| 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 andβ-actin gene fragment. Contrast the difference of expression level in STAT3 gene usingβ-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 andβ-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 geneβ-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, usingβ-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 (P<0.01).2 The results of sequencingBeen blasted in NCBI, the DNA sequence of STAT3 gene DNA sequencing confirms that the RT-PCR product, about 400bp in size, is of STAT3 gene. Also, the DNA sequence of siRNA expression cassettes shows that the component of siRNA expression cassettes is matched with the anticipation. 3 Cells morphology observation3.1 Under invert light microscopeHuman fibroblasts cells in STAT3-SECs and control groups appeared no changes: fusiform shape and pykno-swirl- ing arrangement. The colorectal cancer cells in mismatch-SECs group, transfection reagent and control groups were multiplied and well adherenced. Cells appeared as fusiform shape, spreaded out of the field of vision with clear nucleoli and caryocinesis phase. The colorectal cancer cells of STAT3-SECs group were decreased and bad adherenced.With more floating ones, cells were deranged and inequality of size, shrinkage and vacuoluse, appearing karyoplasmic ratio inver- sion and diminution of karyokinesis phase. There occured effervescencing and descented-deuterium, while cell membrane were integrity.We saw many round micro-bubbles and buddings in cells and losing normal contact inhibition infra- cells. Moreover, the cell debris increased and background got muddy.With the concertration of STAT3-SECs grandualy increased, the change-degree of cells increased correspondingly.3.2 Under fluorescence microscopeStained with Hoechest 33342/PI, all the cells of human fibroblast STAT3-SECs group, SW480 mismatch-SECs group, SW480 transfection reagent group and SW480 control group did not show any visible apoptosis: most normal alive cells were stained into blue ones with integrity cell membrane and common nucleus structure; a small quantity cells were stained into red ones with common nucleus structure.While the majority cells of SW480 STAT3-SECs group were stained into blue ones with highly concertrated chromatin and coagulated into relucent clumping called apoptotic body, some cells got small and anomalo-formed, with nuclear fragmentation; part of the cells were stained into red ones and appeared transparent chromatin coagulation. With the concertration of STAT3-SECs grandualy increased, the ratio of cells apoptosis increased correspondingly.4 The results of RT-PCR after transfectionBy electrophoresis, there wasn′t any visible change in the expression of oncogene STAT3 and geneβ-actin in human fibroblasts STAT3-SECs and control groups; SW480 mismatch-SECs group, SW480 transfection reagent group and SW480 control group could be observed a clear and homogeneous strap at both 400bp and 350bp suggested that there wasn′t any visible change in the expression of STAT3 andβ-actin gene in control groups neither; SW480 STAT3-SECs group could be observed a fuzzy strap at 400bp and a clear strap at 350bp suggested that only the expression of STAT3 gene changed. The data, from 2D image display, treated by statistics show that, contrusted with SW480 control group, the expression of STAT3 gene in SW480 STAT3-SECs group diminished 62.16±12.50%, usingβ-actin as inner reference by gel imaging analysis sofeware. There were significant differences betweeen the SW480 STAT3-SECs and control group(P<0.01); while there were no significant differences between SW480 transfection reagent, SW480 mismatch-SECs group and the SW480 control group; neither in human fibroblasts STAT3-SECs and control group (P>0.05).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. |
