Effect Of RNA Interference Targeting Homeobox Gene A5 On Cell Proliferation And Apoptosis Of Esophageal Squamous Cell Carcinoma

China is a country with the highest morbidity and mortality of esophageal carcinoma in the world. Each year, 300 thousand people develop esophageal carcinoma, while more than half of them are Chinese (167.2 thousand). In China, particularly in Henan Province, the mortality caused by esophageal carcinoma, the majority of which is esophageal squamous cell carcinoma (ESCC), ranks No.4 among that caused by malignant tumor. Thus, the current hotspot falls on studies of early diagnosis, carcinogenesis, prevention and treatment of esophageal carcinoma.The uncontrollable proliferation, caused by abnormal regulation of cell cycle, is the key characteristics of tumor. Furthermore, tumor is linked closely with cyclins. It has been reported that CyclinD1 is the key protein in the proliferation signal at G0/G1, and CyclinB1 is the key factor for the entry into G2/M. Over-expression of them can trigger the occurrence and development of the tumor.The cell apoptosis has a close relation with the occurrence, development, and treatment of malignant tumor. Livin is a new member of the inhibitors of apoptosis protein (IAPs), which have been discovered recently. Recent investigations revealed the abnormal expression of anti-Livin antibody and Livin in blood serum and solid tumor of patients with colorectal cancer, bladder cancer, and lung cancer. Second mitochondria-derived activator caspase(Smac), a promoting-apoptosis gene discovered recently, takes part in the downstream reaction of apoptosis. Accumulated studies displayed that the over-expression of Smac not only promote the apoptosis, but also increase the chemosensitivity of tumor cells.Tumorigenesis, which is related to induction of multifactor, participation of multigene, and development of multiphase, presents a very complex pathologic process. Activation of oncogene and/or inactivation of tumor suppressor gene form the substantial basis for human tumor. The abnormal expression of homeobox gene (HOX) family, a key transcription factor family regulating the development of cells, plays an important role in the occurrence and development of tumor through regulating the cell cycle and/or cell apoptosis. Studies showed that the abnormal expression of HOXA5 has been found in leukemia, nervous system neoplasm, cervical cancer, colorectal cancer and so on.RNA interference (RNAi) is a sequence-specific, posttranscriptional gene silencing mechanism by the introduction of double stranded RNA (dsRNA) homologous in sequence to the targeted gene. It is a multistep process that involves generation of active small interfering RNA (siRNA) in vivo through the action of an RNase III endonuclease, Dicer. The resulting 21- to 23-nt siRNA mediates the degradation of targeting mRNA to silence effectively endogenous target genes expression in mammalian cells. RNAi-induced knockdown of genes of interest is attractive for its speed, specificity, usefulness, and low cost. So RNAi technique is hopeful to become an ideal method for gene therapy of diseases.In order to further explore the relationship between HOXA5 and the occurrence, development, proliferation, and apoptosis of ESCC, the author is to investigate contents in this study as follows:â‘ test the expression of HOXA5, CyclinD1, CyclinB1, Livin, Smac in ESCC;â‘¡construct siRNA eukaryotic expression system targeting HOXA5 mRNA, and transfect into EC9706 cells, then observe the effects of it on the biological behavior of EC9706 cells and the growth of the transplanted tumor in nude mice. This study will offer theoretical and experimental evidences for exploring the occurrence and targeted therapy of esophageal carcinoma.This paper is made up of four parts.Part One The mRNA and Protein Expression of HOXA5, CyclinB1, CyclinD1, Livin and Smac in ESCCMethods1. 62 cases of patiens with esophageal cancer were choosed, and the specimens were obtained from cancer focus, adjacent cancer within 3 cm from cancer focus, and esophageal mucosa of surgical stump, respectively, within 30 min after operation.2. The HE staining assay confirmed that all 62 cancer specimens were ESCC, all specimens adjacented within 3 cm from the cancer focus were precancerous atypical hyperplasia, as well as all specimens of esophageal mucosa obtained from surgical stump were corresponding normal mucosa. Among the 62 cases of ESCC, 15 cases of grade I , 25 cases of grade II, and 22 cases of grade III were found; 20 cases with lymph node metastasis and 42 cases without lymph node metastasis were demonstrated; 7 cases were found to infiltrate in mucosa, submucosa, or shallow layer, 14 cases were observed in deep muscle and 41 cases were discovered in fiber membrane.3. The mRNA and protein expression of HOXA5 in cancer focus, precancerous atypical hyperplasia and normal mucosa of ESCC were detected by fluorescent quantitation-PCR(FQ-PCR),and immunohistochemistry SP staining, respectively.4. The mRNA and protein expression of CyclinB1 and CyclinD1 in cancer focus, precancerous atypical hyperplasia and normal mucosa of ESCC were detected by hybridization in situ and immunohistochemistry SP staining, respectively.5. The mRNA and protein expression of Livin and Smac in cancer focus, precancerous atypical hyperplasia and normal mucosa of ESCC were detected by RT-PCR and immunohistochemistry SP staining, respectively.6. The correlation was analyzed between the mRNA and protein expression of HOXA5, CyclinB1, CyclinD1, Livin, Smac and the clinical pathologic parameters of ESCC, and the correlation was analyzed between expression of HOXA5 protein and mRNA.7. Statistical analysis was performed by chi-square test and ANOVA with SPSS 12.0, and test standardα=0.05.Results1. The positive rates of HOXA5 protein expression in normal esophageal mucosa, adjacent hyperplasia and ESCC were 32.3%, 58.1%, and 64.5%, respectively (X²=14.66, P<0.05); The levels of HOXA5 mRNA were 0.97±0.46, 1.76±1.03, and 2.37±2.11, respectively(F=15.490, P<0.05), and the multiple comparison of HOXA5 mRNA had statistical difference. Furthermore, the levels of HOXA5 mRNA and protein in adjacent hyperplasia and ESCC were higher than those in normal esophageal mucosa (P<0.05).2. The mRNA and protein expression of HOXA5 were related with the pathological grade of ESCC (P<0.05), and it was higher in pathological grade III than those in grade I and grade II. Howere, It was not related with gender, age, the depth of invasion and lymph node metastasis in ESCC (P>0.05).3. The consistency was found between the HOXA5 protein expression and the mRNA expression (r=0.718, P<0.05).4. The protein and mRNA expression of CyclinB1 and CyclinD1 were higher in paracancerous dysplasia and esophageal cancer tissues than those in normal esophageal mucosa, in which the protein and mRNA expression of CyclinB1 and CyclinD1 were undetectable or low expression, and the difference among the 3 groups was statistically significant (P<0.05).5. The mRNA and protein expression of CyclinB1 and CyclinD1 were related with the histological grade, depth of invasion or lymph node metastasis (P<0.05), while it was not related with gender and age in ESCC (P>0.05).6. The consistency was found between the protein expression of CyclinB1 and CyclinD1 and the mRNA expression of CyclinB1 and CyclinD1 in normal esophageal mucosa, adjacent hyperplasia and ESCC.7. The positive rates of Livin protein expression in normal esophageal mucosa, adjacent hyperplasia and ESCC were 38.7%, 61.3%, and 80.6%, respectively, and the difference among the 3 groups was statistically significant (x²=22.801, P<0.05). Likewise, the positive rates of Smac protein expression in normal esophageal mucosa, adjacent hyperplasia and ESCC were 67.7%, 48.4%, and 33.9%, respectively, and the difference among the 3 groups was statistically significant (x²=14.323, P<0.05).8. The protein expression of Livin and Smac were related with the histological grade, depth of invasion or lymph node metastasis (P<0.05), while it was not related with gender and age in ESCC (P>0.05).9. The expression of Livin protein were negative correlation with the expression of Smac protein in ESSC tissues(r=-0.516, P<0.05).10. The protein expression of HOXA5 was positive correlation with CyclinB1, CyclinD1, or Livin in ESSC tissues, while was negative correlation with Smac.Part Two The Construction of pRNAT-U6.1-siHOXA5 Expressin Vector and its Effect on the Biological Characteristics of Esophageal Cancer Cell EC9706Methods1. The sequence of HOXA5 mRNA were taken from GenBank, and three 19-21 bp oligonucleotides were selected as targeting sequences (112-130 nt, 193-211 nt and 726-744 nt), according to the designation principle of siRNA.2. The three pairs of hairpin DNA oligonucleotide with BamH I and Xho I sites targeting HOXA5 were designed and a pair of nonspecific hairpin DNA oligonucleotide was as control, and were cloned into pRNAT-U6.1 expression vector.3. The recombinant expression vector was identified by PCR and sequencing.4. The four siRNA expression systems were transfected into EC9706 cells. Meanwhile, the empty vector was set as control and the cells without transfection as blank control. The expression of HOXA5 mRNA in different cell models was evaluated by RT-PCR to select the most effective siRNA expression system.5. After the vector beening transfected into EC9706 cells, and the alterations of morphological characteristics and proliferative abilities between the cells untransfected and trancefected were investigated by MTT assay, and the variations of cell cycle and apoptosis between the cells untransfected and trancefected were assayed by flow cytometry and DNA Ladder,the changes of HOXA5 mRNA and protein expression between the cells untransfected and trancefected were detected by RT-PCR and Western blotting, respectively.Results1. The bands of designed hairpin DNA of siHOXA5A, siHOXA5B, siHOXA5C and siC were visualized at 50-60 bp by gel electrophoresis, identical with the size designed.2. The results from PCR indicated that the size of PCR product for empty vector was 150 bp, while the recombinant plasmids were 210 bp, suggesting the 60 bp hairpin DNA oligonucleotides had been inserted successfully. The results from sequencing showed that the sequences of inserted fragments in the recombinant plasmid pRNAT-U6.1-siHOXA5A, pRNAT-U6.1-siHOXA5B, pRNAT-U6.1- siHOXA5C and pRNAT-U6.1-siC were identical to the designed sequences.3. The results from RT-PCR displayed that the mRNA Levels of HOXA5 in the EC9706 cells transfected with pRNAT-U6.1-siHOXA5A, pRNAT-U6.1-siHOXA5B, and pRNAT-U6.1-siHOXA5C were obviously lower than that in three control groups. The HOXA5 mRNA in cells transfected with pRNAT-U6.1-siHOXA5B was almost suppressed completely, Compared to the cells transfected with pRNAT-U6.1-siHOXA5A, pRNAT-U6.1-siHOXA5B, and pRNAT-U6.1-siHOXA5C, the cells transfectd with nonspecific siRNA plasmid, empty plasmid and blank cells produced high levels of HOXA5 mRNA.4. The results from light microscope exhibited that the EC9706 cells which transfected with HOXA5B siRNA presented certain suppression of proliferation after 24-96 h. The cell density was lower than that of the cells transfectd with nonspecific siRNA plasmid, empty plasmid and blank cells (P<0.05).5. The results from flow cytometry revealed that the percentages of G0/G1 cells were 77.3%±5.4%, 59.5%±4.9%, and 59.8±5.2% in the cells transfectd with HOXA5B siRNA, nonspecific siRNA plasmid, and blank cells, respectively, after 48 hours. The difference had statistical significance among the 3 groups (F= 11.65, P<0.05).6. The results from flow cytometry revealed that the apoptotic rates were 35.57%±2.40%, 5.39%±1.23%, and 5.77%±1.31% in the cells transfectd with HOXA5B siRNA, nonspecific siRNA plasmid, and blank cells, respectively, after 48 hours. The difference had statistical significance among the 3 groups (F=300.20, P<0.05).7. The results from agarose gel electrophoresis showed that, compared to the cells transfectd with nonspecific siRNA plasmid and blank cells, the "DNA ladder" (the bands of apoptosis ) can been seen in the cells transfected with HOXA5 siRNA after 48 h.8. The result from RT-PCR showed that the mRNA level of HOXA5 in the cells transfected with HOXA5 siRNA was 0.324±0.017, while the mRNA level of HOXA5 was 0.842±0.088 in the cells transfected with nonspecific siRNA, and the mRNA level of HOXA5 was 0.819±0.076 in blank cells. The expression of HOXA5 mRNA in the cells transfected with HOXA5 siRNA was remarkably lower than those in the two control groups (F=55.82, P<0.05).9. The result from Western blotting indicated that the protein expression of the cells transfected with HOXA5 siRNA was clearly lower than those of the cells transfected with nonspecific siRNA and blank cells. Part Three The Effects of Transfection with HOXA5 siRNA on the Expression of CyclinD1, Livin, and Smac in EC9706 CellsMethods1. EC9706 cells transfected with pRNAT-U6.1-siHOXA5B (siRNA transfection group), EC9706 cells transfected with non-specific siRNA, and blank cells were all cultured in the incubator at 37°C, 5%CO₂.2. The cells which cultured on tiny glass sheet were prepared and total RNA were extracted from the three groups, respectively.3. The mRNA and protein expression of of CyclinD1, Livin, Smac were detected by hybridization in situ, RT-PCR, and immunocytochemistry, respectively.Results1. The mRNA and protein expression of CyclinD1 in control EC9706 cells transfected with non-specific siRNA and blank cells were more than those in the EC9706 cells transfected with siRNA, and there were statistical significance in three groups (F=296.38, 257.74, P<0.05).2. The results from RT-PCR showed that the mRNA expression of Livin in control EC9706 cells transfected with non-specific siRNA and blank cells were more than those in the EC9706 cells transfected with siRNA, and there were statistical significance in three groups (F=1 551.74, P<0.05); The mRNA expression of Smac in control EC9706 cells transfected with non-specific siRNA and blank cells were lower than those in the EC9706 cells transfected with siRNA, and there were statistical significance in three groups (F=1 695.56, P<0.05).3. The positive staining of Livin and Smac mainly located in cytoplasm. The results from immunocytochemistry exhibited that the protein expressin of Livin were more in control EC9706 cells transfected with non-specific siRNA and blank cells than those in cells transfected with siRNA, and there were statistical significance in three groups (F=4 452.99, P<0.05). While the protein expressin of Smac were lower in control EC9706 cells transfected with non-specific siRNA and blank cells than those in cells transfected with siRNA, and there were statistical significance in three groups (F=283.97, P<0.05).Part Four The Inhibition of siRNA-mediated HOXA5 Gene Silencing on the Transplantation Tumor in Nude MiceMethods1. The tumor-bearing experiments were completed by inoculating the EC9706 cells transfected with pRNAT-U6.1-siHOXA5B, pRNAT-U6.1-siC and untransfected cells into five nude mice in each group subcutaneously, respectively.2. The long diameter(l) and short diameter(s) were measured every 3 days, and the tumor volumes were calculated depending on the fomula V=l×s²Ã—0.5. Then, the growth curves of tumor were prepared, and the tumor suppression rates were evaluated.3. The nude mice were killed by cervical dislocation after beening inoculated 5 weeks, then tumor were extracted. After tumor volume beening measured by Vernier caliper, one part of tumor was freezed for PCR, the other was used for immunohistochemistry and hybridization in situ.4. The mRNA and protein expression of HOXA5, CyclinD1, CyclinB1, Livin, and Smac in tumor tissues were detected by RT-PCR, hybridization in situ, and Western blotting.Results1. The transplantation tumor grew slowly in the group transfected with pRNA-U6.1-siHOXA5B, and the volumes of transplantation tumor were obviously smaller than those in bank group and nonspecific siRNA group (P<0.05).2. The results from light microscope by HE staining indicated that the tumor cells were mostly round or polygonal epithelioid-like cells. They were aligned closely and showed evident atypia, and some nucleuses were visible clearly, in addition that some pathological karyokinesis can be found. The quantity and size of necrotic focus increased in the order of blank group, non-specific siRNA transfected group and siRNA transfected group.3. The mRNA and protein expression of HOXA5 were negative in tumor tissues from siRNA transfected group. While the mRNA and protein expression of HOXA5 were positive in tumor tissues from the other two control groups. There were significant differences among the three groups (P<0.05). The similar results were showed by RT-PCR.4. The mRNA and protein expression of CyclinD1 and CyclinB1 were low in tumor tissues from siRNA transfected group, While the mRNA and protein expression of CyclinD1 and CyclinB1 were high in tumor tissues from the other two control groups. There were significant differences among the three groups (P<0.05).5. The protein expressin of Livin were lower in tumor tissues from siRNA transfected group than those in tumor tissues from the other two control groups. There were statistical significance in the three groups (P<0.05).6. The mRNA and protein expressin of Smac were more in tumor tissues from siRNA transfected group than those in tumor tissues from the other two control groups. There were statistical significance in the three groups (P<0.05).ConclusionThis study systemically observed the mRNA and protein expression rules of HOXA5 in normal esophageal mucosa, adjacent hyperplasia tissues and ESCC, firstly. Meanwhile, the study explored the mRNA and protein expression of CyclinD1, CyclinB1, Livin and Smac, and investigated the relationship between the mRNA and protein expression of above four parameters and mRNA and protein expression of HOXA5 in the development of ESCC. Furthermore, the study observed the feasibility of gene therapy targeting HOXA5 for ESCC, and conclusions were drawn as follows: 1. Compared to the normal esophageal mucosa, the mRNA and protein expression of HOXA5 increased in ESCC and adjacent hyperplasia tissues, which suggested that the abnormal expression of HOXA5 is an early event in the occurrence and development of ESCC and it play an important role in the course of ESCC progression.2. The mRNA and protein expression of CyclinD1, CyclinB1 and Livin presented over-expression in ESCC, and the mRNA and protein expression of Smac were also more in adjacent hyperplasia tissues.3. The protein expression of HOXA5 has positive relationship with the protein expression of CyclinD1, CyclinB1 and Livin, while it has negative relationship with the protein expression of Smac in ESCC.4. In this study, 3 expression systems targeting HOXA5 mRNA were constructed successfully, and found that pRNAT-U6.1-siHOXA5B is the most potent. Silencing the expression of HOXA5 can inhibit the proliferation of EC9706 cells, impel more cells to arrest at G0/G1 and promote apoptosis.5. The mRNA and protein expression of CyclinD1 and Livin decrease, but the mRNA and protein expression of Smac increase after the EC9706 cells being transfected with expression vector of siRNA targeting HOXA5B.6. The knockdown of HOXA5 expression can directly lead to the down-regulation of the CyclinD1 and Livin expression, while lead to the up-regulation of Smac expression in vitro and in vivo.7. The HOXA5 gene is desired to be a new target in gene therapy of ESCC.