Construction Of SiRNA Expression Vectors For Silencing Calcium Transport Protein Gene And Inhibitory Effect On Cervical Carcinoma Cell Line HeLa

Cervical cancer is one of the most common gynecology malignant tumors . Occupies the second in the global gynecology malignant tumors.There are about 131,500 new cases in our country,which is 28 . 8% of the whole world.There are two million women dies from it every year of the whole world,53,000 of which is in china.At present the main treatment for cervical cancer is surgery combined with chemotherapy and radiotherap,and the most effective chemotherapeutics is cisplatin (DDP),it destroys the structure and the function of DNA to treat cancer,which suits most of the advanced stage cervical cancer and the recrudescence case.But DDP has the obvious poisonous side effects on the bone marrow,liver,kidneys and other tissues and organs,and there is resistance.there are some difficulties in the clinical practice.Calcium transport protein (CaT1) is cloned from the small intestine in 1999,belong to the TRPV family,which is one of the main channels in the cell membrane to regulate the calcium ion into cell.Its main function is to transport calcium ion of the top of the epithelial to the epithelial cells.It is closed to Cell Proliferation.Study showed that the CaT1 plays an important role in the tumor occurrence and development .RNA interference (RNAi) is the process of sequence-specific gene silencing triggered by double-stranded RNAs(dsRNAs),which is a effective treatment to tumors.This mechanism can reduce the specific purpose of gene expression.At present,we have not yet seen related study that block the CaT1 gene by RNA interference technology to discuss its effect to cervical cancer cell proliferation, differentiation and apoptosis.Objective:Using RNA silencing technology block the cell CaT1 channel to observe its effect in chemotherapy drug DDP induced Hela cell apoptosis and proliferation, and to explore the mechanism of RNAi CaT1 effect in enhancing chemotherapy sensitivity.Method:1. Cell culture ;2. PSH1Si-CaT1 expression vector construction and testing of silence;3. The viability of cells was measured by MTT assay ;4. mRNA levels detectting of Bcl-2,Bax,AKT-1,IκB by RT-PCR.Result and Discussion:1. Identification of digestion and DNA sequencing analysis showed that CaT1 gene siRNA vector is correct, RT-PCR results show that CaT1 gene expression levels were significantly lower. 2. MTT assay showed that DDP can significantly decrease the survival rate of Hela cells in a dose-dependent manner,and the survival rate of the transfected cell is deceased compared with the negative control or untransfected group.It is anticipated that reduction of CaT1 levels could provide an advantage in increasing the sensitivity of Hela cells to DDP-mediated apoptosis.3. RT-PCR results show that the use of DDP alone does not alter the AKT-1 and IκBmRNA expression, but only increase the ratio of Bax/Bcl-2 .It indicated that DDP mainly through the apoptosis pathway to inhibite Hela cell proliferation.Bax/Bcl-2 increase obviously in transfected cell compared with the untransfected group. AKT-1 and IκB mRNA level decreased in transfected cell compared with the untransfected group.AKT also known as protein kinase B (protein kinase B, PKB), is a serine / threonine protein kinase, has been defined as oncogenes.It through phosphorylation of mTOR, GSK3, TSC2, Foxo-family and other promote tumor cell growth, proliferation, inhibition of apoptosis and promote cell invasion , metastasis and angiogenesis.It resistant cell apoptosis in the chemotherapy and radiation.After PSH1Si-CaT1 transfection, the expression of AKT-1mRNA is lower.It is indicated that CaT1 RNAi-not only through the promotion of apoptosis, but also by inhibiting cell survival to enhance the DDP sensitivity of the Hela cells. NF-κB system is composed by the NF-κB family and its inhibitor IκB family.IκB is the most important and direct signs of the activation of NF-κB,and its reduction means the activation of NF-κB. It is presumed that NF-κB may also be involved in enhancing the DDP sensitivity of the RNAi-CaT1 Hela cells.Conclusion:1. Our study successfully constructed a recombinant siRNA expression plasmid which can significantly inhibit the CaT1 gene expression of Hela cell.2. CaT1 gene silencing enhanced DDP-induced Hela cell apoptosis.It is that CaT1 gene silencing of Hela cell could provide an advantage in increasing the sensitivity of Hela cells to DDP-mediated apoptosis.3. The effect of CaT1 gene silencing in increasing the DDP sensitivity of Hela cells may be related to inhibiting the expression of AKT and IκB , and to direct or indirect regulation of apoptosis-related proteins of the Bcl-2 family (Bax/Bcl-2) expression.CaT1 gene silencing has synergistic effect in the process of DDP-induced apoptosis.