Small activating RNA induces myogenic differentiation of rat adipose-derived stem cells by upregulating MyoD

ABSTRACTPurpose:RNA activation (RNAa) is a mechanism of gene activation triggered by promoter-targeted small double stranded RNAs (dsRNAs), also known as small activating RNAs (saRNAs). Myogenic regulatory factor MyoD is regarded as the master activator of myogenic differentiation cascade by binding to enhancer of muscle specific genes. Stress urinary incontinence (SUI) is a condition primarily resulted from urethral sphincter deficiency. It is thus expected that by promoting differentiation of adipose-derived stem cells (ADSCs) into myoblasts by activating MyoD gene through RNAa may offer benefits to SUI.Materials and Methods:Rats ADSCs were isolated, proliferated in vitro, and identified by flow cytometry. Purified ADSCs were then transfected with a MyoD saRNA or control transfected. Real-time polymerase chain reaction (RT-PCR) and western blotting were used to detect MyoD mRNA and protein expression, respectively. Immunocytochemical staining was applied to determine the expression of desmin protein in transfected cells. Cell viability was measured by using CellTiter 96® AQueous One Solution Cell Proliferation Assay kit.Results:Transfection of a MyoD saRNA (dsMyoD) into ADSCs significantly induced the expression of MyoD at both the mRNA and protein levels, and inhibited cell proliferation. Desmin protein expression was detected in dsMyoD treated ADSCs 2 weeks later.Conclusion:Our findings show that RNAa mediated overexpression of MyoD can promote transdifferentiation of ADSCs into myoblasts and may help treat stress urinary incontinence (SUI)–a condition primarily resulted from urethral sphincter deficiency.

Evaluation of Tumor Formation of Three Bladder Cancer Cell Lines in Nude Mice / 华中科技大学学报（医学）（英德文版）

This study examined the differences in tumor formation of three bladder tumor cell lines (BIU-87,T24 and E J) after subcutaneously transplanted into nude mice,in order to find the best technique for establishing in vivo bladder tumor model.BIU-87,T24 and EJ cells at logarithmic phase were re-suspended in serum-free medium.The cells suspensions of the identical concentration were subcutaneously transplanted into nude mice and then the success rate and tumor growth were compared among the three cell groups.The results of tumor formation were pathologically evaluated.Lung,liver and kidney tissues were also pathologically examined for distant metastasis.The proliferation of the three cells were determined by immunohistochemically detecting the PCNA expression in the tumors.The results showed that the success rates of EJ and T24 cells were significantly higher than that of BIU-87 cells and no distant metastasis was noted among the three groups.The proliferation levels of EJ and T24 cells was significantly higher than that of BIU-87.But at the later stage of tumor formation,as compared with T24 cells,EJ grew more vigorously,soon resulting in the central necrosis of tumor,which affected the measurement of the actual size of the tumors.Moreover,PCNA staining exhibited that the proliferation of EJ and T24 was significantly higher than that of BIU-87 cells.It is concluded that as compared with BIU-87 cells,EJ and T24 cells had higher success rates,with not significant differences in death rate and distant metastasis found among them.There existed no significant difference in tumor formation between EJ and T24 cells and T24 cells do not rupture easily,which makes it a better cell line for the establishment of in vivo bladder tumor model.

Effect of Silencing LRIG3 Gene on the Proliferation and Apoptosis of Bladder Cancer T24 Cells / 华中科技大学学报（医学）（英德文版）

This study examined the effect of silencing LRIG3 expression on the proliferation and apoptosis of bladder cancer T24 cells and explored the role of LRIG3 in the tumorigenesis of bladder cancer.Bladder cancer T24 cells were routinely cultured and pSilencer plasmids were employed to construct LRIG3 eukaryotic expression vector of LRIG3-siRNA,i.e.,pSilencer-LRIG3-siRNA.After confirmation,the vector was transfected into HEK293 cells to make a replication-deficient adenovirus,pAd-LRIG3-siRNA,which was then introduced into bladder cancer T24 cells.RT-PCR,Western-blotting were performed to detect the levels of LRIG3 mRNA and proteins.Cells number was determined by using MTT test.Hoechst33258 staining,transmission microscopy,flow cytometery were conducted to examine the cell apoptosis.Three groups included a blank control group,a negative control group (containing non-interfering plasmids) and a pAd-LRIG3-siRNA group.Our results showed that the recombinant pAd-LRIG3-siRNA was successfully transfected into the bladder cancer T24 cells.The siRNA formed by the transcription of the recombinant plasmids resulted in significantly reduced expressions of LRIG3 gene and protein and significantly decreased cell proliferation and growth in the pAd-LRIG3-siRNA group as compared with the control group (P＜0.01).The siRNA also caused apoptotic changes of some cells,with the apoptosis rate being (17.69±0.75)％,which was significantly different from that of the control group (P＜0.01).It was concluded that recombinant pAd-LRIG3-siRNA plasmids could effectively decrease the expression of LRIG3 mRNA and proteins and,to some extent,inhibit the proliferation and promote the apoptosis of bladder cancer T24 cells.Silencing LRIG3 gene might be a novel alternative for the treatment of bladder cancer.

The Assessment of Bladder and Urethral Function in Spinal Cord Injury Patients / 华中科技大学学报（医学）（英德文版）

The correlation between the anatomic site of spinal cord injury and real-time conditions of bladder and urethral function was assessed in order to provide a reasonable basis for the clinical treat-ment of neurogenic bladder. A total of 134 patients with spinal cord injuries (105 males, 29 females;averaged 34.1 years old) were involved in this retrospective analysis, including urodynamic evaluation,clinical examination and imaging for anatomical position, and Bors-Comarr classification. The associa-tions between the levels of injury and urodynamic findings were analyzed. The results showed that mean follow-up duration was 16.7 months (range 8-27 months). Complete spinal cord injuries occurred in 21 cases, and incomplete spinal cord injuries in 113 cases. Of the 43 patients with upper motor neu-ron (UMN) injuries, hyperreflexia and (or) detmsor sphincter dyssynergia were demonstrated in 30 (69.8%), 31 (72.1%) suffered low bladder compliance (less than 12.5 mL/cmH2O), 28 (65.1%) had high detrusor leak point pressures (greater than 40 cmH2O), and 34 (79.1%) had residual urine. Of the 91 pa-tients with lower motor neuron (LMN) injuries, areflexia occurred in 78 (85.7%), high compliance in 75 (82.4%), low leak point pressures in 80 (87.9%), and residual urine in 87 (95.6%), respectively. The as-sociations between the anatomical site of spinal cord injury and urodynamic findings were ill defined. In patients with spinal cord injury, this study revealed a significant association between the level of injury and the type of voiding dysfunction. The anatomical site of spinal cord injury can not be predicted in real-time condition of bladder and urethral function. Management of neurogenic bladder in patients with spinal cord injury must be based on urodynamic findings rather than inferences from the neurologic evaluation.

Relationship between Single Nucleotide Polymorphisms in -174G/C and-634C/G Promoter Region of Interleukin-6 and Prostate Cancer / 华中科技大学学报（医学）（英德文版）

The association between the single nucleotide polymorphisms (SNPs) in -174G/C and -634C/G of interleukin-6 (IL-6) promoter region and prostate cancer was examined in the population of Han people in Hubei region. TaqMan PCR was employed for the gene-typing of -174G/C and -634C/G in promoter region of IL-6 gene to compare the prostate cancer patients and normal controls in terms of genotype frequency, allele frequency and risk of prostate cancer. Enzyme-linked immunosorbent assay (ELISA) was used for the detection of IL-6 concentration in peripheral blood of the patients with prostate cancer and the relationship between the IL-6 level and the genotype was studied.Our results showed that in all the subjects, the genotype of genetic locus -174G/C was found to be GG and no CG and CC were observed. There was a significant difference in gene frequency of GG,CG and CC of-634C/G and allele frequency of G and C between prostate cancer patients and normal controls (P<0.05) and the gene frequency of GG+CG increased with the clinical stages and pathological grades of prostate cancer. The IL-6 level in GG+CG group was significantly higher than that in CC group. It was.concluded that no SNP in-174G/C IL-6 promoter region was found in the population of Han people in Hubei region. The SNP in -634C/G was, to some extent, associated with the development and progression of prostate cancer. The population with GG+CG genetype has higher risk for prostate cancer.