MiR-320a Effectively Suppresses Lung Adenocaresistant Cell Proliferation And Metastasis By Regulating STAT3 Signals

Lung cancer has become one of the greatest threats to human health in the world.The treatment for lung cancer,including surgical treatment,radiation therapy,drug chemotherapy.The radiation therapy is an important treatment for lung cancer.However,radiation can also activate various radiation response genes.This leads to the production of various proteins,some of which might change the radiosensitivity of cancer cell and confer survival advantage to cancer cells.The survival cancer cells after radiation contributed to the recurrence and metastasis of cancer.Therefore,exploring how to sensitize lung cancer cells to radiation is the major problem.MicroRNA is closely related to the occurrence and development of lung cancer.In order to study the role of microRNA and its related signal factors in the radiosensitivity of lung cancer,we carried out this study.In this study,we investigated the role of miR-320a in the proliferation of lung adenocarcinoma cells.Our results showed that miR-320a could effectively inhibit the proliferation of lung adenocarcinoma cells and promote more cell apoptosis after irradiation by X-ray in vitro or in vivo.These results indicated that miR-320a could not only inhibit the proliferation of lung adenocarcinoma cells but also enhance the radiosensitivity of lung adenocarcinoma cells.Finally,we found that miR-320a could inhibit the migration of lung adenocarcinoma cells in vitro and in vivo.1.miR-320a inhibits the growth of lung adenocarcinoma cells(1)The expression of miR-320a in lung adenocarcinoma tissues and para-carcinoma tissues.Lung adenocarcinoma tissues(n=18)and para-carcinoma tissues(n=18)were collected after clinical operation,then the expression of miR-320a was detected by qRT-PCR.Our result showed that the expression of miR-320a lower in lung adenocarcinoma tissues than those in para-carcinoma tissues(P<0.05).(2)The effects of miR-320a on the proliferation,apoptosis and radiosensitivity of radioresitant lung adenocarcinomaLTEP-a-2 cells were transfected into miR-320a,mu-320a,negative control group(NC),MTT and apoptosis assay to detect the effect of miR-320a on proliferation and apoptosis of LTEP-a-2 cells.The results showed that miR-320a could inhibit cell proliferation and promote cell apoptosis.LTEP-a-2 cells were transfected into miR-320a,mu-320a,and negative control group(NC)and irradiation by X-ray.The effects of miR-320a on the radiosensitivity of cells were detected by MTT and apoptosis assay.The results showed that miR-320a could enhance the radiosensitivity of LTEP-a-2 cells.Radiation induced radiation resistance in lung adenocarcinoma cell(A549-34R)transfected with miR-320a,negative control group(NC)and irradiation by X-ray,using MTT and apoptosis assay to detect the effects of miR-320a on the proliferation and radiosensitivity of A549-34R cells.The results showed that miR-320a could inhibit the proliferation of A549/R34 cells,promote cell apoptosis and enhance the radiosensitivity of cells.2.The mechanism of miR-320a inhibiting the proliferation of lung adenocarcinoma cells(1)The construction of pcDNA-GFP-TRB2-3’UTR vectorThe specific sequence of STAT3 3 ’UTR was amplificated by PCR,which was cloned into the pcDNA-GFP vector,the results showed that the successful construction of pcDNA-GFP-stat3-3’UTR vector.(2)miR-320a regulates the STAT3 and STAT3-related signalsdPcDNA-GFP-stat3-3 ’UTR vector and miR-320a were cotransfected into lung adenocarcinoma cells,and the pcDNA-GFP-stat3-3’ UTR vector and NC were used as control group.The expression of GFP was detected by fluorescence microscopy and flow cytometry.The result showed that GFP intensity was considerably lower in miR-320a-transfected cells compared with control treatment,and the percentage of GFP positive cells decreased in miR-320a-treated cells.Western blot and immunofluorescence further detected that the expression of STAT3/p-STAT3,BCL-2 were reduced in miR-320a treatment.(3)The effects of STAT3 signaling pathway on cell growth2 siRNAs specific to STAT3 mRNA were synthesized to knockdown of STAT3 expression.The effects of siRNA on proliferation and apoptosis of A549-34R cells and LTEP-a-2 cells were detected by MTT and Annexin V-FITC/PI.The results showed that knockdown of STAT3 could inhibit cell proliferation and promote cell apoptosis.Moreover,it was found that the siRNA group significantly inhibited more cell proliferation and promoted cell apoptosis after irradiation by X-rays with 10Gy.(4)The inhibition of miR-320a on lung cancer cell growth in vivoTransfection of miR-320a,siRNAl and a control group of A549/34R were injected into BALB/c-nu/nu mice to produce xenografts.The maximum and minimum tumor diameters were measured every 3 d.One month after injection,the volumes of xenografts were considerably smaller in miR-320a-treated mice than in scrambled control.STAT3-specific siRNA1 treatment also inhibited A549/34R cell growth and reduced the volumes of xenografts.Moreover,the weights of A549/34R cell xenografts were considerably lower in miR-320a-treated mice or in siRNAl-treated mice compared with those in scrambled control.On the basis of dynamic changes in volumes,we found that the growth rate of A549/34R cell xenografts of was inhibited effectively in vivo by miR-320a or siRNAl compared with scrambled control treatment.After irradiating by X-rays with 10 Gy.Our results showed that 10 Gy X-ray treatment obviously further reduced the volumes of xenografts in the miR-320a-or siRNAl-treated mice compared with those in scrambled control.The weights of xenografts in miR-320a-(n =3)or siRNAl-treated mice(n =3)were decreased by more than 3-fold after 10 Gy X-ray treatment.We then tested the expression of STAT3 and its signals in the xenografts.The results of treatment with or without X-ray showed that the expression of STAT3 and p-STAT3 decreased sharply,whereas that of cleaved Caspase 3 increased in the miR-320a-or siRNAl-treated xenografts compared with that in scrambled control,supporting the roles of miR-320a or siRNA in regulating STAT3 signals in vivo.3.The inhibition of miR-320a on lung cancer cell migration in vivo and in vitro(1)miR-320a inhibited lung cancer cell migration in vitroTranswell chamber experiments showed that miR-320a treatment obviously reduced the number of migrating A549/34R cells in vitro compared with scrambled control treatment.SiRNA1 treatment specific to STAT3 also obviously reduced the number of migrating A549/34R cells.Moreover,we found that 10 Gy X-ray treatment further reduced the number of migrating A549/34R cells by approximately 2-to 3-fold in miR-320a-or siRNAl-treated cells compared with scrambled control treatment.These data suggest that miR-320a or siRNA inhibits A549/34R cell migration in vitro.(2)miR-320a inhibited lung cancer cell migration in vivoTo understand the effects of miR-320a on metastasis of A549/34R cells in vivo,we injected cells transfected with miR-320a or siRNA or controls into male nude mice through their tail vein.Seven weeks after injection,we observed that miR-320a or siRNAl reduced the number of metastatic nodules on the surface of lungs compared with the scrambled control treatment.In addition,HE staining of lung sections showed the significantly reduced migration of miR-320a-or siRNA-treated tumors.CD44,a cell adhesion molecule,increases the migratory and invasive capacity of various cancers.We further detected human CD44 expression in metastatic A549/34R cell nodules,and found that significantly lower levels of CD44 in miR-320a-or siRNA-treated tumors than that in scrambled control treatment.The in vivo results further confirmed the functional effects of miR-320a on tumor migration.(3)Lung adenocarcinoma samples show high STAT3 levelsLung adenocarcinoma tissues(n=18)and para-carcinoma tissues(n=18)were collected after clinical operation,then the expression of STAT3 was detected by qRT-PCR.Our result showed that the expression of STAT3 higher in lung adenocarcinoma tissues than those in para-carcinoma tissues(P<0.05).Data on STAT3 expression data were further downloaded from data link Data Link(s):http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc D GSE3141.Overall survival was determined using Kaplan-Meier survival analysis.Patients showing high STAT3 levels displayed short survival time.In summary1.miR-320a can effectively inhibit the proliferation of lung adenocarcinoma cells,promote cell apoptosis,and enhance the radiosensitivity of lung adenocarcinoma cells in vitro.2.miR-320a,as a novel miRNA,directly targets STAT3 and regulats STAT3 signals.MiR-320a inhibits the proliferation of lung adenocarcinoma through STAT3.3.miR-320a can inhibit the migration of lung cancer cell in vivo and in vitro.