Silencing Of RhoC Gene By RNA Interference Regulates Proliferation, Invasion, Metastasis In Human Inflammatory Breast Cancer

Objective:Inflammatory breast cancer is the most malignant tumor in breast carcinomas, accounting for about one percent to six percent of total breast carcinomas. IBC is also a particularly local advanced breast cancer, which has characters of high invasion, high metastasis and high human mortality. The histology is not special and the main performances of pathological types are advanced ductal cancer, lobular cancer and so on. Lymph vessels and blood vessels are always invaded by cancer embolus. IBC carcinoma cells show low differentiation, with local regional lymph node invasion. Because of high malignancy and rapid development, extensive body metastasis often happens in early stages and the prognosis is very poor. Currently, the therapy strategy of operation, chemotherapy combined with radiotherapy is used to treat IBC. However, the effect is always very limited. Recently, some studies found that oncogene was expressed extensively in most of IBC, which had intimate correlation with growth, invasion and metastasis of IBC. The founds point in the right direction for the molecular targeted therapy of IBC. This research project aims to silence RhoC gene of IBC SUM190 cell lines in order to explore the effects on proliferation, invasion, metastasis with the help of RNA interference technique.Methods:66 cases of breast benign diseases and 94 cases of primary breast cancer were collected. S-P immunohistochemical staining and gray scanning analysis system were applied to detect the expression of RhoC proteins. The correlation between the expression of RhoC and clinical significance was also analyzed. In vitro experiments, human IBC SUM190 cell lines were divided into control group and research group randomly. Cell culture media were added in control group. And no liposome was added. While, lipofectamine2000 and other transfection reagents were given in research group so as to transfect normally. We transfected RhoC-siRNA into human IBC SUM 190 cell lines by lipofectamine2000. After transfected for 48 hours, senti-quantitative RT-PCR assay was made to detect the mRNA expression levels of RhoC,KAI1,MMP9,CXCR4. Western blot was also used to analyze the expression level of RhoC protein. At 24,48,72 hours time point after transfection, MTT was also used to detect growth graph of IBC cell lines respectively. Cell growth speed was expressed with optical density (absorbance A). After transfected for 48 hours, the apoptosis was analyzed by flow cytometry. The ability to invasion and metastasis was analyzed by transwell hole. In invasion assay, the power of penetrating membrane was seemed as the ability to invasion. In metastasis assay, the power of penetrating membrane was seemed as the ability to metastasis. Meanwhile, immunohistochemistry and image analysis system were applied to detect the protein expression levels of KAI1,MMP9,CXCR4.Results:The mean gray value of RhoC protein expression in breast benign disease and breast cancer was (0.051±0.014),(0.328±0.122), there was obvious difference (P<0.01). The mean gray value of RhoC protein expression in breasr cancer was (0.307±0.111),(0.358±0.172) in<45 years old and≥45 years old. There was no obvious difference (P>0.05). The average gray value of RhoC protein was (0.266±0.107) in breast cancer without axillary lymph node metastasis. And the average level of RhoC protein was (0.409±0.187) in breast cancer with axillary lymph node metastasis. There was marked difference (P<0.05). Furthermore, The average gray value of RhoC protein (0.435±0.211) inⅢstage of breast cancer was significantly higher than that (0.237±0.088) inⅠ+Ⅱstage of breast cancer (P< 0.05). In vitro experiments, we used RhoC-siRNA to transfect human IBC SUM190 cell lines and found that the ratio of transfection exceeded to 70%. After transfected for 48 hours, the expression levels of RhoC mRNA in IBC SUM190 cell lines were (0.907±0.387) in control group. The expression levels of RhoC mRNA in IBC SUM190 cell lines were (0.367±0.152) in research group. There were significant differences (P<0.01). The expression levels of RhoC protein in IBC SUM 190 cell lines were (0.914±0.389) in control group. And the expression levels of RhoC protein in IBC SUM190 cell lines were (0.375±0.164) in research group. There were significant differences (P<0.01). At 24,48,72 hours time point after transfection, the growth speed of IBC SUM190 cell lines were (0.43±0.09),(0.57±0.20),(1.32±0.48),(1.88±0.68) in control group. The growth speed of IBC SUM190 cell lines were (0.45±0.12),(0.50±0.17),(1.28±0.40),(1.75±0.57) in research group. There were no obvious differences between two groups in each time point respectively (P>0.05). The percentage of G0/G1 phase in IBC SUM190 cell lines was (52.26±3.68)% in control group, while the percentage of G0/G1 phase in IBC SUM190 cell lines was (55.38±3.85)% in research group, there was no obvious difference (P>0.05). The percentage of S phase in IBC SUM190 cell lines was (23.78±1.42)% in control group, while the percentage of S phase in IBC SUM 190 cell lines was (21.15±1.26)% in research group, there was no obvious difference (P >0.05). The apoptosis rate was (4.77±0.34)% in control group, while the apoptosis rate was (5.25±0.45)% in research group, there was no obvious difference (P>0.05). In invasion assay, the amount of penetrating cells was (352.2±50.6) in control group, which was significantly higher than that (85.6±12.7) in research group (P<0.05). In metastasis assay, the amount of penetrating cells was (46.6±7.7) in research group, which was markedly lower than that (189.6±23.3) in control group (P<0.05). After RhoC-siRNA was transfected into IBC SUM190 cells for 48 hours, The mRNA expression levels of KAI1 in control group, research group were (0.462±0.192),(0.762±0.281), there were obvious differences (P<0.05). The mRNA expression levels of MMP9 in control group, research group were (0.889±0.349),(0.587±0.212), there were also obvious differences (P<0.05). The mRNA expression levels of CXCR4 in control group, research group were (0.873±0.331),(0.620±0.277), there were also significant differences (P<0.05). The protein expression levels of KAI1 in IBC SUM 190 cell lines were (0.54±0.11) in control group, while the protein expression levels of KAI1 in IBC SUM190 cell lines were (1.13±0.39) in research group. There were obvious differences (P<0.05). The protein expression levels of MMP9 in IBC SUM 190 cell lines were (1.28±0.45) in control group, while the protein expression levels of KAI1 in IBC SUM 190 cell lines were (0.68±0.20) in research group. There were obvious differences (P<0.05). The protein expression levels of CXCR4 in IBC SUM190 cell lines were (1.35±0.50) in control group, while the protein expression levels of KAI1 in IBC SUM 190 cell lines were (0.77±0.23) in research group. The protein expression levels of CXCR4 in control group were markedly hgher than those in research group. (P<0.05).Conclusion:RhoC protein is highly expressed in primary breast cancer tissues, which has a strong correlation with tumor pathological characters. When the malignancy of carcinoma is high, the expression of RhoC protein is increased. Furthermore, the expression of RhoC protein had a significantly positive correlation with lymph node metastasis and TNM stages of carcinoma. RhoC is expressed extensively in human IBC SUM190 cell lines. It plays an important role in the carcinogenesis and aggression in IBC. We find that using RNAi technique, we can silence RhoC gene in human IBC SUM 190 cells, which greatly inhibits invasion and metastasis in IBC. However, the proliferation of human IBC SUM190 cells can not be suppressed. RNAi technique perhaps becomes an important gene targeted therapy tool for IBC.