The Interference of Small Interfere RNA ... - Cancer Research Center

[Abstract]

Objective: It is generally acknowledged that STAT3 is an oncogene.It is significant and persisitent over-expressed in many cancer cells,also in colorectal cell.Becacuse of its high performance and accuracy ,as well as its high specificity,RNA interference(RNAi),newly rising technology,is gradually substituting other traditional gene therapeutic tools to become a powerful tool in gene therapy.This study was intended to investigate the influence of cell apoptosis on colorectal cell in which STAT3 is over-expressed,using small interfere RNA (siRNA) targeting STAT3.Methods: 1 Cell culture Choose human colorectal carcinoma cell line SW480 and normal human fibroblast, and then, subcultured these cells respectively for following experiment.2 RNA abstraction and RT-PCR analysis expression level of STAT3 gene The primer pairs for RT-PCR were designed with the primer design software. The total RNA was purified from cultured cells, respectively, with the Trizol reagent, and reverse-transcribed to obtain cDNA. Use the cDNA as template, amplificated STAT3 and?-actin gene fragment. Contrast the difference of expression level in STAT3 gene using?-actin as inner reference by gel imaging analysis sofeware. Recover and purify the gel strap containing STAT3 gene and analysis the sequence of STAT3 gene using automatic DNA sequencing.3 Synthesis the siRNA expression cassettes (SECs) According to general principle, design and synthesis the downstream primer targeting STAT3 gene to generate SECs by PCR. After confirming the size of PCR product fragment, the product was recovered and purified from the gel followed by sequence analysising to identify whether the sequence of the PCR product is identical with the experimental anticipation.4 Transfection According to the transfection protocol for adherent cells, transfect the SECs targeting STAT3 (STAT3-SECs) into human colorectal carcinoma SW480 cells. At the same time, set human fibroblast control, SW480 control, SW480 transfection reagent and SW480 mismatch-SECs groups, respectively.5 Observe the morphologic change of the cells After 48-hour transfection, study the modality change under invert light microscope. And staine the transfected cells by Hoechst33342/PI to observe the morphology changes of the nucleons.6 Analysis the change of mRNA expression within STAT3 by RT-PCR After 48-hours transfection, the total RNA, abstracted respectively from the cells with Trizol reagent was reverse-transcribed into cDNA. Use this cDNA as template, amplify STAT3 and?-actin gene fragment. Then contrast the difference of expression level in STAT3 gene by gel imaging analysis sofeware.7 Analysis the apoptotic peak and aspection of transferred cells by flow cytometry (FCM) The target cell was paleted in 50mm culture flask and prepared into cell suspension after 48-hour transfection. Keep on with some treatment, the cell susupession can be analysised by flow cytometry.Results: 1 The expression lever of oncogene STAT3Human colorectal carcinoma SW480 cells and human fibroblast express both gene?-actin at 350bp and oncogene STAT3 at 400bp. Contrusted with human fibroblast, human colorectal carcinoma expressed more oncogene STAT3 at 400bp, The data, from 2D image display, treated by statistics show that the expression of STAT3 gene in human colorectal carcinoma SW480 cells is 3.10?1.16 times of which in human fibroblast, using?-actin as inner reference by gel imaging analysis sofeware. That is, oncogene STAT3 is over-expressed in human colorectal carcinoma SW480 cells and lower expressed in human fibroblast. There were significant differences among the values (P0.05).5 The result of FCMAfter 48-hour transfection, cells in the SW480 STAT3- SECs group appeared visible apoptosis, and the hypodiploid peak appeared before stage G1 is apoptotic peak, the percen- tage of apoptosis increased from 0.2% to 26.0%, while the ratio of cell number in S stage decreased from 32.3% to 9.2%.Conclusion: 1 STAT3 gene is over-expressed in colorectal cancer cell but is lower expressed in normal human fibroblast cell.2 The small interfere RNA targeting STAT3 (STAT3-siRNA) can perform its interference in colorectal cancer cell over-expressing oncogene STAT3, with the appearance: apoptosis of the most cells, decrease of cell number in S stage and down regulation in the expression of oncogene STAT3.3 The RNA interference of STAT3-siRNA exert only on colorectal cancer cell over-expressing oncogene STAT3, but not on normal human fibroblast.

Title: The Interference of Small Interfere RNA Targeting STAT3 in Colorectal Cancer Cell

Category: Stomach Cancer

Filename: The Interference of Small Interfere RNA Targeting STAT3 in Colorectal Cancer Cell.pdf

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