcinoma cells,and to find out the direct binding mRNA and downstream pathways that regulate the molecular mechanism of Mex3a's regulation of lung adenocarcinoma invasion and metastasis.Method:(1)Screen the up-regulated RBP-Mex3a in lung adenocarcinoma tissue by transcriptome chip sequencing,and verify the expression changes of Mex3a mRNA in lung adenocarcinoma tissue and normal tissue through GEO and TCGA databases.(2)Detect the expression of Mex3a protein in lung adenocarcinoma tissues and adjacent tissues by immunohistochemistry(IHC)and western blot technology,and analyze the expression level of Mex3a protein according to the H score results of IHC.The relationship between the clinicopathological characteristics of patients with lung adenocarcinoma,the analysis of follow-up data of patients with lung adenocarcinoma,the relationship between the expression of Mex3a and the survival time of patients with lung adenocarcinoma was drawn by the Kaplan-Meier method.(3)Detect the expression of Mex3a gene in lung adenocarcinoma cell lines by real-time quantitative reverse transcription PCR(qRT-PCR)and western blot,and screen out endogenous cells with low Mex3a and high Mex3a expression.Select H1299 and A549 cell lines with high Mex3a expression to transfect Mex3a small interference,and construct H1299 and A549 cell lines that stably interfere with Mex3a through lentiviral transfection,and select PC9 cell lines with low Mex3a expression to transfect Mex3a overexpression plasmid.(4)Observe the effect of Mex3a on lung adenocarcinoma cell proliferation through CCK8 and EDU experiments,observe the effect of Mex3a on lung adenocarcinoma cell invasion and migration through wound healing and Transwell experiments,and detect the impact of Mex3a on lung adenocarcinoma cell epithelial mesenchymal transition through western blot.(5)Use packaged Mex3a shRNA and control lentivirus to infect lung adenocarcinoma cells,construct Mex3a stable knockdown lung adenocarcinoma cell lines and control cell lines after puromycin screening,and inject them into nude mice through the subcutaneous and tail veins to observe the effect of Mex3a on the growth,invasion and metastasis of lung adenocarcinoma cells under in vivo conditions.(6)Screen the possible binding mRNA of Mex3a and the downstream signal pathways by transcriptomics sequencing,and verify the interaction between Mex3a and LAMA2 mRNA by qRT-PCR and RNA immunoprecipitation(RIP).The actinomycin experiment proved that the regulation of LAMA2 expression by Mex3a was achieved by affecting the stability of target gene RNA.(7)Use GEO,TCGA database and clinical samples to verify the correlation between Mex3a and LAMA2 and the expression changes of LAMA2 mRNA between lung adenocarcinoma tissues and normal tissues.Detect the expression of LAMA2 protein in lung adenocarcinoma tissues and adjacent tissues by immunohistochemistry,and analyze the relationship between LAMA2 protein expression level and clinicopathological characteristics of lung adenocarcinoma patients according to the H score results of IHC.The follow-up data were sorted and analyzed by Kaplan-Meier method to draw the relationship between the expression of LAMA2 and the survival time of patients with lung adenocarcinoma.(8)Detect the expression of LAMA2 gene in lung adenocarcinoma cell lines by qRT-PCR,select the cell line PC9 with high expression of LAMA2 to transfect LAMA2 small interference,observe the invasion and migration ability of LAMA2 on lung adenocarcinoma cells by wound healing and Transwell experiment.The effect of LAMA2 on the epithelial-mesenchymal transition of lung gland cells was detected by western blot.Rescue experiments in vivo and in vitro further confirmed that Mex3a promotes lung adenocarcinoma metastasis by regulating LAMA2.Result:(1)The expression level of Mex3a in lung adenocarcinoma tissue is increased and is associated with poor clinical prognosisHigh-throughput mRNA expression profiling chip(GSE140797)was used to detect 7 pairs of lung adenocarcinoma tissue and adjacent tissue samples to screen for significantly differentially expressed mRNA.Chip data showed that in the Mex3 family(Mex3a,Mex3b,Mex3c,Mex3d),only Mex3a was expressed in 7 cases of lung adenocarcinoma tissues significantly higher than the adjacent tissues.Through GEO(GSE 19804,GSE 116959)and the TCGA database,it was found that the expression of Mex3a mRNA in lung adenocarcinoma tissue was significantly higher than that in adjacent tissues.Western blot and immunohistochemical staining showed that Mex3a was highly expressed in lung adenocarcinoma tissues,but almost not expressed in adjacent tissues.Correlation analysis of clinicopathological parameters showed that in lung adenocarcinoma tissue,high expression of Mex3a was positively associated with lymph node metastasis(P=0.038),TNM stage(P=0.007),tumor differentiation(P=0.028)and patient survival status(P=0.018),but there was no significant correlation with parameters such as age,gender,tumor size,and metastasis.Kaplan-Meier survival curve analysis found that patients with lung adenocarcinoma with higher Mex3a expression had a shorter overall survival time(OS,P=0.0016).Secondly,we analyzed the lung adenocarcinoma website(KM Plotter)online and found that compared with patients in the Mex3a lower expression group,the overall survival time(P<0.0001)and disease-free survival time(PFS,P<0.0001)significantly shortened.(2)Mex3a promoted LUAD cell proliferation,migration and invasion in vitroThe results of qRT-PCR and western blot showed that Mex3a was expressed in lung adenocarcinoma cell lines(A549,H1299,HCC827 and H1975).The expression of PC9 was the lowest,and the expression of H1299 and A549 was the highest.The qRT-PCR results showed that compared with the control group,the two small interference sequences(Mex3a-sil and Mex3a-si2)and the overexpression plasmid(Mex3a-OE)could effectively reduce and increase the expression of Mex3a in lung adenocarcinoma cells.CCK8 and EDU cell proliferation experiments showed that the proliferation ability of Mex3a was significantly weakened after interference with Mex3a,and the proliferation ability of lung adenocarcinoma cells was significantly increased after overexpression of Mex3a.qRT-PCR and Western blot results showed that transfection of Mex3a siRNA significantly inhibited the expression of Mex3a,while transfection of overexpression plasmids could significantly up-regulate the expression of Mex3a in lung adenocarcinoma cells.Wound healing and Transwell results showed that interference with Mex3a inhibited the migration and invasion of lung adenocarcinoma cells,while overexpression of Mex3a can significantly promote the migration and invasion of lung adenocarcinoma cells.Western blot results showed that after knocking down Mex3a,the expression of interstitial markers(N-cadherin,Snail,Slug)in H1299 cells decreased,and the expression of epithelial markers(E-cadherin)increased,but the opposite result appeared in PC9 cells with Mex3a overexpression.(3)Mex3a promoted LUAD cell proliferation,migration and invasion in vivoFirstly,the Mex3a stable knockdown(shMex3a)and control(shNC)lung adenocarcinoma cell lines were constructed.An engrafted tumor mouse model was established by implanted the H1299 cells subcutaneously in nude mice.the mice that received Mex3a knockdown H1299 cell resulted in a significant inhibition of tumor growth and tumor weight.Therefore,IHC staining proved that the expression of Ki67 and N-cadherin in the shMex3a group was significantly lower than that in the control group,while the expression of E-cadherin in the shMex3a group was significantly higher than that in the control group.We performed tail vein injection with Mex3a knockdown H1299 cells to establish tumor metastasis mouse models.Mex3a knockdown decreased the number of metastasis lesions.Tumor metastasis was confirmed by HE staining.(4)LAMA2 is the downstream target gene of Mex3aTranscriptome sequencing was performed by using Mex3a-silenced H1299 cells and control cells.Pathway analysis showed that Mex3a knockdown had important effects on genes that are mainly related to ECM-receptor interactions and PI3K-AKT pathway Western blot results showed that in H1299 and A549 cell lines,knocking down Mex3a reduced the expression of PI3K and phosphorylated AKT in the PI3K/AKT pathway.In PC9 cell lines,overexpression of Mex3a increased the expression of PI3K and phosphorylated AKT.The results of qRT-PCR indicated that LAMA2,COL5A1,CLDN1 and PIK3R3 are possible downstream target genes of Mex3a.The results of RNA immunoprecipitation(RIP)experiment showed that Mex3a protein can bind to LAMA2 mRNA.Actinomycin test confirmed that Mex3a can reduce the stability of LAMA2 mRNA.(5)LAMA2 expression is reduced in lung adenocarcinoma and is associated with poor prognosisGEO(GSE 19804,GSE 116959)and TCGA database showed that the expression of LAMA2 mRNA was significantly down-regulated in lung adenocarcinoma tissues.At the same time,we found that LAMA2 is lowly expressed in lung adenocarcinoma tissues and highly expressed in adjacent tissues through immunohistochemical staining on tissue microarrays.Correlation analysis of clinicopathological parameters showed that the low expression of LAMA2 in lung adenocarcinoma tissue was significantly positively correlated with the TNM stage and the patient's survival status(P<0.0001),while there is no significant correlation with parameters such as age,gender,tumor size,lymph node metastasis,the degree of differentiation and metastasis.Kaplan-Meier survival curve analysis found that patients with lung adenocarcinoma with low LAMA2 expression had a shorter overall survival time(OS,P=0.0016).Secondly,through the online analysis of lung adenocarcinoma website(KM Plotter),we also found that compared with patients with LAMA2 high expression group,the overall survival time(P<0.0001)and disease-free survival time(PFS,P<0.0001)was significantly shorter in patients with LAMA2 lower expression group.In addition,patients with high Mex3a expression and low LAMA2 expression had a poor clinical prognosis(P<0.0001).In addition,GEO and TCGA databases analyzed the correlation between Mex3a and LAMA2 mRNA in lung adenocarcinoma tissues,and the results showed that the expression of LAMA2 mRNA was significantly negatively correlated with the expression of Mex3a mRNA.Similarly,the results of tissue microarray staining were used to analyze the protein level of Mex3a.The expression was also significantly negatively correlated with the expression of LAMA2.(6)LAMA2 inhibits lung adenocarcinoma cell invasion,migration and epithelial-mesenchymal transitionThe results of qRT-PCR showed that PC9 cells expressed the highest LAMA2.The results of scratch experiment and Transwell migration and invasion experiments showed that PC9 cell invasion and migration ability was inhibited after knocking down LAMA2.Western blot results showed that after knocking down LAMA2,the expression of interstitial markers(N-cadherin,Snail,Slug)in PC9 cells decreased,and the expression of epithelial markers(E-cadherin)increased.(7)LAMA2 participates in Mex3a-mediated malignant biological behavior of lung adenocarcinoma cellsThe results of in vitro cell functional phenotyping rescue experiments show that the small interference of LAMA2 transfection in H1299 and A549 cell lines with stable knockdown of Mex3a,whether it is wound healing experiment,Transwel migration and invasion experiment or western blot experiment,LAMA2 knockdown can reverse the phenomenon that Mex3a knockdown can reduce the invasion,migration and epithelial-mesenchymal transition of lung adenocarcinoma cells and the inhibition of PI3K/AKT pathway.The results of the in vivo metastasis model rescue experiment showed that LAMA2 knockdown can reverse the decrease in the number of lung metastases caused by Mex3a knockdown.Conclusion:(1)The expression of Mex3a is increased in lung adenocarcinoma and the expression of Mex3a is related to lung adenocarcinoma metastasis and poor clinical prognosis.(2)In vitro cell experiments and in vivo animal experiments prove that Mex3a promotes the proliferation,invasion,migration and epithelial-mesenchymal transition of lung adenocarcinoma cells.(3)The low expression of LAMA2 in lung adenocarcinoma and the expression of LAMA2 are related to the high-grade staging of lung adenocarcinoma and poor clinical prognosis(4)Mex3a promotes its degradation by binding to LAMA2 mRNA,thereby affecting the invasion and migration ability of lung adenocarcinoma cells.