The Study Of MiRNA-449a Expression And Biological Function In Lung Cancer Cell Line

Objective： MicroRNA(miRNA) is a kind of highly conservednon-coding small RNA in the length of20~23nt, it could regulate geneexpressions in a variety of eukaryotic organisms through complementing withpartial sequences of one or more mRNAs, and then affect the life activities.Disorders of important process, like cellular differentiation, proliferation andsurvival, may be caused by miRNA’s dysfunction. miRNA plays a role as anoncogene or antioncogene in the tumorigenic process. Having a poorprognosis due to many aspects, lung cancer is one of the malignant tumorswith the highest incidence rate and lethality at present. One of the key issuesinfluencing the curative effects and survival time of patients with lung canceris transfer whose mechanism remains unclear. As a result, the effectivetreatment to lung cancer is still lacking. According to present studies,miRNA-449a in cancers of stomach, bladder and prostate, is a suppressor, butits effect on the biological function of lung cancer is unclear. In this study, wetry to explore the internal mechanisms of development of lung cancer bystudying the biological role of miRNA-449a, in order to provide a new way toimprove the early diagnosis, specific treatment, prognosis and a new specifictarget of lung cancer.Methods：(1) Gene chip analysis: Extract the total RNA separately fromthe human pulmonary giant cell carcinoma95D/95C cell lines with high andlow metastatic potential, detect the differential expression level ofmiRNA-449a in95C/95D by microRNA chip technology;(2) Verification ofthe differential expressed miRNA-449a through real-time PCR technology:Firstly, extract the total RNA from the human pulmonary giant cell carcinoma95D/95C cell lines, then design the reverse transcription primer，upstreamprimer and downstream primer of miRNA-449a, set the reference U6as control, the last, detect the expression level of miRNA-449a in95C/95D cellline by reverse transcription RNA, while identify the products of real-timePCR by the use of agarose gel electrophoresis.(3)Construction of theeukaryotic expression vector pCDNA3.1-miR-449a which over expressesmiRNA-449a: Firstly, determine the sequence of miRNA-449a according tothe miRBase database, design amplified primers of miRNA-449a and amplifyobjective genes, then construct pCDNA3.1-miR-449a which is the eukaryoticexpression vector after identification, the last, identify it again by enzymedigestion and gene sequencing.(4) Establishment of95D-pCDNA3.1-miR-44-9a cell line which over expresses miRNA-449a stably: Transfect95D cell lineby pCDNA3.1-miR-449a which is the eukaryotic expression vector，screen itby hygromycin, then95D-pCDNA3.1-miR-449a which over expressesmiRNA-449a stably is selected, verify it by real-time PCR technology.(5)Assay of95D-pCDNA3.1-miR-449a cell line’s proliferation: Set blank controlgroup and negative control group, detect the proliferation ability changes of95D cell line by MTT method after stable over expression of miR-449a.(6)Assay of95D-pCDNA3.1-miR-449a cell line’s migration: Set blank controlgroup and negative control group, detect the migration ability of95D cell lineby wound healing assay after stable over expression of miR-449a.(7) Assay of95D-pCDNA3.1-miR-449a cell line’s invasion: Set blank control group andnegative control group, detect the invasion ability changes of95D cell line byBoyden chamber experiment after stable over expression of miR-449a.Results:(1)Gene chip analysis showed that miRNA-449a levels weresignificantly higher in human lung giant cell carcinoma cell line95C thanhuman lung giant cell carcinoma cell line95D, real-time PCR again confirmedthe level of miRNA-449a in human lung giant cell carcinoma cell line95Chigher than the level of human lung giant cell carcinoma cell line95D;(2)eukaryotic expression vector pCDNA3.1-miR-449a was successfullyconstructed and confirmed by enzyme digestion and gene sequencing,then wefurther established cell line pCDNA3.1-miR-449a which overexpressingmiRNA-449a and confirmed by real-time PCR;(3)the MTT experiments showed that overexpression of miRNA-449a did not affect the proliferation ofthe95D cell line (P>0.05); wound healing assay showed that after48h culture,the wound healing width of95D-pCDNA3.1-miR-449a cell line (298.3±4.382μm) was significantly greater than the blank control group(106.7±3.753μm) and transfected with empty vector group (89.1±4.017μm)(P<0.01),proved that over-expression of miR-449a could reduce the migration ability of95D line; Boyden chamber experiments showed the invasive quantity of cellstransfected with pCDNA3.1-of miR-449a (53.79±3.972) was significantly lessthan the control group (162.28±3.819) and transfected with empty vectorgroup (150.63±4.625)(P<0.01), proved that overexpression of miR-449acould reduce the invasion ability of95D line.Conclusions:(1)Compared to the human pulmonary giant cell carcinoma95D cell line with low metastatic potential, the expression level of miR-449ain95C cell line with low metastatic potential is significantly higher. It meansmiR-449a may have relation with the transfer of lung cancer and become thetarget gene of treatment.(2)The successful construction of pCDNA3.1-miR-4-49a which is the eukaryotic expression vector and the establishment of95D-pCDNA3.1-miR-449a cell line which over expresses miRNA-449a stably,are bases of studies about miR-449a’s biological function to lungcancer.(3)Over expression of miR-449a reduces the migration and invasion of95D cell line, but is unchangeable in proliferation. It means miR-449a mayinhibit lung cancer’s migration and invasion and have relation with thetransfer of lung cancer. It will also provide a new theoretical basis of earlydiagnosis and target gene therapy to lung cancer.