Effects Of RNA Interference Targeting Four Different Genes On The Growth And Proliferation Of Nasopharyngeal Carcinoma CNE-2Z Cells

Nasopharyngeal cancer (NPC) is a kind of common malignant tumor in Southern China with high incidence. At present, the treatment for NPC mainly depends on radiotherapy and sometimes combines with chemotherapy, immunotherapy, surgical management, traditional Chinese medicine and so on. However, local recurrence and distant metastasis easily occur after radiotherapy, NPC cells are not sensitive to chemotherapeutics and drugs have toxic and side effects, which will affect patients'prognosis. Therefore, to search for a new technology which possesses low toxicity, good therapeutic effect and strong specificity has important clinical significance. RNA interference technology has become a focus in the field of NPC gene therapy.RNA interference refers to post-transcriptive gene silencing. Many experiments have displayed that RNA interference targeting single gene can inhibit the growth and proliferation of tumor cells. Howevwe, interference targeting a single gene has limitations in the prevention and treatment for cancer Little research has been done on RNA interference targeting multiple genes, especislly more than 3 genes.As we know that tumorigenesis is result from multiple gene mutation. Multiple gene silencing may have better therapeutic effects on malignant tumors than single gene silencing. To confirm our speculation, in this study, we built shRNAs together and separately targeting VEGF, C-myc, Survivin and hTERT, and respectively transfected these plasmids into human NPC CNE-2Z cells and xenograft tumors in nude mice to observe the effects of these plasmids on NPC.ObjectiveTo explore the effects of RNAi targeting four different genes (VEGF, C-myc, Survivin, hTERT) on the growth and proliferation of nasopharyngeal carcinoma (NPC) CNE-2Z cells.Methods(1) Fluorescein-labeled shRNA plasmids together and separately targeting VEGF, C-myc, Survivin and hTERT were built and respectively called Plasmid-1,2,3,4, and 5. These plasmids were respectively transfected into human NPC CNE-2Z cells.(2) The expression of plasmids in NPC CNE-2Z cells and xenograft tumors was observed.(3) Cell proloferation was detected with MTT assay.(4) The inhibitory effects on target genes were evaluated with real-time-PCR.(5) Western blot at the levels of mRNA and protein(6) The effects of plasmids on the biological behavior of CNE-2Z cells were observed with Transwell invision chamber models.(7) Apoptosis was determined with flow cytometer. (8) The inhibitory effect of plasmids on xenograft tumors was observed in nude mice.Results(1) successfully established a carrier of four different genes encoding shRNA plasmid vector system.(2) Detection of plasmid transfection. Twenty four hours after plasmids were transfected into cells, green-fluorescent cells were not present in BC group, while in other groups, a lot of green-fluorescent cells were seen under fluorescence microscope. In NC group, cells grew well with a lot of adherent cells expressing green fluorescence. In P1-5 groups, a lot of floating, round, dead green-fluorescent cells were seen, especially in P-1 group. Transfected CNE-2Z cells were observed under confocal microscopy and a lot of cells with green fluorescent were seen, demonstrating that plasmids were successfully transfected into CNE-2Z cells.(3) MTT assay. A values at each time points were significantly lower in P-1, P-2, P-3, P-4 and P-5 group than in BC group with P<0.05. A values at each time points were significantly lower in P-1 group than in P-2, P-3, P-4 and P-5 group with P<0.05. There was no statistical significance in A values at each time point between BC group and NC group with P>0.05. The inhibition ratios of cell growth 96 h after transfection reached 72%,42 %,41%,40% and 45% in P-1, P-2, P-3, P-4 and P-5 group, respectively. MTT assay and in vitro invasive assay indicated that cell proliferation was significantly inhibited.(4) Quantitative expression of mRNA of different genes in each group 48 h after transfection is shown in Table 4. The inhibition ratios of VEGF mRNA in P-1 and P-2 group were 76.43% and 69.75%, respectively. The inhibition ratios of C-myc mRNA in P-1 and P-3 group were 79.73 and 77.43%, respectively. The inhibition ratios of Survivin mRNA in P-1 and P-4 group were 80.89% and 73.92%, respectively. The inhibition ratios of hTERT mRNA in P-1 and P-5 group were 74.60% and 70.47%, respectively. Real-PCR indicated that mRNA expression of single gene were decreased in P-2, P-3, P-4 and P-5 group, while in P-1 group, mRNA expression of four genes all was decreased, demonstrating that the effect of multi-gene silencing is stronger than single gene silencing.(5) Western blot. The inhibition ratios of VEGF protein in P-1 and P-2 group were (66.95±2.42)% and (64.30±3.13)%, respectively. The inhibition ratios of C-myc protein in P-1 and P-3 group were (62.89±2.97)% and (59.18±3.09)%, respectively. The inhibition ratios of Survivin protein in P-1 anf P-4 group were (79.48±1.92)% and (70.51±2.56)%, respectively. The inhibition ratios of hTERT protein in P-1 and P-5 group were (79.60±1.87)% and (76.33±4.11)%, respectively. Wester blot indicated that perotein expression of single gene were decreased in P-2, P-3, P-4 and P-5 group, while in P-1 group, protein expression of four genes all was decreased, demonstrating that simultaneous multi-gene silencing is better than single gene silencing.(6) In vitro invasive assay. The number of cells to penetrate the membrane in P-1 was significantly reduced compared with those in other groups 48 h after transfection (P<0.05). The number of cells to penetrate the membrane was less in P-2, P-3, P-4 and P-5 group than in group BC (P<0.05). There was no statistical difference in the number of cells to penetrate the membrane between NC and BC (P>0.05). Above results indicated that single gene plasmid and multi-gene plasmid all could decrease in vitro invasive ability of CNE-2Z cells, but the reduction of invasive ability was the most obvious in multi-gene group. in vitro invasive ability was significantly decreased after transfection in P-1 group, demonstrating that the plasmid together targeting VEGF, C-myc, Survivin and hTERT is better than the plasmid separately targeting VEGF, C-myc, Survivin and hTERT in inhibiting CNE-2Z cell growth and decreasing invasive ability.(7) Flow cytometer. Forty eight hours after transfection, apoptosis was (5.3±1.26)% in BC group, (7.4±1.02)% in NC group, (37.6±0.94)% in P-1 group, (20.6±1.27)% in P-2 group, (22.6±0.98)% in P-3 group, (23.7±1.09) % in P-4 group, (22.9±1.31)% in P-5 group. There was no statistical significance in apoptosis between BC and NC group (P>0.05). The apoptosis was higher in P-2, P-3, P-4 and P-5 group than in BC group (P <0.05). The apoptosis was higher in P-1 group than in BC group (P<0.01). The apoptosis was higher in P-1 group than in P-2, P-3, P-4 and P-5 group (P<0.05). Flow cytometry indicated that multi-gene silencing was better than single gene silencing in inhibiting tumor cell proliferation and inducing apoptosis.(8) In vivo anti-tumor experiment. NPC CNE-2Z cells were successfully inoculated in all nude mice. One week later, subcutaneous tumor was formed and gradually enlarged. After treatment, tumor growth was obvious in BC and NC group, while in P 1-5 groups, tumor growth was slow, especially in P-1 group. No mice died in each group during the experiment. Under fluorescence microscope, expression of green fluorescence of cancer tissue was seen in P 1-5 groups and NC group, but in BC group, no green fluorescence was seen (Figure 5). The tumor volumes in each group are shown in Table 5. By the end of treatment, tumor growth was significantly inhibited in P-1, P-2, P-3, P-4 and P-5 group compared with BC group. Tumor-inhibiting rate was 82.40% in P-1 group,46.24% in P-2 group, 48.47% in P-3 group,51.93% in P-4 group and 46.78% in P-5 group. In vivo anti-tumor experiment also indicated that simultaneous four-gene silencing effectively inhibited tumor cell growth. The four genes may have synergetic effect during occurrence and development of tumor.ConclosionThis study confirms that RNAi together targeting four different genes can better inhibit NPC CNE-2Z cell proliferation and induce apoptosis. RNA interference targeting multiple genes can effectively inhibit NPC proliferation and induce apoptosis, which provides a experiment basis for NPC gene therapy.