Expression Of Hsa_circ₀025036 In Lung Adenocarcinoma And Its Effects On Cell Proliferation And Apoptosis

Background and objectiveLung cancer has contributed the most to tumor associated death.As a well known public issue,its prevention and therapy has become a pivotal problem in the field of medical health in China.Among various types of lung cancer,lung adenocarcinoma is the most common subtype.Moreover,with a high morbidity and mortality,lung adenocarcinoma has brought about hazardous burden to both the patients and the society.Thousands of researches have pointed out that to seek early diagnostic approaches and therapeutic targets through deep investigation into gene regulation and cancer stem cells is of vital significance towards lung adenocarcinoma.In recent years,many studies have discovered that non-coding RNAs play a significant role in tumor regulation,forming a complex regulatory network.Circular RNAs?circRNAs?are moleculars produced during gene transcription.In latest researches,circRNA has been identified with various roles as regulators in different pathobiological processes.circRNAs are supposed to competitively inhibit miRNA,thus to affect tumorigenesis and development of virous cancers including lung cancer.Although the exploration towards circ RNAs has achieved some progress,the study on their explicit roles in different cancers is still at the very initial stage.More functional circRNAs remain to be discovered,and the relative mechanisms and their detailed roles in the regulatory network is not completely clear.We found via circRNA chip that the expression of hsa_circ₀025036 was significantly increased in lung adenocarcinoma tissues in our previous work,which indicates that hsa_circ₀025036 maybe involved in lung adenocarcinoma.However,its specific roles and the underlying mechanisms have not been exploited yet.In the present study,we first confirmed the existance of hsa_circ₀025036,assessed its expression in lung adenocarcinoma tissues and its relationship with different clinical pathological parameters.Then we investigated the effects of downregulated hsa_circ₀025036 towards lung adenocarcinoma cells A549 and Calu-3.In order to explore potiential interacting mi RNA and downstreamed target genes,bioinformatics analysis and dual-luciferase reporter assay were utilized.In this way,we primarily explored the related function and mechanisms of hsa_circ₀025036.This research was hopefully designed to search some novel potential diagnostic and therapeutic targets for lung adenocarcinoma.This study will be presented in two parts.Part I displays the confirmation process of the existance of hsa_circ₀025036,its expression level in lung adenocarcinoma and its effects on cell proliferation,apoptosis and cell cycle.Part II describes the primary investigations into the underlying mechanisms analyzing the regulatory roles of hsa_circ₀025036 in lung adenocarcinoma.Part I The expression level and biological functions of hsa_circ₀025036 in lung adenocarcinomaMethods1.Lung adenocarcinoma tissues and the adjacent normal lung tissues were collected following designed standards.2.PCR,TA cloning and gene sequencing were applied to confirm the existance of hsa_circ₀025036 in lung adenocarcinoma tissues and different cell lines.3.The expression level of hsa_circ₀025036 in lung adenocarcinoma tissues and paired normal tissues were detected through qRT-PCR.4.The relationships of hsa_circ₀025036 expression with clinicopathological parameters were analyzed.5.Both siRNA specially downregulating hsa_circ₀025036 and irrelevant siRNA sequence were designed and synthesized.Lipofectamine T M2000 was applied to transfect siRNA into lung adenocarcinoma cells,A549 and Calu-3 cells.Blank group was set as control.qRT-PCR was utilized to assess hsa_circ₀025036 expression in different groups.6.Cell proliferation assays including CCK-8 and colony formation assay were conducted to detect cell proliferation ability of A549 and Calu-3 cells with downregulated hsa_circ₀025036.7.Cell apoptosis assays including AnnexinV-FITC/PI flow cytometer and Hoechst staining were performed to investigate the effect of downregulated hsa_circ₀025036 in A549 and Calu-3 cells.8.Cell cycle assay,PI stained flow cytometer was utilized to analyze the effect of downregulated hsa_circ₀025036 on cell cycle.Western blot was applied to detect cell cycle related proteins,including CDK1,Cyclin D1 and Cyclin B1.Results1.A new subtype of hsa_circ₀025036 with a length of 196 nt was confirmed to exsit in lung adenocarcinoma tissues and cell lines which consisted of Exon 5,Exon6 and part of Exon 7 of FOXM1.The BS junction site located between Exon 5 and Exon 7.We teperately named it as hsa_circ₀025036 in the present study.2.Compared with paired normal tissues,in lung adenocarcinoma hsa_circ₀025036 was significantly upregulated?P<0.05?.3.The expression level of hsa_circ₀025036 was related with differentiation,TNM stage and lymph node metastasis?P<0.05?,while it was not related with age and gender of the patients?P>0.05?.4.The assays of detecting the effects of downregulated hsa_circ₀025036 towards cell proliferation showed that the OD value of cells in si-circRNA group was significantly decreased,the colony number was also decreased compared with siNC group and Blank group?P<0.05?,suggesting that downregulated hsa_circ₀025036 could inhibit cell proliferation ability of A549 and Calu-3 cells.5.Cell apoptosis ability represented that the apoptotic rate in si-circRNA group was significantly elevated?P<0.05?,indicating that downregulated hsa_circ₀025036 could enhance cell apoptosis ability of A549 and Calu-3 cells.6.Cell cycle anylsis showed that no difference existed among cell percentages at different cell cycle phases?P>0.05?.Meanwhile,Western blot showed the protein levels of CDK1,Cyclin D1 and Cyclin B1 among three groups possessed no significant difference.These data implied that downregulated hsa_circ₀025036 didn't affect cell cycle progress of A549 and Calu-3 cells.Part II Primary exploration of molecular mechanisms analyzing hsa_circ₀025036 involvement in lung adenocarcinomaMethods1.Bioinformatics analysis was utilized to assess hsa_circ₀025036 sequence and to predict the potential miRNA.2.The interaction between hsa_circ₀025036 and miR-198 was detected via dualluciferase reporter assay.3.The expression level of mi R-198 in lung adenocarcinoma tissues and paired normal tissues were assessed via qRT-PCR followed by analyzation of the relationship of miR-198 level and related clinical parameters.We then explored the correlation between miR-198 and hsa_circ₀025036 expression.4.qRT-PCR was applied to investigate the expression level of miR-198 in cells with downregulated hsa_circ₀025036.5.Specific miR-198 mimic and scrambled miRNA were designed and synthesized.LipofectamineT M2000 was applied to transfect A549 and Calu-3 cells with miR-198 or miR-NC.Blank group was set as control.The expression level of miR-198 in three groups was explored via qRT-PCR.6.We explored the effect of upregulated mi R-198 towards biological behaviors of A549 and Calu-3 cells via the following methods.CCK-8 and colony formation assays were used to detect cell proliferation ability.Flow cytometer was conducted to assess cell apoptosis rate and cell cycle.7.In vivo experiment.Recombinant lentivirus vector containing pre-miR-198 was successfully conducted,then the vectors were applied to infect A549-luc and Calu-3-luc cells.qRT-PCR was conducted to verify the success erection of lung adenocarcinoma cells highly expressing miR-198.Xenografted nude mice model were then perfomed.Small animal imaging system was utilized to monitor growth condition of xenografted tumors.Growth curve was drawn to analyze the tumor size in three groups.8.The potential targeted genes were predicted via bioinformatics analysis.9.Dual-luciferase assays were performed to verify whether mi R-198 could target 3'UTR of SHMT1 and TGF-? mRNA.10.Restore assay of hsa_circ₀025036 was then conducted.mi R-198 inhibitor and hsa_circ₀025036 siRNAs were co-transfected into A549 and Calu-3 cells to observe whether downregulated mi R-198 could restore the effect of hsa_circ₀025036 siRNA towards lung adenocarcinoma cells.CCK-8 and flow cytometer were applied to investigate related data.11.Restore assay of SHMT1 and TGF-? was performed.Recombinant vectors of pcDNA3.1-SHMT1 and pcDNA3.1-TGF-? not containing their 3'UTR were successfully conducted and then transfect A549 and Calu-3 cells respectively or together with mi R-198 mimic.Western blot was used to explore the expression level of SHMT1 and TGF-? proteins.CCK-8 assay was conducted to detect cell proliferation and flow cytometer was utilized to test cell apoptotic rate.12.Western blot was utilized to assess the expression of SHMT1 and TGF-? proteins in cells with downregulated hsa_circ₀025036 or upregulated miR-198.13.The effects of downregulated hsa_circ₀025036 and upregulated miR-198 towards cell biological bebaviors of A549 and Calu-3 cells were compared then.CCK-8 and flow cytometer were applied to explore related parameters.Results1.Bioinformatics analysis showed that interactive regions existed between hsa_circ₀025036 and miR-198.2.Dual-luciferase reporter assay exhibited significantly lower luciferase activity in mi R-198 mimic and pmirGLO-Wt-circRNA co-transfection group?P<0.05?compared with other three groups.After co-transfection with pmirGLO-MutcircRNA and mi R-198 mimic,the luciferase activity showed no significant difference compared with control groups,indicating that hsa_circ₀025036 could interact with miR-198 through the seed region.3.mi R-198 expression in lung adenocarcinoma tissues was decreased compared with normal lung tissues?P<0.05?.Besides,the expression level of miR-198 was related with aggressive clinical parameters including TNM stage and lymph node metastasis.mi R-198 expression in tumor tissues was negatively correlated with hsa_circ₀025036 expression.4.mi R-198 expression in si-circRNA group was upregulated via qRT-PCR?P<0.05?,suggesting that upregulated hsa_circ₀025036 in A549 and Calu-3 cells could increase mi R-198 expression.These data indicate that hsa_circ₀025036 could negatively regulate miR-198.5.The effects of upregulated miR-198 towards lung adenocarcinoma cells were then detected.The cell proliferation rate in miR-198 group was significantly decreased compared with control groups?P<0.05?.The apoptotic rate in miR-198 group was increased?P<0.05?.Besides,the number of cells at S-phase was decreased while the number at G2/M phase was increased?P<0.05?.Western blot displayed that CDK1,Cyclin D1 and Cyclin B1 expression in mi R-198 group was decreased.These results suggest that upregulated mi R-198 could inhibit cell proliferation,enhance cell apotosis and lead to cell cycle arrest.6.Xenografted tumor growth curve displayed that the fluorescent signal in miR-198 group was significantly decreased compared with control groups,suggesting that upregulated mi R-198 could inhibit the growth of xenografted lung adenocarcinoma.7.The interaction regions between miR-198 and SHMT1 or TGF-? were discovered via bioinformatics approaches.8.Dual-luciferase reporter assay exhibited significantly decreased luciferase activity in mi R-198 mimic and pmirGLO-Wt-SHMT1 or pmirGLO-Wt-TGF-? cotransfection group?P<0.05?,indicating that miR-198 could directly target 3'UTR of SHMT1 and TGF-? mRNA.9.Restore assay of hsa_circ₀025036 was performed then.Cell proliferation rate in mi R-198 inhibitor and hsa_circ₀025036 siRNA co-transfection group was significantly increased while the apoptotic rate was decreased?P<0.05?.These results indicate that mi R-198 inhibitor possibly restore the effect of hsa_circ₀025036 siRNA on cell proliferation and apoptosis of lung adenocarcinoma cells.These data suggest that mi R-198 exerted functions as a downsteam factor of hsa_circ₀025036.10.Recombinant vectors pcDNA3.1-SHMT1 and pcDNA3.1-TGF-? not containing their 3'UTR were successfully constructed.Western blot showed that SHMT1 and TGF-? expression were significantly increased in mi R-198 mimic and recombinant vector co-transfection group.Besides,the OD value in co-transfection group was elevated and cell apoptotic rate was decreased?P<0.05?.These data suggest that miR-198 couldn't target SHMT1 and TGF-? without 3'UTR.SHMT1 and TGF-? were proved to be downstream of miR-198.11.The expression levels of SHMT1 and TGF-? proteins were decreased in sicircRNA and mi R-198 group?P<0.05?,suggesting that downregulated hsa_circ₀025036 or upregulated miR-198 probably suppressed the expression of SHMT1 and TGF-? proteins.12.The comparison of the functions of downregulated hsa_circ₀025036 and upregulated mi R-198 exhibited that they possessed similar effects towards cell proliferation and cell apoptosis.However,miR-198 mimics could lead to cell cycle arrest while hsa_circ₀025036 siRNA didn't show any effects on cell cycle.These data indicate that other molecular mechanisms might still need to be discovered to further explain the biological functions of hsa_circ₀025036 in lung adenocarcinoma.Conclusions1.A new subtype of hsa_circ₀025036 with a length of 196 nt was confirmed to exist in lung adenocarcinoma tissues and cell lines which consisted of Exon 5,Exon6 and part of Exon 7 of FOXM1.The BS junction site located between Exon 5 and Exon 7.We temporarily named it as hsa_circ₀025036 in the present study.2.The expression level of hsa_circ₀025036 was upregulated in lung adenocarcinoma tissues and was related with clinical parameters including differentiation,TNM stage and lymph node metastasis.3.Downregulated hsa_circ₀025036 suppressed cell proliferation ability,and enhanced cell apoptosis of lung adenocarcinoma cells.4.hsa_circ₀025036/miR-198/SHMT1&TGF-? axis is probably a significant mechanism analyzing the involvement of hsa_circ₀025036 in lung adenocarcinoma.5.hsa_circ₀025036 exerts cancer promoting effects in lung adenocarcinoma,and will probably serve as a novel potential therapeutic target in clinical application.