Effect Of MiR-663 On Proliferation Invasion And Apoptosis Of Pancreatic Cells By Acting On EEF1A2

Background and PurposePancreatic cancer is a kind of highly malignant cancer which develops extremely rapidly with poor prognosis and high mortality rate, especially pancreatic ductal adenocarcinoma(PDAC), the five-year survival rate of which is under 5%.Clinical diagnosis of pancreatic cancer often depends on image detection. Early detection and differential diagnosis of pancreatic cancer are both difficult due to low accuracy of diagnostic kits specificity. There is an upward trend about pancreatic cancer incidence in recent years. Early symptoms of pancreatic cancer are not obvious and there is no economic and effective clinical method for early detection.Molecular diagnostics is a rapidly developing discipline in recent years and is one of the means of clinical diagnosis. Because of its feature of specific expression in pancreatic cancer, micro RNAs(mi RNAs) can be used as a new diagnostic marker of pancreatic cancer and its related detection techniques also have achieved further development. Relevant studies show the significant influence of mi RNAs upregulation on survives, proliferation, invasion, apoptosis and metastasis of pancreatic cancer and mi RNAs are promising for being the biomarkers or therapeutic targets of pancreatic cancer prediction or prognosis.mi RNAs are found in eukaryotes, with length of about 20 ~ 25 nucleotides,which is endogenous non-coding RNA and has the function of regulation. Matured mi RNAs are generated from longer primary transcription products(~1000 nt)through shear processing of double-strands-RNA-specific RNase, Drosha and Dicer,followed by assembly into RNA-induced silencing complex(RISC) and identify targets by means of complementary base pairing and direct silence complex degradetarget m RNA or repress the translation of the target m RNA depending on the degree of complementary. Relevant study shows that mi RNA-663 inhibits the proliferation, migration and invasion of glioblastoma cells via targeting TGF-β1.Preliminary experiments of this study showed that the expression level of mi R-663 in pancreatic cancer tissue significantly lower than that of adjacent non-tumor tissuesnotably. The predicted results of bioinformatics software Target Scan and DIANA-Micro T showed that eukaryotic elongation factor 1-a2(e EF1A2) is correlative to mi R-663 and probably the target of mi R-663. The objective of our study is to know the expression situation of mi R-663 in pancreatic cancer tissue and corresponding adjacent non-tumor tissues, analyze the correlation of character in patients suffered from pancreatic cancer pathologically and the influence of mi R-663 upregulation on pancreatic cancer cells from aspects of proliferation, invasion and apoptosis. We also identify the target and regulation mechanism of mi R-663 preliminarily.Methods1. 68 cases of cancer tissue and adjacent non-tumor tissues of patients with pancreatic cancer after pancreaticoduodenectomy or distal pancreatectomy at the First Affiliated Hospital of Zhengzhou University and Henan Tumor Hospital,Zhengzhou University, Zhengzhou, China between 2007 and 2009. These tissues were snap frozen in liquid nitrogen. The expression level of e EF1A2 protein was detected through western blot assay; the expression level of mi R-663 and e EF1A2 m RNA were detected by Quantitative Real-Time PCR(q RT-PCR), correlation between which and progression stage, lymph node metastasis situation of pancreaticcancer patients was analyzed. All of the pancreatic cancer patients aforementioned were followed up and Kaplan-Meier analysis was used on all pancreatic cancer samples for evaluating the correlation between clinicopathologic features of pancreatic cancer and survival of patients and the correlation between expression levels of e EF1A2, mi R-663 and survival of patients. q RT-PCR about normal pancreatic cell HPDE6-C7 and pancreatic cancer cell PANC-1, Capan-2, SW1990,Bx PC3 and As PC-1 was conducted for observing the expression level of mi R-663.Western blot assay about normal pancreatic cell HPDE6-C7 and pancreatic cancer cell PANC-1, Capan-2, SW1990, Bx PC3 and As PC-1 was conducted for observing the expression level of e EF1A2 protein in cells.2. The pancreatic cancer cells were divided into three groups according to different kinds of transfection. Group A was mi R-663 group, with transfection of mi R-663 agomir and liposome complex; group B was negative control, with transfection of mi R-663 agomir negative control and liposom complex; group C was blank control, with transfection of only liposome complex. Cells were placed in incubator(37℃、5%CO2) for usage afterwards. After 24 h, the transfection effects of transfected cells were detected using q RT-PCR.3. Proceeded CCK-8 assay and colony formation assay to cells in groups A, B and C in order to observate the influence of mi R-663 on proliferation of pancreatic cancer cells.4. Tumor xenograft transplant assay. Pancreatic cancer cells( PANC-1) of blank group and mi R-663 group were subcutaneously injected in the dorsal scapular region of 6-week-old BALB/c. Calibrate the volume of tumor and calculate it according to formula. Compare the discrepancy of tumor volume.5. Transwell assay was used to scrutinize the effects of mi R-663 on cellular invasive abilities using NC group and mi R-663 group pancreatic cancer cells(PANC-1 and As PC-1).6. Pancreatic cancer cells(PANC-1 and As PC-1)in NC group and mi R-663 group were conducted flow cytomytry assay and hochest staining to detect the influence of mi R-663 on pancreatic cancer cells.7. Software Target Scan and DIANA-Micro T were applied to analyse and predict the target of mi R-663. Vectors of reportor gene of 3’UTR region in e EF1A2(including wild type and mutational type) was constructed, then were co-transfected into pancreatic cancer cell lines PANC-1 and As PC-1 with mi R-663 agomir or mi R-663 agomir negative control. Western blot assay and dual-luciferase assay were conducted for identification of target site of mi R-663.8. Restore experiment. We constructed the expressional vector of e EF1A2 without 3’UTR region, then transfected it or co-transfected it with mi R-663 agomir into pancreatic cancer cells PANC-1 and As PC-1.9. Compared the effect of e EF1A2 downregulation and mi R-663 upregulation acting on pancreatic cancer cells. e EF1A2-si RNAs and mi R-663 agomir were transfected into pancreatic cancer cell PANC-1, then compare effects of both on proliferation, invasion and apoptosis of pancreatic cancer cell PANC-1. Western blot assay was used to detect the expression of relevant genes.Results1. The expression level of mi R-663 in pancreatic cancer tissue was significantly lower than corresponding adjacent non-tumor tissues(P<0.05) and expression level of e EF1A2 m RNA and protein notably higher than adjacent non-tumor tissues(P<0.05). The expression level of mi R-663 gene and e EF1A2 gene were negative correlated. The expression level of e EF1A2 and mi R-663 had a notable relation with TNM stages of pancreatic cancer and lymphnode metastasis of pancreatic cancer patients(P<0.05) The results of Kaplan-Meier showed that tumor diameter(P<0.001), differentiation(P<0.001), clinical stage(p<0.001) and lymph node metastasis(P<0.001) have significant association with the survival of pancreatic cancer patients, but the gender(P=0.975), age(P=0.523) and location(P=0.613) were not significantly correlated with the survival of pancreatic cancer patients. Pancreatic cancer patients with higher level of e EF1A2 had shorter survival periods and pancreatic cancer patients with higher level of mi R-663 had longer survival periods(P < 0.05). e EF1A2 and mi R-663 is related to the survival periodsof pancreatic cancer patients. Result of q RT-PCR about different cell line HPDE6-C7,PANC-1, Capan-2, SW1990, Bx PC3 and As PC-1 showed that compared with the normal cell line HPDE6-C7, the expression level of mi R-663 was generally downregulated in pancreatic cancer cell lines(PANC-1, Capan-2, SW1990, Bx PC3 and As PC-1). A statistically significant difference of expression levels(P<0.05) was observed. Western blot assay suggested e EF1A2 protein expression level in pancreatic cancer cell lines were higher than in the normal pancreatic cell line(P<0.05).2. Detection result after transfection of cells showed a significant higher expression level in mi R-663 group compared with blank group and NC group(P <0.05), indicating mi R-663 expression level upregulation after transfection of pancreatic cancer cells.3. CCK-8 assay demonstrated that compared to the Blank and NC groups, the OD value at 450 nm decreased after 1-day, 2-day and 3-day transfection, respectively,suggesting that both of proliferationrate of the two pancreatic cancer cell lines have significant decline. The difference is statistically significant(*P<0.05). Results of colony formation assay showed that colony numbers of mi R-663 group decreased significantly, compared with blank group and NC group. The difference is statistically significant(*P<0.05).4. Result of tumor xenograft transplant experiment showed the inhibition of tumorous growth in tumor xenograft model of nude mouse with injections of PANC-1 cells overexpressing mi R-663. The difference is statistically significant(P<0.05).5. Transwell assay manifested that the number of cells invading the matrigel notably decreased significantly in mi R-663 group cells compared with NC group.The numerical difference is statistically significant(P<0.05).6. Data of flow cytomytry assay and hochest staining indicated that the number of apoptotic cells, together with apoptotic cells observed through fluorescence microscope have notable increase consistently, compared with NC group. The numerical difference is statistically significant(P<0.05).7. Results of western blot and duel-luciferase assay showed that the target of mi R-663 was e EF1A2.8. Restore experiment presented the fact that mi R-663 regulated e EF1A2 expression negatively by acting on 3’UTR region of it. mi R-663 participated in proliferation, invasion and apoptosis of pancreatic cancer cells.9. Compared the effect of e EF1A2 downregulation and mi R-663 upregulation acting on pancreatic cancer cells and detected the expression of relevatnt genes. The results showed similar effects of e EF1A2 downregulation and mi R-663 upregulation,suggesting the inhibitive effect of mi R-663 on pancreatic cancer was at least partly due to acting on e EF1A2.Conclusions1. The expression level of mi R-663 in pancreatic cancer tissue has a significant downregulation and is related withlymph node metastasis and TNM stages of pancreatic cancer patients.2. Upregulation of mi R-663 effectively repress the proliferation and invasion of pancreatic cancer cells, prompting their apoptosis.3. mi R-663 conversely regulates expression level of e EF1A2 through acting on 3’UTR region thereby exerting its biological function.