| 1.Background:Lung cancer is the most cancer that prevalent worldwide and that is diagnosed with more 1.8 million new cases every year.In 2018 has been reported 2.09 million new cases of lung cancer occurred,which was the top rank among all cancer types globally that year.There are two types of Lung cancer Non Small coding Cell Lung Cancer(NSCLC)and Small Cell Lung Cancer(SCLC).Whereas,NSCLC has the largest share by 80-85% compared to other types of lung cancer and is further divided into three subtypes i.e.adenocarcinoma,squamous cell carcinoma,and large cell carcinoma.The overall prognosis of NSCLC is still very poor i.e.only 15% of patients survive for 5 years after their initial Diagnostic.In recent years,the rapid incidence rate of lung adenocarcinoma among both men and women has imposed a serious threat to human health.Through the genome are determined all the Non-coding RNAs(nc RNAs)that are not yet totally understood their functions.Long non-coding RNAs(lnc RNAs)are a heterogeneous class of RNAs that are defined as non-protein coding transcripts.They are playing a vital role in regulating gene expression through the regulations of transcriptional and posttranscriptional,and epigenetic modification in the cancer cells.Numerous studies strongly indicate that lnc RNAs have considerable and complicated capabilities that contribute chiefly in cancer development.The main function of lnc RNAs in many cancer types is playing as tumor oncogenic or suppressive.MALAT1(Metastasis associated lung adenocarcinoma transcript 1),also known as non-coding nuclear-enriched abundant transcript 2(NEAT2),is a lnc RNA that represents both a promising cancer biomarker and potential therapeutic target to restrict metastatic growth.In recent studies,MALAT1 is found to be involved in cancer metastasis and recurrence,and also is upregulated in several solid tumors,including gastric,bladder,breast,and lung cancers.Several studies reported that MALAT1 drives tumorigenesis through promoting cell proliferation,in previous studies;depletion(knockdown and knockout strategies)of MALAT1 has inhibited in vitro cell motility and in vivo cancer progression.Abnormal fucosylation is catalyzed by the specific fucosyltransferases(FUTs).The FUTs family comprises over four subfamilies i.e.a1,2-(FUT1 and 2),a1,3/4-(FUT 3,4,5,6,7,9,10,11),a1,6-(FUT8)and protein O-FUTs(POFUT1 and POFUT2).FUT4 is a key enzyme that catalyzes the transfer of the Fuc of GDP-fucose to ?-N-acetylglucosamine of sugar chain and regulates tumor-associated carbohydrate antigen Lewis Y(Le Y).Le Y has been found to induce activation and glycosylation of EGFR resulting in in-vitro oral cancer cell migration.Research in tumor glycobiology has clearly indicated that FUT4,an important fucosyltransferase,is highly expressed in various cancer types and plays a vital role in tumor proliferation,apoptosis,metastasis and epithelial-mesenchymal transmission(EMT),but the driving regulators are still not well understood.MALAT1 and FUT4 have an important role in proliferation and metastasis,but it is not clear in lung cancer that needs further investigation.Therefore,in this study,we investigate the role of MALAT1 and FUT4 in the progression of lung cancer and confirmed the correlation between MALAT1 and FUT4 in clinical pathological stages.Moreover,we demonstrate that MALAT1 promotes proliferation and metastasis by regulating FUT4 through PI3K/Akt signaling pathway.This study will create the novel biomarkers for the Diagnostic as well as treatment and therapeutic evaluation in lung cancer.2.Objectives:1.To demonstrate the expression and correlation between MALAT1 and FUT4 in human lung cancer tissue and serum.2.To fortify and comparing MALAT1 and FUT4 expressions in different lung cancer cell lines.3.To detect the role of MALAT1 in proliferation and invasion by regulating FUT4 of lung cancer cell lines.4.To study the effect of MALAT1 on the expression of ?1,3-fucosylation of EGFR.5.To detect the effect of MALAT1 on the PI3K/Akt signaling pathway.3.Material and methods:Clinical samples collection Fifty fresh-frozen tissues of lung cancer,and adjacent non-cancerous tissues,and fifty serum samples classified as(normal,and lung cancer stage I-IV),were obtained between March 2017 to July 2018 at First Affiliated Hospital of Dalian Medical University(Dalian,P.R China).The expression MALAT1 and FUT4 genes,quantitative real time-PCR,ELISA,western blot,immunohistochemistry have been performed,The Spearman correlation analysis was used to analyze the relationship between MALAT1 and FUT4 in the serum using SPSS.Furthermore,to investigate the in-vitro model,A549,H1299,and HBE cell lines were revived from liquid nitrogen tank of Clinical laboratory department of Dalian Medical University,by washing three times with sterile cold phosphate buffer saline(PBS)have been refreshed and cultured with sterile 10% fetal bovine serum(FBS)in sterile 1640 cell culture medium for 24 hours in 5% CO2 at 37?C maintained in RPMI/F12 supplemented with 10% FBS and 100 U/ml of penicillin and 100 ?g/ml of streptomycin.Likewise,the normal HBE cell line in the DMEM has been maintained.Firstly,by using quantitative real time-PCR,western blot,and immunofluorescence the expression MALAT1 and FUT4 genes,to prove the relation between MALAT1 and FUT4,transfection si RNA and c DNA were performed to knockdown or overexpress the expression of MALAT1 and FUT4 Expression,were transfected/co-transfected into A549 and H1299 cells with lipofectamine 2000 reagent.Immunoprecipitation also performed to determine ?1,3-linkage fucosylated glycans on EGFR by LTL and Le Y epitopes.In addition,to detect the proliferation and migration like Cell viability,clonogenic,migration and invasion assays were also applied.4.Results:In human lung cancer tissue and serum,the result had been shown the FUT4 and MALAT1 expression in lung cancer tissue was significantly increased compared with adjacent tissue.Moreover,these data displayed a positive correlation between the expression levels of MALAT1 and FUT4.Besides,we investigated the expression of FUT4 protein in lung cancer tissue and adjacent tissue using Western blot,FUT4 expression was significantly increased in lung cancer compared with adjacent tissues.To further investigate,we have been examined the MALAT1 and FUT4 gene expressions in the serum samples of healthy people,lung cancer patients,and patients who have been received regular chemotherapy using q PCR.The results showed that MALAT1 and FUT4 expressions were significantly increased in lung cancer compared with normal,while the expressions genes of MALAT1 and FUT4 after chemotherapy were significantly dropped compared with lung cancer.Furthermore,we investigated more to confirm that results using ELISA showed that FUT4 expression was significantly increased in the lung cancer compared with normal,while the expression of FUT4 after chemotherapy was significantly dropped compared with lung cancer.These findings are showing a positive correlation coefficient between MALAT1 and FUT4 expressions.Moreover,in-vitro model,after indicating high expression of MALAT1 in serum and tissues of lung cancer patients,we examined the expressions of MALAT1 and FUT4 in A549 and H1299 lung cancer cells comparing with HBE normal lung cells using q PCR.We observed the expressions of MALAT1 and FUT4 were significantly high in cancer cells compared with normal cells.CCK-8,colony formation,and Transwell assays were performed to assess the regulatory role of MALAT1 in cell proliferation and migration using A549 and H1299 cells with si-MALAT1 or FUT4 c DNA transfection,CCK-8 assay,colony formation,and Transwell assays.We indicated that si-MALAT1 significantly decreased the cell proliferation rate of A549 and H1299 cells compared with control cells,as well as significantly increased in the transfected with FUT4 c DNA cells.In additional,immunofluorescent staining,our results revealed that the FUT4 expression in the transfected cells was lower than control.Le Y biosynthesis showed consistent changes.After immunoprecipitation of EGFR,the level of Le Y on EGFR was further analyzed with either LTL lectin or Le Y antibody.We found that transfection with si-MALAT1 reduced the Le Y level on EGFR which contributed to reduced ?1,3-fucosylation.These findings suggest that MALAT1 plays a contributive role in the function of EGFR fucosylation.To further analyze the activation of the signaling pathway of PI3K/Akt was evaluated and activated by MALAT1.5.Conclusion:1.Lnc RNA MALAT1 and FUT4 had high expression and a positive correlation in human lung cancer tissue and serum.2.We reported that the MALAT1 role is up-regulating the FUT4 expression in lung cancer cell lines.3.Lnc RNA MALAT1 by regulating FUT4 promoting cell proliferation and metastasis in lung cancer.4.Lnc RNA MALAT1 regulates ?1,3-fucosylation expression of EGFR in lung cancer and activates the PI3K/Akt signaling pathway.This research will develop a novel biomarker for lung cancer Diagnostic,treatment,and prognosis assessment. |
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