| Background and objectiveLung cancer makes most threats against human’s health and life in all malignant tumors.According to the didfference of pathological characteristic,lung cancer can be divided into non-small cell lung cancer(NSCLC,accounting for 85%)and small cell lung cancer(SCLC).Lung adenocarcinoma,which accounts for more than half of NSCLC,become the highest incidence of lung cancer types in our country.The current treatment of lung adenocarcinoma includes surgery,radiotherapy,chemical medication,targeted therapy and tumor immunotherapy.With the development of the studies on disease biology and tumor mechanism,some selected patients benefited from small molecule tyrosine kinase inhibitors and immunotherapy.However,the overall cure and survival rates for NSCLC remain low,particularly in metastatic disease.Therefore,more researches on the mechanism of lung adenocarcinoma have important practical significance and theoretical value to explore more effective treatments improving outcomes.The results of human genome show that fewer than 2%of whole genomecould encode protein,and the majority of non-coding sequences are transcribed into long non-coding RNA(lncRNA,with the length of more than 200 bases).lncRNAs participate in the regulation of gene expression to promote or inhibit the occurrence and development of various tumors.Therefore,lncRNA is expected to be a new tumor marker and an important target for the prevention and treatment of cancer.In our study,the abnormal expression of LINC01296 in lung cancer tissues was screened by gene chip technology.LINC01296 located in chromosome 14,at present the report of LINC01296 is limited,only relevant bioinformatics database shows LINC01296 abnormal expression in the part of the tumor.LINC01296 may have played a very important role in the development of cancer.The study is the first report of the expression and biological function of LINC01296 in lung adenocarcinoma,and the potential related molecular mechanisms was elucidated.This study includes three parts.In the first part,we detected the expression level of IncRNA and analyzed the correlation between clinicopathological characteristics and LINC01296 in lung adenocarcinoma.In the second part,we studied the role of knockdown of lncRNA LINC01296 in migration,proliferation and invasion of lung adenocarcinoma A549 cells.Finally,we studied that LINC01296 activated the JAK/STAT pathway to promote the development of lung adenocarcinoma.Part One Study of abnormal expression of lncRNA and correlation with clinicopathological characteristics in lung adenocarcinomaMethods1.LncRNA chips was used to detect the expression of lncRNA in 3 samples of lung adenocarcinoma and corresponding adjacent normal tissue samples.2.RT-qPCR was used to detect the expression levels of 4 lncRNAs(LINC01296,CTC429P9,RP11-344B5.2 and RXRG-2)in 60 specimens and verify the reliability of the results of IncRNA microarray from the tissue level.3.We use RT-qPCR to detect expression of lncRNA LINC01296 in the 3 lung adenocarcinoma cell lines(A549,SPC-A1 and Calu-3)and a Normal human embryonic lung fibroblasts.HPF-1 and to validate the reliability of lncRNA chip results from the cells.4.According to the expression level of IncRNA,Chi-square test was used to analysis relationship between LINC01296 and in lung adenocarcinoma patients gender,age and pathological type,differentiation degree,TNM stage,lymph node metastasis and distant metastasis.Results1.Using gene chip in lung adenocarcinoma tissue and tissue adjacent to carcinoma of extract 873 differentially expressed lncRNA,Lung adenocarcinoma tissues were screened for 16 differentially expressed lncRNAs,in which 9(56.25%)lncRNAs are upregulation and 7(43.75%)are downregulation,and IncRNA LINC01296 are highly expressed in lung adenocarcinoma tissue.2.The qRT-PCR results of 60 cases of lung adenocarcinoma tissues and corresponding adjacent normal tissues showed that LINC01296,CTC429P9 and RP11-344B5.2 expression in lung adenocarcinoma tissues were significantly higher than that in adjacent normal tissues(P<0.05)and the expression of RXRG-2 in lung adenocarcinoma was significantly lower than that in adjacent normal tissues(P<0.05),which indicated that results of RT-qPCR was well consistent with IncRNA chip and confirmed the reliability of microarray data.3.The expression LINC01296 in lung adenocarcinoma cell lines A549,SPC-A1 and Calu-3 was significantly higher than Normal human embryonic lung fibroblasts lines HPF-1(P<0.05),which further indicated that the expression level of LINC01296 was significantly increased in lung adenocarcinoma.4.Analysis of Chi-square test showed that expression of LINC01296 significantly correlated with histological differentiation,lymph node metastasis and distant metastasis(P<0.05)and and have no correlation with the patient age,gender,and smoking(P>0,05).Part two Effects of downregulate LINC01296 expression on proliferation,migration,invasion and apoptosis of lung adenocarcinoma cell line A549 Methods1.We constructed lentiviral vector which downregulate LINC01296 expression and transfected it into A549 cells to obtain A549 cells with knockdown LINC012962.CCK-8 experiment was used to detect the effect of downregulate LINC01296 expression on the proliferation of A549 cells.3.colony-forming unit assay was used to detect the effect of downregulate LING01296 expression on the cloning ability of A549 cells.4.Adhesion assay were used to detect the effect of downregulate LINC01296 expression on the adhesion ability of A549 cells.5.scratch assay were used to detect the effect of downregulate LINC01296 expression on the migration ability of A549 cells.6.Transwell assay were used to detect the effect of downregulate LINC01296 expression on the migration ability of A549 cells.7.Flow cell technology were used to detect the effect of downregulate LINC01296 expression on apoptosis of A549 cells.Results1.The pLVTHM-LINC01296 vector constructed successfully and sequencing and BLAST results showed identical;RT-qPCR results showed that the expression of LINC01296 in groups transfected with siLINCl,siLINC2 and siLINC3 group in A549 cells significantly decreased compared with the control groups(P<0.05).2.Results of CCK8 experiments showed that the proliferation of SCR cells groups had no significant change at each time point after transfection(P>0.05),and siLINC1,siLINC2 and siLINC3 groups of A549 cells appeared inhibition after transfection 12h,compared with the control groups(P<0.05),and suppression increasing with time passes.3.Colony-forrming experiment showed that compared with control groups(56.5±9.8),the number of colony-forming units of SCR groups(84.5±8.8)had no significant difference(P>0.05),and the number of colony-forming units siLINCl groups(27.2±7.1),siLINC2 groups(32.2±8.8)and siLINC3 groups(35.0±3.9)the number of clones was decreased(P<0.05).the results showed that when the LINC01296 expression was downregulated,the colony-forming ability of A549 cells was significantly inhibited.4.Adhesion experiments showed that compared to the control groups(51.8±7.3),after the cells planked 90min,the adhesion rate of SCR groups(47.2±5.1)%have no significant difference(P>0.05),and the adhesion rate of siLINCl groups(22.9±3.1),siLINC2 groups(25.7±4.5)and siLINC3 groups(29.1 ±2.4)were significantly decreased(P<0.05).These results showed that downregulate LINC01296 expression induced adhesion ability of A549 cells decrease.5.The results of scratch assays showed that compared with the control groups,Cell mobility of SCR groups had no significant difference(P>0.05),and the Cell mobility of siLIINC1,siLIINC2 and siLINC3 groups was significantly decreased(P<0.05).These results showed that downregulate LINC01296 expression induced migration ability of A549 cells was significantly inhibited.6.Transwell assays showed that compared with the control groups(62.7±10.5)compared with,the transmembrane cells number of SCR groups(56.7±8.5)have no significant difference(P>0.05),and transmembrane cells number of siLINC1,(23.4±4.7),siLINC2(25.7±2.4)and siLINC3 groups(30.7±4.2)was significantly decreased(p<0.05).These results showed that the downregulate LINC01296 expression induced the invasion of A549 cells were significantly inhibited.7.Flow cell technology showed that compared with the control groups(7.3±1.2)%,apoptosis Rate of SCR groups(7.94±1.6)%have no significant difference(P>0.05),and transmembrane cells number of siLINCl,(30.3±3.2),siLINC2(22.6±2.6)%and siLINC3 groups(25.96±6.78)%was significantly decreased(p<0.05).Part Three The study of LINC01296 activated the JAK/STATpathway to promote the development of lung adenocarcinomaMethods1.RT-qPCR technology was used to detect the expression of PIM1 in lung adenocarcinoma tissue and then analyze the correlation between the2.PIM1 and clinical pathological characteristics.2.The lentiviral vector,which induce silencing of PIM1,was transfected into A549 cells and then to detect the effects on proliferation,colony-forming,migration,invasion and apoptosis of lung adenocarcinoma cell line A549 cells by CCK-8 assay and colony-forming assay,adhesion assay,wound healing assay,transwell invasion assay and FCM.3.Western-blot was used to detect the effects of downregulate LINC01296 expression on the expression of proteins of JAK/STAT3 pathway in lung adenocarcinoma cell line A549 cells.4.Western-blot was used to detect the effect of downregulate LINC01296 expression on the expression of JAK2/STAT3 pathway in lung adenocarcinoma cells A549.5.Construct LINC01296 overexpression vector and obtain LINC01296 overexpression A549 cell lin,Western Blotting was used to detect the effects of LINC01296 overexpression and JAK2 inhibitor AG490 on JAK2/STAT3 pathway related proteins in lung adenocarcinoma cells A549.Results1.Compared with adjacent tissue,the expression of PIM1 was significantly increased in lung adenocarcinoma tissues(P<0.05).The Chi-square test showed that the expression of PIM1 in lung adenocarcinoma tissue were significantly related with lung adenocarcinoma tissue differentiation,lymph node metastasis,TNM stage(P<0.05),and have no correlation with the patient age,gender,and smoking(P>0.05).There was positive correlation between LINC01296 and PIM1 expression in lung adenocarcinoma tissues(R2 =0.580),and both of which showed abnormal high expression characteristics.2.Knockdown of PIM1 in A549 cells of lung adenocarcinoma can significantly inhibit cell adhesion,proliferation,,invasion and migration,and promote apoptosis.3.Virus vector with siLINCl sequence was transfected into lung adenocar-cinoma A549 cells,the use of Western blot method was used to test PIM1,the results show that compared with Con group(0.388 + 0.055),the SCR group(0.390 + 0.094)there was no significant difference(P>0.05),siLINCl group(0.221-0.042)PIMl was decreased(P<0.05).The expression of PIM1 was inhibited by knockdown LINC01296 in the A549 cells.4.The silencing of LINC01296 in A549 cells of lung adenocarcinoma significantly reduced the expression of JAK2/STAT3 pathway related proteins.5.Compared with the control group,overexpression of LINC01296 can increase the expression of JAK2/STAT3 pathway related protein(P<0.05),and AG490 can lower the expression of JAK2/STAT3 pathway related protein(P<0.05).Compared to LINC01296 group,LINC01296+AG490 group related protein of JAK2/STAT3 pathway were decreased,and the difference was statistically significant(P<0.05).Conclusions1.Both LINC01296 and PIM1 are highly expressed in lung adenocarcinoma,which are positively relevant.Morever,they are well correlated with tissue differentiation,lymph node metastasis and TNM staging.2.LINC01296 plays an important role in the proliferation,migration,invasion and apoptosis of lung adenocarcinoma,and PIM1 is the main executor of the biological function of LINC01296.3.LINC01296 may regulate the expression of PIM1 in lung adenocarcinoma via JAK2/STAT3 pathway,which is expected to be a new therapeutic target for lung adenocarcinoma. |
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