| Tumor is a worldwide common and frequently-occurring disease, there are many differences on the cell morphology and organization structure between tumor tissues and normal tissues. Physical and chemical factors, viruses, bacteria, genetics and molecular biology are regard as precipitating factor of tumor. It is closely related to the basis of molecular biology and the original abnormal expression of oncogenes or tumor suppressor genes, apoptosis regulation genes and DNA repair genes function disorder. In essence, tumor is a kind of gene disease.microRNA (miRNA), also called small RNA, is a kind of single small conservative noncoding RNA molecules exist in eukaryotes, contains about 21 to 25 nucleotides. It can target mRNA by matching the nucleotides of mRNA, make its degradation or inhibit its translation, to play the function of regulate genes expression. miRNA associated with physiological function in vivo, such as cell growth, differentiation, proliferation and apoptosis and other biologyprocess. In recent years, miRNA has become a hot research issue, has now established a huge database of miRNA. With the development of bioinformatics, various prediction software or online web site which used to find the miRNA target genes provides a quick and easy method for miRNA targets prediction.Produce and application of gene chip has brought the new opportunity for the gene function research, the high-throughput, rapid, sensitive features greatly improve the study efficiency and accuracy, also provide a lot of data for researchers to mining meaningful information. miRNA expression profile chip has become a important tool to analysis the difference of miRNA between several tissues, and widely used in the study of miRNA in tumor. At the same time, with the development of big data, such as gene chip, the database and related analysis software that contain DNA, RNA, miRNA and protein information have also gradually established and perfected. Providing methods for gene function research, regulation and control network analysis.The accuracy and reliability for bioinformatics analysis were improved.Multiple studies have showed that the occurrence and development of tumor are closely related to miRNA regulation mechanism. miR-335 is also associated with multiple tumors for instance ovarian cancer, breast cancer and lung cancer, plays the role of oncogenes or tumor suppressor genes. miR-335 involved in regulation of tumor proliferation, metastasis and other biological processes, it is also related with signal pathways and gene regulation networks. Based on the researches founding of miR-335 has a certain influence in multiple tumors, we speculated that miR-335 may has an important role in tumor pathogenesis mechanism. Using bioinformatics methods to analyze the sequences of miR-335 and its conservation to speculated the role of miR-335 in evolution of species, then we further predict miR-335 target genes considering the regulatory mechanism of miRNA, and then analyze the function and signal pathway of miR-335 target genes, so as to clear the role of miR-335 in tumor, and provides a certain direction for further experimental study.According to the result of miR-335 analysis by use bioinformatics methods, we finally choose cervical cancer as the research object to following experimental research for which the cervical cancer data set is relatively special. Cervical cancer is a high incidence in female that jeopardize women’s health. Cervical cancer is the top 2 mortal malignancies in women globally, more than 0.5 million cases were diagnosed every year. More than85% of these cases diedin developing countries. In China, approximately 50,000 women died from cervical cancer annually. At present, the method of treatment of cervical cancer mainly include traditional therapy such as surgery, radiation and chemotherapy, gene therapy, targeted therapy and other new methods. Looking for the target genes for therapy is still the important direction for protocol of cervical cancer prevention and diagnosis.miRNA mimics can effectively enhance the function of the certain miRNA in the cells, and the miRNA inhibitor can effectively inhibit the certain endogenous mature miRNA function, efficient inhibits the miRNA activity for a long time, mimic NC and inhibitor NC can eliminate the effects of the sequence specificity, thus a comprehensive understanding of the similarities and differences between mimics or inhibitor as well as the background effects. In order to further study the regulation of miR-335 in cervical cancer, we collected the cervical cancer patients’cervical cancer tissues and para-carcinoma formalin-fixed paraffin-embedded (FFPE) specimens, and quantified miR-335 expression by qRT-PCR. We chosen HeLa cervical cancer cells for in vitro cell experiment. The chemical synthesis miR-335 mimics and miR-335 inhibitor and its control group mimics NC and inhibitor NC were transfected into HeLa cells. After artificial changed miR-335 expression level in HeLa cells, we detected HeLa cell line proliferation, cell cycle, invasion and migration and other biological behavior, to reveal the mechanism of miR-335 regulation in cervical cancer. The research we studied before have showed that the expression of G2E3(G2/M phase-specific E3 ubiquitin-protein ligase) in cervical cancer tissues was higher than normal cervical tissues.Down-regulated G2E3 expression could inhibit the proliferation, migrationof Hela cells, and arrest the cells in G2/M phase. The results ofmiR-335 target genes predicting also showed G2E3 may be a target of miR-335. Therefore, experiments were performed to verify the hypothesis.In this research, the outcomes can provide a new direction and train of thought for further studies of miR-335 regulation in tumor. And deepen our understanding of the pathogenesis of cervical cancer; provide new clues for cervical cancer to find the new potential targets for diagnosis and treatment.Methods:1. miR-335 sequence in multiple species were downloaded from miRNA database miRBase, and sequence alignment of miR-335 was used to analyze its conservation in the species.2. The miRNA expression microarray data set of common tumor tissues were downloaded from NCBI databases GEO, choose the data sets which include tumor tissues and normal tissues group and miR-335 expression data as a follow-up study data sets. The data sets information was imported into chip data analysis software Qlucore Omics Explorer (QOE) 3.0 to standardize the data, selecting the appropriate statistical test methods to screening differentially expressed miRNAs between the tumor and normal tissues, and analysis the expression level of miR-335 in different tumor tissues.3. Predicting miR-335 target genes by on-line tools:DIANA-microT, PicTar, miRDB and TargetScanin, and take the intersection of the results to the several predicted results. Quering miR-335 target genes in miRTarBase and research papers that have confirmed by experiments, then take the union set with predicted target genes as miR-335 targets set for the following analysis.4. Uploading miR-335 target genes to DAVID online, choose human genome as the background, then analyze the function (GO-analysis) and signal pathway (KEGG Pathway analysis) of miR-335 target genes.5. Based on the analysis of the bioinformatics, we select cervical cancer as the further research object.19 pairs of cervical carcinoma and para-carcinoma formalin-fixed paraffin-embedded (FFPE) specimens were collected, and quantified miR-335 expression by qRT-PCRto evaluate miR-335 expression level between cervical carcinoma and para-carcinoma specimens.6. Getting the difference information of miR-335 expression level in cervical cancer and its adjacent carcinoma, bioinformatics analysis results, we then chosen HeLa cervical cancer cells for subsequent experiment in vitro, to explore how miR-335 influence the biological behavior of HeLa cervical cancer cells. HeLa cervical cancer cells were transfected with chemical synthesis of miR-335 mimics, miR-335 inhibitor and their controls mimics NC and inhibitor NC respectively, to increase or reduce miR-335 expression level. At the same time, using FAM tag miRNA transfected HeLa cells as the negative control group, to preliminary observe the transfection efficiency. And quantified miR-335 expression in transfected HeLa cells to confirm the transfection efficiency by qRT-PCR.7. The viability of HeLa cells was evaluated by CCK-8 assay after transfection,optical density was measured to reflect the cell viability; cell cycle was analyzed by flow cytometry. Take the results together to discuss the influence of HeLa cells proliferation which made by the change of miR-335 expression.8. The cell invasion and migration capacity was determined by transwell cell matrigel invasion and migration assays. The permeated cell count of each group can reflect the cell invasion and migration capacity. In addition, wound-healing assay was carried out to confirm the transwell cell migration assays. The migration capacity was analyzed by calculating the percent migration of cells.Results:1. miR-335 mature sequences of rats, mice, dogs and other species, a total of 12 species were found in miRBase. Sequence alignment showed that miR-335 sequence were highly similar in the 12 species, suggesting that the sequence of miR-335 was highly conserved among different species.2. According to the retrieval condition,6 miRNA expression profile chip data sets were downloaded, they are liver cancer (GSE31383), lung cancer (GSE48414), breast cancer (GSE38167), intrahepatic cholangiocarcinoma (GSE53870), liposarcoma(GSE45364) and cervical cancer (GSE303656). Importing the datas into Qlucore Omics Explorer (QOE) 3.0, the analysis results showed that miR-335 was low-expression in the tissues of liver cancer, lung cancer, breast cancer, intrahepatic cholangiocarcinoma and liposarcoma compared with the normal tissues, the differences in multiples were 0.32, 0.07,0.19,0.93,0.58(P< 0.05); Meanwhile, miR-335 was significantly higher quantities in cervical cancer tissues than in para-cancer tissues, the differences in multiples was 1.10(P=0.05).3. Using the target prediction on-line tools to predict miR-335 target genes, we got 166,137,251 and 2308 predicted target genes respectively,8 target genes were selected by taking the intersection. The count of miR-335 target genes in miRTarBase and research papers in PubMed that have confirmed by experiments were 15 and 20 genes respectively. Taking the 3 results together, a total of 34 target genes were collected for further study.4. DAVID was used to analyze the function and signal pathway of miR-335 target genes. The analysis results showed that the function of these target genes mainly enriched in cell division, cell migration, regulation of apoptosis, protein binding, regulate transcription factor activity and protein amino acid phosphorylation(P< 0.05). In KEGG pathway, the predicted target genes set was involving in axon guidance signal, melanoma diseases pathway, focal adhesion and TGF-beta signaling pathway(P< 0.05).5. Expression of miR-335 were measured by qRT-PCR in 19 pairs of cervical cancer and para-cancer tissues, which showed significantly lowered quantities of miR-335 in cancer tissues than in para-cancer tissues in 17 pairs, while higher quantities in the rest 2 pairs. miR-335 relative mean expression levels was down-regulated in cervical cancer tissues. The differences in multiples was 0.287(t=-6.169, P< 0.001).6. HeLa cells transfected with FAM tag miRNA after 6 h were observed under fluorescence inverted microscope, visible cells with green fluorescence, coverage rate of about 80%. qRT-PCR assay was performed to calculate miR-335 expression level of HeLa cells that were transfected with chemical synthesis of miR-335 mimics, miR-335 inhibitor and their controls mimics NC and inhibitor NC respectively. The outcomes showed that expressions of miR-335 in mimics group was higher than mimics NC group, the differences in multiples was 180.046 (t=15.825, P=0.004); Expressions of miR-335 in inhibitor group was lower than inhibitor NC group, the differences in multiples was 0.458 (t=-8.292, P=0.030).7. The results of CCK-8 assay indicated that cells proliferation of transfected HeLa cells have significant difference in group and time, time and group interaction effect also existed.Cells in miR-335 mimics group had a decreased proliferation rate after 2nd day (t=-10.252, P=0.001); while cells in miR-335 inhibitors group demonstrated a higherproliferation rate after 2nd day (t=22.396, P< 0.001). Cell cycle alteration in each group was tested by flow cytometry. The consequenceshowed cells of G0/G1 phase gained a lower percentage in miR-335 mimics group than that in mimics NC group(t=-22.734, P=0.002); while S phase gained a higher percentage in miR-335 mimics group than that in mimics NC group (t=6.707, P=0.003).8. In transwell invasion and migration experiments, permeated cell count of miR-335 mimics group was less than that of mimics NC group (P< 0.05). Permeated cell count of miR-335 inhibitors group outnumbered that of ininhibitors NC group (P< 0.05). In wound-healing assay, cell migration rate of miR-335 mimics group was less than that of mimics NC group on 24h,48h (P<0.05), while the migration rate of miR-335 inhibitors group was greater than that of inhibitors NC group on 24h,28h (P<0.05).Conclusion:The sequence of miR-335 was highly conserved among different species. Compared with the normal tissues, miR-335 was low-expression in the tissues of liver cancer, lung cancer, breast cancer, intrahepatic cholangiocarcinoma and liposarcoma. The target genes of miR-335 closely related to multiple biological processes and signal transduction pathways in cancer. miR-335 expression is lowered in cervical carcinoma, HeLa cells had abatements in proliferation, invasion and migration after the transfection of miR-335 mimics, and were arrested in S phase; While HeLa cells had promots in proliferation, invasion and migration after the transfection of miR-335 inhibitor. |
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