Expression Of MiR-155 In Lung Adenocarcinoma Patients And The Regulation Of Proliferation And Apoptosis By InterferingmiR-155 In Adenocarcinoma Cells

Adenocarcinoma is the most common type of lung cancer in lifelong non-smokers. Adenocarcinomas account for approximately 40% of lung cancers. Its incidence has been increasing in many developed Western nations in the past few decades, where it has become the most common major type of lung cancer in smokers (replacing squamous cell lung carcinoma) and in lifelong nonsmokers. According to the Nurses’ Health Study, the risk of adenocarcinoma of the lung increases substantially after a long duration of previous tobacco smoking, with a previous smoking duration of 30 to 40 years giving a relative risk of approximately 2.4 compared to never-smokers.This cancer usually is seen peripherally in the lungs, as opposed to small cell lung cancer and squamous cell lung cancer, which both tend to be more centrally located, although it may also occur as central lesions. For unknown reasons, it often arises in relation to peripheral lung scars. The current theory is that the scar probably occurred secondary to the tumor, rather than causing the tumor. The adenocarcinoma has an increased incidence in smokers, and is the most common type of lung cancer seen in non-smokers and women. The peripheral location of adenocarcinoma in the lungs is due to the use of filters in cigarettes which prevent the larger particles from entering the lung.MicroRNAs constitute a recently discovered class of non-coding RNAs that play key roles in the regulation of gene expression. Acting at the post-transcriptional level, these fascinating molecules may fine-tune the expression of as much as 30% of all mammalian protein-encoding genes. MicroRNAs were first reported in mammalian systems in 2001. In the latest release of miRBase (v.15), more than 14000 microRNAs have been annotated, highlighting the rapid growth of this field of research. However, the functions of most of these microRNAs still remain to be discovered.The challenges of studying microRNAs are two-fold. First, microRNAs are very short. This means that traditional DNA-based methods are not sensitive enough to detect these sequences with any reliability. Second, closely related microRNA family members differ by as little as one nucleotide, emphasizing the need for high specificity and the ability to discriminate between single nucleotide mismatches.The recent research of miR-155 showed that it might be related to several tumor tissues including lung cancer, especially in lung adenocarcinoma, which indicated miR-155 could be a potential biomarker in diagnosing and prognosing the process of this disease.So far, there are no reports about experimental studies on MicroRNA-155 interference in adenocarcinoma in vitro.Object1. Testing the expression levels of both MicroRNA-21and MicroRNA-155 to investigate the regulating role of MicroRNA-155 and its target gene PTEN (phosphatase and tensin homolog)、PI3K (phosphatidylinositol 3-kinase) in lung adenocarcinoma proliferation.2. To observe the expression of microRNA-155in adenocarcinoma lung cancer patients and healthy control group, analysis the differential expression in all stages of patients to reveal the value of miR-155 in early diagnosis.3. Oberve the changes of biological behavior including proliferation, apoptosis, and invasion of adenocarcinoma cell lines as well as the expression of MicroRNA-21, MicroRNA-155, PTEN and PI3K, by down-regulating microRNAs viainfection technique using the lentiviral vector mediated MicroRNA-21 and MicroRNA-155 Inhibitors and western blot protein electrophoresis.Part IExpression of MicroRNA-21 and MicroRNA-155 in Lung Parenchymaof Adenocarcinoma Patients and Healthy ControlMethods1. Lung Parenchyma of 36 lung cancer patients were collected randomly and miRNAs were extracted in both tumor and normal area from the same patient served as control, concentration and purity of miRNAs were tested via Nanodrop(?) spectrophoto meter detection.2. The levels of mir-21 and mir-155 were assessed by real-time reverse-transcription polymerase chain reaction in the tissues of cancer patients.Results1. The expressions of mir-21 and mir-155 could be detected stabley in lung parenchyma both in cancer patients and healthy control.2. miR-21 and miR-155 levels were increased in all adnocarcinoma samples compared with normal control (P< 0.05).3. Expressions of miR-155 could be found stably through all stages of the cancer process, especially a higher level detected in STAGE Ⅱ and STAGE Ⅲb than others (P<0.05), followed with STAGE IV, STAGE Ⅲa and STAGE I the lowest.Part II Expressions of miR-21 and miR-155 in Serum of Lung Adenocarcinoma Patients and Healthy ControlsMethods1. Whole blood from every patient and negative control was collected in regular tubes and immediately processed to prevent contamination by cellular nucleic acids.2. Small RNA molecules with size<200 nucleotides were extracted and purified from 200 μ1 of each serum sample. Concentration and purity of miRNAs were tested via Nanodrop(?) spectrophoto meter detection.3. The levels of mir-21 and mir-155 were assessed by real-time reverse-transcription polymerase chain reaction in the serums of cancer patients.4. Analysis the expression value of mir-155 as a potential biomarker compare with CEA and CA-125 when using in diagnosing adenocarcinoma of lung.Results1. The expressions of mir-21and mir-155 could be detected stabley in serums of both cancer patients and healthy control group.2. miR-21 and miR-155 levels were increased in all adnocarcinoma samples compared with normal control (P< 0.05). Defining miR-155control as the basic foundation "1", The mean fold increase in tumor for mir-155 levels was 2.03 times than it was in normal (P< 0.05), as to miR-21, it reached a high point of4.06 times in tumor than the basic miR-155 control (P< 0.05).3. Testing results of serum miR-155 levels showed a much higher sensitivity (0.722) than that for CA-125 or CEA;4. CEA associated with CA-125 had the highest Youden’s index (0.639) in all terms of combinations;5. Combined with CA-125 testing, miR-155 received a competitive sensitivity (0.889) and specificity (0.688) for diagnosing lung adenocarcinoma (OOP=14.88). Part III Interfering Proliferation and Apoptosis in Adenocarcinoma Cells by Regulating miR-155 ExpressionsMethods1. Three commonly used adenocarcinoma cell lines, A549, SPC-A-1, LTEP-a-2 were involved in this study for observing the changes after interfering. Three groups were founded for the need of this research, they are Mock group, miR-21/miR-155 Inhibitor group and miR-21/miR-155 NC group..2. Using lenti-virus vector mediated miR-21/miR-155Inhibitor to infect each of the adenocarcinoma cell line, the efficiency of infection was observed.The expression levels of miR-21 and miR-155 were defined by real-time reverse-transcription polymerase chain reaction in each of the group as mentioned before:Inhibitor, NC and Mock group.3. Plantcells (2 X 104/ml)in 96-well plates for each group and cultured for 120 hours, 3 wells were taken every 12 hours for cell number counting to draw growth curves according to the records.4. Same procedure were applied for the cells as before, stained the cells with PI and analyzed cell cycle by Flow Cytometry (FCM) at 96-hour point.5. Proliferation differences in cells after infecting lentivirus vector mediated microRNAInhibitor were detected using the EdU cultural staining method; Invasion changes for each group were detected via cell Transwell method.6. After 96-hourcell culture after transfection, early cell apoptosis were revealed by AnnexinV-FITC combined with PI staining method and, late stage cell apoptosis were demonstrated by TUNEL assay method.7. The levels of phosphatase and tensin homolog (PTEN) andPI3K (phosphatidylinositol 3-kinase) protein were tested by Western Blot analysis in Inhibitor, NC and Mock groups.Results1. Expressions of miR-155 in all the three adenocarcinoma cell lines in Inhibitor group were significantly suppressed.miR-155 in NC group was 8.15 times higher compared with Inhibitor group. (P<0.05), there were no differences between Mock and NC groups(P>0.05).2. Compared with Inhibitor group, there were not much change could be found in growth curves in NC group which treated with PBS and Monk group(P>0.05), while growth curves of Inhibitor group was significantly low(P<0.05).3. Adenocarcinoma cells infected with Inhibitors showed an increase at the phases G0/G1 (P<0.05) and a decrease at the phase S (P<0.05), compared with the other two groups, all data were analyzed in the percentage of the cells in each of the phase.4. Cells proliferative detection tested via EdU culture at 96-hour point showed an obvious decrease effect within the Inhibitor group, all positive stained cells in the sightview of the microscope were calculated (P<0.05).5. Either early apoptosis detected by Annexin V-FITC, or late stage apoptosis via TUNEL staining at 96 hours after Infection showed great promise of increase for cell apoptosis in Inhibitor group, compared with NC and Monk group(P<0.05).6. Infected withMicroRNA-155 Inhibitor for 48h, the cell numbers that invased across the membranes were significantly reduced compared withNC and Monk group (P>0.05).7. Expressions of PTEN protein in Inhibitor group was up-regulated compared to microRNA-NC and microRNA-Mock group, while the expressions of PI3K protein were significant down-regulated (P<0.05), no differences were found between Mock and NC groups (P>0.05).Conclusions1. In the tissue of lung Adenocarcinoma patients, there were significant expression levels of MicroRNA-21 and MicroRNA-155 than they were in healthy controls.2. MicroRNA-155 could be stably measured in patient serum with marked differences when compared with health controls. It shows a better specificity than CA-125 and CEA in adenocarcinoma diagnosis.3. Transfection of Inhibitor could inhibit proliferention and apoptosis in lung Adenocarcinoma cells by blocking the target genes of microRNAs. Manually modulate microRNA levels might lead to an alteration in the expression of cancer-related downstream proteins, which are targeted and regulated by the corresponding microRNAs. Along with the changes of PTEN and PI3K protein expressions indicated that the proliferative characteristics of lung epithelial cells were enhanced in patients.