Initial identification of low temperature and culture stage induction of miRNA expression in suspension CHO-K1 cells

This paper describes the first miRNA analysis carried out on hamster cells specifically Chinese hamster ovary (CHO) cells which are the most important cell line for the manufacture of human recombinant biopharmaceutical products. During biphasic culture, an initial phase of rapid cell growth at 37 degrees C is followed by a growth arrest phase induced through reduction of the culture temperature. Growth arrest is associated with many positive phenotypes including increased productivity, sustained viability and an extended production phase. Using miRNA bioarrays generated with probes against human, mouse and rat miRNAs, we have identified 26 differentially expressed miRNAs in CHO-K1 when comparing cells undergoing exponential growth at 37 degrees C to stationary phase cells at 31 degrees C. Five miRNAs were selected for qRT-PCR analysis using specific primer sets to isolate and amplify mature miRNAs. During this analysis, two known growth inhibitory miRNAs, miR-21 and miR-24 were identified as being upregulated during stationary phase growth induced either by temperature shift or during normal batch culture by both bioarray and qRT-PCR. Sequence data confirmed the identity of cgr-miR-21, a novel Cricetulus griseus ortholog of the known miRNA miR-21. This study offers a novel insight into the potential of miRNA regulation of CHO-K1 growth and may provide novel approaches to rational engineering of both cell lines and culture processes to ensure optimal conditions for recombinant protein production.     

Inhibition of endogenous miR-23a/miR-377 in CHO cells enhances difficult-to-express recombinant lysosomal sulfatase activity

Difficult-to-express (DTE) recombinant proteins such as multi-specific proteins, DTE monoclonal antibodies, and lysosomal enzymes have seen difficulties in manufacturability using Chinese hamster ovary (CHO) cells or other mammalian cells as production platforms. CHO cells are preferably used for recombinant protein production for their ability to secrete human-like recombinant proteins with posttranslational modification, resistance to viral infection, and familiarity with drug regulators. However, despite huge progress made in engineering CHO cells for high volumetric productivity, DTE proteins like recombinant lysosomal sulfatase represent one of the poorly understood proteins. Furthermore, there is growing interest in the use of microRNA (miRNA) to engineer CHO cells expressing DTE proteins to improve cell performance of relevant bioprocess phenotypes. To our knowledge, no research has been done to improve CHO cell production of DTE recombinant lysosomal sulfatase using miRNA. We identified miR-23a and miR-377 as miRNAs predicted to target SUMF1, an activator of sulfatases, using in silico prediction tools. Transient inhibition of CHO endogenous miR-23a/miR-377 significantly enhanced recombinant sulfatase enzyme-specific activity by ~15–21% compared to scramble without affecting cell growth. Though inhibition of miR-23a/miR-377 had no significant effect on the mRNA and protein levels of SUMF1, overexpression of miR-23a/377 caused ~30% and ~27–29% significant reduction in endogenous SUMF1 protein and mRNA expression levels, respectively. In summary, our data demonstrate the importance of using miRNA to optimize the CHO cell line secreting DTE recombinant lysosomal sulfatase.     

Profiling highly conserved microrna expression in recombinant IgG‐producing and parental Chinese hamster ovary cells

MicroRNAs (miRNAs) play important roles in global gene regulation. Researchers in recombinant protein production have proposed miRNAs as biomarkers and cell engineering targets. However, miRNA expression remains understudied in Chinese Hamster Ovary cells, one of the most commonly used host cell systems for therapeutic protein production. To profile highly conserved miRNA expression, we used the miRCURY? miRNA array for screening miRNAs in CHO cells. The selection criteria for further miRNA profiling included positive hybridization signals and experimentally validated predicted regulatory targets. On the basis of screening, we selected 16 miRNAs for quantitative RT‐PCR profiling. We profiled miR expression in parental CHO DG44 and CHO K1 cell lines as well as four recombinant DG44‐derived CHO lines producing a recombinant human IgG. We observed that miR‐221 and miR‐222 were significantly downregulated in all IgG‐producing cell lines when compared with parental DG44, whereas miR‐125b was significantly downregulated in one IgG‐producing line. In another IgG‐producing line, miR‐19a was significantly upregulated. miRNA expression was also profiled in two of these lines that were amplified by stepwise increase of methotrexate. In both amplified cell lines, let‐7b and miR‐221 were significantly downregulated. In parental CHO K1, let‐7b, miR‐15b, and miR‐17 were significantly downregulated when compared with DG44. The results reported here are the first steps toward profiling highly conserved miRNAs and studying the clonal difference in miRNA expression in CHO cells and may shed light on using miRNAs in cell engineering. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Next-generation sequencing of the Chinese hamster ovary microRNA transcriptome: Identification, annotation and profiling of microRNAs as targets for cellular engineering

Chinese hamster ovary (CHO) cells are the predominant cell factory for the production of recombinant therapeutic proteins. Nevertheless, the lack in publicly available sequence information is severely limiting advances in CHO cell biology, including the exploration of microRNAs (miRNA) as tools for CHO cell characterization and engineering. In an effort to identify and annotate both conserved and novel CHO miRNAs in the absence of a Chinese hamster genome, we deep-sequenced small RNA fractions of 6 biotechnologically relevant cell lines and mapped the resulting reads to an artificial reference sequence consisting of all known miRNA hairpins. Read alignment patterns and read count ratios of 5' and 3' mature miRNAs were obtained and used for an independent classification into miR/miR* and 5p/3p miRNA pairs and discrimination of miRNAs from other non-coding RNAs, resulting in the annotation of 387 mature CHO miRNAs. The quantitative content of next-generation sequencing data was analyzed and confirmed using qPCR, to find that miRNAs are markers of cell status. Finally, cDNA sequencing of 26 validated targets of miR-17-92 suggests conserved functions for miRNAs in CHO cells, which together with the now publicly available sequence information sets the stage for developing novel RNAi tools for CHO cell engineering.     

Transferability of miRNA-technology to bioprocessing: Influence of cultivation mode and media

The biopharmaceutical industry strives for improvement of their production processes. In recent years, miRNAs have been shown to positively impact the production capacity of recombinant CHO cells, especially with regard to difficult to express proteins. Effective and reliable gene regulation of process relevant target genes by miRNAs is a prerequisite for integrating them into the toolbox of industrial cell engineering strategies. However, most studies rely on transient transfection of miRNA mimics; there is low standardization in evaluation of miRNA function and little knowledge on transferability of effects found during transient expression to stable expression during industry relevant fed-batch cultivation. In order to provide more insight into this topic, we used the pcDNA6.2 vector for stable miRNA overexpression during batch and fed-batch cultivation in CHO DG44 cells, optimized the vector, and compared the miRNA levels and effects with those achieved by transfection of miRNA mimics. We found that miR-1 downregulated TWF1 mRNA in different recombinant CHO DG44 clones in a dose-dependent manner during transient batch cultivation. Cells stably overexpressing miR-1 also showed a TWF1 mRNA downregulation when cultivated in batch mode using in-house medium 1. However, when the cells stably overexpressing miR-1 were cultivated in fed-batch mode using in-house medium 2. Consequently, a change of cultivation mode and medium seems to have an impact on target gene regulation by miRNA. Taken together, our findings highlight the importance to standardize miRNA evaluations and test miRNAs in the final application environment.     

Profiling conserved microRNA expression in recombinant CHO cell lines using Illumina sequencing

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate multiple aspects of cell physiology. The differential expression of conserved miRNAs in two Chinese hamster ovary (CHO) cell lines producing recombinant proteins was examined relative to the CHO-K1 cell line. A total of 190 conserved CHO miRNAs were identified through homology with known human and rodent miRNAs. More than 80% of these miRNAs showed differential expression in recombinant CHO cell lines. The small RNA sequencing data were analyzed in context of the CHO-K1 genome to examine miRNA organization and develop sequence-specific miRNA resources for CHO cells. The identification and characterization of CHO miRNAs will facilitate the use of miRNA tools in cell line engineering efforts to improve product yield and quality.     

The Epstein-Barr virus (EBV)-induced tumor suppressor microRNA MiR-34a is growth promoting in EBV-infected B cells

Epstein-Barr virus (EBV) infection of primary human B cells drives their indefinite proliferation into lymphoblastoid cell lines (LCLs). B cell immortalization depends on expression of viral latency genes, as well as the regulation of host genes. Given the important role of microRNAs (miRNAs) in regulating fundamental cellular processes, in this study, we assayed changes in host miRNA expression during primary B cell infection by EBV. We observed and validated dynamic changes in several miRNAs from early proliferation through immortalization; oncogenic miRNAs were induced, and tumor suppressor miRNAs were largely repressed. However, one miRNA described as a p53-targeted tumor suppressor, miR-34a, was strongly induced by EBV infection and expressed in many EBV and Kaposi's sarcoma-associated herpesvirus (KSHV)-infected lymphoma cell lines. EBV latent membrane protein 1 (LMP1) was sufficient to induce miR-34a requiring downstream NF-κB activation but independent of functional p53. Furthermore, overexpression of miR-34a was not toxic in several B lymphoma cell lines, and inhibition of miR-34a impaired the growth of EBV-transformed cells. This study identifies a progrowth role for a tumor-suppressive miRNA in oncogenic-virus-mediated transformation, highlighting the importance of studying miRNA function in different cellular contexts.     

Engineering CHO cell growth and recombinant protein productivity by overexpression of miR-7

The efficient production of recombinant proteins by Chinese Hamster Ovary (CHO) cells in modern bioprocesses is often augmented by the use of proliferation control strategies. The most common method is to shift the culture temperature from 37 °C to 28-33 °C though genetic approaches to achieving the same effect are also of interest. In this work we used qRT-PCR-based expression profiling using TLDA™ cards to identify miRNAs displaying differential expression 24 h after temperature-shift (TS) from 37 °C to 31 °C. Six miRNAs were found to be significantly up-regulated (mir-219, mir-518d, mir-126, mir-30e, mir-489 and mir-345) and four down-regulated (mir-7, mir-320, mir-101 and mir-199). Furthermore, qRT-PCR analysis of miR-7 expression over a 6 day batch culture, with and without TS, demonstrated decreased expression over time in both cultures but to a significantly greater extent in cells shifted to a lower culture temperature. Unexpectedly, when miR-7 levels were increased transiently by transfection with miR-7 mimic in CHO-K1 cells, cell proliferation at 37 °C was effectively blocked over a 96 h culture period. On the other hand, transient inhibition of endogenous miR-7 levels using antagonists had no impact on cell growth. The exogenous overexpression of miR-7 also resulted in increased normalised (per cell) production at 37 °C, though the yield was lower than cells grown at reduced temperature. This is the first report demonstrating a functional impact of specific miRNA disregulation on CHO cell behavior in batch culture and provides some evidence of the potential which these molecules may have in terms of engineering targets in CHO production clones. Finally, we report the cloning and sequencing of the hamster-specific cgr-miR-7.     

Dysregulated microRNAs in the pathogenesis and progression of cervical neoplasm

MicroRNAs (miRNAs) play an important role in a variety of physiological as well as pathophysiological processes, including carcinogenesis. The aim of this study is to identify a distinct miRNA expression signature for cervical intraepithelial neoplasia (CIN) and to unveil individual miRNAs that may be involved in the development of cervical carcinoma. Expression profiling using quantitative real-time RT-PCR of 202 miRNAs was performed on micro-dissected high-grade CIN (CIN 2/3) tissues and compared to normal cervical epithelium. Unsupervised hierarchical clustering of the miRNA expression pattern displayed a distinct separation between the CIN and normal cervical epithelium samples. Supervised analysis identified 12 highly differentially regulated miRNAs, including miR-518a, miR-34b, miR-34c, miR-20b, miR-338, miR-9, miR-512-5p, miR-424, miR-345, miR-10a, miR-193b and miR-203, which distinguished the high-grade CIN specimens from normal cervical epithelium. This miRNA signature was further validated by an independent set of high-grade CIN cases. The same characteristic signature can also be used to distinguish cervical squamous cell carcinoma from normal controls. Target prediction analysis revealed that these dysregulated miRNAs mainly control apoptosis signaling pathways and cell cycle regulation. These findings contribute to understanding the role of microRNAs in the pathogenesis and progression of cervical neoplasm at the molecular level.     

Differential effects of miR-34c-3p and miR-34c-5p on SiHa cells proliferation apoptosis, migration and invasion

MicroRNAs (miRNA) regulate expression of several genes associated with human cancer. Here, we analyzed the function of miR-34c, an effector of p53, in cervical carcinoma cells. Expression of either miR-34c-3p or miR-34c-5p mimics caused inhibition of cell proliferation in the HPV-containing SiHa cells but not in other cervical cells irrespective of tumorigenicity and HPV content. These results suggest that SiHa cells may lack of regulatory mechanisms for miR-34c. Monolayer proliferation results showed that miR-34c-3p produced a more pronounced inhibitory effect although both miRNAs caused inhibition of anchorage independent growth at similar extent. However, ectopic expression of pre-miR-34c-3p, but not pre-miR-34c-5p, caused S-phase arrest in SiHa cells triggering a strong dose-dependent apoptosis. A significant inhibition was observed only for miR-34c-3p on SiHa cells migration and invasion, therefore implying alternative regulatory pathways and targets. These results suggest differential tumor suppressor roles for miR-34c-3p and miR-34c-5p and provide new insights in the understanding of miRNA biology.     

miRNA profiling of naïve, effector and memory CD8 T cells

microRNAs have recently emerged as master regulators of gene expression during development and cell differentiation. Although profound changes in gene expression also occur during antigen-induced T cell differentiation, the role of miRNAs in the process is not known. We compared the miRNA expression profiles between antigen-specific na?ve, effector and memory CD8+ T cells using 3 different methods--small RNA cloning, miRNA microarray analysis and real-time PCR. Although many miRNAs were expressed in all the T cell subsets, the frequency of 7 miRNAs (miR-16, miR-21, miR-142-3p, miR-142-5p, miR-150, miR-15b and let-7f) alone accounted for approximately 60% of all miRNAs, and their expression was several fold higher than the other expressed miRNAs. Global downregulation of miRNAs (including 6/7 dominantly expressed miRNAs) was observed in effector T cells compared to na?ve cells and the miRNA expression levels tended to come back up in memory T cells. However, a few miRNAs, notably miR-21 were higher in effector and memory T cells compared to na?ve T cells. These results suggest that concomitant with profound changes in gene expression, miRNA profile also changes dynamically during T cell differentiation. Sequence analysis of the cloned mature miRNAs revealed an extensive degree of end polymorphism. While 3'end polymorphisms dominated, heterogeneity at both ends, resembling drosha/dicer processing shift was also seen in miR-142, suggesting a possible novel mechanism to generate new miRNA and/or to diversify miRNA target selection. Overall, our results suggest that dynamic changes in the expression of miRNAs may be important for the regulation of gene expression during antigen-induced T cell differentiation. Our study also suggests possible novel mechanisms for miRNA biogenesis and function.     

XB130, a New Adaptor Protein,Regulates Expression of Tumor Suppressive MicroRNAs in Cancer Cells

XB130, a novel adaptor protein, promotes cell growth by controlling expression of many related genes. MicroRNAs (miRNAs), which are frequently mis-expressed in cancer cells, regulate expression of targeted genes. In this present study, we aimed to explore the oncogenic mechanism of XB130 through miRNAs regulation. We analyzed miRNA expression in XB130 short hairpin RNA (shRNA) stably transfected WRO thyroid cancer cells by a miRNA array assay, and 16 miRNAs were up-regulated and 22 miRNAs were down-regulated significantly in these cells, in comparison with non-transfected or negative control shRNA transfected cells. We chose three of the up-regulated miRNAs (miR-33a, miR-149 and miR-193a-3p) and validated them by real-time qRT-PCR. Ectopic overexpression of XB130 suppressed these 3 miRNAs in MRO cells, a cell line with very low expression of XB130. Furthermore, we transfected miR mimics of these 3 miRNAs into WRO cells. They negatively regulated expression of oncogenes (miR-33a: MYC, miR-149: FOSL1, miR-193a-3p: SLC7A5), by targeting their 3′ untranslated region, and reduced cell growth. Our results suggest that XB130 could promote growth of cancer cells by regulating expression of tumor suppressive miRNAs and their targeted genes.