The Effection Of Radiosensitivity Of HeLa Cells In Cervical Cancer After Silenced TPX2 By SiRNA

Objective Radiation is the main treatment for advanced cervical cancer,which has no way to operate.The radiosensitivity and radiosensitivity-related therapy was connected with post-radiosensitivity DNA damage,rehabilitation and multi-gene regulation in the tumor.Targeting protein for Xenopus kinesin-like protein 2(TPX2)is a microtubule-associated protein,which is mediated by the cell cycle.It plays a central role of the spindle assembly.It has been demonstrated that TPX2 overexpression induces the amplification of centrosomes and results in DNA polyploidy.TPX2 plays a role in the cellular response to DNA double strand breaks induced by ionizing radiation.The level of TPX2 is co-related to the response of the DNA damage.We studied on the expression of TPX2 and the radiosensitivity after silence the TPX2 gene of human cervical cancer,while it provides reference for further investigation on radiosensitivity of human cervical carcinoma.Method This article takes HeLa cells of cervical cancer as research object.On the basis of the principle of si RNA,it was designed and synthesised by Ribo Company that three kinds of si RNA targeting TPX2 gene(si RNA-1,si RNA-2,TPX2-3)and one negative control si RNA.si RNA was transfected into HeLa cells with ribo FECTTMCP.Total RNA was extracted after 48 h.To observe the change of mRNA and protein,RT-PCR and Western Blot was used.4Gy X-rays was exposed to HeLa cells after transfection.With silencing TPX2 gene,radiosensitivity of HeLa cells was observed by CCK8 and FCM.Results After 48 h,total RNA was exacted from transfected HeLa cells.It was RT-PCR that the relative expression of TPX2 mRNA was detected by.We found that three kinds of TPX2-si RNA can silence the expression of TPX2,especially si RNA-2.Compared to the control group(1.000)and negative group(0.981 ± 0.814),the expression of si RNA-2 group(0.133±0.008)is lowest,which has significant difference(P<0.05).The HeLa cells were cultivated about 48 h after transfection.All proteins were isolated from the cells.Western-Blot was used to detect the relative expression of TPX2 protein.Si RNA-2 group(0.518±0.685)represented the lowest expression of protein,comparing with the control group(0.064 ±0.794)and negative group(0.981 ±0.814).The result has significant difference(P<0.05).The radiosensitivity of tumor cells after transfection was detected:(1)According to the OD values,we found the viability of transfected cells was different.The viability of silencing TPX2 group is(0.147±0.003),with negative group(0.224±0.009)and control group(0.353±0.012).With analysis of variance,we discovered silenced TPX2 group has significant difference when compared to the control group and negative group(F=9.243,P=0.0015).(2)We find that the apoptosis rate of every group showed a decrease.In addition,TPX2 silenced group has a highest apoptosis rate(12.68±0.03%)than negative group(9.37±0.11%)and control group(5.81±0.21%).With analysis of variance,we discovered the result was significant(F=10.242,P<0.0001).Conclusion The si RNA targeted at TPX2 can really inhibit the expression of TPX2 in HeLa cells.More important,it can increase the sensitivity of HeLa cells with silencing TPX2 gene.