The Molecular Mechanism Of Long Non-coding RNA LINC00996 Regulating Non-small Cell Lung Cancer Proliferation And Metastasis

Background and objective Recent studies indicated that the long non-coding RNAs had played an important role in the aspects of dosage compensation,epigenetics regulation,cell cycle regulation,cell differentiation,cell apoptosis,cell ageing and tumor genesis.Many types of lnc RNAs has been found with lots of function mechanisms.And with the development of high-throughput transcriptome sequencing technology,more and more long non-coding RNAs will be found in the further.But the detailed mechanisim of long non-coding RNAs for regulating the cell function was not clear.So,it was very important to find the functional long non-coding RNAs from the long non-coding RNA pools.And the aim of this study was to selecte the potential regulating long non-coding RNAs and further discuss its function in the aspects of cell differentiation,cell apoptosis,cell ageing and et al.Methods(1)The different expressed long non-coding RANs was screened and selected from the GEO database(Http://www.ncbi.nlm.nih.gov/geo)according to the SAGE tags.The up or down expression long non-coding RNA more than 1.5 times between lung cancer tissue and normal lung tisusue were selected as the target long nong coding RNA.(2)The different expression of long non-coding RNAs were conrimed by using the real-time polymerase chain reaction between normal bronchial epithelial cells(BEAS-2B)and non small cell lung cancer cell(NL9980,L9981,A549,H1299,H446 and et al).And the long non-coding RNA LINC00996 was selected as the study object.(3)The expression of LINC00996 was arrayed in 30 cases of non small lung cancer patients with their cancer tissue and adjacent lung cancer tissue to confirm the different expression pattern between the cancer and normal lung tissue.(4)The small interfeenc RNAs was designed and synthesized according to the full lenths of the LINC00996 and was transinfected by Lipofect-200 to the non small cell lung cancer cell H1299 as well as no correlative siRNA.(5)The cell cycle changes was arrayed bty flow cytometry between the know down LINC00996 non small cell lung cancer cells(H1299)and normal controls.(6)The cell proliferation ability changes was tested by cell counting instrument between know down LINC00996 non small cell lung cancer cells(H1299)and normal controls.(7)The cell proliferation ability was also arrayed by clone formation assay.(8)The cell migration ability was arrayed by wound healing and transwell test between know down LINC00996 non small cell lung cancer cells(H1299)and normal controls.(10)The proteins associated with cycle pathway were arrayed by western-blot test.Results(1)Forty three different expressed long noncoding RNAs was selected form the GEO databases with the SAGE tags.(2)The long noncoding RNA LINC00996 was selected as the study objects because of its high expressed in the non small cell lung cancer cell.(3)The expression pattern of long non coding RNA LINC00996 was tested in 30 paired non small cell lung cancer patiens with 17 cases up expression of LINC0096 and 3 down expression of LINC00996.(4)The small interference RNA was synthesized and LINC00996 was succesfull knowdown by small interference RNA(si-868).(5)The proliferation ability was decreased(P<0.05),the proportion of G0/G1 cell phase was elevated(P<0.05),the cell apoptosis proportion was elevated(P<0.05)and the cell migration ability was decreased(P<0.05)after know down the LINC00996 expresin in H1299 cell line.(6)The p-Rb and c-myc protein was decreased after knowing down the expression of LINC00996 withou changes of total Rb protein.And the protein of P15 was elevated.The p-Rb,c-myc and P15 changes after knowing down the expression of LINC00996 was incordance with the inhibiration of cell proliferation pathway.Conclusion(1)Long non-coding RNAs had different expression pattern in the lung cancer cell line,normal bronchial epithelial cells,cancer tissue of non-small cell lung cancer patiens and normal tissue of non-small lung cancer patiens.(2)High expression of LINC00996 was detected in several lung cancer cell lines and cancer tissue compared with normal bronchial epithelial cells and normal tissue of non small cell lung cancer patients.(3)Long non-coding RNA LINC00996 may be involved in the procedure of cell prolofiration,migration,apoptosis,cell cycle and cell ageing.(4)The cell proliferation biological phenotype change affected by LINC00996 may through interaction with Rb,P15 and c-myc.