Effect Of HARD1Gene Silence By ShRNA On Growth Of Transplanted Tmour And Apoptosis Of Tissues Cells From Colorectal Adenocarcinoma In Nude Mice

Objective:human arrest defective1(hARDl),which is a kind of N-acetyltransferase (NAT), plays N-a-acetylcatalytic function;In the research of certain human tumors and other diseases, hARD1were studied from the gene to protein level, In recent years, it has gradually become the research hotspot-This study investigated that the effects of hARD1gene silence by shRNA on growth of xenografts and apoptosis of tissues from colorectal adenocarcinoma cells in nude mice. so as to provide experimental and theoretical basis for molecular targeting therapy of colorectal cancer.Methods:1. Cultivation of SW620cell strains.2.Construction and identification of recombinant plasmid:construction of hARD1interference plasmid (pENTRTM/U6-hARDl-shRNA), which is whether or not successfully constructed by sequencing technology.3.Cell transfection、Screening and identification of stably transfected cell clones:pENTRTM/U6-hARDl-shRNA and pENTRTM/U6-NC were transfected into SW620cells.by LipofectamneTM2000,Screening resistant cells from the culture medium by G418,which is extended culture. cell clones of interference plasmid as experimental group, cell clones of unrelated sequence plasmid as the negative control group, untransfected cells as control group; Observation of the situation of cells stably transfection by using inverted fluorescent microscopy.4.Construction of transplantation tumor model of human colorectal adenocarcinoma in nude mice: the nude mice were randomly divided into3groups(10rats in each group):experimental group, negative control group and blank group; injecting the three groups cells into the right scapular subcutaneous,To observe the growth status of transplanted tmour.5.To observe the changes of morphology of the tissue of transplanted tmour. and related organ by.HE staining6.Using the western blot to examine each group of transplanted tmour tissue cells’hARD1protein expression.7.Using the qRT-PCR technology to examine each group of transplanted tmour tissue cells’ hARDl mRNA expression.8.Using the TUNEL to examine each group of transplanted tmour tissue cells’situation of apoptosis.9.Using the transmission electron microscope to observe each group of transplanted tmour tissue cells’situation of apoptosis.10. Statistical analysis:The SPSS17.0statistical software package was used for all analyses. The date were expressed by mean士standard deviation[x士s]and analyzed using the ANVOA. The level of significant difference is a=0.05.Results:1.SW620were cultured successfully and good growth state in vitro.2.The recombinant plasmid(pENTRTM/U6-hARDl-shRNA) was constructed successfully,it was transfected successfully into human colorectal adenocarcinoma cells-SW620,after G418selects the stable expression cell lines,the transfection efficiency was higher than97%in both the experimental group and the negative control group by.inverted fluorescence microscope.3.Effect of hARDl gene silence by shRNA on growth of transplanted tmour from colorectal adenocarcinoma cells in nude mice.All of the xenografts have a good growth status in nude mice, Tumor formation rate of3groups were100%,After inoculation; xenografts volume were measured every3days, continuous25days.rendering the volume growth curve, the nude mice were killed by cervical dislocation after25d weighting there tumor; we found that the experimental group of minimum, it’s volume and weight is significantly less than the others group. The results were as follows:the experimental group compared to the control group at the volume and weight of transplanted tumor were statistically differences (P<0.05); the blank control group compared to the negative control group is no significant difference (P>0.05).4.Changes of the xenograft tumors’Pathological histologyAfter HE staining,there are different size and shape cancer cell mass in the xenografts of nude mice under optical microscopy, with small patchy or focal necrosis.we can see a small amount of fibrous connective tissue which form stroma component in the neoplastic cells of nude mice, Tumor cell unequal in size, most of the circular, hyperchromatic nuclei with occasional punctate nucleoli, marked pleomorphism, pathological mitotic are seen. Relevant viscera showed normal morphological changes after HE staining in these nude mice.5.Western blot examines each group of transplanted tmour tissue cells’hARD1protein expression.Through analysis the gray value of hARDl protein expression in three groups of transplanted tumor tissue The results show that the experimental group compared to the control group at the volume and weight of transplanted tumor were statistically differences (P<0.05); the blank control group compared to the negative control group is no significant difference (P>0.05).6.QRT-PCR technology examines each group of transplanted tmour tissue cells’hARD1mRNA expression.Extracting the total RNA from three groups of transplantation tumor, Designing and synthesizing primer.GAPDH as the reference gene, Homogenization to the hARD1mRNA, to the relative expression of hARD1mRNA was calculated by2-△△T method,The results show that the experimental group compared to the control group at the volume and weight of transplanted tumor were statistically differences (P<0.05); the blank control group compared to the negative control group is no significant difference (P>0.05).7.TUNEL examines each group of transplanted tmour tissue cells’situation of apoptosisUsing the TUNEL to examine each group of transplanted tmour tissue cells apoptosis The nucleus of apoptotic cells is labeled blue in experimental group, the other cells is labeled pale red, there are not positive cells in control group, the experimental group compared to the control group at the apoptosis rate is significantly increased.8.Using the transmission electron microscope to observe each group of transplanted tmour tissue cells’situation of apoptosisUsing the transmission electron microscope to observe three groups of transplanted tmour tissue cells’situation of apoptosis, we found:the nucleus of tumor tissue cells were large and irregular, nucleolus became enlargement and irregular,which is close to nuclear membrane, rich of euchromatin, organelles were reduced, the decrease in the number of mitochondria, we can see a few rough endoplasmic reticulum, nucleus mitotic figures is common, the ratio of nucleus and cytoplasm increases,whch is in the blank control group and negative control group, The above is typical morphological characteristics of malignant tumor cells. In the experimental group,we can see this phenomenon:cytoplasm pyknosis, cell volume became smaller, electron density of cytoplasmic increased, chromatin condense into different shape, which gathered on different parts of the nucleus, nucleus split into pieces, it produce different number and volume of apoptotic bodies; there is no exact apoptotic cells in blank group and negative control group.Conclusion:1.hARD1gene silence by shRNA can downregulate the expression of mRNA and protein of hARD1from xenografts of colorectal cancer cell in nude mice.2.hARD1gene silence by shRNA can inhibit the growth of transplanted tmour of colorectal adenocarcinoma in nude mice, To provide a theoretical basis of a molecular target treatment of colorectal cancer.3.hARD1gene silence by shRNA can induce apoptosis of xenografts tissue cell in vivo. To provides a theoretical basis that further research which roles hARD1plays in pathogenesis of colorectal cancer.