Experimental Study On The Inhibitory Effect Of Specific-HER-2 SiRNA On BT-474 Cell

Background:Malignant tumor has become one of the most frequent diseases which are harmful to humans. Moreover, the morbility of malignant tumor has sharply increased all over the world in recent years. The morbility of breast cancer increases yearly. There are 1, 200, 000 persons suffering from breast cancer every year. China is one of the countries in which the morbility of breast cancer increases sharply. The morbility of breast cancer has increased at the speed of about 3% per year. The age of the patients tends to be young. It has been a key point in the medical field how to effectively prevent and treat breast cancer.In 1985, Coussens found the human epidermal growth factor receptor 2 (HER-2/neu, c-erbB-2), which is one of highly regulated tyrosine kinases (TKs) genes. HER-2 is an important number of the epidermal growth factor receptor family. HER-2 was proved to be tumorigenic oncogene and over-expressed in 25%～30% of the breast cancer. Lately it has been proved in many studies that the overexpression of HER-2 was consistently associated with the onset, growth, metastasis and poor prognosis of breast cancer. So it has become the hotspot in the studies on breast cancer how to effectively inhibit HER-2 overexpressing.RNA interference (RNAi) is a kind of reliable gene silencing techniques, which involves double-stranded RNA-mediated, sequence-specific and highly-efficient mRNA degradation. It could induce specific single strand mRNA degradation post-transcriptionally. RNA interference technologies are current techniques widely utilized in functional genomic and gene-therapy studies.BT-474 cell is one of breast cancer cell lines, in which HER-2 is over-expressed. We selected BT-474 cell as the object of this study. HER-2 in it was silenced by RNA interference technology, at the same time, the anti-tumor effects were observed and the mechanisms were investigated.Objective:To design and construct specific RNA interference plasmid vector targeted to HER-2 gene mRNA, to construct stable transfection cell lines, to investigate the interference efficiency of HER-2 in the cell line and the inhibitory effects on the tumor. To provide the theoretical basis of RNAi technology in prevention and treatment to the breast cancer.Methods:1. Based on the human HER-2 mRNA gene sequencing, 3 sequences targeting HER-2 gene and 1 free correlation sequence were designed and synthesized. pRNAT-U6.1/Neo plasmid was used to construct the specificity RNA interference vector (pRNAT-U6.1/Neo-siRNA-1, pRNAT-U6.1/Neo-siRNA-2 and pRNAT- U6.1/Neo-siRNA-3) aimed at HER-2 gene mRNA and pRNAT-U6.1/Neo- siRNA-neg contains a free correlation sequence. The constructed siRNA expression vectors of positive clones were validated by PCR and DNA sequencing.2. Cotransfect BT-474 cell with pRNAT-U6.1/Neo-siRNA by cationic lipsome. Screen positive stable transfection cell clone. Detect the change of HER-2 mRNA with RT-PCR and HER-2 protein expression with Western blot technology. Determine the change of the cell cycle and cell apoptotic peak with flow cytometry. Measure cell proliferation activity with MTT chromatometry and the assay of colones formation in plate. Measure the mobility of the cell with matrigel mobility assay. 3. Construct nude mouse tumor animal model. Measure the tumor volume and draw the tumor growth curve. Execute the nude mouse 30 d after injection, to strip and weigh the neoplasm block and calculate the inhibition rate of the anti-tumor. Determine tumor tissue HER-2 protein expression with Western blot technique and HER-2 gene level with RT-PCR.Results:1. The constructure and identification of the specific-HER-2 siRNA expression vectors. The constructed siRNA expression vectors of positive clones were confirmed by PCR and DNA sequencing. The results showed that inserted fragments of positive clones were consistent with what expected.2. In vitro study of specific-HER-2 siRNA in BT-474 cell2.1 The constructed siRNA expression vectors were transfected into BT-474 cell by cationic lipsome. The cell clones of positive stable transfection were screened.2.2 The results of RT-PCR showed that the mRNA expression level of HER-2 decreased significantly in siRNA-1 and siRNA-2 groups compared with that in control group(P＜0.05), The inhibitory rates of HER-2 mRNA level were 61.04% and 41.05%, respectively, but there was no significant difference between siRNA-neg group and control group.2.3 The result of Western blot showed that the expression level of HER-2 protein decreased significantly in siRNA-1 and siRNA-2 groups compared with that of control group(P＜0.05).The inhibitory rates of HERo2 protein level were 59.34% and 38.01%, respectively, but there was no significant difference between siRNA-neg group and control group.2.4MTT assay showed that BT-474 cell proliferation in siRNA-1 group was significantly inhibited compared with that in control group(P＜0.05), but there was no significant difference between siRNA-neg group and control group.2.5 Cell cycle assay measured by flow cytometry showed that the cell proportion of siRNA-1 group increased at G₀/G₁ stage and decreased at S stage significantly compared with control group(P＜0.05), but there was no significant difference between siRNA-neg group and control group.2.6 By flow cytometry, it showed significant apoptosis in BT-474 cell of siRNA-1 group compared with that of control group(P＜0.05). There was no significant difference between siRNA-neg group and control group.2.7 The assay of colones formation in plate showed the formation rate of siRNA-1 group had been significantly decreased compared with that of control group(P＜0.05). There was no significant difference between siRNA-neg group and control group.2.8 The matrigel mobility assay showed the mobility of siRNA-1 group had been significantly decreased compared with that of control group(P＜0.05), while there was no significant difference between siRNA-neg group and control group.3 In vivo study of HER-2-speeific siRNA in BT-474 cell3.1 The subcutaneous tumor model in nude mice was successfully established.3.2 The tumor growth curve showed that the tumor of siRNA-1 group grew significantly slowly compared with control group(P＜0.05). There was no significant difference between siRNA-neg group and control group.3.3 The weight of the neoplasm block of siRNA-1 group was significantly lower than control group(P＜0.05), the anti-tumor inhibition rate was 51.61%. There was no significant difference between siRNA-neg group and control group.3.4 The result of Western blot showed that HER-2 protein level of siRNA-1 group was significantly decreased compared with control group(P＜0.05), and the inhibition rate was 50.64%. There was no significant difference between siRNA-neg group and control group.3.5 The result of RT-PCR showed that HER-2 mRNA level of siRNA-1 group was significantly decreased compared with control group(P＜0.05), the inhibition rate of HER-2 mRNA was 54.69%. There was no significant difference between siRNA-neg group and control group. Conclusion:1. Using pRNAT-U6.1/Neo plasmid, we successfully designed and constructed specific RNA interfere plasmid vector aimed at HER-2 gene mRNA.2. The BT-474 cell was cultured and transfected stably with specific-HER-2 siRNA interference plasmid.3. In vitro, specific-HER-2 siRNA could downregulate HER-2 mRNA and protein expression in BT-474 cell, make the cycle of BT-474 cell arrest at G₀/G₁ stage, significantly suppress the function of growth, proliferation and invasiveness; promote apoptosis of the cells.4. In vivo, specific-HER-2 siRNA could significantly downregulate HER-2 mRNA and protein expression, inhibit growth of the subcutaneous tumor.5. It provided some evidence for RNAi technology in prevention and treatment to the breast cancer.