The Effect Of Smad4Small Interfering RNAs In Injured Skeletal Muscle After Acute Contusion

TGF β was very important in the process of fibrosis after skeletal muscle injured, which not only induced fibrosis in skeletal muscle, but also played an important role in the process of fibrosis in lung, kidney, hepatic and skin. TGF β signals within the cell through the Smad family of transcriptional activators. As being the only co-Smad, Smad4is the key protein in the TGF β-Smad signal transduction pathway. The aim of this study was to suppress the expression of Smad4in the injured mouse skeletal muscle and to block TGFβ-Smad signal transduction pathway, then inhibit the fibrosis and improve the healing process. First of all, we constructed a lentiviral-mediated Smad4siRNA and found that Smad4-siRNA could efficiently knock down the gene and protein expression of Smad4in the C2C12myoblast cells and in the contunded mice gastrocnemius muscle on a long-term basis. Then we found that suppressing Smad4expression using lentiviral-mediated RNA interference (RNAi) in vivo may improve the functional recovery of the muscle by inhibiting fibrosis and scar tissue formation after acute contunded. Part One The Construction and Identification of Lentiviral Vectors Carrying Smad4siRNAObjective To construct and identify the lentiviral vectors-mediated small interfering RNA (siRNA) targeting Smad4.Methods5siRNAs and1scramble RNA as control were designed based on the sequence of the mouse Smad4cDNA. They covered different regions of the Smad4CDS and showed no homology with non-Smad4sequences. The oligonucleotides were synthesized, annealed and cloned into the pshRNA-H1-GFP lentivector. SiRNA1, siRNA2and siRNA3were chosen for study. NIH3T3cells were infected with the3siRNAs using Lipofectamine2000. The relative Smad4mRNA and protein levels were determined by quantitative Real-time PCR and western blotting analyses after infection respectively. Among the3siRNAs examined, siRNA1was selected as the most efficient siRNA for use. Lentivirus was then packaged in293TN cells. After concentration of lentivirus, the titer of virus was detected with gradient dilution methods.Results3Smad4siRNA were successfully constructed whose DNA sequences were completely compatible with the design. After NIH3T3cells were infected with the3siRNAs, the transfection efficiency observed under fluorescence microscope was about75%by regarding the green fluorescence as a reference. Real-time PCR and western blotting analysis revealed that all3siRNAs led to remarkable suppression of Smad4expression in NIH3T3cells (P<0.05). As siRNAl exerted a powerful and specific knock-down effect on Smad4expression up to more than80%(p<0.01), we selected siRNAl as a tool and used it in the following study. Lentivirus was then packaged in293TN cells. After concentration of lentivirus, the titer of virus was detected with gradient dilution methods, which showed that the titer is as high as1*104ifu/μ1and is good enough for infection. Then we stored them at-70℃for use.Conclusion Lentivirus-mediated Smad4siRNA was constructed successfully, and could suppress the expression of Smad4successfully in NIH3T3cells both at gene and protein level. Part Two Lentivirus Mediated Smad4siRNA Tranfected into C2C12Myoblast Cells and its Effect on Suppressing the Expression of Smad4Objective To determine the transfection efficiency of lentivirus mediated Smad4siRNA into C2C12myoblast cells and their capability of suppressing the expression of Smad4both at gene and protein level.Methods We infected the C2C12myoblast cells with lentivirus mediated Smad4siRNA.72hours after infection, the green fluorescent protein expression and distribution were observed under the inverted fluorescence microscope, and then the cells were counted after trypsinization to calculate the optimal MOI value of infection. The flow cytometer was further used to testify the transfection efficiency. The expression level of Smad4in C2C12cells were detected by Real-time PCR and western blotting respectively.Results After the lentivirus-mediated Smad4-siRNA transfecting into C2C12myoblast cells, the efficiency highest up to99%in a MOI of6was verified by both immunofluorescence counting and flow cytometic analysis. Both real time-PCR and Western blot analysis showed that C2C12cells passages3maintained the siRNA silencing effects against Smad4with the efficiency up to more than85%.Conclusion Lentivirus mediated Smad4siRNA could transfect C2C12myoblast cells in vitro successfully and suppress the expression of Smad4both at gene and protein level. Part Three Lentivirus-mediated siRNA Transfected into the Acute Contunded Mice Skeletal Muscle and its effect on Suppressing the Expression of Smad4Objective To determine the efficiency of lentivirus-mediated Smad4siRNA transfection into the acute contunded mouse skeletal muscle and its capability of suppressing Smad4both at gene and protein level on the injured skeletal muscle.Methods A muscle acute contusion model was utilized to hit the mouse’s right gastrocnemius muscle for our experiment.72female C57b1mice (20-25g) were randomly assigned to1of3groups:PBS control group, Lv siRNA injection group and Lv Negative injection group (24each).0.1ml of culture medium containing PBS, Lv Negative or Lv siRNA was injected into the contunded area10days after injury. Six mice for each group were sacrificed7、14、21、28days respectively after contusion, and the right gastrocnemius muscle and the heart, the liver, the kidney and the brain were isolated and prepared for use. The green fluorescent protein expression and distribution in each group were observed under the fluorescence microscope. Real-time PCR and Western blot were used to analyze Smad4expression at the contunded area and he heart, the liver, the kidney and the brain in vivo both at gene and protein level.Results The GFP detected after injury demonstrated that transfection of the lentivirus-mediated Smad4siRNA was successful. Meanwhile, the expression of GFP in the liver, heart, brain and kidney of Smad4siRNA injected mice was negative, indicating that siRNA was stably transfected only into the local injection area and not into other parts of the animals. Local injection of lentivirus mediated Smad4siRNA could suppress the expression of Smad4both at gene and protein level in acute contunded skeletal muscle.Conclusion Lentivirus mediated Smad4siRNA was successfully transfected only into the local injection area and not into other organs of the animals, which indicates that gene’s transfection into the mice contunded skeletalmuscle is specific and can play a long term role of suppressing Smad4expression both at gene and protein level so that the safety can be guaranted. Part Four The Effect of Lentivirus Mediated Smad4siRNA Gene Silencing on the Fibrosis and Functional Recovery of Injured Mice Skeletal MuscleObjective To determine the effect of lentivirus-mediated Smad4siRNA transfection on the fibrosis, scar formation and functional recovery of the acute contunded mice skeletal muscle.Methods A muscle acute contusion model was utilized to hit the mouse’s right gastrocnemius muscle for our experiment.72female C57bl mice (20-25g) were randomly assigned to1of3groups:PBS control group, Lv siRNA injection groupand Lv Negative injection group (24each).0.1ml of culture medium containi ng PBS, Lv Negative or Lv siRNA was injected into the contunded area10days after injury. Six mice for each group were sacrificed7、14、21、28days respectively after contusion, and the right gastrocnemius muscle was isolated and prepared for Masson trichrome staining, immunohistochemical analysis and western blot analysis of Vimentin, a SMA and collagen I to identify the fibrosis at local injured area. Moreover, we tested the fast-twitch and tetanus strength of the gastrocnemius muscle to determine the functional recovery of injured muscle.Results Masson trichrome staining revealed a smaller collagenous area in the Lv siRNA group compared with the Lv negative group and PBS control group with statistical significance14,21and28days after acute contusion. Immunohistochemical and western blot analysis showed that in Smad4siRNA injection group there was a significant decrease significantly in the vimentin a SMA and collagen I positive area compared with the other two groups. These results showed that the fibrosis area significantly decreased in injured muscle treated with lentivirus-mediated Smad4siRNA, which will alleviate the scar formation. The results of tetanic strength and fast-twitch testing after21and28days after acute contusion demonstrated that Smad4siRNA-injected muscle was stronger than Lv Negative and PBS control-injected muscle with significant differences observed between the groups (P<0.05). In summary, lentivirus-mediated Smad4siRNA improved the functional recovery of the injured skeletal muscle. Conclusion The injection of lentivirus-mediated Smad4siRNA into the acute contunded mice skeletal muscle displayed a beneficial effect in suppressing skeletal muscle fibrosis, alleviating scar formation and promoting the functional recovery of the muscle.