Expression And Effect Of LncRNA RGMB-AS1 On The Biological Behavior In Lung Adenocarcinoma

Background and objective Lung cancer is one of malignant tumors.Based on the clinic standard treatment,most of lung cancer is non-small cell lung cancer（NSCLC）,about 80%.Another is small cell lung cancer（SCLC）.Environmental pollution and smoking are more serious in recent years.The morbidity and mortality of lung cancer is rising year by year.Although the treatment methods keep in progress,the 5-year survival rate is less than 15% because of the proliferation,invasion and metastasis of lung cancer cell.In order to raise the survival rate and reduce the mortality of lung cancer patients,we must further study its pathogenesis,and intervene the progression of lung cancer in early stage.The biological formation mechanism and function of non-coding RNA（nc RNAs）have been drawn public attention.Mi RNA has been verified to play an important role in cell biological behavior,such as cell growth,differentiation,apoptosis and cell cycle by regulating the target gene at the transcription and post-translation levels.Long non-coding RNA（lnc RNA）is a diverse class of nc RNA with >200 nucleotides in length.So far,expression of several lnc RNAs have been reported in many kinds of tumors,and involved in regulating expression of gene by a series of molecular pathways,which resulted the change of cell biological behavior.Therefore,it is conducive to provide new and potential target for tumor therapy to study the function and mechanism of lnc RNAs in-depth.Lnc RNA RGMB-AS1 is located on human chromosomal band 5q21.1.No report was found about lnc RNA RGMB-AS1 in lung cancer at home and abroad.Combined with the results of preliminary experiment about the high expression of lnc RNA RGMB-AS1 in lung adenocarcinoma tissues and its correlation with cancer tissue differentiation and lymph node metastases,we think it is worthy of exploring the function and mechanism of lnc RNA RGMB-AS1 in the development of lung adenocarcinoma.Therefore,our study synthetically analysed the expression and function of lnc RNA RGMB-AS1 in lung adenocarcinoma and discussed its potential mechanism.Our study included four parts.The first part is expression and analysis of lnc RNA RGMB-AS1 in lung adenocarcinoma tissues;the second part is biological effects of downregulating potential lnc RNA RGMB-AS1 expression in A549 and SPC-A-1 cell;the third part is biological effects of downregulating potential lnc RNA RGMB-AS1 expression;the fourth part is preliminary study of mechanism of lnc RNA RGMB-AS1.Part One: Expression and analysis of lnc RNA RGMB-AS1 in lungadenocarcinoma tissuesMethods: 1.110 paired lung adenocarcinoma tissues and adjacent normal tissues（≥3 cm away from tumor）were obtained from patients who received surgical resection of lung adenocarcinoma.2.The expression of lnc RNAs was detected in 5 cases lung adenocarcinoma tissues and adjacent normal tissues by lnc RNA chip.3.q RT-PCR method was used to detect expressions of 9 lnc RNAs in 110 cases of lung adenocarcinoma tissues,including 5 up-regulated lnc RNAs（LOC284801,KIAA1908,XLOC₀08466,LINC00665 and RGMB-AS1）and 4 down-regulated lnc RNAs（MAGI2-AS3,LOC100505495,LOC400550 and XLOC₀01412）.The aim is to test and verify the reliability of lnc RNA chip.4.Beased on lnc RNA RGMB-AS1 expression,correlation analysis was used to verify the correlation between lnc RNA RGMB-AS1 expression and gender,age,differentiation status,lymph node metastases and TNM stage.Results: 1.Compared with adjacent normal tissues,lung adenocarcinoma tissues exhibited 907 differentially expressed ln RNAs,including 272 lnc RNAs upregulated（30%）and 635 lnc RNAs downregulated（70%）.Lnc RNA RGMB-AS1 is upregulated in lung adenocarcinoma tissue（P < 0.05）.2.q RT-PCR results showed that expression level of 9 lnc RNAs were consistent with the results of ln RNA chip.3.Lnc RNA RGMB-AS1 has a significant upregulation in lung adenocarcinoma tissues（P < 0.05）.And its expression in lung adenocarcinoma tissues was associated with differentiation status,TNM stage and lymph node metastases（P < 0.05）.There was no statistically significant correlation between lnc RNA RGMB-AS1 expression and either gender or age（P<0.05）.Part Two Effect of down-regulated lnc RNA RGMB-AS1 on growth,migration and invasion of A549 and SPC-A-1 cellsMethods: 1.Small interferring RNA（si RNA）against lnc RNA RGMB-AS1,and control oligonucleotides（negative control,NC）were synthesized and transfected into A549 and SPC-A-1 cells using LipofectamineTM2000.2.q RT-PCR was used to detect the change of lnc RNA RGMB-AS1 expression in every group to select the best si RNA oligonucleotides against lnc RNA RGMB-AS1.3.CCK8 and colony formation assay experiment were used to detect proliferation and growth ability of A549 and SPC-A-1 cells in each group.4.Flow-cytometric analyses were used to assess cell cycle progression of A549 and SPC-A-1 cells in each group.5.Wound healing assay was performed to assess migration ability of A549 and SPC-A-1 cells in each group.6.Transwell invasion assay was performed to assess invasion ability of A549 and SPC-A-1 cells in each group.7.SPSS 21.0 was used to analyze all the dates.Results: 1.Compared with Blank and NC groups,lnc RNA RGMB-AS1 expression was significantly decreased in si RNA1 group（P < 0.05）.2.Results of CCK8 showed that the proliferation of A549 and SPC-A-1 transfected with si RNA was markedly decreased with time passing when compared to the control groups（NC and Blank group）（P<0.05）.3.Results of the colony formation number of si-lnc RNA group had a significant reduce in A549 and SPC-A-1 cells when compared to the control groups（NC and Blank group）（P<0.05）,which revealed that down-regulation of could affect the proliferation capacity of A549 and SPC-A-1 cells 4.Results of flow cytometry assay showed a marked increase in the percentage of cells in the G0/G1 phase and a reduction in the percentage of cells in the S phase in the si-lnc RNA group in A549 and SPC-A-1 cells when compared to the control groups（NC and Blank group）（P<0.05）.5.Results of wound healing assay showed that the migratory ability of A549 and SPC-A-1 cells in the si-lnc RNA group was lower at 24 h post-wounding compared with both NC and Blank groups（P<0.05）.6.Results of transwell invasion assay showed that the number of invading cells was significantly lower in the si RNA group than in the two control groups in both A549 and SPC-A-1 cells（P<0.05）.Part Three Effect of down-regulated lnc RNA RGMB-AS1 on the growth of xenograft tumor in nude miceMethods: 1.sh RNA against lnc RNA RGMB-AS1,and negative control sh RNA were synthesized.And construct recombined lentiviral vector about down-regulated lnc RNA RGMB-AS1 and negative control,which were infected Luciferase-tagged A549 and SPC-A-1 cells（A549-Luc and SPC-A-1-Luc cells）.2.q RT-PCR was used to test the expreesion of lnc RNA RGMB-AS1 in infected cells.3.Infected A549-Luc and SPC-A-1-Luc cells were implanted in BALB/c nude mice by subcutaneous injection and tumor xenograft model were constructed.The growth of tumor xenograft models were observed at 1,2,3 and 4 week.4.Immunohistochemistry and western blot were used to detect Ki67 expression.Results: 1.Lentiviruses with lnc RNA RGMB-AS1 sh RNA and NC were successfully constructed.Stably infected A549-Luc and SPC-A-1-Luc cells were constructed.2.The growth of xenograft tumors was assessed to further examine the tumor-suppressing effect of si RNA against lnc RNA RGMB-AS1 on lung adenocarcinoma.In A549 and SPC-A-1 cell tumors,the luciferase signal was clearly lower in sh RNA1 mice than in the control groups at 2,3,and 4 weeks.The signal intensity in the sh RNA1 group decreased with time（P < 0.05）.3.Results from immunohistochemistry and western blot showed that significant differences in Ki67 expression were observed between the sh RNA1 group and the control groups（P < 0.05）.There were no differences in Ki67 expression between NC group and Blank group（P > 0.05）.Part Four Preliminary study about mechanism of lnc RNA RGMB-AS1Methods: 1.UCSC database transcription information was used to predict the potential target gene of lnc RNA RGMB-AS1.2.Based on the analysis results,p EGFP-N3-Exon1,p EGFP-N3-Exon2 and p Silencer 3.1-Lnc-Exon3 were constructed respectively.And then they were transfected into A549 cells using LipofectamineTM2000 respectively.Cells were subdivided into six groups as follows: For Exon1,Exon1-I: cells transfected with p EGFP-N3-Exon1 only;Exon1-II: cells co-transfected with p EGFP-N3-Exon1 and p Silencer 3.1;Exon1-III: cells co-transfected with p EGFP-N3-Exon1 and p Silencer 3.1-Lnc-Exon3;for Exon2,Exon2-I: cells transfected with p EGFP-N3-Exon2 only;Exon2-II: cells co-transfected with p EGFP-N3-Exon2 and p Silencer 3.1;Exon2-III: cells co-transfected with p EGFP-N3-Exon2 and p Silencer 3.1-Lnc-Exon3.3.Inverted fluorescence microscope and fluorescence photometer were used to observe and test the fluorescence in every group.4.q RT-PCR method was used to detect expressions of RGMB in 110 cases of lung adenocarcinoma tissues,and correlation analysis was used to verify the correlation between RGMB expression and gender,age,differentiation status,lymph node metastases and TNM stage.Western blot was used to detect expression of RGMB in protein level.And the relation of lnc RGMB-AS1 and RGMB was analyzed by the Spearman correlation analysis.5.pc DNA3.1（+）-RGMB recombinant vector was constructed and transfected into A549 and SPC-A-1 cells by lipofectamineTM2000 to further verify the potential relationship between lnc RGMB-AS1 and RGMB in lung adenocarcinoma cells.Results: 1.The UCSC database transcription information indicated that lnc RNA RGMB-AS1 is located on human chromosome 5 and has two interaction sites with the first exon and second exon of RGMB in the third exon region.2.RGMB has a significant downregulation in lung adenocarcinoma tissues（P < 0.05）.And its expression in lung adenocarcinoma tissues was associated with differentiation status,TNM stage and lymph node metastases（P < 0.05）.There was no statistically significant correlation between lnc RNA RGMB-AS1 expression and either gender or age（P<0.05）.3.Western blot analysis showed that the expression of RGMB was higher in the RGMB and si-lnc RNA groups than in the Blank groups（P < 0.05）.4.The changes of fluorescence in the different groups were analyzed at 48 h after transfection.The fluorescence intensity of the p EGFP-N3-Exon1 group was used as a control to analyze the relative fluorescence intensity in the other groups.For Exon1,the results of fluorescence microscopy and fluorescence microplate reader showed that the fluorescence intensity of cells co-transfected with p EGFP-N3-Exon1 and p Silencer 3.1-Lnc-Exon3 did not differ from that of cells transfected with p EGFP-N3-Exon1 only or that of cells co-transfected with p EGFP-N3-Exon1 and p Silencer 3.1（P>0.05）.For Exon2,the fluorescence intensity of cells co-transfected with p EGFP-N3-Exon2 and p Silencer 3.1-Lnc-Exon3 was weaker than that of cells transfected with p EGFP-N3-Exon2 only or that of cells co-transfected with p EGFP-N3-Exon2 and p Silencer 3.1（P < 0.05）.There was no difference between the fluorescence intensity of cells transfected with p EGFP-N3-Exon2 only and that of cells co-transfected with p EGFP-N3-Exon2 and p Silencer 3.1（P > 0.05）.5.Results of CCK8 showed that the OD450 values in RGMB cells on days 2,3 and 4 were significantly decreased compared with those in Blank cells,and the decreasing trend was more obvious in si-lnc RNA cells（P < 0.05）.6.Flow cytometric analysis of cell cycle progression indicated that the proportion of cells in S phase was reduced in the si-lnc RNA and RGMB groups,and thereduction was greater in the si-lnc RNA group than in the RGMB group（P < 0.05）.7.Transwell invasion assays demonstrated that the invasive ability was higher in the si-lnc RNA group than that in the RGMB group（P < 0.05）.Conclusions 1.907 lnc RNAs in lung adenocarcinoma tissues have abnormal expression,including 272（30%）up-regulation and 635（70%）down-regulation.Lnc RNA RGMB-AS1 expression level increased significantly when compared to corresponding adjacent normal lung tissues.Lnc RNA RGMB-AS1 expression level in lung adenocarcinoma tissues was associated with differentiation status,TNM stage and lymph node metastases（P < 0.05）.2.Down-regulation of lnc RNA RGMB-AS1 in lung adenocarcinoma cells in vitro can significantly inhibit cells growth,reduce the migration and invasion ability of cells promote cells apoptosis.The animal experiment indicated that the down-regulation of lnc RNA RGMB-AS1 can significantly inhibit the transplant tumor growth in nude mouse models.3.Lnc RNA RGMB-AS1 negatively regulated RGMB expression,thus exert its biological function.4.Lnc RNA RGMB-AS1 can play a role in cancer promotion and is expected to become new targets for lung adenocarcinoma gene therapy.