MicroRNA-19b Regulates Nasopharyngeal Carcinoma Growth,Progression And Chemosensitivity By Targeting KRAS

BackgroundNasopharyngeal carcinoma(NPC)Nasopharyngeal carcinoma is one of the high incidence of malignant tumors in China,the incidence of which is the head of the head and neck malignant tumor.According to the WHO statistics,80% of the world’s nasopharyngeal cancer patients are in China,and the incidence is on the rise.Currently in clinical,the preferred radiotherapy for nasopharyngeal carcinoma,with the wide application and progress of IMRT radiotherapy technology,the limitations of the nasopharyngeal lesion control rate increased significantly,but due to the onset of deep location,early diagnosis,patients in the hospital has been part of distant metastasis,although the combination of chemotherapy and targeting drug therapy and surgical treatment can be chosen,the prognosis of patients has been improved to some extent,but the overall effect is still unsatisfactory.At present,the mechanism of nasopharyngeal carcinoma is not clear.KRAS is a member of the RAS gene family,encoding the K-ras protein.Studies have shown that 30% of human tumor is related to the mutation of RAS gene.The products after mutation can be activated continuously,and participate in the survival,proliferation,migration,proliferation and angiogenesis of tumor cells.MiRNAs(microRNAs)is a class of non coding single stranded RNAs encoded by endogenous genes,with a length of about 22 nucleotides,which mainly regulates gene expression at translation level.The mechanisms of miRNAs are divided into 2 categories: the translation of the target gene or the promotion of the degradation of the target genes.At this stage,with the application of high-throughput sequencing technology in the field of cancer research,researchers found that there was a significant difference between the expression level of many miRNA in nasopharyngeal carcinoma tumor tissues and adjacent normal tissues.These results suggest that miRNA may play a role in the occurrence and progress of pathological process of nasopharyngeal carcinoma.MiR-19 b belongs to miRNA.In recent years,studies have confirmed that miR-19 b is highly correlated with the occurrence of various kinds of tumors.However,its expression and mechanism in nasopharyngeal carcinoma are not yet clear.Chemotherapy drugs cisplatin(cisplatin,CDDP)in clinic is mainly used for the treatment of solid tumor,anticancer spectrum is wide,it is now known as one of the most effective anti-tumor drugs,also is a line middle-late stage of nasopharyngeal carcinoma chemotherapy drugs.However in the process of using cisplatin chemotherapy,the clinical patients are prone to resistance,this may not be the specification of drug use,tumor cell gene instability and high mutation rate and so on.Drug resistance has become the advanced nasopharyngeal carcinoma cells and the main reason of recurrence chemotherapy failure.In recent years,many studies focused on the mechanism of cisplatin resistance,and actively seek the effective way to overcome the cisplatin resistance.Studies have shown that the miRNA plays an important role in cancer drug resistance,drug resistance in the cells and the amount of expression in cells that are sensitive to drugs.Therefore,further study of nasopharyngeal carcinoma pathogenesis and oncogene changes is of great significance for finding new therapeutic targets and new anti nasopharyngeal carcinoma drugs.ObjectiveThis study aimed to analyze the clinical tissue samples of patients with nasopharyngeal carcinoma and further to explain the role of miR-19 b in in the development of human nasopharyngeal carcinoma,through the research of miR19 b targeted expression of KRAS gene,the regulation of drug sensitivity of nasopharyngeal carcinoma cells,provides a new perspective for the further screening of the pathogenesis of nasopharyngeal carcinoma and further reveal the new anti nasopharyngeal carcinoma drug.cer.MethodsRT-qPCR was used to detect the miR-19 b expression in 37 pairs of tissues in NPC compared with adjacent normal esophageal tissues.In addition,RT-qPCR was used to detect the expression of miR-19 b in normal esophageal epithelial cells in vitro and in 4 kinds of nasopharyngeal carcinoma cell line.The bioinformatics search software(TARGETCAN)is used to predict miR-19 b target gene of miR-19 b in NPC..The human KRAS gene and its mutation control were cloned to pimr-GLO.293 T cells were cotransfected wt-pmirglo or its mutant control with miR-19 b.Double luciferase reporter gene detection was used to verify the effect of miR-19 b target KRAS gene.Western blot was used to detect protein expression of KRAS.And RT-qPCR was used to detect the expression of 37 pairs of KRAS mRNA in patients with normal nasopharyngeal carcinoma in cancer tissues and adjacent tissues.CNE1 and HNE1 cells were transfected miR-19 b precursor,scramble control.RT-PCR was used to detect mRNA expression of miR-19 b.CNE1 and HNE1 cells were transfected miR-19 b precursor,scramble control.CCK8 assay was used to detect the cell proliferation.The invasive ability of CNE1 and HNE1 cells transfected to miR-19 b precursor in nasopharyngeal carcinoma was tested by transwell.CNE1 cells transfected with miR-19 b precursor,scramble control,and KRAS.Scratch assay was applied to detect the cell invasion ability.CCK8 assay was used to detect the cell proliferation of CNE1 cells treated with CDDP.FACs and caspase assay were used to dectect cell apoptosis of CNE1 cells treated with CDDP.The tumorigenicity of CNE1 cells transfected with miR-19 b precursor in nude mice was tested.The tumor volume of nude mice was measured at 14 D,17 D,20 D,23 D,and 26 D after transplantation.After26 D,the tumor were separated and weighted.ResultsThe expression of miR-19 b is significantly lower than that of adjacent normal tissues.From patients(n=37,p=0.041).In the four esophageal cancer cell lines,the expression of miR-19 b was decreased,especially in CNE1 and HNE1 cells.MiR-19 b was found to have one seed region matching 3’-UTR KRAS gene showed that: nucleotide 342-348.Furthermore,luciferase assay found that cotransfected with wild type miR-19 b KRAS 3’-UTR construction to produce significant luciferase activity in the inhibition of 293 T cells compared with the negative control group.The mutant KRAS miR-19 b binding site 3 ’-UTR repealed the ability of miR-19 b to regulate the expression of luciferase.The levels of endogenous proteins that overexpress the strongly reduced miR-19 b KRAS gene are compared with these levels of control.In clinical nasopharyngeal carcinoma specimens,there is a negative correlation between the mRNA level of KRAS gene and the expression level of miR-19 b.Overexpression of miR-19 b markedly attenuated cell proliferation in CNE1 and HNE1 cells.Then,overexpression of miR-19 b significantly inhibited the scratch detection ability of CNE1 and HNE1 cells.In addition,overexpression of miR-19 b significantly reduced cell proliferation,cell invasiveness and CDDP cell induced apoptosis.However,excessive miR-19 b significantly promotes this behavior.The tumor model of nude mice was transplanted to develop smaller tumor expression and miR-19 b CNE cell CNE1 cells compared with miR-19 b.ConclusionsMiR-19 b expression decreased in nasopharyngeal carcinoma tissues and 4 nasopharyngeal carcinoma cell lines cultured in vitro,and the expression level was closely related to tumor stage in nasopharyngeal carcinoma patients.MiR-19 b can inhibit the proliferation,invasion and tumor cell formation of nasopharyngeal carcinoma by targeting KRAS,thus inhibiting the occurrence and development of nasopharyngeal carcinoma.The expression of the target gene KRAS in nasopharyngeal carcinoma cells can improve the sensitivity of tumor cells to cisplatin in chemotherapeutic drugs.