The Mechanism On The Regulation Of Mir-451a,which Is Based On GEO,on The Biological Behavior Of Small-cell Lung Cancer

BackgroundAccording to GLOBOCAN 2018,there is an estimated 18.1 million new tumor patients and 9.6 million patients died in 2018.One of cause of global tumor mortality is lung cancer,and 2.1 million new patients and 1.8 million patients died in 2018[1-2].Each year,250,000 new patients around the world are diagnosed with small cell lung cancer（SCLC）,and at least 200,000 patients worldwide died of SCLC[1].Non-small cell lung cancer（NSCLC）accounts for a majority of lung cancers.In addition to NSCLC,SCLC is more aggressive and represents 15%of lung cancer cases[3].The 5year survival rate of SCLC is less than 7%due to rapid growth and easy early diffusion,metastasis,and recurrence[4].Although radiotherapy has made clinical progress,most SCLC cancer will recur,and the median survival time is less than six months[3].Lines of studies have demonstrated that the molecular pathogenesis of SCLC is very complex,and the underlying mechanisms remain ambiguous,which significantly restricts the advancements of SCLC therapy.MicroRNA（miRNA）is non-coding RNA with a length of about 21-24 nucleotides,which can inactivate or degrade mRNA by binding to the 3’-untranslated region（3’UTR）of genes[5].And up to 60%of mammalian genes are regulated by various miRNA[6].Notably,many investigations revealed that dysregulation of miRNA is common in cancer patients,including NSCLC.It has been reported that several up-regulated miRNAs（such as miR-210 and miR-182）act as tumor promoters[7].Several downregulated miRNAs（such as miR-451a and miR-126）as tumor suppressors in NSCLC compared to normal tissues[8].Among them,miR-451a has been proved to be downregulated and modulate cell biological behaviors in multiple cancers,such as acute myeloid leukemia[9],glioma[10],stomach[11],and NSCLC[12].miR-451 comprises two homologies;miR-451a and miR-451b,and miR-451a attracted more attention in cancer.miR-451a expression was negatively associated with an unfavorable outcome for NSCLC[13].And miR-415a could intensify chemosensitivity of NSCLC to cisplatin by regulating Mcl-1[14].These investigations highlight the inhibition of miR-451a despite NSCLC with different target genes;however,whether miR-451 a roles the same way by targeting the particular gene in SCLC.Lymphocyte-specific helicase（HELLS）belongs to the Sucrose nonfermenting（SNF2）family.SNF2 is the core subunit of the Switch（SWI）/SNF chromatin remodeling complex,and SNF2 belongs to the SF2 subfamily of helicase[15].However,SNF2 has no helicase activity and can use the energy of ATP hydrolysis for chromatin remodeling.Studies have shown that SWI/SNF family proteins are closely related to human health[16],and SWI/SNF chromatin remodeling complex mutations are found in about 20%of cancer patients[17].Previous studies have found that there is a positive correlation between HELLS and cancer development.HELLS is up-regulated in many kinds of tumors,such as colorectal cancer[18],glioma[19],and retinoblastoma[20],and is related to poor prognosis.For example,HELLS is highly expressed in hepatocellular carcinoma（HCC）.It regulate chromatin remodeling and epigenetic silencing of genes,promote the progression of HCC,and is an indicator of poor prognosis of HCC[21].The expression of HELLS and intercellular adhesion molecules in NSCLC is increased and negatively associated with the prediction of lung adenocarcinoma patients[22].In vitro experiments confirmed that HELLS promoted the invasion of NSCLC[23].However,whether miR-451a roles the same way by targeting certain gene in SCLC is ambiguous.AimsFirstly,we analyze the existing data about SCLC in the public database GEO and study bioinformatics analysis;then,we put forward a research hypothesis:miR-451a regulates the progression of SCLC by targeting HELLS to regulate the mTOR/Bcl2 signaling pathway.Then,we construct in vitro cell models by gene transfection technology to verify the differential miR-451a,HELLS,and the predicted regulation of AKT/mTOR and Bcl-2 signal pathways in SCLC.Finally,we collected samples from lung cancer patients,detected the expression of HELLS protein in cancer tissues and adjacent tissues,and analyzed the relationship between expression of HELLS and pathological grade and lymph node metastasis of lung cancer patients.We propose the feasibility of miR-451a mimic as an anti-SCLC treatment and HELLS as an auxiliary diagnosis of SCLC.Methods1.Part I:Bioinformatics study on miRNA and mRNA related differences in SCLC（1）Bioinformatics study of miRNA related differences in SCLC① We downloaded the data of SCLC by the GEO database and used R language to analyze the data and screened out miRNA with a different expression between SCLC tissues and normal tissues adjacent to cancer,and visually analyze the miRNA with different expression through R language.② GO function enrichment analysis and biological pathway enrichment analysis on the differentially expressed miRNA genes using online tools.③Using the online website to predict the target gene of miR-451 a.（2）Bioinformatics study of mRNA related differences in SCLC① We downloaded the data of SCLC by the GEO database,and used R and Perl language to analyze the data and screened out mRNA with a different expression between SCLC tissues and normal tissues adjacent to cancer,and visually examine the differentially expressed mRNA by using R language.② GO function enrichment analysis and KEGG enrichment analysis was performed on differentially expressed mRNA using the R language.③ We intersected the up-regulated mRNA with the target-genes predicted by the miR451a,the we obtained common genes（POU3F2 HELLS,KIF24,SPC25）.GSEA was used for single gene analysis to screen possible cell signaling pathways.Finally,according to the data analysis results,we developed a research hypothesis.2.Part Ⅱ:MiR-451a regulates the cell function of SCLC cells（1）We construct Vitro cell models of SCLC by gene transfection.（2）Cell counting kit-8（CCK-8）assay,cell scratch test,Trans-well test,and plate colony formation assay were used to study the changes of malignant biological behaviors such as proliferation,migration,and invasion ability of NCI-H1688 and NCI-H446 cells in different treatment groups.3.Part III:To explore the molecular mechanism of the regulation of miR-451a on the cell function of SCLC.（1）The expression of miRNA-451a in two SCLC cell lines（NCI-H1688 and NCI-H446 cells）and human lung bronchus epithelial cell line（BEAS2B cells）was detected by Real-time quantitative PCR.The expression of miRNA-451a in SCLC and adjacent tissues of the GSE19945 data set was analyzed.（2）The expression of HELLS-mRNA in NCI-H1688 and NCI-H446 cells and BEAS2B cells was detected by Real-time quantitative PCR.The expression of HELLS-mRNA in SCLC and adjacent tissues of the GSE43346 data set was analyzed.（3）We verify the targeting relationship between miR-451a and HELLS by a double luciferase report experiment on cancer cells.（4）We construct Vitro cell models of SCLC by gene transfection.① The expression of HELLS-mRNA in NCI-H1688 and NCI-H446 cells of different treatment groups was detected by Real-time quantitative PCR.②The expression of HELLS protein in NCI-H 1688 and NCI-H446 cells of different treatment groups was detected by western blot assay.③ The fluorescence intensity of HELLS protein in nuclei of NCI-H 1688 and NCIH446 in different treatment groups was detected by cell immunofluorescence technique.④.The expression of AKT/mTOR signaling pathway and Bcl2/Bax apoptosis signaling pathway protein in NCI-H1688 and NCI-H446 cells of different treatment groups were detected by western blot assay.4.Part Ⅳ:To analyze the expression of HELLS in lung cancer and the clinicalcharacteristics of lung cancer patients（1）We purchase a lung cancer tissue chip from Shanghai Outdo Biotech Company.Item No:HLugC120PT01.The tissue chip includes 60 cases（pairs）of lung cancer and matched adjacent tissues,with age,sex,symptoms,location of disease,pathological diagnosis and grading,number of lymph nodes metastasis and H＆E staining results.The diameter of tissue spots on the chip is 1.0mm.Helsinki Declaration was complied with,and tissue chip is allowed to be used for commercial and research purposes.Clinicians with professional and technical qualifications give pathological diagnoses to each tissue according to the classification and grading specified by WHO.Approved by the Ethics Committee of Shanghai Xinchao Biotechnology Co.,Ltd.（2）The fluorescence intensity of HELLS protein in the nuclei of lung cancer tissues and adjacent tissues was detected by tissue immunofluorescence technique.The fluorescence intensity of nuclear HELLS protein was analyzed with lung cancer patients’ clinical and pathological characteristics.（3）The online database Kaplan-Meier plot（http://kmplot.com/analysis/）was used to analyze the predictive value of HELLS for the prognosis of lung cancer patients.Results1.Part ⅠTo find possible diagnostic markers and therapeutic biomarkers for SCLC,we use the public database GEO to analyze the existing data on SCLC and obtain the following results.（1）There are many miRNAs differentially expressed in SCLC.One hundred sixtyseven miRNAs were obtained,of which 64 were up-regulated,and 103 were downregulated.（2）GO function enrichment analysis and biological pathway enrichment analysis of differentially expressed miRNA were carried out using FunRich software.In addition,miRNA-451a was selected as the following research object based on the literature.（3）The target genes of miR-451a were predicted by TargetScan,which provided a possible direction for further research.（4）There were many mRNAs differentially expressed in SCLC.Eight hundred fortyfour differentially expressed mRNA were obtained,of which 448 were up-regulated,and 396 were down-regulated.（5）GO function enrichment analysis and KEGG enrichment analysis was performed on differentially expressed mRNA using R language.GO function enrichment mainly involved biological function such as cell growth regulation,cell adhesion,division,DNA synthesis,repair and transcription regularity,and protein translation regulation;The molecular functions mainly focus on DNA replication origin binding,enzyme binding,kinase binding,protein kinase binding,and RNA binding.KEGG is primarily concentrated in the cell cycle.（6）Four common genes were obtained.Single gene GSEA enrichment analysis showed that HELLS was significant.The pathways enriched in the high expression group included the mTOR signal pathway,SCLC,and cell cycle pathway.In contrast,those enriched in the low expression group had complement and coagulation-related pathways,lipid metabolism,and apoptosis.According to the literature,HELLS was selected as the target,and the mTOR pathway and apoptosis were its downstream pathways.（7）Through obtaining patients’ clinical data and survival information,we concluded that HELLS had diagnostic value for SCLC.The sensitivity and specificity were 95.7%and 71.4%,respectively.2.Part Ⅱ（1）CCK-8 assay,cell scratch test,plate colony formation assay,and Trans-well chamber confirmed that miR-451a mimic could inhibit the proliferation,migration,and invasion NCI-H446 and NCI-H1688 cells.（2）Overexpression of HELLS promoted the proliferation,migration,and invasion of NCI-H446 and NCI-H1688 cells.（3）In addition,the OD values of CCK-8,the number of cell colonies formed by plate colony formation assay,cell migration rate by scratch test,and invasive cell capability by Trans-well in mimic+HELLS group elevated compared to the mimic group,which suggests that HELLS attenuated the suppression of SCLC proliferation,migration,and invasion induced by miR-451a.3.Part Ⅲ（1）The expression of miRNA-451a in SCLC cells of NCI-H446 and NCI-H1688 was significantly lower than that in human lung bronchus epithelial cell line BEAS2B.It is consistent with the result of lower expression of miRNA-451a in cancer tissue of GEO database GSE19945 data set.（2）The expression of HELLS-mRNA in SCLC cells of NCI-H446 and NCI-H1688 was significantly higher than that in human lung bronchus epithelial cell line BEAS2B.It is consistent with the result of higher expression of HELLS-mRNA in cancer tissue of GEO database GSE43346 data set.（3）The binding sites and the complementary base pairs between miR-451 a and 3 ’-UTR of HELLS were predicted by TargetScan.The transfection of miR-451a mimics dramatically reduced the luciferase activity in the WT-HELLS group,whereas in the Mut-HELLS group,the luciferase activity in NCI-H1688 cells co-transfected with miR451a mimic presented no significant difference from that in miR-451a NC cotransfected cells.（4）We construct Vitro cell models of SCLC by gene transfection.① MiR-451a negatively modulated the expression of HELLS-mRNA of NCI-H446 and NCI-H1688 cells.Over-expression of HELLS could partially reverse the negative regulation of HELLS-mRNA caused by miR-451a.② The results of western blot and cellular immunofluorescence showed that miR-451 a negatively modulated the expression of HELLS protein of NCI-H446 and NCI-H 1688 cells.Over-expression of HELLS could partially reverse the negative regulation of HELLS protein caused by miR-451a.③ mTOR and apoptosis pathways were respectively enriched in HELLS high-and low-expressed group by GSEA.And the subsequent western blot assays confirmed the results.In the mTOR signaling pathway,no remarkable change was observed in the protein expression of AKT and mTOR.In contrast,the expression levels of phosphorylated AKT and mTOR were remarkably altered.It was evident that compared to NC groups,the over-expression of miR-451a inhibited the phosphorylation of AKT and mTOR.4.Part Ⅳ（1）This tissue chip contained 60 patients.Twenty-six cases were lung adenocarcinoma（LUAD）（mean ages,57.5 years;SD,7.5;men,50%）.The tumor sizes were different,but the pathological grades were Ⅰ-Ⅱ with lymph node metastasis.Eight cases were SCLC（mean ages,60.9 years;SD,11.6;men,100%）,and the pathological grades wereⅢ with lymph node metastasis.Fourteen cases were squamous cell carcinoma of lung（LUSC）（mean ages,63.0;SD,7.7;man,85.7%）.All patients were accompanied by lymph node metastasis,and the pathological grade was grade II.Twelve cases were large cell lung cancer（not included in the study）.All patients with lung cancer had no distant metastasis.The age of tumor onset is primarily middle-aged and older men.Among them,two patients with lung adenocarcinoma were confirmed to be off-target after multiple rechecks.The off-target data were removed when analyzing the data.（2）HELLS expression in lung cancer and its clinicopathological features.① Compared with the adjacent tissues,the expression of HELLS is higher in both SCLC and LUAD/LUSC.,and the expression of HELLS in SCLC and LUAD with lymph node metastasis is raised than that without lymph node metastasis.All patients with LUSC were accompanied by lymph node metastasis.However,there was no significant difference between pathological grade and HELLS expression in both SCLC and LUAD/LUSC.②Kaplan-Meier plot online database analysis showed that the total survival time and progression-free survival time of patients with high expression of HELLS in LUAD were significantly shorter than those with low expression,and there was no positive correlation between the expression of HELLS in LUSC and the prognosis.At present,Kaplan-Meier plot online database lack relevant data on SCLC.Conclusions1.The occurrence and development of SCLC is accompanied by the inhibition of miR451a expression and the up-regulation of HELLS expression.2.MiR-451a can inhibit proliferation,migration and invasion of SCLC cells.HELLS can promote the proliferation,migration and invasion of SCLC.3.MiR-451a inactivates mTOR and activates apoptosis signal pathway by targeting HELLS,which inhibits proliferation and invasion of SCLC cells.Up-regulation of miR-451a can induce invasion inhibition of SCLC,which is expected to provide a promising biomarker and target for diagnosing and treating SCLC.