Fig 1.
In vivo treatment show no adverse effect with PANC-1 xenograft model.
Average body weight of animal with the same grouping as Fig 4A.
Fig 2.
Dual luciferase system to identify the most optimum mutant selective constructs.
Coding sequences for the first 17 amino acids of KRAS wild-type (wt) and mutant (mu) were inserted into the amino terminus of the psiCHECK2’s hRluc (renilla) and hluc (firefly) coding sequence, respectively. Knockdown of wt vs. mu sequence is compared by renilla to firefly intensity ratio. A. Schematic of the sequence insertion into psiCHECK2 for reporter constructs. B. Bar graph show comparison of reporter constructs relative light unit (RLU) intensity ratio of renilla (RL) to firefly (FF). Y-axis is RL/FF RLU ratio. X-axis is reporter test vectors and parent reporter vector. Standard deviation bar represents measurement from quadruplet samples of independently transfected cells in 96-well format and assayed simultaneously post transfection. C. Positional effect of G12D knockdown constructs; panel a: Table illustrate each constructs guide strand sequence in relation to G12D mutation site. 1st column indicates position of G12D mutation in guide strand of each construct. 2nd column is the code for each construct. The guide strand sequence is shown as the complement of target sequence at 3’ to 5’ orientation; panel b: Bar graph show comparative plot of FF/RL RLU ratio (mu/wt) for each knockdown construct. Sample C is the control without knockdown vector. The red bar represents average control sample value for visual enhancement. Standard deviation bar represents measurement from quadruplet samples of independently transfected cells in 96-well format and assayed simultaneously post transfection. Two-tailed student T-test indicates ρ-value ≤ 0.05 between control and samples 86, 87, 88, 75, 76 and 77. D. G12D, G12V, G12R and G12C knockdown constructs (with mutation nucleotide at position 3 or 4 of the guide strand) were tested against test reporter vectors of all four mutations. Bar graph shows the summary of relative average FF/RL RLU ratio (mu/wt). * indicate the most effective constructs for each mutation. X-axis is the knockdown constructs for G12D, G12V, G12R and G12C. P3 indicate knockdown construct with mutated nucleotide at position 3 of the guide strand. P4 indicate knockdown construct with mutated nucleotide at position 4 of the guide strand. Y-axis is the FF/RL RLU ratio.
Fig 3.
Triple mutant knockdown constructs and mutant allele selective knockdown.
A. Schematics shown expression unit sequence arrangement of triple knockdown constructs in either miR30a backbones or miR-17-92 cluster backbones. B. Bar graphs show analysis of the specificity of triple knockdown constructs by reporter vectors. For each bar graph, the Y-axis is FF/RL RLI ratio (mu/wt) and the X-axis is knockdown vectors tested. At the top of each panel indicate individual mutant targeting vectors tested. Samples from left to right: C = control, G12D = G12D knockdown vector (three independent knockdown vectors with different efficiencies; G12D specific knockdown vector with mutated nucleotide at position 2, position 3 and position 6 of the guide strand, respectively), G12V = G12V knockdown vector with mutated nucleotide at position 4 of the guide strand, G12C = G12C knockdown vector with mutated nucleotide at position 3 of the guide strand, G12R = G12R knockdown vector with mutated nucleotide at position 4 of the guide strand, DVR = triple knockdown vectors for G12D, G12V and G12R in miR30a backbone (code named 129), CDV = triple knockdown vectors for G12C, G12D and G12V in miR30a backbone (code named 130), DVR = triple knockdown vectors for G12D, G12V and G12R in miR17-92 backbone (code named 131), CDV = triple knockdown vectors for G12C, G12D and G12V in miR17-92 backbone (code named 132). The mutated nucleotide position at the guide strand of the triple constructs for G12D, G12V, G12C and G12R were guide strand position 3, 4, 3 and 4, respectively. Standard deviation bar represents measurement from quadruplet samples of independently transfected cells in 96-well format and assayed simultaneously post-transfection. C. Elecropherogram show RFLP of KRAS mRNA in PANC-1 cells stably transformed with triple knockdown vectors. Each individual lane is labeled with their respective % of mutant vs. wild KRAS transcripts. % of mutant vs. wild-type was assessed by electrophaerogram band intensity scan. Samples PANC1 = non-transformed parent PANC-1 cells, Empty vector = non-transformed parent PANC-1 cells transfected with empty vector (pUMVC3 with no insert), Triple DVR (129) = PANC-1 cells transformed with DVR triple knockdown vector in miR-30a backbone (code named 129), Triple CDV (130) = PANC-1 cells transformed with CDV triple knockdown vector in miR-30a backbone (code named 130), Triple DVR (131) = PANC-1 cells transformed with DVR triple knockdown vector in miR-17-92 backbone (code named 131), Triple CDV (132) = PANC-1 cells transformed with CDV triple knockdown vector in miR-17-92 backbone (code named 132), G12V = PANC-1 cells transformed with G12V knockdown vector.
Fig 4.
A. Average tumor volume measurement of PANC-1 tumor xenograft. Group 1: no treatment (blue line), Group 2: vehicle treated (red line), Group 3: 5 μg of 131 (DVR triple knockdown in miR17-92 backbone, green line), Group 4: 25 μg of 131 (DVR triple knockdown in miR17-92 backbone, light blue line), Group 5: 5 μg of 132 (CDV triple knockdown in miR17-92 backbone, purple line), Group 6: 25 μg of 132 (CDV triple knockdown in miR17-92 backbone, dark red line). B. Bar graph show average copy number of plasmids per 100 ng of genomic DNA found in tumor samples. The same treatment grouping as for panel A, samples A, B, or C represents three different tumors from three different animals of the same treatment group.
Fig 5.
Molecular analysis of in vivo tumor samples.
A. Electropherogram analyze % of mu and wt KRAS transcripts in in vivo treated tumor samples. Tumors were removed from animals after four weeks of various treatments, proportions of mu and wt KRAS transcripts were analyzed by RFLP and assayed by Experion. Sample designation is the same as indicated for Fig 4B. % mu and wt KRAS transcripts show at the bottom of the figure was determined by Experion software. B. Western transfer shows protein expression in various tumor samples. Numbers on each sample indicate treatment groups as presented in Fig 4. Two independent isolated tumors are analyzed for each treatment group. Panel a shows RAS protein in various treated tumor samples normalized against GAPDH. Panel b shows p-EGFR at position Y1068 quantitatively normalized against total EGFR protein. Panel c shows total EGFR protein for various treatment groups. C. Bar graphs summarize fold intensity difference from various groups of in vivo samples. Sample groupings are the same as shown on Fig 4. Panel a is for p-EGFR at Y1045 normalized to total EGFR protein. Panel b is for p-EGFR at Y1068 normalized to total EGFR protein. Panel c is for p-EGFR at Y1125 normalized to total EGFR protein. Panel d is total EGFR protein normalized to GAPDH. For each sample n = 3. Bar graphs shown are data obtained from approximately half of tumor of three independent animals. Standard deviation bar represents measurements of tumor from three animals. With one tailed, equal variances, student T-test, the following samples show statistical significant ρ-value ≤ 0.05: Panel a between samples 1 and 6, Panel c between samples 1 and 5, Panel d between samples 1 and 4, 2 and 4, 2 and 6.
Table 1.
Physical properties of reconstituted material comparison.