The Role And Mechanism Of SCARA5 In Non-small-cell Lung Cancer

Object:Non-small-cell lung cancer(NSCLC)is a tumor occurring on bronchial epithelial tumors.Its incidence rate ranks second in all malignant tumors,and mortality is the top.At present,the treatment of non-small-cell lung cancer mainly depends on the stage of the disease.Most patients with early lung cancer choose surgical resection,and the five-year survival rate is as high as 80%-90%.But patients with advanced lung cancer can not be surgical resection,and the five-year survival rate is only 15%-20%.Although the development of targeted therapeutic drugs for different targets has been rapid and significantly prolonged the survival of patients,there are still challenges such as drug resistance.Therefore,it has become the focus of current research that elucidate the mechanism of lung cancer development and develop new molecular diagnostic markers and potential therapeutic targets.Our previous work found that scavenger receptor class I member 5(SCARA5)is a gene with differential DNA promoter methylation between lung cancer and adjacent tissues,which is expected to become a potential diagnostic marker and therapeutic target.This study will further verify the expression difference of SCARA5,and focus on the role and mechanism of SCARA5 in NSCLC.Methods:1.Promoter methylation and gene expression of SCARA5 in non-small cell lung cancer(NSCLC)1.1 The databases,cell lines and tumor tissues used in this studyThe main databases used in this study were: TCGA database,Kaplan Meier plotter database,and Meth HC database.The promoter methylation of SCARA5 in lung adenocarcinoma(LUAD)and squamous cell carcinoma of lung(LUSC)was analyzed by querying Meth HC database,and the gene expression of SCARA5 in lung cancer and adjacent tissues was analyzed by querying TCGA database,KM Plotter database was used to analyze the effect of SCARA5 on the prognosis of lung cancer patients.The cell lines used in this study included BEAS-2B human normal lung epithelial cell line,H1299 lung adenocarcinoma cell line,A549 lung adenocarcinoma cell line,H460 large cell lung cancer cell line,H2122 lung adenocarcinoma cell line,H358 small cell lung cancer cell line.The lung cancer tissue samples(n = 64)and paired paracancerous tissues,which was confirmed as normal lung tissues by pathology(n = 25)used in this study were all from the Department of thoracic surgery,the First Affiliated Hospital of Chongqing Medical University.All tumor samples were pathologically and histologically identified,and the proportion of tumor cells was more than 70%.The clinicopathological information of patients was collected.This study complied with the declaration of Helsinki and obtained the following results Approved by the institutional ethics committee of the First Affiliated Hospital of Chongqing Medical University(Approval notice: 2016-75).1.2 Analysis of SCARA5 gene expression,methylation and prognosis in lung cancer by online database1)By downloading the data sets of LUAD and LUSC in TCGA database,querying the probe number of SCARA5,extracting the expression data of cancer and normal tissues for analysis.2)The methylation data and paired gene expression data of LUAD and LUSC samples were downloaded from Meth HC database,and the correlation between methylation and gene expression was analyzed.3)Analysis of the effect of SCARA5 on the survival of patients with lung cancer by online database KM plotter4)The online database Oncomine was used to select the GEO gene set,and then the corresponding gene set was downloaded through the GEO database.The mechanism of SCARA5 affecting the prognosis of lung cancer was preliminsarily explored through GSEA.1.3 Collect clinical samples and verify the online database1)DNA was extracted from 64 cases of lung cancer tissues and 25 cases of paired paracancerous tissues,and DNA methylation of SCARA5 in lung cancer and paired paracancerous tissues was detected.2)The m RNA expression of SCARA5 in lung cancer and adjacent tissues was detected by reverse transcription and Q-PCR.3)16 pairs of lung cancer tissue samples were collected,fixed and paraffin embedded.The protein expression of SCARA5 in lung cancer and adjacent tissues was detected by immunohistochemistry(IHC).4)Lung cancer cell lines A549 and H1299 were treated with demethylation drug 5-Aza to detect the m RNA expression of SCARA5 after demethylation2.Function of SCARA5 in inhibiting malignant proliferation of non-small-cell lung cancer2.1 Construction of stably overexpressing-SCARA5 lung cancer cell lines1)Screening: A549 and H1299 with low background expression were selected and transfected with SCARA5 overexpression plasmid with genetic resistance.After screening for 2 weeks with G418,the lung cancer cell lines stably overexpressing SCARA5 were obtained,which were named with SCARA5-A549 and SCARA5-H1299 respectively,and Vector-A549 and Vector-H1299 were used in the control group.2)Validation:The m RNA and protein of control group and overexpression group were extracted and verified at gene level and protein level respectively.2.2 Effect of overexpression of SCARA5 on malignant proliferation of lung cancer cell line in vitro1)Participants: A549 and H1299 cell lines2)Group setting: Control group(Vector-)and SCARA5 overexpression group(SCARA5-)3)Methods: The experiment was carried out(1)CCK8 kit was used to detect the cell viability in 24,48,72 hours(2)Colony formation assay was used to detect cell proliferation.(3)Cell cycle and apoptosis were detected by flow cytometry.2.3 Effect of overexpression of SCARA5 on the proliferation of subcutaneous lung cancer xenografts in nude mice1)Experimental model: 4-week-old immunodeficient female mice(BALB/c-nude mice)2)Participants:Control group(Vector-A549)and SCARA5 overexpression group(SCARA5-A549)3)Methods:(1)A549 cells were subcutaneously injected into nude mice.The cells in the control group were located in the left back,and the cells in the overexpression group were located in the right back.The tumor size was observed and recorded every two days.(2)After 19 days,the mice were sacrificed,measured and weighed.(3)HE staining was used to observed Nuclear morphology and immunohistochemistry(IHC)were used to detect Ki67.3.Mechanism of SCARA5 inhibiting malignant proliferation of non-small-cell lung cancer3.1 Mechanism of SCARA5 regulating A549 cell cycle1)Participants:Vector-A549 and SCARA5-A5492)Detection indexes: Gene expression of cyclins and kinases such as Cyclin B1,CDC25 C,CDK1,CHK13)After overexpression of SCARA5,the m RNA and protein expressions of cyclins and kinases were detected in A549 cell line,and then the transcription factors regulating the cell cycle were predicted by bioinformatics for further study(as shown in 3.2).3.2 SCARA5 regulates FOXM1 expression in A549 cells1)These results suggest that SCARA5 could down regulate the expression of FOXM1.In this part,we will first verify this prediction by detecting its m RNA and protein.2)The luciferase reporter gene assay was used to further detect whether FOXM1 could bind to the promoters of Cyclin B1,CDC25 C and CHK1 in A549 cells,and then regulate the transcription of these downstream target genes.3)FOXM1 was overexpressed in Vector-A549(Control group)and SCARA5-A549(Overexpression group)cells to investigate whether SCARA5 could reverse the inhibition of cyclins and kinases.3.3 The mechanism of SCARA5 regulating FOXM11)Transcriptome sequencing was performed in Vector-A549 and SCARA5-A549 cells.2)Go analysis and KEGG analysis were carried out to find out the most relevant genes among the differentially expressed genes,which are related to SCARA5 inhibiting the malignant proliferation of NSCLC3)According to the above go analysis and KEGG enrichment analysis,heat shock protein 70(HSP70)family is the most significant gene related to the inhibition of malignant proliferation of NSCLC.In this part,the expression of HSP70 family members m RNA and protein in A549 cell line was detected after overexpression of SCARA5.4)KEGG analysis showed that the DEGs were related to endoplasmic reticulum function.Therefore,immunofluorescence(IF)was used to explore whether SCARA5 protein was Co located with endoplasmic reticulum,so as to further clarify whether the regulation of downstream gene expression was mediated by SCARA5 through affecting endoplasmic reticulum function.3.4 Effect of SCARA5 on chemosensitivity of A549 cell line1)The inhibition rate of Vector-A549 and SCARA5-A549 cells was detected under different concentrations of chemotherapy drugs,and IC50 was calculated.2)At medium concentration,Vector-A549 and SCARA5-A549 cells were treated with 5-FU for 48 hours,and cell apoptosis was detected by flow cytometry.Results:1.In NSCLC tissues,promoter of SCARA5 is hypermethylated,and its m RNA and protein levels are lower than those in adjacent tissues.The expression level of SCARA5 is positively correlated with the prognosis of patients.1.1 The methylation of SCARA5 in lung cancer tissues was higher than that in adjacent tissues1)The methylation of SCARA5 promoter in lung adenocarcinoma and squamous cell carcinoma was significantly higher than that in adjacent tissues(P < 0.001);2)In 64 cases of clinical lung cancer tissue samples,57 cases had high methylation of SCARA5 promoter(89%),while none of 25 paired adjacent tissues had methylation of SCARA5 promoter(0%),which was consistent with the prediction of the database,indicating that the methylation of SCARA5 promoter in lung cancer was higher than that in adjacent tissues.1.2 The expression of SCARA5 in lung cancer tissues was lower than that in adjacent tissues1)The expression of SCARA5 in lung adenocarcinoma and squamous cell carcinoma was significantly lower than that in adjacent tissues(P <0.001)2)The m RNA expression of SCARA5 in 12 pairs of clinical lung cancer tissues was lower than that in adjacent tissues(P < 0.01);The expression of SCARA5 in most lung cancer cell lines was lower than that in normal human Lung epithelial cell line BEAS-2B.3)The expression of SCARA5 protein in 16 pairs of clinical lung cancer was lower than that in adjacent tissues(P < 0.01),which was also consistent with the database prediction,indicating that the expression of SCARA5 was downregulated in lung cancer tissue.1.3 SCARA5 promoter methylation was negatively correlated with its gene expression1)Analysis of the Meth HC database showed that there was a negative correlation between SCARA5 promoter methylation and gene expression in lung cancer tissues(r = 0.3121,P = 0.0012)2)The expression of SCARA5 was up-regulated in A549 and H1299 cell lines treated with 5-Aza,which proved that the down-regulation of SCARA5 expression in lung cancer was caused by promoter hypermethylation.1.4 The expression of SCARA5 was positively correlated with the prognosis of lung cancer patients1)Online database KM plotter showed that SCARA5 was positively correlated with prognosis(P < 0.001).2)GSEA analysis showed that SCARA5 may be associated with G2/M cell cycle arrest in lung cancer tissue.2.SCARA5 inhibited malignant proliferation of lung cancer cell lines in vitro and in vivo2.1 Construction of SCARA5 stable overexpression cell lineThe results of m RNA and Western blot showed that A549 and H1299 cell lines with SCARA5 stably overexpressed were successfully constructed.2.2 SCARA5 inhibited malignant proliferation of lung cancer cell lines in vitroAfter overexpression of SCARA5,the cell viability of A549 and H1299 cell lines detected by CCK8 kit was lower than that of the control group(P< 0.01);the results of colony formation assay showed that the single cell proliferation ability of A549 and H1299 cell lines was lower than that of the control group(P < 0.01)2.3 SCARA5 induced apoptosis and cell cycle arrest in NSCLC cell linesThe number of apoptotic cells(early and late apoptosis)in SCARA5-A549 and SCARA5-h1299 groups was significantly increased compared with the control group(P < 0.001),and their cell cycles were blocked,in which SCARA5-A549 cells were arrested in G2 / M phase and SCARA5-H1299 cells were arrested in S phase.2.4 SCARA5 inhibited subcutaneous tumorigenesis of lung cancer cells in nude mice1)In SCARA5-A549 group,the size and weight of subcutaneous tumor in nude mice were significantly lower than those in control group(P < 0.001).2)HE-staining results showed that SCARA5-A549 group had more deeply stained nuclei and more irregular nuclei,suggesting that the number of apoptosis was higher than that of the control group;at the same time,the proportion of Ki67 positive cells was lower,that means,the proliferation ability of the cells was lower than that of the control group.This part of the results showed that SCARA5 could inhibit the proliferation of NSCLC cells in vitro and in vivo by inducing apoptosis and promoting cell cycle arrest.3.SCARA5 inhibited the expression of cyclins and kinases by suppressing FOXM1,which led to G2/M cell cycle arrest.3.1 SCARA5 inhibited the expression of cyclins and kinases involved in G2/M phase in A549 cell line1)After overexpression of SCARA5,the protein and m RNA expressions of Cyclin B1,CDK1 and CDC25 C were down regulated compared with the control group in A549 cell line(P<0.05)2)Overexpression of SCARA5 up-regulated the expression of DNA damage marker γ-H2 AX and down regulated the protein and m RNA expression of CHK1 in A549 cells(P < 0.001)3)Venn map shows that the two genes FOXM1 and FOS1 in differential expression genes of GEO data set GSE12667 were common transcription factors that regulated the expression of Cyclin B1,CHK1 and CDC25 C,suggesting that SCARA5 inhibited cyclins and kinases by regulating the expression of FOXM1 and FOS1.3.2 SCARA5 blocked cell cycle progression by inhibiting the expression of FOXM11)After overexpression of SCARA5,m RNA and protein expression of FOXM1 were down regulated compared with the control group(P < 0.01);2)Luciferase reporter assay showed that FOXM1 could bind to the promoters of CDC25 C and Cyclin B1(P < 0.05)3)Overexpression of FOXM1 could reverse the expression of Cyclin B1,CDC25 C and CHK1 in A549 cell line overexpressing SCARA5 and control group,suggesting that SCARA5 could block the G2/M cell cycle progression of A549 cells by inhibiting FOXM1.These results suggested that SCARA5 could down regulate the expression of Cyclin B1,CDC25 C and CHK1 by inhibiting FOXM1 expression,which led G2 / M cycle arrest of A549 cells through this mechanism.4.SCARA5 located in endoplasmic reticulum and induced up regulation of HSP70 expression4.1 SCARA5 protein co localization with endoplasmic reticulum1)Transcriptome sequencing showed that 509 genes were up-regulated and 310 genes were down regulated after overexpression of SCARA5 in A549 cell line.2)Go analysis showed that 509 upregulated genes were mainly related to UPR.3)KEGG analysis of 509 up-regulated differential genes showed that the up-regulated differential genes were related to endoplasmic reticulum function(ER).4)Immunofluorescence confirmed that SCARA5 protein was co localized with endoplasmic reticulum.These results suggested that SCARA5 protein could bind to endoplasmic reticulum and mediate UPR in A549 cells.4.2 SCARA5 induced up regulation of HSP70 family proteins1)Among the 509 up-regulated genes,HSP70 family protein was associated with Go analysis and KEGG analysis.2)By q PCR and Western blot,we found that HSPA1 A,HSPA1B and HSPA5 were up-regulated in HSP70 family members.These results suggested that SCARA5 could up regulate the expression of HSP70 family proteins in A549 cells.Because HSP70 is the main effector protein in UPR process,combined with the result of 4.1,we speculated that the mechanism of SCARA5 up regulating HSP70 is that SCARA5 locates in endoplasmic reticulum and then mediates UPR.5.SCARA5 increased chemosensitivity of lung cancer cell line A549 to DNA damage drugs1)After overexpression of SCARA5,the inhibitory rate of A549 cells in different drug concentrations was higher than that in the control group(P <0.05),and the IC50 was significantly lower than that in the control group;2)Under the same drug concentration,the apoptosis rate of SCARA5-A549 cells was higher than that in the control group(P < 0.001),which proved that SCARA5 affected the drug resistance of lung cancer A549 cell line.These results suggested that SCARA5 could enhance the sensitivity of lung cancer cell line A549 to chemotherapeutic drugs,and may be a potential therapeutic target.Conclusion:1.The down-regulation of SCARA5 expression in NSCLC is mainly due to hypermethylation of gene promoter;the expression level of SCARA5 in lung cancer is positively correlated with the prognosis of lung cancer patients.2.SCARA5 can inhibit the growth and proliferation of NSCLC in vivo and in vitro,showing the function as a tumor suppressor gene;the inhibitory effect is related to inducing cell apoptosis and cell cycle arrest.3.SCARA5 inhibits the expression of cyclins and kinases by inhibiting FOXM1 expression,which mediates G2 / M cell cycle arrest in A549 cells.4.SCARA5 co localizes with endoplasmic reticulum and induces up regulation of HSP70 expression.The mechanism of up regulation of HSP70 by SCARA5 may be related to its localization in endoplasmic reticulum and subsequent UPR.5.SCARA5 enhances the sensitivity of NSCLC to DNA damage chemotherapeutic drugs.These results suggest that the promoter methylation of SCARA5 gene may be a potential marker for prognosis of NSCLC,and SCARA5 gene itself may be a potential target for combined chemotherapy of NSCLC.