Expression Pattern And Functional Study Of Long Non-coding RNA Loc101928809 In Non-small Cell Lung Cancer

Background:Lung cancer is one of the common malignant tumor,and also it causese serious damage to human body health,its incidence and mortality has been ranked first of various tumors.Epidemiological studies have shown that,the main risk factors includes:smoking,passive smoking（such as second-hand smoking）,chronic bronchitis,pulmonary tuberculosis,emphysema,family history of tumor,family history of lung cancer,biofuel pollution,ionizing radiation,air pollution and heavy metal pollution and so on.the5-year survival rate of lung cancer is less than 18%At present,the most common and effective treatment strategy is pneumonectomy adjuvant chemotherapy,the therapeutic stratage of lung cancer is searching for effective early diagnosis and prognosis biomarkers.it can provide effective scientific basis for the treatment of lung cancer.the treatment of lung cancer is renewed every day,different types of lung cancer have different strategies,and the main means of diagnosis is pathological section.It is accurate but easily affected by sampling and it is hard to diagnose the accurate subtypes of lung cancer,which affects the choices of the strategies for the treatment of patients,so we need effective classification of biomarkers for lung cancer.In recent years,long non-coding RNA（lncRNAs）emerged in tumor expression patterns,biological effects,and more and more literatures revealed the biological mechanisms of lncRNAs in the process of tumor.According to the latest literature,there is only about 2%protein-coding RNA in the human genome,more than 98%belong to the non-coding RNA genes.Those non-coding RNA was once considered as the"noise"in the transcription,but now more and more scientific research shows that non-coding RNA is involved in a variety of biological processes in the body,including the transcription,mRNA splicing,even the translation can be regulated by non-coding RNA.Long non-coding RNA（lncRNAs）is a kind of non-coding RNA family,which is more and more popular in the scientific research.LncRNAs is longer than 200 nt and roughly divided into:（1）Sense lncRNAs,（2）antisense lncRNAs,（3）bidirectional lncRNAs,（4）Intergenic lncRNAs and（5）Intronic lncRNAs.There are many reports about the lncRNAs,which is associated with lung cancer,such as lncRNA MALAT1（metastasis associated in lung adenocarcinoma transcript 1）,which is highly expressed in lung cancer tissues,and lowly expressed in the adjacent tissues in the carcinoma,the biological function reveals that lncRNA MALAT1is associated with the metastasis and prognosis of lung cancer,and it may be as a sign of lung cancer diagnosis and targeted therapy.In addition,lncRNA HOTAIR（HOX transcript antisense intergenic RNA）which is highly expressed in lung cancer tissues can promote the invasion and metastasis of lung cancer cells in the bodyWe use 8 pairs of lung cancer tissue（cancer tissues and adjacent normal tissues）to do RNA-sequence detection,we discover that loc101928809 is lowly expressed in lung cancer tissues.So we detected the expression level of Loc101828809 in 276 pairs of lung caner tissues in our lab.And we comformed the functions of Loc101828809 through overexpression and shRNA technology,these expriments help us to reveal the biological role of Loc101828809 in the development of lung cancer,and at the same time we treated the stable transfeced cells by chemical drugs to explore whether loc101928809would work in chemical treatment,we hope to reveal that loc101928809 could provide a scientific basis on the lung cancer diagnosis and treatment strategies.AIM:Analysis the expression patern and biological functional of long non-coding RNA loc101928809 in non-small cell lung cancerMethod:1.Detect the relative expression of loc101928809 in the lung cancer tissues and its corresponding adjacent tissues through qRT-PCR technology,at the same time analysis the relation between the expression of loc101928809 and clinical features of patients with lung cancer.2.Build loc101928809 over-expression and shRNA lentivirus vetors and the corresponding expression control vector,using lentivirus packaging technology to transfect lung cancer cell lines A549 and PC-9 cells,Detect the function of loc101928809through stable transfectd cell lines,using CCK8,transwell migaration and invasion,colony formation,colony formation in soft agar,flow cytometry and nude-mice model.3.Treated the stable transfected A549 and PC-9 cell lines with DDP,to explore the possible clinical biological effect4.“RESCUE”assay is comformed to explore the biological association bewteen loc101928809 and CXCL-9.RESULT1.Through the qRT-PCR technique,we found that the relative expression of66.30%（183/276）of lung cancer tissue is lowly expressed,33.70%（93/276）of lung cancer tissue is highly expressed（Mean±SD,0.002298±0.003469 vs 0.001355±0.001523,P=0.028）.2.loc101928809 was majorly located in the nucleus other than cytoplasm,indicating a role as transcriptional regulator of the LncRNA.3.loc101928809 lower expression in patients with lung cancer is associated with clinical progress（Ⅰ+Ⅱvs.Ⅲ+Ⅳ,P=0.0002）.Lymph node metastasis（N0 vs.N1+N2+N3,P=0.0033）,Distant metastases（M0 vs.M1,,P=0.0019）.Further analysis with the survival data,we found that the high expression of loc101928809 in patients caused a high MST than loc101928809 lowly expressed in patients(MST:18.0月vs.11.0月,P_logrank<0.05)4.By filtrating two stable transfected cell line A549 and PC-9:loc101928809 lowly expressed cell lines,loc101928809 highly expressed cell lines and the corresponding high or low expression control cell lines.The CCK8 assay datas tell us,in the cell line A549 and PC-9,the cell growth rate of up-regulated loc101928809 is lower than the control group,and the cell growth rate of down-regulaed loc101928809 is higher than the control group（P<0.05）.5.Colony formation and colony formation in soft agar is uesd to detect the clonogenesis ability of loc101928809 in stable tranfected cell lines.Both the assays tell us,the counts of the loc101928809 highly expressed group is lower than the control group,and the loc101928809 lowly expressed group is higher than the conrtol group.（P<0.05）.6.By flow cytometry instrument,we detected the cell cycle and apoptosis of stable transfected cell lines.Over expression of loc101928809 made more cells stop in the phase of（G0-G1）,（P<0.05）and less cells in the phase of（G2-M）（P<0.05）,thus inhibiting the proliferation of cell division.Apoptosis tests have shown that over-expression of loc101928809 can promote a higher cell apoptosis.（P<0.05）7.By Transwell Migration and Invasion assay,we discovered that both in stable transfected A549 and PC-9 cell lines,the migrated cells was significantly lower in loc101928809 over-expression cells than the lower expression cells.（P<0.05）8.By treating the stable transfected A549 and PC-9 cell lines with DDP.First we found IC50%of A549 cells is 8 ug/ml,IC50%of PC-9 is 32 ug/ml,respectively using IC50%to treat the stable transfected A549 and PC-9 cell lines,using flow cytometry instrument to detect cell cycle and apoptosis after drug treatment.Over expression of loc101928809 made more cells stop in the phase of（G0-G1）,（P<0.05）Apoptosis tests have shown that over-expression of loc101928809 can promote a higher cell apoptosis.（P<0.05）,suggesting that over-expression of loc101928809 can increase the sensibility of DDP during the cisplatin therapy.By using different concentrations of cisplatin to treat the different group of cells,we discovered that the over-expressed cells had a lower proliferation than the lower-expressed cells.also,we use the IC50 to treat the stable tansfected cells,and we found that the IC50 of over-expression loc101928809 was ahead of the controls and low-expression of I50 was after the controls（P<0.05）.Nude mice cisplatin model was comformed to comfirm the cisplatin sencitivity,we found that the up-regulated loc101928809 had a higher medium survival time,which was 53 days than the controls that is 36 days,and the down-regulated loc101928809 had a lower MST than the control（down-regulation VS.Control:28 days VS.40 days,P<0.05）9.The nude mice experiments showed that the volume of the injected cells with over-expression of loc101928809 is smaller than the control vectors and which with lower-expression of loc101928809 is larger than the control vector both in A549 and PC-9 cell lines.（P<0.05）.The nude mouse metastasis model through tail vein injection tells us,both in stable transfected A549 and PC-9 cell lines,the low-expression of loc101928809 has a higher metastasis compared to the control vector,and the HE staing comfirmed the metastasis was tumor cells.10.Through bioinformatics analysis,loc101928809 is associated with CXC motif chemokine ligand 9（CXCL-9）.we found that CXCL-9 was lowly expressed in the lung cancer tissues and highly expressed in their corresponding adjacent tissue by the qRT-PCR data（P<0.05）,the expression of CXCL-9 was positively correlated with loc101928809（r=0.3286,P=0.0014）.Also we we detected the concentration of CXCL-9 in cell culture medium,we found that both in the transfected A549 and PC-9cell lines,the over-expression of loc101928809 has a higher concentration of CXCL-9compared to the control group,and the lower expression of loc101928809 has lower concentration of CXCL-9.The“RESCUE”assay,which we co-transfected the A549 and PC-9 cells with over-regulation vector of loc101928809 and down-regulation vector of CXCL-9.The CCK8 assay datas tell us,in the cell line A549 and PC-9,the cell growth rate of up-regulated loc101928809 is lower than the group with loc101928809up-regualtion and CXCL-9 down-regualtion（P<0.05）,suggesting that loc101928809 is associated with CXCL-9.Conclusion:1.loc101928809 was lowly expressed in lung cancer tissues;the high expression of loc101928809 can obviously promote the clinical progress of lung cancer,prolong the survival time in patients with lung cancer.2.loc101928809 can inhibit lung cancer cell growth,proliferation,metastasis and invasion,promote cell apoptosis both in vivo and in vitro.3.loc101928809 can increase the sensibility of DDP during the cisplatin therapy.Summary:loc101928809 was down-regulated in lung cancer tissues,and loc101928809 can influence the expression of CXCL-9,inhibit the lung caner cells proliferation,metastasis and invasion,indicating that loc101928809 could surve as a potential progress and prognosis biomarker of lung cancer.