MiR-96 Gene Regulation HERG1 Experimental Pancreatic Cancer Proliferation And Metastasis Inhibition

Pancreatic cancer, the fourth most common cause of cancer-related death, with the characteristics of early detection difficult, high degree of malignancy, poor sensitivity to radiotherapy and chemotherapy, and low five-year survival rate. The foreign studies report that the peak incidence of pancreatic cancer is more than60years old, but in China, the peak incidence is advanced to40-59years old, and the morbidity and mortality is increasing year by year. It has become a serious harm to the health of our population. In addition, despite extensive research efforts, the prognosis for pancreatic cancer is the worst among all cancers due to minimal improvements in its prevention and treatment. Therefore, the quest for new associated factors and novel therapeutic targets in pancreatic cancer remains an imperative clinical issue.HERG1, a human inward rectifier in the voltage-gated potassium channel family, which is a special potassium channel. Lot’s of experiments showed that HERG1expressed in several tumor cells, and associated with tumor’s proliferation, apoptosis, differentiation and invasive power. Nevertheless, studies examining the role of HERG1in human pancreatic cancer are rare.MicroRNAs (miRNAs) are highly conserved endogenous small20-25nucleotide non-coding RNAs. miRNAs regulate the expression of targetd gene, leading to the degradation of the target mRNA or post-transcriptional inhibition of translation levels, mainly through binding the target gene mRNA in the3’end non-coding region (3’-UTR) combined with the degree of complementarity. Now a large number of studies have shown that the miRNA plays an important role in the development, diagnosis and treatment of cancer.In the present study, we aimed at taking a comprehensive picture of HERG1role in human pancreatic cancer through the analysis of its expression and function in vitro and in vivo. It may suggest novel therapeutic strategies in pancreatic cancer.Methods:The study of HERG1promotes the proliferation and metastasis of pancreatic cancer1. To test the HERG1expression in pancreatic cancer and adjacent normal tissues, immunohistochemistry was performed in78pairs of pancreatic tissue samples, and then we analyzed the clinicopathological characteristics in78pancreatic tumors;2. We investigated HERG1expression by RT-PCR and western blot in pancreatic cancer cell lines;3. Blocking of HERG1was carried out by siRNA technology, and then we transfected it into pancreatic cancer cell lines;4. The cellular growth activity was measured by MTT assay, and the cellular migration and invasion ability were detected by transwell assay;5. The cell cycle and apoptosis were tested by flow cytometry;6. The tumorigenicity and metastasis were tested by nude mice model.miR-96affects the proliferation and metastasis of pancreatic cancer via downregulates HERG11. We used three microRNA (miRNA) target prediction programs, and information from the existing literature to predict the miRNAs which targets HERG1,3’-UTR luciferase reporter assay was used to filter the potential miRNAs;2. The expression of miR-96in pancreatic cancer tissues and cells was detected by TaqMan real-time PCR;3. Transfection in vitro with mimics or inhibitors of miR-96was used to observe the banding relationship of miR-96with HERG13’-UTR via luciferase reporter assay test, RT-PCR and western blot were detected HERG1mRNA and protein levels;4. Construct Pre-miR-96plasmid, and then transfected it into pancreatic cancer cells, after transfection, the cellular growth activity was measured by MTT assay;5. The cellular migration and invasion ability were detected by transwell assay;6. The cell cycle and apoptosis were tested by flow cytometry;7. The tumorigenicity and metastasis were tested by nude mice model;8. The tumor tissues from nude mice were detected by immunohistochemistry again to verify the relationship between miR-96and HERGl.Results:The study of HERG1promotes the proliferation and metastasis in pancreatic cancer1. We find that HERG1expression is dramatically increased both in pancreatic cancer tissues and cell lines, and is also related with clinic stage;2.Blockade of HERG1could inhibit the ability of proliferation in PANC-1and CFPAC-1cells;3. Blockade of HERG1could make the PANC-1and CFPAC-1cells tend to accumulate in G1phase, and increase apoptotic cell population;4. Blockade of HERG1could inhibit the ability of invasion and migration in PANC-1and CFPAC-1cells;5. Blockade of HERG1could inhibit the ability of tumorigenicity and metastasis of CFPAC-1cells in nude mice model.miR-96affects the proliferation and metastasis of pancreatic cancer via downregulates HERG11. We found that miR-96, miR-105,miR-152, miR-199a-5p, miR-220, miR-329, miR-331-5p, miR-592, let-7c and let-7g may affect the expression of HERG1. After the luciferase reporter assay test, we suggested the miR-96is the most potent regulator of HERG1gene levels in vitro;2. The miR-96expression was significanty lower in pancreatic cancer tissues and cells compared to the adjacent non-tumor tissue;3. We demonstrated that HERG1is the direct target of miR-96via transfecting miR-96mimics, inhibitor and luciferase reporter assay test.4. Highexpression of miR-96could inhibit the ability of proliferation, invasion and migration in PANC-1and CFPAC-1cells;5. Highexpression of miR-96could inhibit the ability of tumorigenicity and metastasis of CFPAC-1cells in nude mice model. We verify that HERG1is the direct target of miR-96via detecting the expression of HERGl in the tumor tissues from nude mice by immunohistochemistry again.Conclusions:1. HERG1was high expressed in pancreatic cancer, and plays a oncogene role in the development of pancreatic cancer;2.HERG1is a direct target gene of miR-96;3. miR-96was lowexpression in pancreatic cancer, reduces the degradation of HERG1, and then improve the development of pancreatic cancer;4. After highexpression of miR-96, the malignant biological activity in pancreatic cancer cells become weaker, and plays a role of Tumor suppressor gene. miR-96may be considered as a novel molecular therapeutic target in pancreatic cancer treatment.