| Objectives:1. To construct heparanase short hairpin RNA(shRNA) interference recombinant lentivirus-based vectors, and screen siRNA sequence of inhibiting HPSE gene expression in breast cancer cells in vitro experiment.2. Effection of invasive capacity of breast cancer cells was measured after inhibiting HPSE gene expression by use of transwell-room.3. To construct breast cancer model, HPSE-shRNA recombinant lentivirus injected nude mice transplant tumour. To study the feasibility of silencing of heparanase gene to therapy breast cancer by real-time PCR and Western blot testing the expressing for HPSE gene. This finding could provide an experimental basis for breast cancer gene therapy by using HPSE-targeted.Methods:1. Using lentivirus-based vectors, using a technique called RNA interference, screening siRNA sequence of inhibiting HPSE gene expression in breast cancer cells in vitro experiment. Accoding to the designing principle of RNA interference sequence, designed and composed five HPSE-shRNA sequence, and at the same time set up negative control sequence. Using pGPSV lentivirus-based vectors system constructed the expressing for HPSE-shRNA lentivirus-based vectors, and then identified by double enzyme cutting and sequencing. Packaging HPSE-shRNA recombinant lentivirus with 293T cells, and then conserved in the icebox of -70℃. Before transfection, virus titre checkedby ues of adopting limited dilution method, and recombinant lentivirus HPSE-602-shRNA1, HPSE-984-shRNA2, HPSE-1360-shRNA3, HPSE-1667-shRNA4, HPSE-1123-shRNA5, negative control transfected to each group of breast cancer MDA-MB-231 cell, respectively, but blank control group did not make whatever trestment. The expressions levels of HPSE mRNA and protein was measued in breast cancer MDA-MB-231 cells by real-time PCR and Western blot at 72 hours after transfection.2. Invasive capacity of breast cancer cells was measued by use of transwell-room: silencing of expressing for HPSE gene sequence of HPSE-602-shRNA1,HPSE-1667-shRNA4 were screened by real-time PCR and Western blot. Packaged to recombinant lentivirus, and transfected breast cancer MDA-MB-231 cells at 72 hours. And setting up negative control and blank control group,Then measuring the invasive capacity of breast cancer cells by use of transwell-room.3. To measure the expressing of HPSE after constructing breast cancer model and injecting recombinant lentivirus by real-time PCR and Western blot. Take 1×10~9 breast cancer MDA-MB-231 cells/ml single cell suspension 0.2ml injecting to the subcutaneous fat tissue of the offside armpit on female nude mice. Divided to four groups randomly with 32 nude mice when the diameter of the tumour was 1cm. Using multiple spot injected method, took recombinant lentivirus (titre 1×10~8TU/ml) carried by HPSE-602-shRNA1, HPSE-1667-shRNA4 and HPSE-negative control injecting each tumour, respectively. 400μl of the total dose each nude mice, 1 sequence every other day and four sequences in all. Recombinant lentivirus injected after 1 month, tumour tissue was cut off by operation. Every tumour was divided into two parts, one conserved into icebox at -70℃, and expression of HPSE mRNA was detected by real-time PCR. The other was fixed by formaldehyde, embed by paraffins, definited the pathology type of every sample by HE staining, and detected expression of HPSE protein by immunohistochemical method.Results:1. Five HPSE-shRNA sequence and negative control group sequence was designed and composed. It is proved that recombinant lentivirus-based vectors was successfully constructed. Transfecting breast cancer MDA-MB-231 cells at 72 hours, about 70% of cells emited green fluorescence in experimental group and negative control group. And the expressions levels of HPSE mRNA and protein was measured by real-time PCR and Western blot. Results showed:①The relatively expressions levels of HPSE gene of HPSE-602-shRNA1 and HPSE-1667-shRNA4 groups were effectively decreased much more than negative and blank groups(P<0.05) .②In contrast, those in negative control group were not significantly different from those in blank control group(P>0.05). It is showed that HPSE-602-shRNA1 and HPSE-1667-shRNA4 groups could effectively inhibit the expressions levels of HPSE of MDA-MB-231 cells in vitro.2. Invasive capacity of breast cancer cells was measured by use of transwell-room at 72 hours after HPSE-shRNA recombinant lentivirus-based vectors transfection. Results showed:①T he relative number of invasive cells in HPSE-602-shRNA1(6.2±0.73) and HPSE-1667-shRNA4(8.7±0.41) groups was significantly lower than negative control(27.1±2.14) and blank control(24.7±1.83) groups(P<0.05).②In contrast, those in negative control group were not significantly different from those in blank control group(P>0.05). It is showed that HPSE-602-shRNA1 and HPSE-1667-shRNA4 were effectively decreased the invasive capacity of breast cancer cells after inhibting the expressions levels of HPSE gene.3. Constructed breast cancer nude mice model successfully, cut off tumour tissue after injecting HPSE-shRNA recombinant lentivirus-based vectors, it is proved that 32 samples were all breast cancer tissue by use of HE staining. Expression of HPSE gene mRNA and protein were detected by real-time PCR and immunohistochemical method. Results showed:①The relatively expressions levels of HPSE gene mRNA and protein of HPSE-602-shRNA1 and HPSE-1667-shRNA4 groups were effectively decreased much more than negative and blank groups(P<0.05).②Those in negative control group were not significantly different from those in blank control group(P>0.05). It is showed that HPSE-602-shRNA1 and HPSE-1667-shRNA4 groups could effectively inhibit the expressions levels of transplanted tumour HPSE gene in vivo.Conclusion:1. The results indicate that the HPSE shRNA recombinant lentivirus-based vectors was successfully constructed, and the breast cancer cells were successfully transfected. And screened the HPSE-602-shRNA1, HPSE-1667-shRNA4 sequence inhibiting HPSE gene by real-time PCR and Western blot.2. It is proved that inhibiting expression of HPSE gene significantly weakened the invasiveness of breast cancer cells by transwell-room experiment after HPSE-shRNA recombinant lentivirus transfecting breast cancer cells.3. Successfully constructed breast cancer model, then after injecting HPSE-shRNA recombinant Lentivirus, it was inhibited the expression of HPSE gene of tumour tissue. It is showed that the feasibility of silencing of expression of HPSE for treatment breast cancer in vivo. |
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