Analysis of the effects of gene expression on cell proliferation response.

1, immune imprinting confirmed that SMU1 affected cell DNA DSBs injury response as a result of confirming immunofluorescence, and further used the Western blot method to detect the changes in the expression level of the protein of each group of cells after the treatment of DNA DSBs damage.
and IF results are basically consistent, SMU1 silent group cell endogenity of the H2AX protein expression level is significantly higher than the control siRNA group cells, and after the formation of DSBs, its gamma H2AX protein fade significantly slower than the control group cells.
2. Discuss the thousands of DNA damage that occurs every day in every cell of the human body, of which DNA DSBs are the deadliest DNA damage in the body, and if the cells are not repaired in time, they will eventually lead to serious consequences such as cell death or malignant transformation.
Higher gynthetic cells have evolved a series of mechanisms to repair DNA DSBs damage, and when DSBs are formed, the DNA damage response path is activated, and a variety of repair proteins are recruited to the fracture point to initiate the DNA damage repair process.
H2AX (histamine H2A variant) is an earlier protein involved in DNA damage response, can be recruited to double-stranded break point within 30 min, its 139-bit serine is upstream kinase ATM/ATR phosphorylation to form pH2AXs139 (H2AX), 30 to 60 min within its activation up to the apex.
H2AX, in collaboration with other repair proteins, gradually completes the repair process of damaged DNA, and with the completion of DSBs repair, the H2AX gradually fades from the damage site.
the number of foci is closely related to the number of cell DSBs, so the number of foci is closely related to the number of cell DSBs, so the H2AX is a sensitive indicator for detecting cell DSBs and can evaluate the dynamic change process of cell DNA DSBs injury response by detecting their dynamic changes.
SMU1 is a protein containing WD40 repeating domain, WD40 domain can mediat the interaction between proteins, in the function of protein plays an important role. The temperature-sensitive mutant type of
tsTM18 cells is caused by the 489th arginine replacement (G489R) of the SMU1 WD40 repeating region, which causes the cells to exhibit increased chromosomal fractures, abnormal spindle assembly, DNA single-stranded fractures, and decreased cell proliferation vitality at non-permissible temperatures.
this study used siRNA-mediated gene silencing to find that lower expression of SMU1 gene significantly affected cell proliferation, which is basically consistent with the above findings.
X-ray radiation is one of the most important causes of DNA double-stranded fractures. The required dose and sensitivity of damage for
H2AX analysis vary from cell to cell, tumor cell line often uses doses above 0.5 Gy to analyze changes in gamma H2AX, reference to relevant literature and pre-experiments have found that 2 Gy X-ray not only induces cells to produce significant expression of gamma H2AX, but also changes in their repair process are more obvious.
therefore, we used 2 Gy X-ray as the DSBs injury inducer, and the evaluation index of DSBs with the gamma H2AX as the evaluation index, systematically studied the effects of SMU1 on the DNA DSBs injury response response of cells.
Our results show that only a very small number of cells without exposure were formed by gamma H2AX, but SMU1 silent group cells showed significantly more foci; after X-ray treatment of 1 h, both groups of cells reached maximum value and no significant difference; and from 1 h to 8 h after treatment, the control group cells H2AX foci decreased sharply, while the SMU1 silent group cells H2AX foci decreased significantly.
further immunoprinting studies have found that the dynamic change process of the expression level of the gamma H2AX is basically the same as that of the immunofluorescent gamma H2AX foci.
these phenomena suggest: (1) SMU1 functional defects may cause genomic instability resulting in more endogentic DSBs, leading to endogenic gamma H2AX active; (2) Cells produce a large number of DSBs in a short period of time (1 h) after X-ray treatment, the active cell DSBs repair signal, under normal circumstances can quickly repair damaged DNA within a few hours, while SMU1 defective cells due to damaged repair function resulting in the long-term existence of DSBs.
, this study explores the role of SMU1 in cell proliferation and DNA DSBs injury response through cell counting, immunoprinting, and immunofluorescence. Preliminary clarification of siRNA-mediated SMU1 knock-downs can cause cell proliferation defects and changes in the dynamics of DNA DSBs injury response process, and we will further study and determine the key gene segments involved in cell DNA DSBs injury response by expressing a series of mutations.
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