The Effect And Mechanism Of Mtdh On Biological Behavior, Chemosensitivity And Radiosensitivity Of Cervical Cancer

Cervical cancer is one of the most common cancers in women worldwide, second only to breast cancer. There are approximately500,000new cervical cancer cases in the world each year, and more than80%of these cases occur in developing countries. Our incidence of cervical cancer ranks second in the world. Although high-risk HPV infection is the main cause of cervical cancer, progression to cervical cancer generally takes place over a period of10to20years. The development of cervical cancer is a complex process involving multiple factors and multiple molecules. The current management for cervical cancer consists primarily of surgery, radiation and chemotherapy. Chemotherapy and radiation are two important methods to treat cervical cancer, mainly for the advanced, recurrence cases and patients who can't accept operation, or joint application with surgery. While these methods above has high cure rates in early stage disease, the survival rates in locally advanced cervical cancer cases remains poor. The exact mechanism of cervical cancer development has not been fully elucidated, and the treatment effect of it is needed to be improved. Therefore, looking for the molecular markers related to development and progression of cervical cancer and target molecules which could enhance chemotherapy and radiotherapy efficacy is the problem that we need to solve urgently.Metadherin (MTDH, also known as AEG-1) was originally reported as a protein induced in primary human fetal astrocytes infected with HIV-1or treated with either HIV gp120or TNF-α. MTDH has emerged in recent years as a potentially crucial mediator of tumor malignancy and a key converging point of a complex network of oncogenic signaling pathways. Recently, clinical studies have revealed that MTDH is overexpressed in various malignancies, including breast, prostate, glioma, hepatocellular, and esophageal cancer and is associated with disease progression and poor clinical outcomes. Moreover, MTDH has been found to promote cancer metastasis, invasion, chemoresistance and angiogenesis, suggesting that it may function as an oncogene. The role of MTDH in cervical cancer has not been reported. Furthermore, MTDH knockdown has been found to sensitize breast cancer and neuroblastoma cells to cisplatin. The mechanisms of radiotherapy and cisplatin to kill cancer cell are similar:causing DNA damage. So we speculate that MTDH may impact the radiosensitivity of cancer cells.Our study aimed to investigate the effect of MTDH on biological behavior, chemosensitivity and radiosensitivity of cervical cancer cell, in order to seek the new target gene which could improve therapy efficacy of cervical cancer. Our study was consisted of two parts. The first part:We knocked down endogenous MTDH using the pSuper-retro-puro vector containing short hairpin RNA (shRNA) against MTDH, and then investigated the effect of MTDH on proliferation, invasion, apoptosis, cell cycle distribution, chemosensitivity and radiosensitivity of cervical cancer cell. The second part:In order to provide new strategies that could improve radiosensitivity of cervical cancer, we studied the mechanism of MTDH regulation the sensitivity of radiotherapy in cervical cancer. PART I THE EFFECT OF MTDH ON BIOLOGICAL BEHAVIOR, CHEMOSENSITIVITY AND RADIOSENSITIVITY OF CERVICAL CANCERObjective: To investigated the effect of MTDH on biological behavior, chemosensitivity, and radiosensitivity of cervical cancer cell.Methods:(1) Expression of MTDH in cervical cancer cell lines SiHa,C-33A and HeLa was detected by quantitative reverse transcription and polymerase chain reaction(qRT-PCR) and Western Blot.(2) Construction of plasmid to knockdown MTDH:Endogenous MTDH was knocked down using the pSuper-retro-puro vector containing short hairpin RNA (shRNA) against MTDH.Construction of stably transfected cell lines:The cells were transfected with either shRNA against MTDH(prp-MTDH) or a control vector(prp-N), and mixed resistant clones were collected and cultured in medium containing puromycin. qRT-PCR and Western blot was used to detect the expression of MTDH in all of the cell lines described above.(3) Effect of MTDH on cell survival of SiHa cells was determined by MTT assay.(4) Cell cycle distribution and the proportion of apoptotic cells were detected by flow cytometry.(5) Effect of MTDH on invasion of SiHa cells was determined by Transwell cell invasion assay.(6) Effect of MTDH on chemosensitivity of SiHa cells was determined by MTT assay.(7) Effect of MTDH on chemosensitivity of SiHa cells was determined by clonogenic assay.Results: (1) The expression of MTDH in SiHa and HeLa was equal. And the level of MTDH in C-33A was lower than in SiHa and HeLa.(2) Stable cell lines that knockdown MTDH (SiHa/MTDH shRNA) and negative control(SiHa/shRNA Vector) was selected. Cells transfected with MTDH shRNA had significantly decreased levels of MTDH compared to the control groups at both the mRNA level and the protein level.(3) Knockdown of MTDH reduced the proliferation of SiHa cells.(4) MTDH had no effect on apoptosis and cell cycle distribution of SiHa cells.(5) MTDH had no effect on invasion of SiHa cells:the number of cells that pass through the Transwell membrane in SiHa/shRNA Vector and SiHa/MTDH shRNA was equal.(6) Knockdown of MTDH increased the sensitivity of SiHa cells to cisplatin, paclitaxel and5-Fu.(7) Knockdown of MTDH increased the sensitivity of SiHa cells to X-ray.Conclusion:MTDH had no effect on apoptosis, cell cycle and invasive ability of cervical cancer. Knockdown of MTDH reduced the proliferation of SiHa cells, and increased the sensitivity of SiHa cells to cisplatin, paclitaxel,5-Fu and X-ray. MTDH could be regarded as specific target molecules to potentiate the effectiveness of radiation and chemotherapy in the cervical cancer. PART Ⅱ THE MECHANISM OF MTDH REGULATION THE SENSITIVITY OF RADIOTHERAPY IN CERVICAL CANCERObjective:To investigate the mechanism of MTDH regulation the sensitivity of radiotherapy in cervical cancer. Methods:(1) The effect of MTDH on cell cycle distribution of SiHa cells with/without X-ray was detected by flow cytometry.(2) The effect of MTDH on apoptosis of SiHa cells with/without X-ray was detected by flow cytometry.(3) The effect of MTDH on expression of protein related with apoptosis in SiHa cells with/without X-ray was analyzed by Western blot.(4) The foci of y-H2AX caused by radiation was tested by immunofluorescence.(5) The effect of MTDH on expression of DNA damage repair proteins in SiHa cells with/without X-ray were analyzed by Western blot.Results:(1) Knockdown of MTDH decreased G2phase arrest induced by X-ray radiation. At10h post-radiation,38.11±0.75%of SiHa/shRNA Vector cells were arrested in G2phase when receiving2Gy of X-ray radiation, while only33.45±0.94%(p<0.05) of SiHa/MTDH shRNA cells arrested in G2phase at the same time after being treated with an equivalent dose of X-rays. The proportion of SiHa/shRNA Vector and SiHa/MTDH shRNA cells that arrested in G2phase without radiation was equal.(2) Knockdown of MTDH increased radiation-induced apoptosis. We evaluated the frequency of apoptotic cells at24h after X-ray radiation. The percentage of apoptotic cells was9.90±0.28%and12.59±0.52%(p<0.05) in SiHa/shRNA Vector and SiHa/MTDH shRNA cells, respectively, after exposure to2Gy of X-ray radiation. The proportion of apoptotic cells was3.51%and4.75%(p>0.05) before and after silencing of MTDH, respectively.(3) MTDH modulated the levels of Bcl-2after radiation:the expression of Bcl-2(an anti-apoptotic protein) in both SiHa/shRNA Vector and SiHa/MTDH shRNA cells was equal(p>0.05). Delivery of2Gy induced a significant upregulation in Bcl-2expression in SiHa/shRNA Vector cells. By contrast, radiation-induced upregulation of Bcl-2was not observed in SiHa/MTDH shRNA cells. (4) MTDH enhanced the repair of DNA double-strand break (DSBs) after irradiation: After the delivery of a single fraction of0.5Gy, the y-H2AX foci disappeared faster in the SiHa/shRNA Vector cells, with the vast majority disappearing at4h post-radiation, in comparison to the SiHa/MTDH shRNA cells, where the foci persisted for up to8h post-radiation.(5) Knockdown of MTDH resulted in a downregulation of Ku70. This difference was more obvious after radiotherapy, whereas no change was observed in the expression of the other repair proteins (Ku86, DNA-PKcs and Rad51).Conclusion:The mechanism of that MTDH knockdown increased the radiosensitivity of cervical cancer cells may include:(1) Knockdown of MTDH decreased G2phase arrest induced by X-ray radiation.(2) Knockdown of MTDH increased radiation-induced apoptosis.(3) MTDH decreased the repair of DSBs after irradiation.