Characterization of Functionally Associated miRNAs in Glioblastoma and their Engineering into Artificial Clusters for Gene Therapy

This protocol allows the simultaneous modulation of many micro RNAs in eukaryotic cells and is based on a very simple and flexible method which minimizes molecular cloning steps. This technique has implications for the study of micro RNA functional interactions in vitro, but more importantly it provides a platform for a more effective use of micro RNAs in therapy. We have mainly developed and used it in the context of brain cancer, but it applies to any diseases where multiple alterations of gene expression are involved.

We have tested our artificial trans genes in other models such as breast and thyroid cancer lines showing the reproducibility of this technique. Basic knowledge of bioinformatics and micro RNA biology is helpful for this protocol, but not indispensable. A bioinformatics approach is important to define relevant micro RNA combinations and the TRK-11, while familiarity with the micro RNA processing measurably is important to understand how these different micro RNAs can be assembled into artificial DNA clusters that can be successfully processed once stable to the specific cells.

To coat dish, first dissolve poly-D-lysine in water at a concentration of 100 micrograms per milliliter. Pour the solution into the dish at the amount of one milliliter of solution per 25 square centimeters surface. Swirl the dish to make sure of coverage of the whole dish.

After six hours aspirate the poly-D-lysine solution and rinse twice with PBS. Plate human neural stem cells in the amount of 100, 000 cells per five milliliter of the medium, either in stem cell medium, astrocytic differentiation medium or neuronal differentiation medium. After cell plating, place the dish in an incubator at 37 degrees Celsius and five percent carbon dioxide for one week and proceed to perform micro RNA expression analysis according to the manuscript.

To culture GBM-34 glioblastoma stem-like cells, start with one times ten to the fifth cells and five milliliter of neurobasal medium in a 25 square centimeter low attachment flask. Place the flask into an incubator at 37 degrees Celsius and five percent carbon dioxide. 48 hours after plating, add doubling concentrations of DNA alkylating agent temozolomide every seven days.

Start with addition of five micromolar TMZ supplemented in five milliliters medium for five days. After five days, remove the medium and replace with fresh medium without temozolomide to allow the surviving cells to recover. After 48 hours, add 10 micromolar temozolomide and incubate for another five days.

Repeat the doubling temozolomide treatment until concentration of at least 100 micromolar is reached and cells become resistant to the drug. After the induction of resistance, lie cells using 1 milliliter of Lysis reagent. Extract total RNA and analyze the expression of the specific micro RNAs according to the manuscript.

To obtain the predicted targetome of each micro RNA previously defined, use micro RNA targeting prediction tools. From the front page of TargetScan, select the micro RNA of interest from the pre-populated drop down menu. Click the submit button.

Download the resulting list of targets as a spreadsheet using the download table link. To decrease the chances of false positive predictions, include only targets within the conserved sites column and downstream analysis. Additional micro RNA prediction programs obtain further stringency and only include targets that are common to all algorithms.

Then, open ToppGene suite 20. To evaluate for the enrichment of pathways that are common to each micro RNA, paste the list of targets obtained in the training gene sets window. Click on HGNC Symbol as the entry type.

Click submit and start. The program provides an output table showing the most significant GO categories for the entered list of genes. To finally establish the contribution of each micro RNA to the regulation of a common pathway or cellular process, open the venn-diagram function provided in the Bioinformatics and Evolutionary Genomics website.

Check each obtained targetome against the full list of genes involved in the specific cellular process. If target messenger RNAs for each micro RNAs of interest are in the respective windows, upload of copy-paste the list. Name each list with a unique identifier.

Click submit. The program provides a visual output of a venn-diagram with numbers of genes in each sector as well as a full list of messenger RNAs for each subset and intersection combinations. To group the selected micro RNAs into a functional trans gene, obtain a transgenic scaffold based on the miR-17-92 cluster locus.

Obtain the nucleotide sequence from the ensembled genome browser. Select the approximately 800 base pair core sequence encompassing all six encoded micro RNA hairpins of the locus and at least 200 nucleotide flanking sequences both upstream and downstream of the core sequence. Paste the sequence into any word editing program.

Define the sequence of each one of the six native hair pins by retrieving them in miR base. Mark each one of these sequences within the span of previously identified core sequence. Any sequences between each hairpin represent spacer sequences.

Next, obtain the hairpin sequences of micro RNAs intended to be over expressed in the trans gene from miR base. Carefully note the specific nucleotides that are at the sites of micro processor cleavage. Delete the native hairpin sequences from the core sequence with the exception of three to five nucleotides at both the five and thee ends of each hairpin which will serve as an acceptor for the new hairpins.

Paste the hairpin sequences of the desired micro RNAs to replace the previously deleted hairpins. Add desired restriction sequences at both flanking regions of the trans gene to facilitate sub-cloning into delivery vectors of choice. Verify that the chosen restriction sequences are not present within the sequence itself.

To verify the two dimensional structure of the trans gene, copy the full transgenic sequence into the RNA structure prediction software program RNA Webfold. Select standard program settings and click proceed. Analyze the graphical output, particularly for the presence of well defined hairpins in presence of double stranded stem structures that are at least 11 nucleotides proximal to the micro processor cleavage site.

Also, look for the absence of branching points within the hairpin sequences. At this point, the trans gene is ready to be produced by gene synthesis and cloned into the desired delivery vectors. This method allowed characterization of a module of three micro RNAs that are consistently down regulated in brain tumors, which are co-expressed specifically during neuronal differentiation.

Co-expression patterns of micro RNA modules during genotoxic stress were confirmed and suggested a strong synergistic activity among these three micro RNAs. The designed trans gene could simultaneously recapitulate the expression of the three micro RNAs in glioblastoma cells, both in vitro and in vivo, with significant interference in tumor biology and promising translational applicability. Additionally, the transgenic cluster was also functional in a breast cancer model.

The crucial requirements of this protocol are maintaining the original size of processor cleavage in the trans gene. And make sure that the stem loop structure of chimeric micro RNA hairpins is preserved. With this method, we can concentrate multiple biologically active micro RNA genes into very short DNA sequences which can literally be vitroly be fitted into any delivery vectors for gene therapy use.

This method is ideal to study the functional interaction among different micro RNAs and to interfere with complex multifactorial cellular pathways which will otherwise require multiple drug combinations.