Influence of phosphatidylinositol-3-kinases P85α silence by RNA interference on cell cycle and apoptosis of colorectal cancer cells in vitro / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the effect of phosphatidylinositol- 3-kinases(PI3K) P85α silence on cell cycle and apoptosis of colorectal cancer cells.</p><p><b>METHODS</b>Four shRNA vectors(shRNA/89, 324, 1073 and 1123) and one negative control vector were designed and stably transfected into SW480 cells. Western blotting was used to determine the expression level of P85α. Flow cytometry was used to determine the PI-labeled cycle after stable transfection. Annexin V-FITC kit was used to determine the apoptosis.</p><p><b>RESULTS</b>Western blotting analysis showed that the expression level of PI3K P85α protein was significantly decreased in cells transfected with shRNA/324 vector. The inhibition rate was 90%. The group was selected for the following experiments. G1 phase cells in the interference group and the control group were (62.4±2.7)% and (51.2±3.5)%, respectively. S phase cells in the interference group and the control group were (23.9±1.7)% and (34.1±3.4)%, respectively. Apoptosis cells induced by 5-FU of interference group and control group were(11.1±3.7)% and (1.4±0.6)%, respectively. The differences were all significant (P<0.05).</p><p><b>CONCLUSIONS</b>Depletion of PI3K P85α can significantly induce SW480 cell cycle arrest and sensitize SW480 cells to 5-FU induced apoptosis. PI3K P85α may be a new therapeutic target for colorectal cancer cells.</p>

Cloning of hsa-miR-148a and construction of its retroviral expression vector / 南方医科大学学报

<p><b>OBJECTIVE</b>To clone hsa-miR-148a and construct its retroviral expression vector.</p><p><b>METHODS</b>The pre-miR-148a amplified by PCR was inserted to pMSCV to construct the recombinant retroviral expression plasmid pMSCV-miR-148a, which was confirmed by restriction endonuclease analysis and DNA sequencing. The retroviral expression vector pMSCV-miR-148a and PIK packaging plasmid were cotransfected into 293FT packaging cells by calcium phosphate-mediated transfection to produce the retrovirus, and the retrovirus titer was measured by infection of NIH3T3 cells.</p><p><b>RESULTS</b>Restriction enzyme digestion and DNA sequencing demonstrated that the retroviral vector pMSCV-miR-148a was constructed successfully, and the virus titer was 5x10(8) CFU/ml after infection of NIH3T3 cells.</p><p><b>CONCLUSION</b>The successful construction of the retroviral expression vector MSCV-miR-148a allows the production of high-titer retrovirus to facilitate further study of the molecular functions of miR-148a.</p>