RNAi Targeting STAT3 And ErbB2 Combined With Radiotherapy For The Treatment Of Glioma

Cerebral tumor,also known as intracranial tumor,often occurs slowly and aggravates gradually.The most common type of primary intracranial tumor is glioma(40%).At present,the major approaches to treat the disease are surgery, radiation therapy and chemotherapy.Since single method usually could not obtain an ideal therapeutic effect,combination of surgery,radiation therapy and chemotherapy is a routine choice.And the effect of radiation therapy and chemotherapy will influence greatly on the prognosis of the patient.So how to increase cell sensitivity to radiation and chemotherapy is a research focus.Since normal tissue surrounding the tumor will be damaged unavoidably during the course of routine treatment,an ideal approach to treat cerebral tumor is to selectively kill tumor cell with little effect on normal tissue.RNAi can silence specific gene expression in highly sequence-specific manner. RNAi may have promising therapeutic effect on diseases caused by upregulation of certain gene.Tumorigenesis usually correlate with the overexpression of some protoncogenes,RNAi targeting these genes may specificly affect tumor cells with no harm to normal cells.Determination of eligible target gene is key to ideal therapeutic effect.STAT3(signal transducers and activator of transcription 3)belongs to STATs protein family.STATs are a family of 7 transcription factors activated by polypeptide ligand,such as cytokine and growth factor.STAT3 is consistly activated in many tumors with important roles in development and aggravation of tumor.In addition, STAT3 can affect therapeutic effects of radiotherapy and chemotherapy on human pulmonary adenoma and rectal carcinoma.ErbB2 is a member of the ErbB receptor tyrosine kinase family.ErbB family are located on cell surface and also includes EGF-R(ErbB1),ErbB3 and ErbB4.Many studies have suggested that ErbB2 was highly expressed in tumor cells,and its expression level is used as pathological classification parameter and prognostic indicator of breast cancer.Ricardo et al suggested that downregulation of ErbB2 could increase radiosensitivity of tumar cells. It is known that tumor growth result from increased cell proliferation ability and cell apoptosis result in decreased tumor mass.Changes in tumor volume reflect dynamic equilibrium between cell proliferation and apoptosis.It has been shown that STAT3 and ErbB2 could maintain cell proliferation of many tumors.Based on tumor specific expression of STAT3 and ErbB2,their facilitation of cell proliferation,and their relationship with tumor radiotherapy and chemotherapy,STAT3 and ErbB2 were selected as targets of RNAi combined with radiotherapy for the experimental treatment of glioma.Data are as follows:1.To construct and screen RNAi plasmid vector,and to verify the silencing effectWe designed the target sequence of STAT3,ErbB2 and GFP control and constructed pSilence2.1-U6-STAT3,pSilence2.1-U6-ErbB2 and pSilence2.1-U6-GFP plasmids using pSilence2.1-U6-H1 vector,and then transfected them into U251 cell,a kind of human brain glioma cell line.The results of western blot assay and Real-Time PCR assay suggested that there is a 8-fold to 14-fold decrease in STAT3 and ErbB2 protein and mRNA expression.2.The synergism effect of STAT3 RNAi and ErbB2 RNAi and the combining effect of RNAi with radiotherapyColony formation assay is the golden standard to assess the survival state and reproductive activity of cells.Cloning efficiency assay is often used for study of cell radiosensitivity.In this study,to determine the dose ofγ-ray used in this study,the cloning efficiency of cells was firstly analyzed after the cells were exposed to ⁶⁰Coγ-ray of different doses.According to the result of the cloning efficiency assay,the dose of 2Gy was defined as the dose of treatment.The result of MTT which was conducted to assess the ability of cell proliferation after exposure of ⁶⁰Coγ-ray of different dose further verified the result of cloning efficiency assay.Next,the study was divided into 10 groups,including the following:control,pSilence2.1-GFP,pSilence2.1-STAT3, pSilence2.1-ErbB2,pSilence2.1-STAT3+pSilence2.1-ErbB2,control+2Gy, pSilence2.1-GFP+2Gy,pSilence2.1-STAT3+2Gy,pSilence2.1-ErbB2+2Gy and pSilence2.1-STAT3+pSilence2.1-ErbB2+2Gy group.The cloning efficiency assay was conducted to determine the effect of different treatments on cell proliferation ability. The Result of the study including the following:(1)Both STAT3 RNAi and ErbB2 RNAi could inhibit the proliferation of U251 cell, and they could take effect synergistically (2)Both STAT3 RNAi and ErbB2 RNAi could enhance the radiosensitivity of U251 cell,and the U251 became more radiosensitive when STAT3 RNAi and ErbB2 RNAi was used simultaneously.3.The effect of STAT3 RNAi and ErbB2 RNAi on the U251 apoptosis and cell cycleIt is well known that the tumorigenesis is mainly due to unlimited cell proliferation following cell cycle disturbance.Then,what was the cause that led the proliferation of U251 cell under control after transfecting the pSilence2.1-STAT3 and pSilence2.1-ErbB2 into U251 cell? Would there be the same effect after transfecting the plasmids into normal astrocyte cell? To clarify the two problems,the primary astrocyte cell(named NA cell)was isolated from the brain mantle of new born(within 24h)rat,and cultured to prepare for study.Firstly,Hoechst 33258 stain assay was conducted,and apoptosis-specific nuclear condense phenomenon was observed in the study after the U251 cell was transfected with pSilence2.1-STAT3 or pSilence2.1-ErbB2,and the phenomenon became more aggravated in a time-dependent manner.But the nuclear condense phenomenon didn't appear after the plasmids were tansfected into NA cell.The DNA ladder phenomenon was also observed after transfection.At the same time,the Annexin-V kit was used for quantifying the cell apoptosis.The result suggested that:U251 cell appeared significant apoptosis phenomenon in a time-dependent manner after it was transfected with pSilence2.1-STAT3,and rate of the apoptosis cell achieved the peak 48 hours after transfection with a rate of about 50%.The U251 cell transfected with pSilence2.1-ErbB2 didn't appear significant cell apoptosis until 48 hours after transfection.Any plasmid couldn't induce the NA cell apoptosis at any time after transfection.The cell cycle was also examined by flow cytometry.The result suggested that:the U251 cell was arrested in S and G2-M phase at 12 hours point after the cell was transfected with pSilence2.1-STAT3 or pSilence2.1-ErbB2;the U251 cell was arrested in G0-G1 phase at 24 hours point after the cell was transfected with pSilence2.1-STAT3 or pSilence2.1-ErbB2;the U251 cell was arrested in s phase at 36 hours point after the cell was transfected with pSilence2.1-STAT3 or pSilence2.1-ErbB2,Interestingly,there was no cell existing in G2-M phase at this time point after transfecting pSilence2.1-STAT3 or pSilence2.1-ErbB2 which indicated the U251 cell stopped dividing.4.The research of STAT3 RNAi and ErbB2 RNAi apoptosis mechanism To clarify the apoptosis mechanism,the levels of caspase3/7,caspase8 and caspase9 in U251 cell and NA cell were examined at different time point after transfection.The effect of Z-VAD-FMK(broad-spectrum caspase inhibitor)and AC-DEVD-CHO(caspase3/7-specific inhibitor)was also observed after adding them to the transfected cell.Mitochondria membrane potential was examined with or without Z-VAD-FMK and AC-DEVD-CHO after transfetion.ROS and MDA levels were also examined after transfection.Comet assay was conducted to detect the DNA double-strand breaks after transfection.The apoptosis-related protein was examined by western blot.The data of these studies were as fllowing:(1)Caspase3/7 were activated during the cause of apoptosis induced by STAT3 RNAi and ErbB2 RNAi, and Mitochondria membrane potential declined at the same time;Z-VAD-FMK could inhibit the activation of caspase3/7 significantly and most of the cell apoptosis,and it could weaken the decline of mitochondria membrane potential.All of the above result suggested that the apoptosis induced by STAT3 RNAi and ErbB2 RNAi was caspase-dependent.(2)Mitochondria apoptosis pathway and death receptor pathway were all involved in the apoptosis induced by STAT3 RNAi and ErbB2 RNAi.The level of caspase8 and caspase9 increased after transfection and the increase could be inhibited mostly by Z-VAD-FMK and AC-DEVD-CHO.(3)STAT3 RNAi could induce the increase of ROS level,and ErbB2 RNAi could induce the increase of MDA level.Not only STAT3 RNAi but also ErbB2 RNAi could induce DNA double-strain breaks.These results indicated that ROS and DNA double-strain breaks were involved in the cell apoptosis induced by RNAi.(4)The down regulation of bcl2 and survivin protein was observed after pSilence2.1-STAT3 and pSilence2.1-ErbB2 transfection.The innovation of the study include the following:knockdown of 2 target genes, STAT3 and ErbB2 whose expression were high in tumour cell but low in normal cell,combined with radiotherapy to enhance the radiosensitivity of U251 cell,which was a combining therapy and more targeted and effective than single therapy.