| BackgroundColorectal cancer is one of the most common malignant tumors.The incidence of colorectal cancer for men and women are all ranked the third number in malignant tumors around the world.In our country,the incidence of colorectal cancer showed an upward trend by the average increase of 4.2%year by year along with the continuous improvement of living standards,changes in diet.At present,the main treatment for colorectal cancer are surgical resection and supplemented by chemotherapy,radiotherapy treatment,et al.Colorectal cancer is a systemic disease, although the tumor was completely removed,the primary tumor may metastasis before the noticeable symptoms occur,recurrence or metastasis may occurre in half of cases after 2 years of operation.Neo-adjuvant chemotherapy has been used as an important supplementary means in the adjuvant treatment of colorectal cancer since the end of the eighties 20th century.The significance of neoadjuvant chemotherapy is to reduce recurrence,distant metastasis after surgery and improve the resection and cure rate,prolong survival time and improve the postoperative quality of life.Its positive significance has been recognized by most scholars.The biggest obstacle in chemotherapy is the multi-drug-resistant of tumor cells,especially the high-expression of multidrug resistance(MDR) gene in primary and secondary colorectal cancer,which made colorectal cancer multi-drug resistance was significantly higher than other tumors,so the effect of chemotherapy in colorectal cancer was significantly lower than that of in other tumors.People's awareness of colorectal cancer has reached the level of molecular biology.Colorectal cancer is a genetic disease from the perspective of genetics and molecular biology.It is caused by Oncogene activation and inactivation of tumor suppressor genes,as well as abnormal regulation of gene expression.The molecular basis of colorectal cancer's occurrence is accumulation process of a variety of longterm, multi-stage,multi-step gene mutations.Gene therapy for colorectal cancer has become a popular research and it has developed into clinical trials from the basic theory stage.At present,the basic researches of gene therapy for colorectal cancer include:①Cytokine gene therapy;②Gene therapy that target oncogenes and tumor suppressor genes;③Antisense gene therapy;④Suicide gene therapy;⑤multi-drug resistance gene therapy;⑥Anti-tumor angiogenesis the formation of gene therapy;⑦Radiation gene therapy et al.The base pairing rules and nucleic acid hybridization are the basic principles Of antisense oligonucleotide technology.By designning the antisense oligonucleotide (ASODN) fragment that complement the specific region of target genes,the expression and function of the target gene will be inhibited when ASODN and specific sequence of DNA and mRNA combined,while the other gene transcription and expression were not changed.The concept of antisense was first described by Stephenso and Zamecnik in 1978,Many studies confirmed that ASODN had the ability to inhibit gene expression specificly.People began to recognize the huge potential of ASODN on treatment.ASODN inhibits gene expression through a variety of different mechanisms:①Inhibit gene transcription,ASODN integrates with the specific DNA double helix molecule then it becomes a DNA triplex which can prevent gene transcription;②Inhibit RNA processing,ASODN mainly inhibit mRNA precursors' splicing;③Inhibit the translation of RNA,if ASODN sequence and mRNA translation initiation sequence complement with each other,the two hybrid combination can prevent mRNA to be the ribosome,while the translation initiation is inhibited;if ASODN sequence and mRNA initiation site downstream sequence complement with each other,a combination of both can terminate translation by preventing ribosomes' movement on mRNA;④Induce RnaseH(RNA enzyme H)-mediated RNA degradation,this may be the most important antisense inhibition mechanism.RnaseH can crack RNA chain which in DNA-RNA hybrid double-stranded. A hybrid double-strand is formed when ASODN combines with mRNA which can start RnaseH pyrolysis function then protein production is inhibited after the mRNA degraded.The advantages of antisense therapy include:①The targets of antisense compounds are the oncogenes that caused tumor and antisense compounds play the therapeutic effects by regulating the expression of gene products.Antisense therapy is a more effective treatment when a large number of pathogenic proteins exist and the traditional medicines for proteins can not play a role.②Antisense compounds can cure the genetic diseases that traditional medicines can not.③Antisense therapy is more secure and efficient than the expression vector in gene therapy.④the costs for antisense therapy is probably cheaper than traditional medicines.Antisense therapy is popular in cancer therapy studies because of its high specificity and low toxic side effects.Research on antisense therapy progresses rapidly and shows the brilliant future.Antisense therapy has been gradually entering the clinical with the continuous improvement.So far,there are some researches on treatment of some malignant tumor by the application of antisense oligonucleotide, such as chronic lymphocytic leukemia,neuroblastoma,bladder muscle cancer, multiple myeloma,breast cancer,stomach cancer,colon cancer,mesothelioma and lung cancer,researchers have achieved a certain degree of efficacy.c-myc gene is located in chromosome 8q24 and it belongs to myc oncogene family.Gene amplification,chromosome's translocation,rearrangement are the primary ways to activate c-myc,c-myc play an important role in some tumors' occurrence,development and prognosis,c-myc oncogene is a nuclear protein gene,it has the ability to transform cells.Combining with the chromosomal DNA is a characteristic of it.c-myc also plays a role in regulating cell growth,differentiation and malignant transformation.Expression activity of c-myc is closely related to cell growth and splitting speed,c-myc is also a potential of apoptosis-inducing factor.The over-expression of c-myc has been found in many different human tumor cell lines, including myeloid leukemia cells,retinoblastoma cell lines,some neuroblastoma cell disease cell lines,breast cancer cell lines and certain lung cancer cell lines,c-myc's amplification was also observed in human colon cancer cell lines,c-myc gene involves in tumor angiogenesis and plays a role in controlling blood vessel growth in the process of tumor formation.This discovery also makes c-myc a new hope to treat tumors,c-myc ASODN have been used in cardiovascular disease,breast cancer, osteosarcoma ovarian cancer,liver cancer,lung cancer,kidney cancer and other related research at home and abroad.Using c-myc ASODN has better effects not only at lowering drug resistance of breast cancer cells but also at inducing apoptosis and inhibiting cell growth such as gastric cancer cells,ovarian cancer cells,bladder cancer cells,liver cancer cells,lymphoma cells,lung cancer cells,et al.Some studies have shown that c-myc's inhibition temporarily is sufficient to stop tumor growth permanently.Currently the research on systemic adjuvant chemotherapy for colorectal cancer is very popular,and it has made remarkable progress.At present,the clinical first-line chemotherapy drugs used in colorectal cancer is mainly platinum and fluorouracil,but their shortcomings such as resistance and side effects also limit their application. United ASODN and traditional chemotherapeutic drugs has become more common strategy in cancer research.In recent year,apoptosis mechanism studies have confirmed that anti-apoptosis factor played an important role in tumor cell growth and drug resistance,c-myc is an important apoptosis regulatory gene,and it is closely related to drug resistance.ASODN has the function of enhancing the effect of chemotherapy.ASODN and chemotherapy drugs such as 5-Fu,oxaliplatin are used in colorectal cancer at the same time to inhibit the proliferation of colorectal cancer, which makes organic combination of two types of anti-tumor mechanism for the purpose of promoting tumor cells' apoptosis.The studies of Sun have shown that c-myc ASODN can not only reduced c-myc expression but also can reverse ovarian cancer's resistance to DDP.Nita increased colon cancer chemotherapy sensitivity to 5-Fu by applications of Bcl-x(L) ASODN.Based on the research at home and abroad and with the mature of Antisense oligodeoxynucleotide technology,we proposed synthesis a number of c-myc antisense oligonucleotide chains for the purpose of inhibiting tumor cells' growth, differentiation.c-myc ASODN will suppress or close c-myc gene expression when it is transfected into colorectal cancer HT29 cells by liposomes.Further more,we will use ASODN and oxaliplatin at the same time to test whether it can improve eolorectal cancer's chemosensitivity to oxaliplatin.This tumor treatment method has great clinical trial research value by makeing perfect combination of antisense oligonucleotide technology and chemotherapy.ObjectivesTo investigate the effects of c-myc antisense oligodeoxynucleotides(ASODN) on the proliferation,apoptosis and chemosensitivity to human colorectal cancer cell line HT-29 in vitroMethods1.c-myc ASODN was transfected into human colorectal cancer cell line HT29 by lipofectamineTM2000;2.The expression of c-myc mRNA in human colorectal cancer cell line HT29 was detected by semi quantitive reverse transcription-polymerase chain reaction(RT-PCR);3.The expression of c-myc protein in human colorectal cancer cell line HT29 was detected by Western blot;4.The effects of the c-myc ASODN on the apoptosis of HT29 cell line was studied by FCM assay.5.The effects of the c-myc ASODN on the proliferation inhibit and sensitivity to oxaliplati of HT29 cell line were studied by MTT assay.Results1.After the transfection of 24 hours,the expression level of c-myc mRNA in HT29 cell was significantly lower in antisense group than that in the control group. Compared with low concentration of c-myc ASODN group,c-myc mRNA expression is lower in high concentration of c-myc ASODN group(P＜0.05).2.Western blot test showed that the protein expression of c-myc gene in HT29 cells was confirmed and it was clear that specific band on PVDF membrane in ASODN group was obviously weaken and there were significant differences between the ASODN group and the control groups.3.MTT assay showed that c-myc ASODN has inhibitory effect on HT29 cells by different concentrations.Cell inhibitory rate of value-added is significantly different between four different times(P＜0.05).With the drug concentration increasing,the inhibition of c-myc ASODN on HT29 cells is also increased,which showed a dose-time dependent manner(P＜0.05).4.FCM assay showed that the proportion of HT29 cells apoptosis increased after 72h transfection with c-myc ASODN(transfection concentration was 1200nmol/L).Apoptosis rate were significantly different between the liposome control group and c-myc ASODN group(P＜0.05).5.When oxaliplatin and c-myc ASODN were using simultaneously,the inhibitory rate of HT29 cell by c-myc ASODN and oxaliplatin was increased significantly as the drug concentration and time increased.There was significant difference about inhibition rate at the same concentration of oxaliplatin between the experimental group(oxaliplatin +c-myc ASODN+ lipofectamineTM2000) and control group(oxaliplatin + lipofectamineTM2000)(P＜0.05).According to 0～4d MTT test results,we can see from the growth curve of HT29 cell(Figure 7) that the HT29 cells' rate of proliferation in experimental group was decreased significantly than the control group(P＜0.05).Conclusions1.The c-myc ASODN can inhibit c-myc mRNA and protein expression effectively in colonic cancer HT29 cells in vitro.2.Blocking c-myc gene expression in HT29 cells not only can inhibit the proliferation of HT29 cells but also can promote apoptosis of HT29 cells.3.Combination using of c-myc ASODN and oxaliplatin can reverse resistance of HT29 cells to oxaliplatin and improve the sensitivity of HT29 cells to oxaliplatin. 4.Antisense technology has great research and clinical trial research value,c-myc ASODN might improve the treatment outcome of colorectal carcinoma and it may be a potential target of gene therapy for human colorectal cancer.
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