Lentivirus-mediated Downregulation Of MiRNA-1246 And Its Biological Role In Cervical Squamous Cell Carcinoma Cells

Objective To construct and identify a lentiviral vector for inhibiton.Methods:The gene sequences of mature micro RNA-1246 Antisenseolignuclleotides were chosen,and the antisense sequences from mature micro RNA-1246 were designed and linked into linearized GV280 vector.The recombinants were screened and identified by colony PCR and DNA sequencing,the success of recombinant amplifiled by competente coli DH5 ?.The GV280-mir-246 down,Helper1.0 ? Helper2.0 were mixed in suitable proportion and then transfected into the cell of 293 T to get recombinant lentiviral vector.The concentrated and purified of virus bulk were detected the drops of it,indeed the virus was used to infect the carcinoma cell of Cervical squamous of Si Ha.The GV280-Micro RNA-1246 down,s expression in the she cell tested by testing the GFP and PCR.Results(1)The corrected of recombination lentiviral vector was screened from the bacterial which was anthenticated by PCR,DNA testing proved that the inserted gene sequence was correct.(2)The cellsof293 T which wastransfected GV280-Micro RNA-1246 down ? Helper1 ? 0 and Helper2.0 produced recombinant lentiviral vector.(3)The target gene se-quences were successfully inserted into Si Ha cell by GV280 lentiviral vector,the recombinant lentiviruses which carring mir-246 down could infect and deliver mir-246 down and GFP genes to Si Ha cells.The infection efficiency was almost100%.Conclusion: The lentiviral micro RNA inhibitor vector has been successfully constructed,which provides a stable tool for further study of therole of micro RNA-1246 on cervical cancer.1.porpose of researchcervical cancer is the most common malignant tumor of female reproductive system,the cervical squamous cell carcinoma is the main pathological pattern,the expression of Micro RNAs is closely related to the happening and development of cervical cancer.but it is rare reported the relation of the expression of mi R-1 246 in the tissue of cervical cancer between the metastasis and infiltration of cervical cancer.the expression of mi R-1 246 in the tissue of cervical cancer and the normal tissue were deteted by fuorogenic quantitative PCR.The relation of the expression of mi R-1 246 in the tissue of cervical cancer and clinical and pathological chacteristics were discussed to explit the clinical value of the expression of mi R-1 246 in the tissue of cervical cancer tissue.2.The research methodEighty patients of cervical cancer tissue ho were received operation in the hospital from march 2014 to December 2015 were chosed as the object of study(Agroup),and eighty normals cervical tissue were chosed as the normal control(Bgroup),the tissue was collected before the primary treatment.The cervical tissue which was the pathological diagnosis was the opration sample from our hospital.The expression of Micro RNA-1246 of the cervical tissue was detected by fluorescent quantitation PCR,therelation of clinical pathological parameters between Micro RNA-1246 and cervical squamous cell carcinoma would be analysised.3.The results of researchThe expression of Micro RNA-1246 in the cervical cancer tissue were obviously higher than the normal cervical tissue,and the higher Micro RNA-1246 were more likely indicated lymphatic metastasis(P<0.01)?we analyzed the relation of clinical pathological parameters between Micro RNA-1246 and cervical squamous cell carcinoma we found that the expression of Micro RNA-1246 were relevant with the size of tumor and the classification of tumor.the consequence was that the higher expression of Micro RNA-1246 the biger size of the tumor and the higher the malignant degree.The expression of Micro RNA-1246 had no correlation with the age of patients.4.conlusionThe expression of Micro RNA-1246 in the cervical cancer tissue was up-regulated,the happeness and the development of cervical cancer cells were suppressed by down-regulated theexpression of Micro RNA-1246.1.porpose of researchInfitration and metastasis were the two characteristics of malignant tumor,including the cervical cancer.Therefore the study of infitration and metastasis of the cervical cancer was of great significance.A great deal of research work showed that Micro RNAs participated in signal path through the expression of the target gene during transcription or after transcription,Micro RNAs would be form a complex and hugeness regulatory network which could affected of the happness and development of tumor,Micro RNAs would be work as the tumor suppressor gene.The study found the Micro RNA-1246 were up regulation in a great quanlity tumors that indicated Micro RNA-1246 was a oncogene.This study was the further of the previous studies.We had done vast original work,especially,we had ensure that Micro RNAs in the serum could be used to forecast the metastasis of the cervical cancer,Micro RNA-1246 was a serum tumor marker,and we also had foud that THBS2 is target gene of Micro RNA-1246.This studies were good basic for the study of target gene and signal path of Micro RNA-1246.This study was that the de-regulated expression of the Micro RNA-1246 through established de-expression of Micro RNA-1246 vector.Meanwhile,the stably transfected Si Ha cells would be constructed by lentiviral vector,and we would study the variation of de-expression of Micro RNA-1246 of Si Ha cells of prolifera migration apoptosis and cycle.the de-regulated expression of the Micro RNA-1246 could suppress the happeness and development of cervical squamous cell carcinoma.2.The research methodThe Si Ha cells were infected by virus bulk,the expression of GV280-Micro RNA-1246 downin the Si Ha cells was detacted bygreen fluorescent protein(GFP)and PCR,the stable cells strain was filted by puromycin.And the expression of micro RNA-1246 GV280-Micro RNA-1246-down in the stable cells strain was detected by PCR?The biological function of the de-expression of Micro RNA-1246 of Si Ha cells was detected by CCK8?transwell?apoptosis and cycle.3.the results of researchThe target gene of Micro RNA-1246 down was transfected into the Si Ha cellssuccessfully,simultaneously it could get express stably and the transfection efficiency can be 100%,the GFP could be observed through fluorescene microscope,the Micro RNA-1246 and the antisense olionuleotides of Micro RNA-1246 could be detected by PCR.the result of PCR showed that the expression of Micro RNA-1246 in the group of mir-246 down was lower than the the group of NC and the differences between the two groups were significant(P?0.05),the result of CCK8?transwell?apoptosis showed that silent of Micro RNA-1246 could suppress Si Ha cells,proliferation,invasion and metastasis.4.conlusionThe Si Ha cells of Stable transfected mir-246 down were established,the silent of Micro RNA-1246 could suppress Si Ha cells,proliferation,invasion and metastasis.