Research On Drug Resistant Function Of The Novel Gene HA117 In Cell Lines And In Nude Mice

ObjectivesTo research on the drug resistant function of the novel HA117 gene in human lymphoma cell lines (Raji cells) and in human colon carcinoma cell line(CW-2 cells) through gene transfection and RNA interference. And to investigate initially the possible mechanism how the HA117 gene could affect on the multi-drug resistance of CW-2 cells. As well to investigate the drug resistant function of HA117 gene further in vivo through establishing the nude mice subcutaneou transplanted carcinoma model.Methods1 Selecting the optimal siRNA target sites of HA117 gene through constructing the double clone recombined plasmid pSOS-HUS-HA117-HAi The pSOS-HUS DNA were extracted and digested by enzymes. Then it linked with HA117 gene to get the recombined plasmid pSOS-HUS-HA117;Five pairs siRNA target sites of HA117 gene were designed and synthesized and cloned them into recombined plasmid pSOS-HUS-HA117 respectively to construct five double clone recombined plasmid pSOS-HUS-HA117-HAi. Then the five kinds of double clone plasmids were transfected into 293 cells respectively. The optimal siRNA target sites were selected and validated through detecting and comparing intensity of green fluorescence by fluorescent microscope and flow cytometry, as well expression of HA117 gene mRNA was detcted by RT-PCR.2 Constructing RNA interferential vector pSES-HUS-HAi5 and Ad- HAi5 targetting to HA117 gene by recombinant DNA technology. The pSES-HUS DNA were extracted and digested by enzymes. Then it linked with the optimal siRNA target sites to get the RNA interferential recombined vector pSES-HUS-HAi5 targetting to HA117 gene. The plasimid pSES- HUS-HAi were transducted into the adenoviral homologous frame plasimid BJ-Adeasy to produce the adenoviral plasimid Adeasy-HAi. Then the Adeasy-HAi was transfected into HEK293 cells , and the recombined adenoviral Ad-HAi5 with high titer were gained through ping-pong infection.3 Investigating the drug resistant function of HA117 gene in Raji,CW-2 cells through gene transfection. The Raji cells were divided into three groups: the experimental group ( the Raji/HA117 cells with HA117 gene transfected), the RNA interferential group(the Raji/ HA117/HAi cells with HA117 gene and it's RNA interferential vector transfected), the blank control group (the untransfected Raji cells). The groups of CW-2 cells were same as Raji cells'. The gene transfection to Raji cells and CW-2 cells were carried out by transfected the reombind plasmid with lipidosome mediated and infected directly the recombind adenovirus, respectively. The transfection or infection efficiencies of cells in every groups were detected by fluorescence and flow cytometry. The HA117 gene mRNA expression levels of cells in every group were detected by RT-PCR. The drug sensitivities to daunorubicin(DNR), adriamycin(ADM), methopterin (CTX), vincristine (VCR), 5-fluorouracil(5-FU)of cells in each group were detected by MTT. The apoptosis rates of CW-2 cells in every group were detected by AnnexinV-FITC and flow cytometry. 4 Investigating drug resistant function of HA117 gene in vivo through establishing the nude mice subcutaneou transplanted carcinoma model. The transplanted mice colon carcinoma models were established by subcutaneous injecting CT26 cells which had HA117 gene drug resistance to the Balb/c nude mice. Seven days after injection when the diameter of the tumor reached about 5-10mm, 20 nude mice were divided randomly into exprimental group (10 mice) and control group (10 mice) . Nude mice in exprimental group were directly injected the recombined adenovirus Ad-HA117 suspension to the bearing-colon carcinomas. While the nude mice in control group were directly injected blank adenovirus Ad-null suspension to the bearing-colon carcinomas. After injected adenovirus 24 hours, nude mice in each group were received chemotherapy with intraperitoneal(i.p.) injection of 5-FU. After two weeks, the diameters of the bearing-colon carcinomas were measureed by type-B ultrasonic. The HA117 gene mRNA expressions in the bearing-colon carcinoma tissue of nude mice in each group were detected by RT-PCR. The effect of HA117 gene on the drug senstivity of mouse colon tumor cells in vivo was investigated through comparing the subcutaneou transplanted carcinomas volumes of nude mice in expremental group to of which in control group.Results1 Five double clone recombined plasmid pSOS-HUS-HA117-HAi were constructed successfully. The optimal siRNA target sites HAi5 was selected and validated from five siRNA sites targetting to HA117 gene by by fluorescent microscope , flow cytometry and RT-PCR.2 The RNA interferential recombined plasmid pSES-HUS-HAi5 targetting to HA117 gene was constructed successfully. After homologous recombined with BJ-Adeasy, it was pakaged to be the recombined adenovirus HAi5 in 293 cells. And the HA117 gene's RNA interferential recombind adenovirus Ad-HAi5 with 2.3×1011pfu/ml were gained through ping-pong infection.3 The drug resistance to DNR,ADM,VCR,CTX and 5-FU of the Raji/HA117 and CW-2 /HA117 cells with HA117 gene transfected were increased(p<0.05) 3～7 times than of which untransfected Raji and CW-2 cells respectively. Campared to of which untransfected Raji and CW-2 cells, the drug resistance to the five kinds of drugs above-mentioned of Raji/ HA117/HAi cells and CW-2/HA117/HAi cells with HA117 gene and it's RNA interferential vector transfected were no significant difference (P>0.05), respectively.4 The apoptosis rate of CW-2 /HA117 cells with HA117 gene transfected was 6.77% and lower than the untransfected CW-2 cells'apoptosisi rate 11.47%(P<0.05).Campared to of untransfected CW-2 cells the apoptosis rate of CW-2/HA117/HAi cells with HA117 gene and it's RNA interferential vector transfected was 12.06%, and no significant difference (P>0.05).5 After injected CT26 cells seven days, the inoculated positions of nude mice developed subcutaneous nodules which diameters were between 5mm and 10mm, and the subcutaneou transplanted carcinoma models of nude mice were established successfully. There was no significance difference between tumor volume of carcinoma-bearing nude mice in experimental group and of that in control group before chemo(P>0.05). After used chem tow weeks , the tumor volume of carcinoma-bearing nude mice in control group exceeded than of which in control group(P<0.05). Conclusions1 The optimal siRNA target sites HAi5 that had strongest RNA interferencial effect tagetting to HA117 gene was selected and validated from five pairs siRNA target sites of HA117 gene, which pave the way for silencing HA117 gene expression successfully using RNA interference technology.2 The RNA interferential recombined plasmid pSES-HUS-HAi5 and recombined adenovirus Ad-HAi5 which express the selected optimal siRNA sites targeting to HA117 gene were constructed successfully. The titer of the abtained recombined adenovirus Ad-HAi5 was 2.3×1011pfu/ml.3 The multi-drug resistance to DNR,ADM,VCR,CTX and 5-FU of the Raji and CW-2 cells expressing highly exogenous HA117 gene increased, while the multidrug resistance of Raji and CW-2 cells were disappeared after the exogenous HA117 gene expression silenced, Which indecated that the novel gene HA117 the multi-drug resistance of Raji cells and CW-2 cells and HA117 gene might have multi-drug resistant function. And the possible mechanism how the HA117 gene could affect on the multi-drug resistance of CW-2 cells might be through inhibitting CW-2 cells apoptosis.4 The novel gene HA117 could increase the resistance of the nude mice subcutaneou transplanted carcinoma to 5-FU in vivo, which indicated further that the HA117 gene might have drug resistant function and be one of important genes which regulated development of tumor cells'drug resisitance.