Reversion The Multi-drug Resistance Of Human Renal Cell Carcinoma By RNA Interference Targeting MDR1 Gene

Multi-drug resistance(MDR)is the major cause for the failure of chemotherapy and the renal cell carcinoma(RCC)is the most resistant tumor of the urinary tumors.Numerous study confirmed that the MDR mainly correlated with the P-glucoprotein(P-gp)which situated at the tumor cellular membrane.It's an ATP-energy transmembrane protein functioned like an excretion pump,thus the chemicals concentration inner the cell depressed.P-gp is encoded by gene MDR1 which situated at the human chromosome 7q21.1.Since the gene MDR1 and its product P-gp was discovered,scientists had studied their structure,function and the distribution in the normal or tumor tissues.It was revealed to be excess expressed on the endepidermis of some tissues such as kidney proximal tubule,prostate,adrenal,normal bile capillary,colon,small intestine,pancreas,bronchus and mammary gland.The molecular mass of P-gp is 170000 and it consists of two repeated parts of 1280 amino acids.The P-gp,similar as the transport protein of bacterial,consumes ATP and pumps the chemicals out of the cell which depressed the intracellular concentration.Therefore,the high expression of P-gp is the mainly reason for the multi-drug resistance of tumor cell.RNA interference(RNAi)is a newly rising and increasingly full-grown technique for gene silencing,which is now considered to be a high-performance research tool in gene engineering fields.With this technique,we could effectively,stably and specially block the expression of target gene,thereby,the gene was 'silenced'.Nowadays,double-strand RNA (dsRNA),especially small interfering RNAs(siRNA)have already been widely utilized in the research of gene function and gene therapy.RNAi can precisely suppress the proto-oncogene in the gene therapy strategy.Just microdosis of siRNA would depress the level of its virulent gene product more than 70～90%,sometimes it could even ahieve the effect of gene knock-out. In addition,RNAi also possesses a strictly series-specificity and definit target,which made it could depress different genes simultaneously without any mutual interference.At the same time,RNAi could be cascaded amplified and had high penetrability.All of these characters suggests it is suitable to the therapy research of cancer gene.Thereafter,the mechanisms of RNAi was proved existing in majority of tumor cells.Exogenous RNAi mediated by chemosynthesis,plasmid/virus or other mode could also effectively depress the endogenous RNA expression.This study will design a stable and high performance expressed MDR1-shRNA lentivirus recombination vector and test its interference effect for the gene MDR1 in the RCC. Furthermore,drug sensitivity of the cell before and after interference will also be identified, then we will determine whether the gene MDR1 is suitable for the target to reverse the drug resistance of RCC.Part 1.Preparation of vshRNA recombinants targeting MDR1 and effective vshRNA screening(1)Preparation of vshRNA recombinants targeting MDR1 and standardization of virus titerOperating the RNAi designing software downloaded from website of Ambion company, we designed three different shRNA arrays aiming directly at hMDR1 based on RNAi structure principle,which were labeled as 1#/2#/3# shRNA.Then these shRNAs were inserted into lentivirus vectors,building up three kinds of recombinants.These three products were transferred into E Coli competent cells and clones of these different infected cells were collected for RT-PCR and sequence analysis.Results showed that these three recombinants were successfully constructed.At the same time,three recombinants and two lenvirus-packaging helpers were extracted.All of these things were infected into 293T cells separated by three groups and the supemate that rich of virus particles were then collected and concentrated.The virus MOI levels were determined in 293T cells.(2)Effective interfering vshRNA screeningThree recombinants were then separately operated to infect the objective cells(ACHN cell series),transfection effect was then detected in order to find the most effective vshRNA among these three.Objective cells were cultured in good condition and planted in 12-well plate one day before virus infecting.According to group designing,vshRNA recombinants of different MOI levels were separately added into the plate for infection.Fluorescence microscope were employed to observe GFP expression three days after the transfection.Five days later,we collected the cells and detected the MDR1 mRNA level of these cells with Realtime PCR to inspect the depressing results of different vshRNA.From photofluorogram,we can see the efficiency of virus infection reached more than 90%.The Realtime PCR outcome showed that of all these three vshRNA,3# was the effective target.It could decrease the expression of target gene about 40%in high MOI level comparing with blank control group.So the 3# was the best sequence of these three shRNA.In addition,the effect of gene knock-out was better in high virus MOI level than in low MOI level.In this part,we successfully built up and packaged three specific targeting hMDR1 vshRNA recombinants,virus MOI were also demarcated in 293T cells.Additionally,we managed to pick out the most effective vshRNA of these three,and clarified the non specific virus toxicity of different MOI.These results provided us reliable evidence to carry on RNAi experiment targeting MDR1 in ACHN cells.Part 2.Investigation of RNAi effect and change of the biological behaviour after RNAiWe had successfully built up three specific vshRNA recombinants targeting hMDR1 and evaluated the 3# vshRNA as the best sequence with high infecting efficiency and silencing effect.In this part of experiment,we employed the 3# vshRNA to infect ACHN cells to identify the knock-down ability of this recombinant.Objective cells(ACHN cells)were cultured in good condition and planted in 6-well plate one day before virus infecting.According to group designing,vshRNA recombinants were separately added into the plate for infection.Fluorescence microscope were employed to observe GFP expression three days after the transfection.Five days later,the cells were collected and the P-gp were extracted.We detected the MDR1 mRNA and P-gp level of these cells with Realtime PCR and western blot methods to inspect the depressing results.Results showed that the 3# vshRNA could swimmingly infect ACHN cells.Realtime PCR data suggested that this recombinant could depress MDR1 mRNA level of ACHN cells obviously to about 60%and the western blot method proved the obviously reduction of P-gp in protein level.So we thought the gene MDR1 was greatly silenced in RNA and protein level after successfully interference.To further determine the change of multi-drug resistance of RCC cells,the generally used chemotherapeutics VCR was given in the cell culture fluid. CCK-8 kit was used to examine the cell growth and the sensitivity to VCR.Due to the unexpected observation of growth inhibiting after MDR1 gene interference in ACHN cells,we added the detection of apoptosis with FCM and the expression of gene mtp53 and surviving with Realtime PCR.Following is the brief introduction.(1)Detect the level of cell proliferation and the change of tolerance with the CCK-8 kit when using the chemicals after the gene MDR1 was silenced.Culture the interfered ACHN cells of good condition in the 96-well cultivation plate.4 hours before termination of culture, 10ul CCK-8 solution was admoved to every well and continued to culture in the incubator for 3-4 hours.Thereafter,the absorbance was detected and determined the level of cell proliferation and the change of drug tolerance of ACHN cells after the gene MDR1 was silenced.(2)FCM was used to detect the level of apoptosis after the gene MDR1 was silenced. Culture the ACHN cells of good condition in the 6-well cultivation plate one day before infection.Admoveantur the RNAi lentivirus particles to the ACHN cells according to the designed groups on the day of infection.Five days later the cells were collected and PI dyeing FCM was used to detect the level of apoptosis after the gene MDR1 was silenced.(3)Using the Realtime PCR similar to the part of target screening to detect the expression of p53 and survivin after the gene MDR1 was silenced.Result:the growth curve of interfering group appeared conspicuous depression than the other two groups(blank group and NC group).The cells were obviously lack of vigor.This result revealed that the reproductive activity of ACHN cells weakened obviously after the gene MDR1 was silenced and the energy for growth of cancer cells was inhibited obviously,too. The cells induced tolerant to VCR changed its sensitivity to VCR after interference which means decreased drug resistance.Through the FCM we can gain the ratio of different period of cells,and it's discovered that the ratio of apoptosis cells in the ACHN cell colony obviously increased in the interfering group.Coherently,the realtime PCR outcome revealed the decreasing expression of p53 and survivin in ACHN cells of interference.Numerous research revealed the occurrence and development of the majority of disease owed to the gene mutation and modification,so how to repair or cut out the virulence gene specially has become the hot spots in the research of gene therapy.Due to the high specificity and high performance of RNA interference which the previous tools can not provide,it becomes the most frequently used means in the gene research increasingly.At the same time, similar to the previous researching tools,it's difficult to maintain the stable and long-term effect of intracellular RNA interference.In our research,we adopted the lentivirus vector aimed to the gene MDR1 and synthetized a shRNA recombination vector.The silencing effect in the human renal cell carcinoma cell line is specially,stable and high performance.Aider the gene MDR1 is silenced, we can find many changes in the biological behaviour such as the response to the chemicals enhanced significantly,reproductive cycle prolonged and ratio of apoptosis increased,etc.On account of the experimental result,we can really deem the gene MDR1 as the key gene in the renal cell carcinoma's multi-drug resistance.It may be the effective target for reversal MDR of renal cell carcinoma.At the same time,we also find the knockdown of MDR1 expression by siRNAs was associated with decreased expression of apoptosis inhibitor mtp53 and survivin, which suggests that MDR1 stimulates tumor growth and MDR at least in part by regulating mtp53 and survivin.At last,the system of lentivirus vector can assist the intracellular stable expression of shRNA fragment.It's an ideal vector for gene therapy.