| Objective: Lung cancer is one of the malignant tumors with the highestincidence rate and lethality at present. Due to the high malignancy of lungcancer, most patients were already in advanced stage and lost the opportunitiesof surgical treatment when they were diagnosed. Radiotherapy andchemotherapy do not specifically kill tumor cells and may cause severe sideeffects, so seeking new approaches of diagnosis and treatment are still thehotspots. Lung cancer metastasis is a multi-step and multi-period process andit depends primarily on the invasive ability of lung cancer cell which involvestumor cell proliferation, adhesion, migration, invasion, cell matrix degradationand so on. Multiple tumor suppressor gene inactivation and overexpression ofoncogenes are involved. To date, a large amount of research has covered thedevelopment and progression of tumor, while the molecular mechanism ineach step is still not elucidated, therefore, searching novel genes andmolecules associated with lung cancer invasion and metastasis andinvestigating their mechanism are considered to be of great importance for theimprovement of survival rate and the rational design of therapeuticagents.MicroRNA (miRNA) is a kind of highly conserved non-coding smallRNA in the length of22~25nt, it could regulate gene expressions in a varietyof eukaryotic organisms through complementing with partial sequences of oneor more mRNAs, and then affect the life activities. Disorders of importantprocess, like cellular differentiation, proliferation and survival, may be causedby miRNA’s dysfunction. miRNA plays a role as an oncogene orantioncogene in the tumorigenic process. According to present studies,miRNA-128in 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 by studying the biological role of miRNA-128, 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 carcinoma L9981/NL9980cell lines with highand low metastatic potential, detect the differential expression level ofmiRNA-128in NL9980/L9981by MicroRNA chip technology;(2)Verification of the differential expressed miRNA-128through real-time PCRtechnology: Firstly, extract the total RNA from the human pulmonary giantcell carcinoma L9981/NL9980cell lines, then design the reverse transcriptionprimer，upstream primer and downstream primer of miRNA-128, set thereference U6as control, the last, detect the expression level of miRNA-128inNL9980/L9981cell line by reverse transcription RNA, while identify theproducts of real-time PCR by the use of agarose gel electrophoresis.(3)Construction of the eukaryotic expression vector pCDNA3.1-miR-128which over expresses miRNA-128: Firstly, determine the sequence ofmiRNA-128according to the miRBase database, design amplified primers ofmiRNA-128and amplify objective genes, then construct pCDNA3.1-miR-128which is the eukaryotic expression vector after identification, the last, identifyit again by enzyme digestion and gene sequencing.(4) Establishment ofL9981-pCDNA3.1-miR-128cell line which over expresses miRNA-128stably:Transfect L9981cell line by pCDNA3.1-miR-128which is the eukaryoticexpression vector，screen it by hygromycin, then L9981-pCDNA3.1-miR-128which over expresses miRNA-128stably is selected, verify it by real-timePCR technology.(5) Assay of L9981-pCDNA3.1-miR-128cell line’sproliferation: Set blank control group and negative control group, detect theproliferation ability changes of L9981cell line by MTT method after stableover expression of miR-128.(6) Assay of L9981-pCDNA3.1-miR-128cellline’s migration: Set blank control group and negative control group, detectthe migration ability of L9981cell line by wound healing assay after stableover expression of miR-128.(7) Assay of L9981-pCDNA3.1-miR-128cell line’s invasion: Set blank control group and negative control group, detect theinvasion ability changes of L9981cell line by Boyden chamber experimentafter stable over expression of miR-128.Results:(1)Gene chip analysis showed that miRNA-128levels weresignificantly higher in human lung giant cell carcinoma cell line NL9980thanhuman lung giant cell carcinoma cell line L9981, real-time PCR againconfirmed the level of miRNA-128in human lung giant cell carcinoma cellline NL9980higher than the level of human lung giant cell carcinoma cell lineL9981;(2) eukaryotic expression vector pCDNA3.1-miR-128was successfullyconstructed and confirmed by enzyme digestion and gene sequencing,then wefurther established cell line pCDNA3.1-miR-128which overexpressingmiRNA-128and confirmed by real-time PCR;(3)the MTT experimentsshowed that overexpression of miRNA-128did not affect the proliferation ofthe L9981cell line (p>0.05); wound healing assay showed that after48hculture, the wound healing width of L9981-pCDNA3.1-miR-128cell line(195.1±3.819μm) was significantly greater than the blank controlgroup(89.3±2.592μm) and transfected with empty vector group(121.7±4.623μm)(p<0.01), proved that over-expression of miR-128couldreduce the migration ability of L9981line; Boyden chamber experimentsshowed the invasive quantity of cells transfected with pCDNA3.1-of miR-128(37.01±2.859) was significantly less than the control group (126.83±4.316)and transfected with empty vector group (93.25±4.316)(p<0.01), proved thatoverexpression of miR-128could reduce the invasion ability of L9981line.Conclusion:(1)Compared to the human pulmonary giant cell carcinomaL9981cell line with low metastatic potential, the expression level of miR-128in NL9980cell line with low metastatic potential is significantly higher. Itmeans miR-128may have relation with the transfer of lung cancer andbecome the target gene of treatment.(2)The successful construction ofpCDNA3.1-miR-128which is the eukaryotic expression vector and theestablishment of L9981-pCDNA3.1-miR-128cell line which over expressesmiRNA-128stably, are bases of studies about miR-128’s biological function to lung cancer.(3)Over expression of miR-128reduces the migration andinvasion of L9981cell line, but is unchangeable in proliferation. It meansmiR-128may inhibit lung cancer’s migration and invasion and have relationwith the transfer of lung cancer. It will also provide a new theoretical basis ofearly diagnosis and target gene therapy to lung cancer. |
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