| Objectives: Heat shock protein 70 (HSP70) is highly expressed in various tumors, cervical cancer is also included, idicating that the gene plays an important role in the development of cancer. Studies demonstrate that the continuous proliferation of tumor cells require large amounts of HSPs as a"molecular chaperone"to regulate and stabilize the abnormal proliferation. HSPs participated in cell cycle regulation and DNA damage repair are closely related with cell cycle, concerned with tumor occurrence, development, immunity and tolerance to the body of the cancer treatment,as well as the prognosis. To further understand the role of HSP70 in the occurrence and development of cervical cancer, this paper observes the effects of HSP70 on proliferation and apoptosis of HeLa cells by using siRNA sequence of gene-specific HSP70 to clarify the role of HSP70 for the treatment of cervical cancer, and to provide a new therapeutic target in clinic. Methods: 1.Design of siRNA against HSP70 was cloned into the vetor pTZU6+1 transformed into DH5αstrain, and then the recombinant was identified by restriction enzyme analysis and sequencing. 2.The recombinant was transiently transfected into HeLa cells by LipofectamineTM2000, the cells were divided into three groups:the first group transfected the recombinant of pHSP70-siRNA as the experimental group, the second group transfected empty vector pTZU6+1 as the negative control group, the third group was without transfenction reagent and plasmid as the blank control group. HeLa cells were extracted total RNA and protein of each group after 48h of transfection respectively, the expression of HSP70 was detected by RT-PCR and Western blot. 3. After transfection of 24h,48h,72h, the cell inhibitory rates were detected by MTT. The transfected cells of each group were collected after 48h, using AO/EB double staining method to detect the morphological changes of cells, and using flow cytometry to detect the recombinant of pHSP70-siRNA effects on apoptosis and cell cycle distribution of the cells.Results:1.The recombinant of pHSP70-siRNA was identified by restriction enzyme digestion and sequencing analysis showed that the recombinant was successfully constructed and named pHSP70-siRNA. 2. After transiently transfected the recombinant into HeLa cells, the two bands were available using RT-PCR and 1% agarose gel electrophoresis. The ratio of HSP70/GAPDH of the experimental group was(0.35±0.03),while the negative group and the blank control group were (0.76±0.05),(0.66±0.07). The statistical analysis showed that the expression of HSP70 mRNA in the experimental group compared with the negative control group was statistically significan(tP<0.01), while the blank control group and the negative control group were not statistically significant(P>0.05). The results reflected that the recombinant of pHSP70-siRNA transient transfection of Hela cells can specifically reduce the HSP70 mRNA expression in Hela cells. The three groups of HSP70/β-actin intensity ratios were (2.46±0.39),(4.68±0.32),(4.57±0.22)through Western blot. Statistical analysis stated clearly that the experimental group was significantly compared with the negative control group(P<0.01), and the blank control group and the negative control group were not statistically significant ( P > 0.05 ) . The results showed that recombinant of pHSP70-siRNA transient transfection of cervical cancer cells can downregulate the HSP70 protein expression in the cells. 3. After transfection of 24h,48h,72h, the absorption values of cells were detected by MTT, calculating the cell inhibition rate of each group, at 24h, the cell inhibition rates of the experimental group,the negative control group and the blank control group were 10.35±0.42,4.11±0.16,3.58±0.31 respectively, at 48h, the inhibition rates of cells were 17.99±0.44,4.00±0.19,3.56±0.23 respectively, at 72h, the inhibition rates of cells were 15.97±0.61,3.97±0.16,3.63±0.33, these results showed that the cells of the experimental group were significantly inhibited compared with the negative control group(P<0.01), and at 48h, the cell inhibition rate of the experimental group was the highest, and the control groups were no significant difference(P>0.05). The MTT results showed that the recombinant of pHSP70-siRNA significantly inhibit the cell proliferation. The results of AO/EB double staining showed that the cells of the experimental group have had apoptotic morphology changes but the control groups did not change significantly. Flow cytometry detected cell cycle distribution of each group, the cells of the experimental group GO/G1,S,G2/M phase accounted for (51.76±0.80)%,(42.16±0.64)%,(6.09±0.16)% respectively, the negative control group GO/G1,S,G2/M phase accounted for (61.18±0.29)%,(36.76±0.21)%,(2.06±0.09)% respectively, the blank control group GO/G1,S,G2/M phase accounted fo(r60.70±0.35)%,(37.44±0.65)%,(1.85±0.30)% . These dates showed that the recombinant of pHSP70-siRNA resulted in the cells at the stage of G2/M and S increased and the cells at the stage of GO/G1 decreased, indicating that the recombinant of pHSP70-siRNA may block the cell mitosis and make the cells accumulate in the stage of G2/M and S to prevent the progress of cell cycle and inhibit the proliferation of the cells. Flow cytometry detected cell apoptotic rate, the apoptotic rate of each group were 37.07±0.75%,3.06±0.09%,3.04±0.07% respectively, and there was significantly apoptotic cells diploid peak in the flow chart of the experimental group.Conclusions: 1. The recombinant of pHSP70-siRNA was successfully constructed and identified by restriction enzyme digestion and sequencing analysis. 2. The recombinant of pHSP70-siRNA transient transfection of HeLa cells,①The recombinant of pHSP70-siRNA can inhibit the expression of HSP70,②The recombinant can inhibit the growth of the cells,③The recombinant makes large numbers of HeLa cells in S and G2/M phase, prevent the progression of cell cycle and have diploid apoptotic peak, indicating that The recombinant of pHSP70-siRNA may inhibit the growth and proliferation of HeLa cells through blocking mitosis. 3. Summary, HSP70 as a"molecular chaperone"plays a role of adjustment and stabilization in the proliferation of tumor cells, and it is related with tumor development,tumor immunity and tolerance to the body of the occurrence of cancer treatment and prognosis.
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