Regulatory Mechanism Of MiR-885-5P In Tumor Growth And Metastasis

Part I Regulatory mechanism of miR-885-5p in metastasis of hepatocellular carcinomaBackground:Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide and the third leading cause of cancer death in China. The clinical symptoms of HCC patients are not obvious, and advanced HCC is diagnosed. Treatments of HCC include surgery, embolism and chemotherapy. However, HCC shows a higher resistance to chemotherapy. Therefore, exploring the biomarker and targeting molecules will be significant to clinical therapy.miR-885-5p is generated from cleavage of miR-885at5prime, and located at3p25.3. Our previous study found, circulating miR-885-5p is present at higher levels in patients with cirrhosis than with HCC patients and control individuals. Other studies found that miR-885-5p suppressed the metastasis of neuroblastoma and glioma. However, how miR-885-5p is involved in HCC metastasis is not reported. This study applied for various experiments to analyse the roles of miR-885-5p in HCC to prove that miR-885-5p contributes to the metastasis of HCC and the molecular mechanisms. Our results provided theoretical basis in clinical therapy of HCC.Methods:1. We used In situ hybridization and Real-time qPCR to test the expression of miR-885-5p in different grades of malignant HCC tissues and cell lines;2. We used In situ hybridization to examine expression of miR-885-5p in55HCC tissues form55patients and did Kaplan-Meier survival analyses;3. We overexpressed miR-885-5p in HCCLM3and SK-Hep-1, and used Transwell and Wound healing assays to identify the regulation of miR-885-5p in migration and invasion; we knocked down miR-885-5p in HepG2cells, and studied the regulation of miR-885-5p in growth; 4. We performed orthotopic HCC models, and identified the regulation of miR-885-5p in HCC in vivo; we utilized a subcutaneous HCC xenograft model to demonstrate the regulation of miR-885-5p in growth in vivo;5. We used the bioinformatics methods to explore the target genes of miR-885-5p; we used dual-luciferase assays to confirm the bioinformatics results; we employed western blot to test the expression of target proteins.Results:1. Patients in stage Ⅱ (n=84) exhibited higher levels of miR-885-5p than patients in stage Ⅲ (n=147; p<0.001). miR-885-5p was down-regulated in higher metastasis HCC cell lines, and up-regulated in non-metastatic HCC cells;2. Higher miR-885-5p levels in the HCC tissues significantly correlated with the markedly prolonged overall survival of these HCC patients;3. Overexpression of miR-885-5p in HCCLM3and SK-Hep-1cells inhibited migration and invasion in vitro; knocking down of miR-885-5p promoted the proliferation of HepG2cells in vitro;4. Overexpression of miR-885-5p inhibited the metastasis of HCC. And injection of miR-885-5p agomir suppressed the growth of HCC and promoted tumor necrosis;5. miR-885-5p inhibited the expression of β-catenin and TCF4via binding to P-catenin and TCF43’UTRs, and suppressed the activity of Wnt/β-catenin signaling pathway and the expression of target proteins.Conclusions:1. miR-885-5p levels were negatively correlated with the clinical stages, and positively correlated with prognosis of HCC patients;2. Overexpression of miR-885-5p inhibited the metastasis of HCC in vitro and in vivo, and knockdown of miR-885-5p promotes the growth of HCC;3. miR-885-5p suppressed the activity of Wnt/β-catenin signaling pathway via inhibiting the expression of β-catenin and TCF4. Part Ⅱ Regulatory mechanism of miR-885-5p in proliferation of DLBCLBackground:Diffuse large B cell lymphoma, with high mortality, is one form of non-Hodgkin’s lymphoma and difficult to cure. Identifying the new molecule used for targeting therapies will greatly benefit to clinical treatment of DLBCL. Aberrant expression of microRNAs (miRNAs) has a key role in regulating progression of diffuse large B cell lymphoma (DLBCL), but exactly how miR-885-5p is involved in remains unclear.Methods:1. This study used Real time qPCR to test the expression of miR-885-5p in human B lymphoblastoid cells as well as DLBCL cell lines;2. We overexpressed miR-885-5p in OCI-LY3cells, and used the CCK8Kit to test the function of miR-885-5p in proliferation of DLBCL;3. We knocked down miR-885-5p in VAL, and used the CCK8Kit to test the function of miR-885-5p in proliferation of DLBCL;4. We used the bioinformatics methods to predict the target gene of miR-885-5p; we used dual-luciferase assays to confirm the bioinformatics results; we employed western blot to test the expression of target proteins.5. We knocked down lymphocyte cytosolic protein1and tested the proliferation of OCI-LY3and OCI-LY19cells.Results:1. miR-885-5p levels in DLBCL cells were lower than that of human B lymphoblastoid;2. Overexpression of miR-885-5p suppressed the proliferation of OCI-LY3cells;3. Knockdown of miR-885-5p promoted the proliferation of VAL cells;4. miR-885-5p directly binded to3’UTR of L-plastin and suppressed its expression;5. Knockdown of L-plastin inhibited the proliferation of OCI-LY3cells and OCI-LY19cells.Conclusions: 1. miR-885-5p suppressed the proliferation of DLBCL cells;2. miR-885-5p suppressed the expression of L-plastin;3. Knockdown of L-plastin inhibited the proliferation of DLBCL cells.