| Purpose: In our previous study,it was confirmed that TSZH3 is a tumor suppressor in CRC.In this study,we further synthesized a small activating RNA(saRNA),which was complementarily paired with the promoter region of TSHZ3,to upregulate TSHZ3 gene expression in CRC cells SW1116 and HT29.Our investigation of the RNA activation properties mediated by saRNA and the altered biological behaviours of saRNA-treated CRC cell lines provides new ideas for the precision treatment of colorectal cancer.Methods: 1.Three nucleic acid sequences(dsTSZH3-1802,dsTSZH3-1795 and dsTSZH3-930)were screened from the promoter region of TSZH3 according to the design rules reported in the literature,and ds Control,which has no homology with human genome,was used as the negative control.Three nucleic acids were transfected into CRC cell lines SW1116 and HT29.Then,qRT-PCR and Western blot were performed to measure the level of TSHZ3 in order to identificate the saRNA that effectively upregulate TSHZ3 expression.2.The biologically active saRNA(dsTSHZ3-930)was transfected into SW1116 or HT29 cells at concentrations of 10,20,50 and 100 n M for 72 h.Up-regulated of TSHZ3 mRNA or protein expressions was determined by qRT-PCR or Western blot.Then,SW1116 or HT29 cells were treated with 50 n M of dsTSHZ3-930 for different periods(1-6 days).The levels of TSHZ3 were determined at each day.3.We synthesized si-TSHZ3,a small interfering RNA targeting TSHZ3,for the following experiments.MTT assay,Edu assay,Hoechst assay,scratch test and Transwell assay were performed to measure the effects of dsTSHZ3-930 or si-TSHZ3 on altering biological behaviours of CRC cells in terms of proliferation,apoptosis,migration and invasion.Meanwhile,the effect of co-transfection with si-TSHZ3 and dsTSHZ3-930 on CRC cells was measured.Results: 1.The TSHZ3 mRNA and protein expressions were significantly up-regulated by dsTSHZ3-930 in CRC cells.2.The gene regulatory capacity of dsTSHZ3-930 enhanced with increasing transfection concentration3.TSHZ3 expressions were gradually up-regulated from day 2 or 3 in CRC cells,followed by a gradual decline on day 4 or 5.4.dsTSHZ3-930 significantly inhibited the proliferation,migration and invasion ability,and promoted apoptosis of CRC cells.Tumor suppression induced by TSHZ3 could be eliminated by si-TSHZ3.Conclusion: Transfection of dsTSHZ3-930 could significantly elevated TSHZ3 expression in CRC cells.The TSHZ3 activating effects of dsTSHZ3-930 were concentration-dependent and time-dependent.dsTSHZ3-930 inhibited the malignant biological behaviors of CRC cells to some extent,and co-transfection with si-TSHZ3 resulted in restoration of malignant capacities of CRC cells,excluding the off-target effect of dsTSHZ3-930 and confirming that dsTSHZ3-930 acts as a suppressor of CRC cells via targeting TSHZ3. |
