Experimental Study On Inhibition Of Hepatocellular Carcinoma Growth Mediated By Oncolytic Adenovirus With RNA Interference

Background and ObjectionPrimary Hepatocellular Carcinoma is a common malignant tumor. The drug treatment is one of the main treatment methods. Lots of Clinical Practice[1-12] and experiments [13-17] have showed that the Compound Kushen Injection could improve the anti-cancer effects in the course of treating hepatocellular carcinoma [1,8,9,17]. The recent research has approved[18] that the Compound Kushen Injection has the function of inhibiting proliferation, inducing the differentiation and improving apoptosis of cancer cells [3-16,18-23]. Although, the Compound Kushen Injection has good effects in cancer therapy, its efficiency components are not clearly, the standards of controlling quality is complex, its therapy effects are not same in different factories and batches products as well as most Chinese Medicines[24-28].With the developing of modern biology technology, human beings can analyze the medicine function mechanism into molecular level and develop into Systemic Biology slowly. Relative research of its molecular mechanism is not countable because the Compound Kushen Injection have good effects in cancer therapy. From the research results[29-31] of gene microarray, immunohistochemical, analyzing results[13,21,32,35] of key genes in gene transcription and translation level showed that the anti-cancer mechanism of the Compound Kushen Injection can be concluded in the following aspects:1. The effect of directly killing cancer cells:the Compound Kushen Injection can inhibit the expression of Cyclin El, Survivin and B-cell lymphoma/Leukemia-2in cancer cells. Result in expression of Caspase-3climb and active, so many cancer cells undergo apoptosis. Lastly the effect of directly killing cancer cells is realized [13,21.32]2. The effect of indirectly killing cancer cells:the Compound Kushen Injection can up-regulate expression of death receptor such as Fas on the surface of cancer cells. Then realize in the effect of indirectly killing cancer cells by organism immunity system, such as cytotoxicity of specific T cell[13,30,31].3. Inhibiting invasion and metastasis of the tumor:the Compound Kushen Injection can improve the expression of nm23[16] in the cancer cells[2], it can also inhibit the expression of CD44v6, at last it inhibits the invasion and metastasis of the tumor[1,19].4. Inhibiting the growth of tumor vessel:the Compound Kushen Injection can inhibit the expression of VEGF and CXCR4, maybe it is the main molecular mechanism reason of inhibiting the growth of tumor vessel[33,34,36-38].5. The Compound Kushen Injection have the function of reducing the pain, improving the quality of life and organism immunity ability[39-54].According to this, I designed a recombinant oncolytic adenovirus with RNA interference. The main molecular mechanism of the Compound Kushen Injection was imitated by RNA interference to inhibit the expression of Cyclin E1, Survivin and Bcl-2in liver cancer cells. It can not only avoid many fixed shortcomings of traditional Chinese Medicine, but also play full of gene therapy advantages. So the anti-cancer ability of recombinant oncolytic adenovirus with RNA interference can be improved. And the recombinant oncolytic adenovirus may be become a kind of new gene therapy medicine for cancer.The Compound Kushen Injection realizes its directly killing effects mainly by inhibiting the expression of Cyclin El, Survivin and Bcl-2in cancer cells. The three proteins play important roles in the process of cell carcinogenesis, proliferation and apoptosis of cancer cells. The overexpression of Cyclin El and Survivin are the key factors[55-64] that cause abnormal of cancer cell cycle-regulation and hyperproliferation of cancer cell. Bcl-2and Survivin are the key factors[63-69] that cause abnormal of cancer cell apoptosis. The overexpression of Cyclin El would lead to shorten G1phase, hyperproliferation, lastly cell carcinogenesis. The expression of Cyclin E1inhibited in cancer cell can cause cell-cycle frozen in GO/G1phase, prevent cancer cell from stepping into S phase, to inhibit cancer cell hyperproliferation. Survivin has double functions[55,70] that regulate cell-cycle and inhibit cell apoptosis. It is one of the key bridges that connect cell-cycle and cell apoptosis. If the expression of survivin was inhibited, the proliferation of liver cancer cells would be inhibited and be pushed into apoptosis[71-75]. Bcl-2also play an important role in anti-apoptosis course of cancer cells, and it has effect in combination with survivin [65,76-79]. So the cancer cell would be easier stepped into apoptosis when the expression of survivin and Bcl-2are inhibited at the same time.RNA interference has been applied early in cancer therapy as a method that inhibit the expression of oncogene[80,1]. Generally, there are two ways to transfer the medicine, one is inject directly after synthesis RNAi, the other is transfer a RNAi expression vector into cell to express RNAi sequence to therapy the cancer. There are many shortcomings in the first method, such as huge immune response, RNAi sequence decompose greatly, low efficiency, poor treatment effects, slowly be quitted[82].In the second method, the key is choosing a vector. If use a common vector, such as pGene-sil, efficiency of transfer will be low, inhibition function will last short time, immune response would be great. So the treatment effect is not good in clinical application[83]. So I chose oncolytic adenovirus as a vector for RNAi. Oncolytic adenovirus could specific proliferate in cancer cells, and spread into tumor organism by itself[84-86]. If put RNAi sequence into oncolytic adenovirus genome, the RNA interference therapy would last longer time, achieve good efficiency and has good targetability.It would be improve the targetability of oncolytic adenovirus if set the El A gene that is the essential gene for adenovirus proliferation under a cancer specific promoter[87]. The cancer specific promoter can be classified into three kinds:①General cancer specific promoter, such as survivin promoter, hypoxia promoter and hTERT promoter and so on[88-90],②Promoter exist in special cancer type, such as DF3/MUC-1promoter, AFP promoter and so on[91,,92].③Promoter exist in special organism, such as PSA promoter[93]. Targeting activity and promoting efficiency should be considered as choose tumor special promoter for El A. For example, when construct an oncolytic adenovirus for liver cancer, general tumor special promoter could be chosen, or AFP promoter could be chosen too. Because of the lower efficiency of AFP promoter, it decline the proliferation ability of adenovirus in liver cancer greatly and the ability of anti-cancer. So it is not a good choice. Survivin promoter is a better one for liver cancer cell base on the results of other research[94].Above all, the study adopt oncolytic adenovirus to transfer RNAi sequence, it overcome many traditional RNAi therapy shortcomings, improve oncolytic adenovirus’ability of anti-cancer as the same time. So the constructed oncolytic adenovirus with RNA interference would be developed into a new medicine for treatment of cancer. The recombination oncolytic adenovirus with RNA interference can inhibit the expression of Cyclin E1, Survivin and Bcl-2to imitate the main molecular mechanism of the Compound Kushen Injection. This method tries a new way for gene therapy. It can not only avoid many fixed shortcomings of traditional Chinese Medicine, but also play full of gene therapy advantages.Methods and materialsA. The construction of expression box with E1A gene and RNA interference1. Clone survivin promoter, construct a T-vector.2. Clone E1A gene, construct a T-vector.3. Construct RNA interference to inhibit the expression of Cyclin El, Survivin and Bcl-2.4. Construct an expression box with E1A gene and RNA interference.B. Construct the recombination adenovirus1. Construct the recombination adenovirus vector with the expression box (pAxcwit2-E1A-RNAi).2. Pack the recombination adenovirus in293T cell.3. Identify the achieved recombination adenovirus.4. Measure the titer of the achieved recombination adenovirus.C. Measure the inhibition effects of the recombination adenovirus to Cyclin E1, Survivin and Bcl-2in liver cancer.1. Measure the inhibition effects of the recombination adenovirus to Cyclin E1, Survivin and Bcl-2mRNA in liver cancer by RT-PCR.2. Check the results of RT-PCR by Real time PCR.3. Measure the inhibition effects of the recombination adenovirus to Cyclin E1, Survivin and Bcl-2protein in liver cancer by Western Blot.D. The study of killing effects of the recombination oncolytic adenovirus for liver cancer.1. Optical microscopic observation of hepatocellular carcinoma cells.2. Measure the killing effects of the recombination oncolytic adenovirus for liver cancer by MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. Measure the absorbance value of24h,48h,72h and96h separately, draw the growth curve. Statistical MethodsMeasurement Data can be expressed as X±SD. Multifactors experimental data were analyzed by factorial analysis; Use2-independent-samples T test to analyze difference between two groups; Use one-way ANOVA to analyze multi-groups difference. Use Welch to revise the unhomogeneity of variance. Multiple comparisons can be used on the premise that there are significant differences among groups. LSD method can be used when the variance is homogeneous. Dunnett’s T3method can be used when the variance is unhomogeneous. The difference (P<0.05) was statistically significant. Use soft of SPSS13.0for windows to analyze results of data.ResultsA. The construction of expression box with E1A gene and RNA interference1. Survivin promoter (276bp) was cloned successfully for the genome of HepG2.2. E1A (1004bp) as cloned successfully for the genome of293T.3. RNA interference was constructed to inhibit the expression of Cyclin E1, Survivin and Bcl-2with restriction site separately.4. The expression box with E1A gene and RNA interference was constructed successfully.B. Construct the recombination adenovirus1. The recombination adenovirus vector with the expression box (pAxcwit2-E1A-RNAi) was constructed successfully.2. The recombination adenovirus was packed successfully in293T cell.3. The achieved recombination adenovirus was identified by PCR and sequence. The results showed that the recombination adenovirus with the expression box was constructed successfully.4. Use the Endpoint Dilution Method to measure the titer of the recombination adenovirus. Its titer is2.1X106. C. Measure the inhibition effects of the recombination adenovirus to Cyclin E1, Survivin and Bcl-2in liver cancer.1. The results of RT-PCR showed that the expression of Cyclin E1, Survivin and Bcl-2mRNA declined deeply by the recombination adenovirus.2. The results of Real time PCR showed that same trend with the RT-PCR.3. The results of western blot showed that the expression of Cyclin El, Survivin and Bcl-2protein declined deeply too.D. The study of killing effects of the recombination oncolytic adenovirus for liver cancer.1. In Ad-PEAS group, the killing effect to liver cancer cell is significant.2. The results of MTT showed that the recombination oncolytic adenovirus has significant killing effect to liver cancer cell.ConclusionThe recombination oncolytic adenovirus with E1A gene and RNA interference can inhibit the expression of Cyclin E1, Survivin and Bcl-2significantly, can inhibit the growth of liver cancer cell efficiently, can improve the apoptosis of liver cancer cell greatly and has killing effects to liver cancer cell clearly.Innovative PointsThe results of the study showed that there are several advantages of oncolytic adenovirus vector, such as high efficiency of inhibition, much time of inhibition.The use of RNA interference inhibit the expression of Cyclin E1, Survivin and Bcl-2clearly, reached association and cooperation of three gene therapy, enhanced the oncolytic adenovirus’killing effects to liver cancer cell significantly. So it can guide clinical treatment of liver cancer by RNA interference significantly.This study imitated the main molecular mechanism of the Compound Kushen Injection to try a new way for gene therapy. It can not only avoid many fixed shortcomings of traditional Chinese Medicine, but also play full of gene therapy advantages.