| BackgroundLung cancer is a serious threat to human health and is associated with significant morbidity and mortality.It is the first and secondary in fatality rate among men and women respectively Non-small cell lung cancer accounts for at least 80%of lung cancers and the incidence has been increasing in recent years.Most patients with lung cancers failed to receive either early diagnosis or individualized treatment due to the low sensitivity and specificity of traditional diagnositic methods(such as X-rays,bronchoscope sputum and cytology examination)and traditional evaluation methods(such as CT scans and detection of tumor markers CEA,CY211).It is reported that the five year survival rate of NSCLC is only 15%.So how to achieve the early diagnosis,individual therapy and effect of monitoring has great significant..MicroRNAs(miRNAs)are small(20-24-nt)single-stranded noncoding RNA molecules.These microRNAs function by directly binding to their potential target site in the 3' untranslated region(3' UTRs)of specific target mRNA,leading to the repression of mRNA translation or the degradation of target mRNAs.They involved in the regulation of numerous traits,including developmental timing,apoptosis,immune function,and neuronal development.Many studies show that microRNAs could act as both oncogenes and tumor suppressor which could play a significant role in the The formation and development of cancer.Many microRNAs such as miR-21,miR-27a,miR-29,miR-34,miR-122,miR-145 and miR-150 have been the subjects of intense research.It was demonstrated that the aberrant expression of miR-27a might be involved in human diseases,including metabolic diseases of bone,cancer and cardiovascular disease.It was concluded that miR-27a could act as oncogene and relates with esophageal cancer,gastric cancer,liver cancer,colorectal cancer and pancreatic cancer.Thus,studying the relation between miR-27a and lung cancer,exploring its clinical application value in lung cancer both have great significance and draw more and more attention.ObjectiveThe aim of this study was to explore the expression changes of miR-27a both in human lung adenocarcinoma cell line SPC-A1 after Cisplatin chemotherapy in vitro and in patients with advanced lung cancer by tracking the whole treatment.The correlation of miR-27a levels with cell apoptosis was determined and whether miR-27a could be a sensitive indicator in curative effect prediction and prognosis evaluation was discussed.MethodsSPC-A1 cells were cultured in vitro and treated with DDP at 2.5?g/mL for 12,24,48 and 72h.1.Cell morphology was observed with phase contrast microscope,as well as cell apoptosis was detected by flow cytometry.2.The total RNA of cultural supernatants and cells was extracted by QIAzol reagent.3.Reverse transcription PCR(RT-PCR)was performed based on the steam-loop structure accompany to the internal control of cel-miR-39 for the cultural supernatants and U6 snRNA for cells,respectively.4.The expression changes of miR-27a both in cultural supernatants and SPC-A1 cells were analyzed by real-time PCR.Twenty-two patients with newly diagnosed lung cancer from the department of oncology of The First Affiliated Hospital of Nanjing Medical University were enrolled in this study between March 2010 and May 2012.The patients consisted of 16 males and 10 females,with a median age of 61 years(range:31?77).All patients were first-visit without radiotherapy or chemotherapy,and were finally diagnosed by histopathology.Each patients received first-line or second-line chemotherapy.The main regimens of first-line chemotherapy were the GP(Gemcitabine and Cisplatin)regimen and the DC(Docetaxel and Cisplatin)regimen.The second-line chemotherapy was Docetaxel.1.Blood samples were withdrawn from a peripheral vein from seven patients received 3-4 cycles before initiation of the first treatment 1?7 days)and then after medication of every chemotherapeutic cycle(1?2 days),as well as from nineteen additional patients before initiation of the first treatment,and 1?2 days after the first cycle of chemotherapy.2.Total RNA was extracted from serum samples by QIAzol reagent.3.Reverse transcription PCR(RT-PCR)was performed based on the steam-loop structure accompany to the internal control of cel-miR-39.4.then analyzed for the amounts of miR-27a by real-time PCR.5.Tumor response was assessed every cycle of chemotherapy using the Response Evaluation Criteria in Solid Tumors(RECIS)to evaluate chemotherapeutic efficacy.In this study,survival data was obtained until March 2013.Results1.The study of apoptosis of SPC-A1 cells in vitro compared to the control group demonstrated that,(1)SPC-A1 cells presented morphological changes of the apoptosis in DDP(2.5?g/mL)group.Numerous round cells,shrinkage of the cellular membrane and cell debris were observed under phase contrast microscopy.(2)There was a higher apoptotic rate of SPC-A1 cells at 48h,72h in DDP(2.5?g/mL)group[(59.3±2.54)%and(76.4±3.11)%,respectively]than control group(P<0.05).(3)Both in cultural supernatants and SPC-A1 cells,PCR analysis demonstrated that the level of miR-27a significantly increased in DDP(2.5?g/mL)group than control group(P<0.05).(4)In DDP(2.5?g/mL)group,miR-27a level increased gradually at 12h,24h,48h and 72h in cultural supernatants,which was statistically significant difference compared with each phase(P<0.05).(5)Likewise,miR-27a level increased gradually at 24h,48h and 72h in SPC-A1 cells,which was statistically significant difference compared with,12h phase(P<0.05).2.All measurement data is in the form of a median(interquartile range).(1)In 26 patients,the median value of miR-27a quantification before and after chemotherapy was 8.362E-4(1.372E-4?5.920E2)and 7.375E-4(1.752E-4?7.043E2),respectively.No significant difference were found between groups(Z=-0.498,P=0.619).(2)After first-line chemotherapy,among 26 patients:no patient achieved complete response(CR),12 patients achieved partial response(PR),10 patients achieved non-remission(NR)[including 4 patients with stable disease(SD),6 patients with progressive disease(PD)],4 patients dropped out.(3)Before chemotherapy,the miR-27a content level of PR group and NR group was 5.058E-4(9.405E-5?2.450E-3)and 8.362E-4(3.441E-5-2.617E-3),respectively.There was no significant difference between the PR group and NR group(Z=-0.040,P=0.968).(4)After chemotherapy,the miR-27a content level of PR group was 5.505E-3(3.450E-4-7.989E-3),which showed a significant increase compared with the level of NR group[3.085E-5(2.135E-5?9.144E-4)](Z=-3.315,P=0.001).(5)The miR-27a content level of 12 patients with partial response(PR)after chemotherapy showed a significant increase compared with the level before chemotherapy(Z=-3.180,P=0.001).Similarly,significant miR-27a content level difference was found in the NR group from 10 patients with lung cancer before and after chemotherapy(Z=-1.992,P=0.046).(6)The survival data was obtained until March 2013,13 cases with the survival time more than or equal to 12 months,while 9 cases with the survival time less than 12 months,the 1-year survival rate was 59%(13/22).Survival analysis showed a statistically better suvival time in patients who had elevated miR-27a level after the first cycle of chemotherapy(?2=8.241,P=0.004).(7)The results of monitoring miR-27a level during the therapy showed that the patients with a continued upward trend in their miR-27a content levels were expected to have an ideal prognosis.On the contrary,the patients with remianing lower miR-27a levels were expected to have a poor prognosis.ConclusionThe level of miR-27a rised in human lung adenocarcinoma cell line SPC-A1 after Cisplatin chemotherapy in virto,which is involved in regulating the cell apoptosis.The miR-27a content levels on the 3rd to 5th days after the end of a circle of chemotherapy can reflect the sensitivity of patients with lung cancers to the current chemotherapies with the superiority of higher sensitivity and specificity.The results of this study show that quantitative monitoring miR-27a can be used as an effective short-term efficacy indicator for patients with lung cancer,and may be used in guiding the formulation individualized treatment plan for patients with lung cancer. |
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