| Backround:Nasopharyngeal carcinoma(NPC)is a malignant epithelial tumor of the nasopharyngeal area.From the spatial distribution of cancer,the geographical distribution of nasopharyngeal carcinoma has its unique characteristics.Cancer cases are mainly distributed in East and Southeast Asian countries.It is worth noting that my country’s Guangdong and Guangxi regions are also nasopharyngeal Areas where cancer is prone to occur.Nasopharyngeal cancer has a great impact on the incidence of certain populations.For example,in high-risk areas in southern China,the incidence of men and women are27.2/100,000 and 11.3/100,000,respectively,while the incidence of other populations is <1/100,000.Nasopharyngeal cancer has many well-known environmental risk factors,including tobacco,alcohol,and preserved foods,the most important of which is EBV infection.Nasopharyngeal carcinoma is divided into three pathological subtypes: keratinizing squamous cell carcinoma,nonkeratinizing carcinoma and basal-like squamous cell carcinoma.Among them,non-keratinizing carcinoma can be divided into differentiated and undifferentiated cancer.The type of cancer with a high incidence of cancer is mainly related to EBV.Persistent EBV infection and genomic alterations promote the NPC formation process,and persistent EBV infection of genetically mutated epithelial cells and hyperproliferation of infected EBV cells are considered prerequisites for initiating oncogenic transformation of nasopharyngeal epithelial cells in the organism.Nasopharyngeal carcinoma is highly metastatic,and its first choice of treatment mainly depends on the type of tumor lymph node metastasis(TNM)stage.Patients with early nasopharyngeal carcinoma receive radiotherapy,and patients with advanced nasopharyngeal carcinoma receive chemotherapy and radiotherapy.Although patients with nasopharyngeal cancer are sensitive to radiotherapy,about 30% of patients still have no obvious response to the treatment and develop local recurrence and distant spread of cancer cells.But the cause of treatment failure is still unclear to us at present.Therefore,there is an urgent need to find new molecular markers and therapeutic targets for NPC.The human PVT1 gene is located in the 8q24 region of chromosome.PVT1 is able to influence tumor cell epithelial mesenchymal transition(EMT)and distant metastasis by changing the adhesion between tumor cells and surrounding normal tissues,prompting tumor cell detachment from the primary tumor tissue,accelerating extracellular matrix degradation,modifying the cytoskeleton to enhance tumor cell motility,and promoting angiogenesis in tumor tissues.It is found that PVT1 gene contains multiple micro RNAs(miRNAs)response elements,and specific micro RNAs can bind to it,thereby silence these micro RNAs and up-regulate the expression of certain proteins,which ultimately affect tumor proliferation,invasion and metastasis,and resistance to chemotherapeutic drugs.PVT1 itself can be spliced into 6 different micro RNAs,namely miR-1204,miR-1205,miR-1206,miR-1207-5p,miR-1207-3p and miR-1208,which have either tumor suppressor or cancer-promoting functions.At present,research on molecular markers and targets of NPC has always been a research focus.We found that micro RNA-1205(miR-1205)can inhibit the expression of EGLN3 protein by targeting to promote the migration of NPC cells.Our study may provide a new target site and ideas for early treatment of NPC.Objective:(1)To investigate whether miR-1205 affects the development of nasopharyngeal carcinoma.(2)How to find and verify the downstream target genes of miR-1205 in nasopharyngeal carcinoma.(3)Explore the molecular mechanism of miR-1205 and its downstream target genes in nasopharyngeal carcinoma.Experimental methods:(1)We found that the expression level of miR-1205 in cancer tissue specimens of nasopharyngeal cancer patients was significantly higher than that of normal tissues.The expression levels of miR-1205 in NPC cell lines were examined using real-time PCR.(2)In order to determine the effect of miR-1205 on the invasion of NPC cell lines,the p SIFmiR1205 and p CDH-miR1205-sponge plasmids were first transfected into NPC cell lines using transfection reagents,and five fields were randomly selected in each experimental group under a 4x objective lens.Healing area of each experimental group corresponding field at 0 h and 24 h was photographed,and then the area of the corresponding field in each group was calculated.(3)To further determine the effect of miR-1205 on the migration of NPC cell lines,lipofectamine 3000 was used to transfect NPC cells to conduct Transwell experiment.Each experimental group was set up with 3 replicate wells.After 24 hours of migration,they were stained with crystal violet.And five fields were randomly selected and photographed under a 10 x objective lens for images.(4)To explore whether EGLN3 is a target gene of miR-1205,we further analyzed the NPC database using three micro RNAs databases to predict miR-1205 target genes,and found that the expression level of EGLN3 was significantly lower in cancer tissue samples from NPC patients than in normal tissue samples.Western blot assay also suggested that miR-1205 inhibited EGLN3 protein expression in NPC cells.(5)To determine the effect of EGLN3 on NPC cell lines migration,EGLN3 overexpression and knockdown stable cell lines were constructed,followed by wound healing assay in the stable cell lines.The experimental results showed that overexpression of EGLN3 inhibited the migration of NPC cells.(6)To further analyze the regulation of EGLN3 by miR-1205 in promoting migration,Transwell experiments were performed following transfection of p SIF-miR1205 plasmid in stable cell lines with EGLN3 overexpression by SUNE1 as well as HONE1 and p CDH-miR-1205 sponge plasmid in stable cell lines with EGLN3 knockdown by HNE1 and CNE2.Results:(1)The expression of miR-1205 was up-regulated in clinical samples of NPC and cell lines.(2)miR-1205 promotes the migration of NPC cells.(3)miR-1205 has no effect on the proliferation of NPC cell lines.(4)The expression level of EGLN3 in cancer tissue of NPC patients is significantly lower than that of normal tissue.Mi R-1205 can inhibit the expression of EGLN3 protein.(5)EGLN3 inhibits the migration of NPC cells.(6)Overexpression of EGLN3 inhibits miR-1205-mediated migration,and knockdown EGLN3 promotes miR-1205-mediated migration.Conclusion:(1)miR-1205 promotes the migration of NPC cells.(2)EGLN3 inhibits the migration of NPC cells.(3)miR-1205 targets the 3’UTR of EGLN3 and inhibits the expression of EGLN3 protein.(4)miR-1205 promote the migration of NPC cells by inhibiting the expression of EGLN3. |
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