**3.1 Vascular endothelial growth factor**

Vascular endothelial growth factor (VEGF) is a heparin-binding growth factor specific to vascular endothelial cells, and can induce angiogenesis in vivo. VEGF and anti-VEGF coexist in normal tissues and remain in a relatively balanced state. In the presence of tumor growth, multiple carcinogenic factors trigger dramatic increase in VEGF, the amount and function of which are by far greater than those of anti-VEGF, thus causing massive growth

RNA Interference for Tumor Therapy 67

VEGF, survivin and MYC were down-regulated, while caspase3 and p53 were up-regulated in STAT3-shRNA group. Huang (Huang *et al,* 2011)studied the effect of RNAi using STAT3 as the target gene on pancreatic cancer, and found that STAT3 RNAi not only inhibited tumor growth but inhibited angiogenesis in the tumors. They also found that VEGF and MMP-2 mRNA and protein levels decreased significantly in SW1990 cells treated with RNAi, indicating that STAT3 plays an important role in the course of tumor development

Human telomerase reverse transcriptase (hTERT) is a rate-limiting enzyme maintaining the activity of telomerase. However, it is found that hTERT not only maintains telomerase but stabilizes cell genomes, promotes cell proliferation, inhibits cell apoptosis, and plays a role in regulating intracellular signaling pathways. A recent study(de Souza Nascimento *et al,*  2006)transfected Hela with shRNA expressing carrier-encoded target shRNA, and found that short telomerase-related hTERT mRNA was reduced dramatically, cell growth was inhibited, and cell apoptosis was accelerated. A similar study(Gandellini *et al,* 2007)showed that transfection of hTERT specific siRNA with lipofectamine 2000 could inhibit the expression of hTERT for a prolonged time, thus affecting the proliferation and

Survivin is a recently discovered tumor-specific inhibitor of the apoptosis protein family. There is no or micro expression in well differentiated tissues, and high expression in embryonic and most human malignant tumor tissues. It inhibits apoptosis of tumor cells, thus promoting cell growth and regulating cell division. In addition, it is closely associated with infiltration and metastasis of tumor cells. Survivin is found expressing in common malignant tumors such as breast cancer, gastric cancer, kidney cancer, melanoma, intestinal cancer, neuroblastoma cancer and ovarian cancer. In their retrospective analysis of tumor treatment, Kanwar (Kanwar *et al,* 2010)demonstrated that survivin-targeted molecular therapy could inhibit the growth of tumor cells. Recently, Chen (Chen&Deng,2008) injected gastric cancer MGC-803 cells treated with plasmids expressing survivin-siRNA and controltreated plasmids, and those without intervention into nude mice subcutaneously. Four weeks later, the mean tumor volume in the intervention group, control group and nonintervention group was 831mm3, 2617mm3 and 2536mm3 respectively, indicating that the apoptosis rate in siRNA intervention group was significantly higher than that in the control and non-intervention groups (27.63% vs 2.15% vs 2.31%). In their experiment study with survivin-target RNAi for the treatment of urinary bladder cancer, Seth (Seth *et al,* 2011) reported the similar result and further confirmed that survivin-target RNAi could inhibit

ERK-MAPK is activated in many malignant tumors, and plays a primarily important role in cell growth, differentiation and survival. siRNA specific to extracelluar signal regulating kinase 1/2 (ERK1/2) could inhibit the proliferation of ovarian cancer cells and

and progression, and can be used as a target for tumor gene therapy.

**3.4 Human telomerase reverse transcriptase** 

tumorigenesis of prostate cancer PC-3 cells.

tumor cell growth effectively.

**3.6 ERK-MAPK** 

**3.5 Survivin** 

of vessels (mainly blood vessels) and promoting tumor growth and metastasis. In addition, VEGF interferes with and inhibits dendritic cells and blocks antigen presentation of B and T cells, which further induces immune escape of tumors, interferes with the normal immune function of the body, and causes resistance to anti-tumor therapy. He (He *et al,* 2009) used CaCO3 as the matrix and form nanoscaled complexed with the plasmid to expresses siRNA targeting VEGF. In their siRNA transfection against colon cancer cells in vitro, they found that this complex could significantly reduce the expression level of VEGF-C in LoVo cells. In the experiment with transplanted colon cancer in nude mice, the mean tumor size treated by the complexes was decreased by 50% as compared to that in the control group. Metastasis of the cancer cells in lymph nodes and lymphatic ducts was also inhibited.

#### **3.2 Vimentin**

Cell structures and morphologies are maintained through a series of complex structural proteins, including microtubules, microfilaments and intermediate filaments. Vimentin is a type III intermediate filament. Studies in recent years have demonstrated that vimentin is closely associated with tumor development and metastasis, and participates in adhesion, migration, invasion and cell signal transduction of tumor cells and tumor-related endothelial cells and macrophages. Vimentin possesses a highly dynamic balance between polymerization depolymerization and a very complex phosphorylated form, which serve as the basis for it to participate in tumor metastasis and intercellular actions. Vimentin is found to be homological to most sequences of miR-17-3p in the miRNA gene cluster 17-92 family. miR-17-3p participates in regulating the expression of vimentin. miR-17-3p is a known tumor-inhibiting gene. It remains at a low level in highly tumorigenic and metastatic cell lines and at a high level in cell lines of low tumorigenesis. Zhang (Zhang *et al,* 2009) used plasmds expressing miR-17-3p to intervene transplanted prostate cancer in mice and found that the tumor size was only 50% of the control group 31 days after the experiment. They also found that vimentin expression was negatively correlated with miR-17-3p expression, and the rate of tumor growth was also negatively correlated with miR-17-3p. Paccione RJ (Paccione *et al,* 2008)used vimentin as the target to perform RNA interference3, and found that the ability of cell proliferation, metastasis and invasion in cellRNAi group decreased by 3 fold as compared with the control group. In addition, tumorigenesis of cells treated with vimentin-RNAi decreased significantly, and the size of tumors injected subcutaneously with vimentin-RNAi was 70% smaller than that of the control group.

#### **3.3 STAT3**

Signal transducer and activator of transcription 3 (STAT3) is a member of the STAT family, participating in signaling pathways closely related to cell proliferation, differentiation and apoptosis. Its abnormal activity may promote cell differentiation and proliferation and inhibit cell apoptosis, causing tumor development and promoting metastasis of tumor cells. It is found to be activated persistently in hepatocellular carcinoma (HCC), breast cancer and esophageal squamous cell carcinoma. Li (Li *et al,* 2009) injected STAT3-target shRNA, negative shRNA and normal saline (NS) into transplanted HCC tumors in nude mice and removed the tumors after observation for a period. It was found that the tumor tissue in STAT3-shRNA, negative shRNA and NS groups was 0.18g, 0.6g and 0.67g respectively. It was also found that the expression of STAT3, phosphorylation of STAT3, and expressions of

of vessels (mainly blood vessels) and promoting tumor growth and metastasis. In addition, VEGF interferes with and inhibits dendritic cells and blocks antigen presentation of B and T cells, which further induces immune escape of tumors, interferes with the normal immune function of the body, and causes resistance to anti-tumor therapy. He (He *et al,* 2009) used CaCO3 as the matrix and form nanoscaled complexed with the plasmid to expresses siRNA targeting VEGF. In their siRNA transfection against colon cancer cells in vitro, they found that this complex could significantly reduce the expression level of VEGF-C in LoVo cells. In the experiment with transplanted colon cancer in nude mice, the mean tumor size treated by the complexes was decreased by 50% as compared to that in the control group. Metastasis of

Cell structures and morphologies are maintained through a series of complex structural proteins, including microtubules, microfilaments and intermediate filaments. Vimentin is a type III intermediate filament. Studies in recent years have demonstrated that vimentin is closely associated with tumor development and metastasis, and participates in adhesion, migration, invasion and cell signal transduction of tumor cells and tumor-related endothelial cells and macrophages. Vimentin possesses a highly dynamic balance between polymerization depolymerization and a very complex phosphorylated form, which serve as the basis for it to participate in tumor metastasis and intercellular actions. Vimentin is found to be homological to most sequences of miR-17-3p in the miRNA gene cluster 17-92 family. miR-17-3p participates in regulating the expression of vimentin. miR-17-3p is a known tumor-inhibiting gene. It remains at a low level in highly tumorigenic and metastatic cell lines and at a high level in cell lines of low tumorigenesis. Zhang (Zhang *et al,* 2009) used plasmds expressing miR-17-3p to intervene transplanted prostate cancer in mice and found that the tumor size was only 50% of the control group 31 days after the experiment. They also found that vimentin expression was negatively correlated with miR-17-3p expression, and the rate of tumor growth was also negatively correlated with miR-17-3p. Paccione RJ (Paccione *et al,* 2008)used vimentin as the target to perform RNA interference3, and found that the ability of cell proliferation, metastasis and invasion in cellRNAi group decreased by 3 fold as compared with the control group. In addition, tumorigenesis of cells treated with vimentin-RNAi decreased significantly, and the size of tumors injected subcutaneously with

Signal transducer and activator of transcription 3 (STAT3) is a member of the STAT family, participating in signaling pathways closely related to cell proliferation, differentiation and apoptosis. Its abnormal activity may promote cell differentiation and proliferation and inhibit cell apoptosis, causing tumor development and promoting metastasis of tumor cells. It is found to be activated persistently in hepatocellular carcinoma (HCC), breast cancer and esophageal squamous cell carcinoma. Li (Li *et al,* 2009) injected STAT3-target shRNA, negative shRNA and normal saline (NS) into transplanted HCC tumors in nude mice and removed the tumors after observation for a period. It was found that the tumor tissue in STAT3-shRNA, negative shRNA and NS groups was 0.18g, 0.6g and 0.67g respectively. It was also found that the expression of STAT3, phosphorylation of STAT3, and expressions of

the cancer cells in lymph nodes and lymphatic ducts was also inhibited.

vimentin-RNAi was 70% smaller than that of the control group.

**3.2 Vimentin** 

**3.3 STAT3** 

VEGF, survivin and MYC were down-regulated, while caspase3 and p53 were up-regulated in STAT3-shRNA group. Huang (Huang *et al,* 2011)studied the effect of RNAi using STAT3 as the target gene on pancreatic cancer, and found that STAT3 RNAi not only inhibited tumor growth but inhibited angiogenesis in the tumors. They also found that VEGF and MMP-2 mRNA and protein levels decreased significantly in SW1990 cells treated with RNAi, indicating that STAT3 plays an important role in the course of tumor development and progression, and can be used as a target for tumor gene therapy.
