The Inhibitory Effect Of RNAi Silencing Survivin Gene On The Growth Of Stomach Cancer And Leukemia Cells

Stomach cancer, a malignant tumor in the epithelial tissue of the stomach, is one ofthe most common cancers, ranking first in alimentary canal tumors, often withoutsymptom at the early stage, easy to miss diagnosis, easy metastasis and recurrence andbad prognosis. There is now an upward trend in the incidence of stomach cancer in Chinaand a higher mortality. It is a common disease that seriously harms health of people in ourcountry, so we should pay more attention to it.At present, surgical operation is still a preferred method of treatment of stomachcancer and surgical operation and assisted preoperative and postoperative radiotherapyand chemotherapy have become a standard mode for the treatment of stomach cancer. Butstomach cancer at the progressive stage is still a difficult problem that needs to be solvedin the treatment, so the search for promising gene therapy technology has an importantsignificance.The key to gene therapy is the selection of the target gene. With the completion ofthe Human Genome Project, many genes have been discovered and cloned, giving greatpromise to the gene therapy of tumors. Survivin is a recently discovered apoptosisinhibiting protein, belonging to the family of apoptosis protein inhibiting factors, withunique structure, only expressed in the embryonic tissue and most tumorous tissues, suchas colon cancer, lung cancer, breast cancer, stomach cancer and liver cancer, and is relatedto the progress of disease and bad prognosis. The specific distribution of surviving makesit become a gene therapy target.RNAi, a post-transcriptional gene silencing, is capable of triggering a certainpost-transcriptional regulation program, resulting in the degradation of specific mRNAand its corresponding gene is inhibited. RNAi is widely found in the nature. Thephenomenon of RNAi is found in the plant, fruit flies, fungi, embryonic cell of mice andcell of mammals. RNAi, characterized by specificity, high efficiency and durability, isapplicable for the analysis of the unknown functional gene in the post-genome era,opening up a road to clinically specific gene interference therapy.In this work, the apoptosis inhibiting gene survivin highly expressed in malignanttumors but no expressed in normal mature tissues was selected as the target gene. TheRNAi technology was used to silence survivin gene to observe its effect against stomachcancer and leukaemia and its action mechanism was preliminarily examined.Methods:1. Expression of survivin in the stomach cancer tissue and its clinical significance54 cases of stomach cancer, 27 cases of peri-tumor tissues and 10 cases of normalgastric mucosa tissues were detected for survivin protein expression by using theimmunohistochemical method and prognosis was analyzed in combination with theclinical indicators.2.Construction and identification of recombinant plasmid pGCsilencerU6/GFP/survivin siRNAThe target sequence that accords with the characteristics was searched in SurvivinmRNA (NM001168)of Genebank, and two DNA oligonucleotide strands targeting the394-413 and 166-185 base sequence in its coding region was synthesized, which werenamed Survivin-1 and Survivin-2 respectively. And RNA secondary structure predictionwas conducted using the RNA structure 3.7 software. Each sense strand contained 5' endBamH I enzyme cutting site and two reversed consistent target sequences (19bp),separated by 9 nonhomological sequences (TTCAAGAGA). TTTTT and Hind III enzymecutting sites were added to 3' end. The two synthesized complementary oligonucleotidestrands were annealed in the annealing buffer (100 mmol/L kalium acetate, pH 7.4, 30mmol/LHEPES-KOH, 2 mmol/L magnesium acetate), forming a double-strandedstructure. pGCsilencer U6/GFP was digested respectively with the BamH I and Hind IIIrestriction enzymes. Under the action of T4 DNA ligase, the two were in a joiningreaction at 16 ℃ overnight, joining products converted to competent E. coli, andaminobenzylpenicilin resistance screening was conducted and the positive colonies werecultured for amplification and a small quantity of plasmids were extracted and sequencingwas conducted to identify the recombinant plasmid.3. Transfection of recombinant plasmid3.1 Transfection of SGC-7901 cell: when the adhesive cell reached 80%-90% fusion,they were washed thrice with serum free medium and divided into three groups: liposomecontrol, negative control and recombinant plasmid. The dosage of the negative controlgroup and recombinant plasmid group were 2ug/5X105 cells. For the details, see theinstructions of the Invitrogen transfection reagent kit. 24-72h after transfection, the cellswere collected and the total protein and total RNA were extracted with the protein lysissolution and Trizol reagent.3.2 Transfection of K562 cell: A bottle of K562 cells well grown was taken out froma CO2 incubator. The cells were counted and inoculated in a 6-hole plate, with 3×105 cellsin each hole (2 ml), and put into a thermostat for 1 h and then transfected. The cells weredivided into three groups: liposome control, blank plasmid control and recombinantplasmid control. The transfection method was the same as that for SGC-7901 cell. 24-72hafter transfection, the cells were collected.4. Detection of cell growth inhibition with the MTT methodThe cells were cultured in a 96-hole culture plate, with the cell density of 2×104/100μl in each hole. The 68-h transfected cells in each group were observed andphotographed under a phase-contrast microscope, then 10μl of MTT at the concentrationof 5g/L was added to each hole and they were incubated in a thermostat for 4 h. Theculture solution was centrifuged away and 100μl of dimethylsulfoxide (DMSO) wasadded and oscillated for 10 min. Then the enzyme marks instrument was used todetermine the absorbency (A) value at 570 nm. The cell survival rate was calculated basedon the A value. The cell survival rate=A experimental group/ A control group×100%. Theexperiment was repeated thrice.5. Detection of apoptosed cells5.1 Detection of cell apoptosis with the flow cytometer: The cells well grown weredivided into three groups: the liposome control group, negative control group andexperimental group and made into the solution of single cell suspension respectively afterthey were cultured for 72 h. Then they were fixed with 70% ethanol for over 12 h andwashed twice with PBS. The cell concentration was adjusted to 1×106/ml. After Rnasedigest, 1.5ml of PI(5mg/100ml)was added and stained for 1h. The cells were detectedafter filtration and then a single parameter analysis was conducted with the flowcytometer after they were mixed. 1×104 cells were detected in each sample. Thesubdiploid peak that appeared before G1 peak was apoptosis peak5.2 Acridine orange stained apoptosed cells: 0.1% acridine orange was added to asmear of 95 ul of suspended SGC-7901 cells after trypsin digestion and the color andshape of the cell were observed under a fluorescence microscope.6. Western blot analysis of survivin expression50 μg of total protein of the cells in each group was extracted for sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred tothe PVDF membrane using a gene pulser and Western blot analysis of the proteins wasconducted respectively using the survivin rabbit antihuman multiclonal antibody (1:500)and β-actin rabbit antihuman multiclonal antibody (1:500).7. Detection of survivin mRNA expression using RT-PCR method3μg of total RNA of the cells in each group was extracted for reverse transcription andsurvivin gene and inner controlβ-actin gene were enlarged using PCR. The length of theenlarged fragment was 415bp. Reaction conditions: 94℃ 30s, 59℃ 30s and 72℃ 60s,72℃ extended for 10min after 30 cycles.Results1. Survivin expression in the stomach cancer tissue: The positive rates of survivinexpression in the peri-tumor tissue and cancer tissue were 14.8% and 38.7% respectively,0 in the normal stomach mucosa, with significance of difference in the positive expressionrates between various groups(P<0.05). Survivin expression was not related to gender,age, lymph node metastasis and clinical staging of patients(P>0.05), but related to thetumor differentiation degree and survival prognosis. Survivin expression significantlyincreased in the low differentiation group compared with that in the high differentiationgroup(P<0.05)and the 3-year survival rate in the in the high differentiation group wassignificantly lower than that in the low differentiation group in the low differentiationgroup(P<0.05).2. Construction of pGCsilencerU6/GFP/survivin siRNA recombinant plasmid:Sequencing demonstrated successful construction.3. Determination of the effect of recombinant plasmid on growth inhibition ofSGC-7901and K562 cells by MTT: siRNA-surviving-1 recombinant plasmid couldsignificantly inhibit the proliferation of SGC-7901 and K562 cells and showed timedependence. The highest inhibition rate was up to 64% and 60%. The blank control groupand negative control group didn't show significant difference in vitro cell proliferationactivity(P>0.05). Observation under the phase-contrast microscope showed that the cellsin the control group well grew adhesively, most in shuttle shape, moderately sized, clearnucleolus, nuclear division phase found;the number of SGC-7901 cell on which siRNA-surviving-1 acted significantly decreased, in irregular shape, shrunk cells, increasedgranules and cell fragments. The above results show that siRNA-survivin-1 has theactivity of inhibiting human stomach cancer cells and siRNA-survivin-2 has a weaker cellinhibiting effect than siRNA-survivin-1. In addition, observation of K562 cells under thephase-contrast microscope showed that the cells in the control group well grew, most in acircle, moderately sized, clear nucleolus, nuclear division phase found;the number ofcells in the siRNA-surviving-1 transfected group significantly decreased, in irregularshape, shrunk cells, increased granules and cell fragments. siRNA-survivin-2 has aweaker cell inhibiting effect than siRNA-survivin-1.4. Apoptosis peak of SGC-7901and K562 cells detected using the flow cytometerIn the siRNA-survivin-1 recombinant plasmid transfected group, SGC-7901 andK562 cells showed significant apoptosis(P<0.01)at 72 h after transfection, and thesubdiploid peak that appeared before G1 phase was apoptosis peak.5. Survivin protein analysis-Western blot: The results showed no significantdifference in surviving expression in the liposome control group and blank plasmidcontrol group, but under the action of siRNA-Survivin-1, surviving expression ofSGC-7901and K562 cells significantly decreased compared with that in the controlgroups(P<0.01), with the inhibition rate of 78.25% and 77.4%. Under the action ofsiRNA-Survivin-2, surviving expression also slightly decreased, with the inhibition rate of58.17%. There was no significant difference inβ-actin expression between groups,showing that the inhibiting effect of Survivin-siRNA on Survivin was specific.6. Survivin mRNA analysis-RT-PCR: The results showed that in the transfectedrecombinant plasmid group, survivin mRNA cells were significantly lower than those inthe blank plasmid group(P<0.01), with the inhibiting rate of 88.75%, but in the blankplasmid transfected group and liposome group no significant difference (P>0.05)wasfound. There was no significant difference inβ-actin expression between groups, showingthat the inhibiting effect of Survivin-siRNA on Survivin was specific.Conclusions1. High survivin expression in the stomach cancer tissue is related to the malignancydegree and prognosis, suggesting that survivin plays an important role in the incidenceand development of stomach cancer and can be used as an indicator for judging stomachcancer prognosis.2. Inhibition of survivin gene expression can inhibit the growth of tumor cells andinduce apoptosis of tumor cells.3. The mechanism of pGCsilencerU6/GFP/survivin siRNA inhibiting tumor cellgrowth is the downregulation of survivin gene expression.In a word, silencing survivin with the RNAi technology shows a significant effectagainst stomach cancer and leukaemia.