MicroRNA-203 Inhibits Tumor Growth And Metastasis Through PDPN

Hypopharyngeal cancer is the worst of all head and neck cancers,with the poorest prognosis.High rates of metastasis in hypopharyngeal cancer patients is the major obstacle to cure the disease.Previously,research mainly focuses on genes,growth factors and signaling pathways related to hypopharyngeal cancer metastasis,including chromosomal region 3q27.3 and hepatocyte growth factor.More recently,comprehensive microRNA profilings have revealed that miRNAs play an important role in regulating hypopharyngeal cancer cell proliferation,invasion,and migration.For instance,miR-1 acts as a tumor suppressive factor in hypopharyngeal cancer.In addition,it has been reported that miR-203 possesses anti-tumor capability in many types of cancers,including human melanoma and colorectal cancer.However,miR-203 is not well studied in hypopharyngeal cancer.The location of hypopharyngeal cancer is destined to have a profound impact on the quality of life and social communication.If the tumor recurrence,the patient's survival,life,family,economy and other aspects will have a huge negative impact.Therefore,it is important to investigate the roles of miR-203 in hypopharyngeal cancer,to understand the association between miR-203 expression and hypopharyngeal cancer metastasis,and to elucidate the involved molecular mechanism.Podoplanin/lymphatic endothelial cell protein(PDPN)is a target gene in this experiment.Podoplanin is a mucin-type protein,and its upregulation has been frequently implicated in multiple cancers.PDPN is able to induce tumor cell-induced platelet aggregation via platelet C-type lectin-like receptor 2 activation,thus enhancing tumor metastasis.Interestingly,PDPN also can play a tumor-supportive role in tumor microenvironment.PDPN-positive cancer-associated fibroblasts(CAFs)act as a negative prognosticator,promoting local invasion of cancer cells in lung adenocarcinoma.Due to limited studies on PDPN in hypopharyngeal cancer,better understanding of the expression and functions of PDPN is needed.In our study,we utilized human specimens and pharynx FaDu cancer cells to investigate the roles of miR-203 and PDPN in hypopharyngeal cancer.Part1.The role of miR-203 in hypopharyngeal carcinoma.Objective:1.To explore the expression of miR-203 in hypopharyngeal carcinoma,and its relationwith tumor stage and lymph node metastasis.2.To explore the role of miR-203 in hypopharyngeal carcinoma FaDu cell proliferation,migration,and invasion.Methods:Hypopharyngeal cancer(n=49)specimens were acquired from cancer patients,while normal tissue specimens were acquired from non-cancer patients undergoing surgical procedures in Qilu Hospital of Shandong University.The expression of miR-203 in specimens was detected by Real Time PCR.Hypopharyngeal carcinoma FaDu cell line was treated with miR-203 and miR-NC.Then Real Time PCR was employed to detece the extpession of miR-203 in each group of FaDu cells.Cell proliferation was examined by Cell Counting Kit-8 assay;Cell invasion and migration assays were performed using Transwel chambers and wound healing assay.Results:miR-203 levels are downregulated in human hypopharyngeal cancer tissues.Our results showed that the relative miR-203 expression levels in normal specimens were much higher than those in hypopharyngeal cancer specimens.Our further analysis of the human specimens revealed that decreased expression of miR-203 was associated with increased cancer grades and lymph node metastasis.miR-203 overexpression inhibits cell viability,migration and invasion of hypopharyngeal cancer cells.Our study found that the expression levels determined by qRT-PCR confirmed the significantly upregulation of miR-203 in miR-203-overexpressing cells.The results indicated significant lower cell viability of miR-203-overexpressing cells compared with miR-NC-overexpressing ones by CCK-8 assay in every 24 h.Furthermore,wound healing assay and Matrigel invasion assay demonstrated that miR-203-overexpressing cells had slower cell migration and less cell invasion than miR-NC control.Conclusion:MiR-203 was low expressed in human hypopharyngeal cancer and its expression related with the tumor stage and lymph node metastasis;miR-203 overexpression inhibits cell viability,migration and invasion of hypopharyngeal cancer cells.Part2.The relationship of MicroRNA-203 and PDPN in hypopharyngeal cancer.Objective:1.To explore whether miR-203 was directly targeting PDPN.2.To explore the role of PDPN in hypopharyngeal cancer.3.To explore the role of miR-203 in hypopharyngeal cancer through PDPN.Metheds:Through in silicon analysis using TargetScan Release 7.1 13,we identified putative seed-matching sites(underlined)for miR-203 in the 3'-UTR of PDPN,and constructed luciferase reporters containing wild type and mutant sites,respectively.Luciferase assay was performed on FaDu cells to detect the relative luciferase activities of wild type and mutant PDPN reporters.The expression levels of PDPN were analyzed by qRT-PCR in normal tissues and human hypopharyngeal cancer specimens.Western blot was further employed to detect the PDPN expression levels in miR-203-overexpressing cells and miR-NC control.CCK-8 cell viability test,wound healing assay,and Matrigel invasion assay were used to evaluate the cell proliferation,migration,and invasion in miR-NC+vector and miR-203+PDPN groups.Results:Our results showed that the wild type PDPN reporter has significantly lower levels of luciferase activities in miR-203-overexpressing cells,indicating miR-203 directly targets and inhibits PDPN expression.FaDu cells transfected with PDPN shRNA showed decreased PDPN expression compared with cells with control vector.CCK-8 cell viability test indicated significant lower cell viability of PDPN-knockdown cells compared with cells with control vector.Furthermore,wound healing assay and Matrigel invasion assay demonstrated that knockdown of PDPN in FaDu cells caused slower cell migration and less cell invasion than the control group.CCK-8 cell viability test showed significant lower cell viability of miR-203+vector group compared with miR-NC+vector group and miR-203+PDPN group,respectively.Consistently,wound healing assay and Matrigel invasion assay demonstrated that miR-203+vector group had slower cell migration and less cell invasion than miR-NC+vector and miR-203+PDPN groups.Taken together,our results suggest overexpression of PDPN in miR-203-overexpressing FaDu cells is able to reverse miR-203 inhibitory effects on viability,migration and invasion.Conclusion:PDPN overexpression effected of hypopharyngeal cancer cell viability,migration and invasion.MiR-203 directly targets oncogene PDPN and its expression could reverse the effects of miR-203 on cancer proliferation and metastasis.