PMC:5056896 / 8424-15131
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/5056896","sourcedb":"PMC","sourceid":"5056896","source_url":"https://www.ncbi.nlm.nih.gov/pmc/5056896","text":"Results\n\nScreening of miRNAs and genes associated with solid tumor\nFrom the literature, 23 miRNAs were found to be involved in seven solid tumors namely; lung, breast, colorectal, pancreatic, prostate, stomach, and bladder [62]. Again through exploration of online databases 64 different target genes (oncogenes and tumor suppressor genes) were observed to be regulated by these 23 miRNAs (Supplementary Table 1). Again out of these seventeen miRNAs are suspected to be effectively involved in controlling the expression pattern of 45 target genes associated in seven solid tumors (Table 1) [67891011121314151617181920212223242526272829303132333435363738394041]. Further, binding affinity was explored using miRTarbase web server. Fifteen miRNAs and 23 target genes were selected as per the availability of MFE score (Supplementary Table 2) and subjected to further study. The involvement of these 23 genes in different biological functions are explored and inspected from UniProt web server (http://www.uniprot.org/). It was observed only PTEN, TGFBR2, and VEGFA genes mostly regulating angiogenesis, apoptosis, cell cycle, cell proliferation, and other biological functions (Table 2, Fig. 1). Further, the conservation in binding pattern among selected miRNAs and mRNAs (Table 3) were studied through sequence similarity algorithm using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/) web server. The study revealed for the existence of quite less portion of conservation at sequence level among chosen 23 mRNAs and also in 15 miRNAs (Fig. 2), is not sufficient to throw light on binding patterns. Therefore, the binding affinity was evaluated between mRNA and miRNA at secondary structural level. Good MFE score suggested, the target genes PTEN, TGFBR2, and VEGFA are having a strong affinity towards miR-106a, miR-21, and miR-29b-2 (Table 3). Hence those genes and miRNAs were taken into consideration to study their interaction at molecular level.\n\nStudy of binding affinity between miRNA-mRNA duplex\nDuplex sequences between miR-106 and PTEN, miR-21 and TGFBR2, miR-29b-2 and VEGFA were extracted from miRTarbase web server and prediction of secondary folding pattern was performed in RNA fold. The secondary folding patterns along with their binding energy value are reported (Table 4). The predicted binding energy suggested for a high affinity among selected miRNA-mRNA duplexes. Predicted three dimensional models [63] of the duplex structures between miR-106 and PTEN, miR-21 and TGFBR2, miR-29b-2 and VEGFA also supported for strong molecular interaction between them.\n\nStructure preparation of AGO protein\nThe three-dimensional crystallized structure of AGO protein, with 685 amino acid residues was extracted from Protein Data bank (PDB ID: 3F73). The structure of AGO protein contains guide DNA and target RNA duplexes. All water molecules and ligands were removed from the structure. As, the AGO protein is a homo dimer, out of two chains, only chain 'A' of AGO protein was considered and refined before docking. The necessary correction in bond order and bond length of all atoms in the structure was performed using prepare protein and clean geometry protocol of Discovery Studio 3.5.\n\nStudy of molecular interaction between miRNAs and AGO protein\nThe AGO protein is a key player in the formation of the RNA-induced silencing complex, a major component of RNA interference. The three dimensional structures of miRNA-mRNA duplexes were prepared using RNA COMPOSER web server (Fig. 3). The first round of docking was performed between the miRNAs and AGO (PDB ID: 3F73, chain A) protein to inspect the binding affinity between the complexes. Out of 10 different poses resulted for docking complexes in Patch Dock server, the pose with highest geometrical shape complementary score [64] is considered as the best docked complex. The resulted geometrical shape complementary score and atomic contact energy scores are reported (Table 5), implicated a strong binding affinity between the miRNAs and AGO protein. The binding affinity between the AGO protein and miRNAs (miR-106a, miR-21, and miR-29b-2) is established through the observation of amino acids (miR-106a: LEU 132, ALA 133, VAL 152, LEU 153, ALA 170, ILE 173, LEU 267, LEU 279, ALA 479,VAL 620, VAL 663, VAL 666, and ILE 671; miR-21: ILE 173, VAL 264, LEU 267, LEU 279, ALA 354, ALA 414, ILE 434, ALA 644, ALA 648, and VAL 685; miR-29b-2: VAL 152, LEU 153, ALA 170, LEU 279, ALA 414, ALA 479, LEU 596, VAL 620, ALA 648, LEU 652, ALA 659, LEU 662, and VAL 663) which are strongly hydrophobic in nature and also amino acids with aromatic rings (miR-106a: TYR 135, TYR 171, TRP415, and PHE 487; miR-21: TYR 135, TYR 171, TRP 415, TYR 642, PHE 647, and PHE 649; miR-29b-2: TRP 156, TRP 415, TYR 642, PHE 647, and PHE649) within a distance of 3.5Å (Table 6, Fig. 4). The presence of hydrogen bonding pattern during interaction (Table 7, Fig. 5) also supported the fact of AGO protein driven miRNA based gene regulation.\n\nStudy of molecular interaction between AGO protein and miRNA-mRNA duplex\nThe second round docking was performed between AGO protein and miRNA-mRNA duplexes (miR-106 and PTEN; miR-21 and TGFBR2; and miR-29b-2 and VEGFA) separately in PatchDock web server, and scores are reported (Table 8). Amino acids which are strong hydrophobic in nature and amino acids with aromatic rings which are relatively hydrophobic generally contribute a lot towards the stability of binding during molecular interaction between two macro molecules. Hence the molecular interaction between miRNA-mRNA duplexes and AGO protein was studied by inspecting close amino acid residues of AGO protein within a distant of 3.5 Å. Strong hydrophobic amino acids of AGO like VAL 152, LEU 153, VAL 264, LEU 265, LEU 267, LEU 277, ALA 278, LEU 279, ALA 414, ILE 434, ALA 479, VAL 663, and VAL 685 in miR-106a and PTEN duplex; LEU 45, LEU 46, ALA 47, VAL 49, ALA 50, ALA 80, ILE 173, LEU 265, LEU 267, LEU 277, ALA 278, LEU 279, LEU 281, ALA 479, ALA 644, ALA 648, LEU 652, and VAL 663 in miR-21 and TGFBR2 duplex; and ALA 80, LEU 132, VAL 152, LEU 153, ALA 170, ILE 173, VAL 264, ALA 479, VAL 606, ALA 644, ALA 648, VAL 663, and VAL 685 in miR-29b-2 and VEGFA duplex are observed within a distant of 3.5 Å. Similarly, amino acids like TYR 43, TRP 415, PHE 487, and TYR 642 in miR-106a and PTEN duplex; TYR 43, TRP 202, TRP 415, TYR 642, PHE 647, and PHE 649 in miR-21 and TGFBR2 duplex; and TYR 43, TYR 86, TYR 135, TRP 415, TYR 171, TYR 642, PHE 647, and PHE 649 in miR-29b-2 and VEGFA duplex, with aromatic ring are also found as participating in interaction within the binding pocket of AGO protein around distance of 3.5 Å (Table 9, Fig. 6).","divisions":[{"label":"Title","span":{"begin":0,"end":7}},{"label":"Section","span":{"begin":9,"end":1961}},{"label":"Title","span":{"begin":9,"end":66}},{"label":"Section","span":{"begin":1963,"end":2589}},{"label":"Title","span":{"begin":1963,"end":2014}},{"label":"Section","span":{"begin":2591,"end":3211}},{"label":"Title","span":{"begin":2591,"end":2627}},{"label":"Section","span":{"begin":3213,"end":4996}},{"label":"Title","span":{"begin":3213,"end":3274}},{"label":"Title","span":{"begin":4998,"end":5070}}],"tracks":[{"project":"2_test","denotations":[{"id":"27729841-18372920-44843560","span":{"begin":592,"end":593},"obj":"18372920"},{"id":"27729841-25084400-44843561","span":{"begin":592,"end":594},"obj":"25084400"},{"id":"27729841-19826040-44843562","span":{"begin":592,"end":595},"obj":"19826040"},{"id":"27729841-23095762-44843563","span":{"begin":592,"end":596},"obj":"23095762"},{"id":"27729841-24912422-44843564","span":{"begin":592,"end":598},"obj":"24912422"},{"id":"27729841-22704331-44843565","span":{"begin":592,"end":600},"obj":"22704331"},{"id":"27729841-24795757-44843566","span":{"begin":592,"end":602},"obj":"24795757"},{"id":"27729841-26063204-44843567","span":{"begin":592,"end":604},"obj":"26063204"},{"id":"27729841-25422179-44843568","span":{"begin":592,"end":606},"obj":"25422179"},{"id":"27729841-22402125-44843569","span":{"begin":592,"end":608},"obj":"22402125"},{"id":"27729841-20686687-44843570","span":{"begin":592,"end":610},"obj":"20686687"},{"id":"27729841-24130168-44843571","span":{"begin":592,"end":612},"obj":"24130168"},{"id":"27729841-24324762-44843572","span":{"begin":592,"end":614},"obj":"24324762"},{"id":"27729841-18836476-44843573","span":{"begin":592,"end":616},"obj":"18836476"},{"id":"27729841-23202912-44843574","span":{"begin":592,"end":618},"obj":"23202912"},{"id":"27729841-24022342-44843575","span":{"begin":592,"end":620},"obj":"24022342"},{"id":"27729841-22821565-44843576","span":{"begin":592,"end":622},"obj":"22821565"},{"id":"27729841-22510476-44843577","span":{"begin":592,"end":624},"obj":"22510476"},{"id":"27729841-24312276-44843578","span":{"begin":592,"end":626},"obj":"24312276"},{"id":"27729841-24823697-44843579","span":{"begin":592,"end":630},"obj":"24823697"},{"id":"27729841-20237410-44843580","span":{"begin":592,"end":632},"obj":"20237410"},{"id":"27729841-25341040-44843581","span":{"begin":592,"end":634},"obj":"25341040"},{"id":"27729841-18174313-44843582","span":{"begin":592,"end":636},"obj":"18174313"},{"id":"27729841-22453030-44843583","span":{"begin":592,"end":638},"obj":"22453030"},{"id":"27729841-24152184-44843584","span":{"begin":592,"end":640},"obj":"24152184"},{"id":"27729841-21779448-44843585","span":{"begin":592,"end":644},"obj":"21779448"},{"id":"27729841-20797623-44843586","span":{"begin":592,"end":646},"obj":"20797623"},{"id":"27729841-22716183-44843587","span":{"begin":592,"end":648},"obj":"22716183"},{"id":"27729841-19407485-44843588","span":{"begin":592,"end":650},"obj":"19407485"},{"id":"27729841-18676839-44843589","span":{"begin":592,"end":652},"obj":"18676839"},{"id":"27729841-23617834-44843590","span":{"begin":592,"end":654},"obj":"23617834"},{"id":"27729841-21106054-44843591","span":{"begin":592,"end":656},"obj":"21106054"},{"id":"27729841-24156637-44843592","span":{"begin":592,"end":658},"obj":"24156637"},{"id":"27729841-22912877-44843593","span":{"begin":592,"end":660},"obj":"22912877"},{"id":"27729841-22539264-44843594","span":{"begin":2434,"end":2436},"obj":"22539264"},{"id":"27729841-15980490-44843595","span":{"begin":3806,"end":3808},"obj":"15980490"}],"attributes":[{"subj":"27729841-18372920-44843560","pred":"source","obj":"2_test"},{"subj":"27729841-25084400-44843561","pred":"source","obj":"2_test"},{"subj":"27729841-19826040-44843562","pred":"source","obj":"2_test"},{"subj":"27729841-23095762-44843563","pred":"source","obj":"2_test"},{"subj":"27729841-24912422-44843564","pred":"source","obj":"2_test"},{"subj":"27729841-22704331-44843565","pred":"source","obj":"2_test"},{"subj":"27729841-24795757-44843566","pred":"source","obj":"2_test"},{"subj":"27729841-26063204-44843567","pred":"source","obj":"2_test"},{"subj":"27729841-25422179-44843568","pred":"source","obj":"2_test"},{"subj":"27729841-22402125-44843569","pred":"source","obj":"2_test"},{"subj":"27729841-20686687-44843570","pred":"source","obj":"2_test"},{"subj":"27729841-24130168-44843571","pred":"source","obj":"2_test"},{"subj":"27729841-24324762-44843572","pred":"source","obj":"2_test"},{"subj":"27729841-18836476-44843573","pred":"source","obj":"2_test"},{"subj":"27729841-23202912-44843574","pred":"source","obj":"2_test"},{"subj":"27729841-24022342-44843575","pred":"source","obj":"2_test"},{"subj":"27729841-22821565-44843576","pred":"source","obj":"2_test"},{"subj":"27729841-22510476-44843577","pred":"source","obj":"2_test"},{"subj":"27729841-24312276-44843578","pred":"source","obj":"2_test"},{"subj":"27729841-24823697-44843579","pred":"source","obj":"2_test"},{"subj":"27729841-20237410-44843580","pred":"source","obj":"2_test"},{"subj":"27729841-25341040-44843581","pred":"source","obj":"2_test"},{"subj":"27729841-18174313-44843582","pred":"source","obj":"2_test"},{"subj":"27729841-22453030-44843583","pred":"source","obj":"2_test"},{"subj":"27729841-24152184-44843584","pred":"source","obj":"2_test"},{"subj":"27729841-21779448-44843585","pred":"source","obj":"2_test"},{"subj":"27729841-20797623-44843586","pred":"source","obj":"2_test"},{"subj":"27729841-22716183-44843587","pred":"source","obj":"2_test"},{"subj":"27729841-19407485-44843588","pred":"source","obj":"2_test"},{"subj":"27729841-18676839-44843589","pred":"source","obj":"2_test"},{"subj":"27729841-23617834-44843590","pred":"source","obj":"2_test"},{"subj":"27729841-21106054-44843591","pred":"source","obj":"2_test"},{"subj":"27729841-24156637-44843592","pred":"source","obj":"2_test"},{"subj":"27729841-22912877-44843593","pred":"source","obj":"2_test"},{"subj":"27729841-22539264-44843594","pred":"source","obj":"2_test"},{"subj":"27729841-15980490-44843595","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#d393ec","default":true}]}]}}