The Expression Of EphA7 In Hepatocellular Carcinoma And The Study Of Its Inhibition In Vitro

Primary hepatocellular carcinoma is one of the malignant tumors with the highest incidence and mortality in the world, which ranking sixth in cancer incidence and third in mortality. Primary hepatocellular carcinoma seriously affects the health of people's lives because of its high recurrence rate, metastasis rate and poor prognosis after surgery. China is an area with a high incidence of liver cancer, more than 300,000 new cases each year, accounting for more than half of the world. In recent years, as the concern and attention about liver cancer increasing and along with the gradually intensive researches, the etiology and risk factors of liver cancer has been definite basically. The pathological changes involved in the development and progression of liver cancer has basically clear. Furthermore, the early screening method for the high-risk patients has been continuously improved. But the survival time of the patients with liver cancer have not been improved significantly. This is mainly due to the obscurity of the condition of the patients with liver cancer, resulting in the difficulty in diagnosis. So, patients are mostly advanced liver cancer and most of them can only receive palliative surgery, chemotherapy, radiotherapy and other therapies. Only a few patients can be cure by surgical resection. Although liver transplantation is a new alternative method of treating liver cancer, its development is limited by the shortage of donor. The patients with unresectable liver cancer can only be treated with the other treatment, such as transcatheter arterial chemoembolization, percutaneous ethanol injection, radiofrequency ablation, microwave coagulation and hyperthermia, etc. But the recurrence of liver cancer often occurs after these treatments and the long-term effects of the treatments are not satisfactory. Therefore, to study the mechanism of metastasis and recurrence and explore appropriate treatment measures of liver cancer are still the focus of current cancer research.With regard to new treatment of liver cancer is complex treatment that combined with a variety of treatment methods. Studies has confirmed that the combined therapy have obvious advantages in the treatment effect compared with monotherapy. In this regard, gene therapy can be considered as a potential auxiliary treatment. To this day, clinical trials have been completed showed that the side effects of gene therapy were at an acceptable range in most cases. As the mechanism of gene therapy is different from the traditional treatment, so the rational combination of gene therapy and traditional treatment can achieve synergistic therapeutic effect. In addition, the improved traditional treatment such as TACE and PEI can deliver the gene therapy vectors into the interior of the liver cancer, thereby increasing the effective dose and minimize side-effects of non-target cells. Today, people use these gene tools to study the factors known or suspected that play a role in tumor formation, to explore the new regulation factor in the process of tumor formation, thus providing new options for the treatment of the disease.Eph receptor family is the largest sub-tribe of tyrosine kinase family, which involved in many important physiological processes such as embryonic development, cell growth, tissue molding and signal transduction in the nervous system, etc. As a key member of the family, EphA7 gene located on chromosome 6q16.1, close to the breaking point. EphA7 gene is widely distributed in the human body with high homology. EphA7 receptor can bind with its Ephrin ligand and form a a bi-directional signal transduction, which involved in the early vertebrate embryo development, axon guidance and many important physiological processes. In the development of central nervous system, the formation of brain connections, neural crest cells orientation and the formation process of motor nerve axon, EphA7 gene plays a key role.In the past, most studies of EphA7 in the past focused on the physical context. In recent years, the role of EphA7 gene in the development of cancer had also caught the attention of researchers. Many studies indicate that abnormal expression of EphA7 was found in lung cancer, colon cancer, gastric cancer, prostatic carcinoma, pleomorphic malignant glioma and other human malignancies. In addition, there is evidence that EphA7 play an important role in tumor angiogenesis, tumor cell proliferation, invasion and metastasis. But overall, the research of EphA7 in tumor is still in the exploration of information and knowledge accumulated phase. At present, few studies about EphA7 have reported at home and abroad and many of them are only initial screening, failed to develop a systematic study. Furthermore, the studies were usually based on a small number of clinical specimens, which may affect the reliability of the results and still need further studies to confirm.Therefore, detect the expression of EphA7 in human hepatocellular carcinoma and explore the effect of EphA7 gnen on biological behavior of hepatocellular carcinoma cells, which would help us to understand the mechanism of EphA7 in the genesis and development of hepatocellular carcinoma, as well as guide the clinical work.In this study, by means of detecting the expression of EphA7 in human primary liver cancer tissues, adjacent liver cancer tissues and normal liver tissues and analyzing he relations with its clinical pathological factors, we intend exploring the relativity and clinical significance of it. After that,the siRNA eukaryotic expression vector targeting EphA7 gene was build and transfected the hepatoma cells. The effects of RNA interference targeting EphA7 gene on the biological behavior of hepatoma cell line in vitro were observed. Then, the nude mice xenograft model was established by injection of this transfected hepatoma cells in order detect the growth of hepatoma cells after the expression of EphA7 gene expression is blocked by siRNA. This study is divided into the following four parts:Part I The expression and clinical significance of EphA7 gene and protein expression in hepatocellular carcinomaObjective To investigate the expression of EphA7 gene and protein in primary hepatocellular carcinoma and analyze the relation with the clinical pathological features of it in order to explore its clinical significance.Methods40 cases of HCC tissues and 40 cases of adjacent liver tissues and 10 cases of normal liver tissues were collected. Among the HCC tissues,23 cases were male and 17 cases were female. The range of age is from 41 to 58 and the average age is 48.3. With tumor grade,16 cases were gradeⅠ～Ⅱ,24 cases were gradeⅢ～Ⅳ. With clinical stage,26 patients were stageⅠ～Ⅱand 14 cases were stageⅢ. RT-PCR, immunohistochemistry, Real-time fluorescence quantitative PCR and Western blot were used to detect the expression of EphA7 mRNA and protein in the specimens mentioned above respectively. The, relation between the expression o f EphA7 and the clinical pathological features of HCC were analyzed too.Results1. The expression of EphA7 mRNA was detected in 40 cases of HCC tissues their adjacent liver tissues and 10 normal liver tissues. The expression level of EphA7 mRNA were 20.0711±32.0232,4.5184±9.4738 and 4.1764±4.7193 (F= 5.399, P<0.05). Statistical analysis showed that the expression of EphA7 mRNA was significantly higher than that in the adjacent liver tissues (P=0.015) and normal liver tissues (P=0.013), while there was no significant difference between the adjacent liver tissues and normal tissues (P=0.998).2. The expression of EphA7 mRNA was unrelated to the age, tumor size, clinical stage, the levels of AFP and other clinical pathological parameters (P>0.05) except the degree of tumor differentiation, portal vein tumor thrombus and lymph node metastasis (P<0.05). The expression of EphA7 mRNA was significantly higher in patients with low degree of differentiation, portal vein tumor thrombosis and lymph node metastasis than those with the higher degree of differentiation (P=0.030), without portal vein tumor thrombosis (P=0.017) and lymph node metastasis (P=0.013). 3. The expression of EphA7 protein was mainly located in the cytoplasm of liver cells and hepatoma cells, as well as endothelial cells of vessels in fiber septa. The expression of EphA7 protein was strongly positive and over stain in hepatoma cells. While in the adjacent liver tissues and normal liver tissues, it was general lighter. Western blot analysis showed that the expression of EphA7 protein in HCC tissues, adjacent liver tissues and normal liver tissues were 0.5752±0.2590, 0.4019±0.2234 and 0.3169±0.1594 (F=7.826, P<0.05). The expression of EphA7 protein in HCC tissues was significantly higher than that in the adjacent liver tissues (P=0.001) and normal liver tissues (P=0.003), while there was no significant difference between the adjacent liver tissues and normal tissues (P= 0.309).4. The expression of EphA7 protein was unrelated to the age, tumor size, clinical stage and other clinical pathological parameters (P> 0.05) except the tumor differentiation, portal vein tumor thrombus, lymph node metastasis and the level of AFP (P<0.05). The expression of EphA7 protein was significantly higher in patients with low degree of differentiation, portal vein tumor thrombosis, lymph node metastasis and lower AFP level than those with the higher higher degree of differentiation (P=0.001), without portal vein tumor thrombosis (P=0.008) and lymph node metastasis (P=0.033) and higher AFP levels (P=0.013).ConclusionEphA7 is involved not only in the growth and development of normal liver cell but also with the malignant transformation of liver cells. It may play an important role in malignant transformation, invasion and metastasis and other biological behavior of liver cancer.Part II Construction and Identification of interference vector pRNA-siEphA7Objective To construct the eukaryotic expression vector pRNA-siEphA7 for the tyrosine kinase receptor EphA7 geneMethodsAccording to the nucleotide sequence of the human EphA7 gene in Genbank and the principles of siRNA design,3 segment sequences were chosen:638-656nt, 713-731nt and 1456-1474nt. All of the three sequences were combined with the corresponding plasmid vector pRNA-u6.1/Neo restriction recognition sites and transcription terminator at the end of them. By this way, we got the double-stranded siRNA for EphA7. After annealing, endonuclease reaction and reconstitution in vivo, the siRNA expressing vectors pRNA-siEphA7 targeting EphA7 gene was constructed. The three vectors:pRNA-siEphA7-1,2 and 3 were transfected into E. coli DH5a. After cloning and selection, PCR and gene sequencing analysis was used to verify the accuracy of recombinant vector.Results1. The sense and antisense oligonucleotide can form the double-stranded after annealing process. The DNA fragments were electrophoresed in 1.5% agarose gel after purification and recovery and analyzed in the UVP image acquisition and analysis system. A clear band around l00bp was found and which is coincided with the 118bp oligonucleotide designed originally.2. By using the empty vector pRNA-u6.1/Neo as control, PCR technique was used to identify whether the vector was recombined with the double-stranded siRNA by using certified primer P1 and P2. We finally found the amplified fragment about 310bp of the positive clones which proved that the pRNAT-U6.1/Neo vector was inserted successfully.3. The positive recombinant plasmids were sequenced and the sequence of the inserted oligonucleotide is proved to be correct, which confirmed that the recombinant plasmid was constructed successfully.Conclusion The siRNA expressing vectors targeting EphA7 gene:pRNA-siEphA7-1,2 and 3 were constructed. After cloning, screening and identification, it was confirmed that the inserted oligonucleotide is correct and the plasmids were constructed successfully.Part III Effects of RNA interference targeting EphA7 gene on the biological behavior of SMMC-7721 hepatoma cell line in vitoObjectiveTo investigate the changes of proliferation activity, cell cycle and invasive ability of SMMC-7721 hepatoma cell line after transfected with recombinant vector in vivo. To select the most effective recombinant vector pRNA-siEphA7.MethodsBy using the SMMC-7721 cells transfected with empty vector as control, the vectors:pRNA-siEphA7-1,2 and 3 were transfected into SMMC-7721 hepatoma cells by using liposome. G418 screening method was used to obtain the stable transfection SMMC-7721/siEphA7 hepatoma cells.Then, Real-time PCR and Western blot were used to detect the changes of EphA7 gene and protein in the stable transfection SMMC-7721 cells. MTT assay was used to detect the proliferative activity of the stable transfection SMMC-7721 cells. The changes of cell cycle after transfection was detected by flow cytometry assay. The changes of invasion and migration abilities after transfection were measured by Transwell chamber invasion assay.Results1. Green fluorescence could be seen from the SMMC-7721 cells that transfected with vectors pRNA-siEphA7-1,2,3 and transfected with empty vector through the inverted fluorescence microscope, which proved that the transfection is well. Green fluorescence could not be observed from the SMMC-7721 cells without transfection.2. Real-time fluorescence quantitative PCR results showed that the expression of EphA7 gene in the SMMC-7721 cells that transfected with interference vector pRNA-siEphA7-1,2,3, empty vector and non-transfected SMMC-7721 cells were as follows:0.1911±0.0153,0.1488±0.0199,0.0874±0.0123,0.4293±0.0533 and 0.4311±0.0410. The statistical analysis showed that the expression of EphA7 gene in the SMMC-7721 cells that transfected with recombinant interference vector were significantly lower than that in empty vector transfected group and non-transfected group, the difference was statistically significant (P <0.05). In the three groups that transfected with interference vector, the expression of EphA7 gene in group that transfected with pRNA-siEphA7-3 was the lowest. The difference compared with the other two groups was statistically significant (P<0.05). The expression of EphA7 gene had no significance between the two groups that transfected with pRNA-siEphA7-1 and 2 (P> 0.05), as well as between the groups that transfected with empty vector and non-transfected group (P> 0.05).3. Western blot results showed that the expression of EphA7 protein in the SMMC-7721 cells that transfected with interference vector pRNA-siEphA7-1,2 and 3, as well as empty vector and non-transfected SMMC-7721 cells were as follows:0.2786±0.0392,0.3011±0.0372,0.2142±0.0147,0.5822±0.0291 and 0.5832±0.0154. The statistical analysis showed that the expression of EphA7 protein in the SMMC-7721 cells that transfected with interference vectors were significantly lower than that in empty vector transfected group and non-transfected group, the difference was statistically significant (P<0.05). In the three groups that transfected with interference vector, the expression of EphA7 protein in group that transfected with pRNA-siEphA7-3 was the lowest. The difference compared with the other two groups was statistically significant (P<0.05). The expression of EphA7 protein had no significance between the two groups that transfected with pRNA-siEphA7-1 and 2 (P> 0.05), as well as between the groups that transfected with empty vector and non-transfected group (P> 0.05).4. MTT assay showed that the speed of cell proliferation among the five groups had no significant difference on the first day (P> 0.05). The speed of cell proliferation of SMMC-7721 cells that transfected with three different interference vectors was slower than that of empty vector transfected group and non-transfected group on the second day (P<0.05). But among the three different interference vectors transfected SMMC-7721 cells, it had no significant difference (P> 0.05). The speed of cell proliferation of SMMC-7721 cells that transfected with pRNA-siEphA7-3 was slower than that of the pRNA-siEphA7-1 and 2 transfected SMMC-7721 cells since the third day and continued to the seventh day (P<0.05). The speed of cell proliferation of SMMC-7721 cells that transfected with pRNA-siEphA7-1 was slower than that of the pRNA-siEphA7-2 transfected SMMC-7721 cells since the fifth day and continued to the seventh day (P<0.05). The speed of cell proliferation between empty vector transfected group and non-transfection group had no statistical difference all the time(P> 0.05).5. Flow cytometry results showed that among the SMMC-7721 cells that transfected with three different interference vectors, as well as empty vector transfected group and non-transfected group, the distribution of the proportion of cells in G1, G2 and S phase are basically similar. The differences among them had not statistically significant (P> 0.05).6. Transwell chamber invasion assay showed that the SMMC-7721 cells that transfected with pRNA-siEphA7-1,2 and 3 passed through the artificial basement membrane were:23.6±3.65,27±5.57 and 13.4±3.43. The number of the SMMC-7721 cells that transfected with pRNA-siEphA7-3 passed through the membrane was lower than that of the pRNA-siEphA7-1 and 2 (P<0.05). But between the pRNA-siEphA7-1 and 2 transfected SMMC-7721 cells, it had no significant difference (P> 0.05). The number of SMMC-7721 cells that transfected with pRNA-siEphA7-1,2 and 3 passed through the membrane were lower than those of the empty vector transfected group (68.6±6.50) and non-transfected group (72±9.30), the difference was statistically significant (P <0.05). Between the empty vector transfected group and non-transfected group, it had no significant difference (P> 0.05). ConclusionThe expression of EphA7 gene and protein in SMMC-7721 was significantly inhibited after transfected with interference vector pRNA-siEphA7-1,2 and 3. The down regulation of EphA7 can reduce the proliferation activity, weaken the invasive ability in vitro of SMMC-7721 cell, but bring little change in the cell cycle. Among the three interference vectors, the interference effect of pRNA-siEphA7-3 (1456-1474nt) is best.Part IV Effects of RNA interference targeting EphA7 gene on the growth of transplanted tumor in nude miceObjectiveTo investigate the growth of SMMC-7721 cell after the expression of EphA7 gene expression is blocked by siRNA in nude mice transplanted tumor model.MethodsThe nude mice tumor model was established by injection of liver cells in the subcutaneous layer of left upper limb. The 32 nude mice were randomLy divided into four groups. According to the different cells injected, the nude mice were divided into the following groups:SMMC-7721 group (SMMC-7721 cells were injected); SMMC-7721/siEphA7-3 group (SMMC-7721 cells transfected with interference vector pRNA-siEphA7-3 were injected); SMMC-7721/Vector group (SMMC-7721 cells transfeceted with empty vector were injected); PBS group (PBS was injected). After five weeks, the nude mice were executed and the size and weight of transplanted tumor was compared. Real-time PCR, immunohistochemistry and Western blot were used to detecte the expression of EphA7 gene and protein in tumor tissues.Results1. About 9-12 days after the injection of cells, the formation of transplanted tumor can be observed at the injection site in addition to PBS group. It was proved that SMMC-7721 cells, SMMC-7721 cells transfected with interference vector and empty vector had the tumorigenic activity in nude mice. The nude mice liver tumor model could be established successfully by using these cells.2.35 days after the model established the tumor volume of SMMC-7721 group, SMMC-7721/siEphA7-3 group and SMMC-7721/Vector group were 2.19±1.18cm3,0.84±0.32cm3 and 2.33±0.97cm3 respectively, the weight of the three groups were 1.78±0.58g,0.79±0.14g and 1.83±0.72g respectively. The tumor volume and weight of SMMC-7721 group and SMMC-7721/Vector group were significantly larger than those of SMMC-7721/siEphA7-3 group, the difference had statistically significant (P<0.05). While between the SMMC-7721 group and SMMC-7721/Vector group, it had no significant difference (P>0.05). The inhibition rate of transplanted tumor was 55%.3. Real-time fluorescence quantitative PCR showed that the expression of EphA7 gene of SMMC-7721/siEphA7-3 group, SMMC-7721/Vector group and SMMC-7721 group were 0.1911±0.0204,0.3900±0.0863 and 0.4219±0.0595. The expression of EphA7 gene of SMMC-7721/siEphA7-3 group was lower than that of SMMC-7721 group and SMMC-7721/Vector group, the difference had statistically significant (P<0.05). While between the SMMC-7721 group and SMMC-7721/Vector group, it had no significant difference (P> 0.05).4. Immunohistochemistry showed that in SMMC-7721/siEphA7-3 group, the cells of positive staining of EphA7 protein was less and the staining was lighter. But in SMMC-7721 group and SMMC-7721/Vector group, those were more and deeper. Western blot showed that the expression of EphA7 protein of SMMC-7721/siEphA7-3 group, SMMC-7721/Vector group and SMMC-7721 group were 0.2044±0.0153,0.4582±0.0631 and 0.4816±0.0607. The expression of EphA7 protein of SMMC-7721/siEphA7-3 group was lower than those of SMMC-7721 group and SMMC-7721/Vector group, the difference had statistically significant (P<0.05). While between the SMMC-7721 group and SMMC-7721/Vector group, it had no significant difference (P> 0.05). ConclusionUsing siRNA to suppress the expression of EphA7 gene and its protein of SMMC-7721 hepatoma cell line, the growth of SMMC-7721 cells in vivo could be slowed.