Effects Of Small Interfering RNA Interference Of Connexin37 On Subcutaneous Gastric Tumours In Mice

Section One Srceenig of efficacy Cx37-si RNA interfering sequencesObjective: To construct lentiviruses targeting mouse connexin 37(Cx37) small interfering RNA(si RNA).Methods: Design si RNAs according to Cx37 gene sequences, cotransfected with overexpression vector in HEK293 cells, detect the protein expression levels of Cx37 gene,and confirm the effective Cx37-si RNA sequences.Results: Western-blot analysis showed that two Cx37-si RNA sequences can effectively inhibit the protein expression of Cx37 gene. Cx37-si RNA-3 was choosed for further si RNA interfering sequences for lentiviruses expression vetors and lentiviruses constructions.Conclusions: Cx37-si RNA interfering sequences was successfully screened.Section Two Construction of lentiviruses targeting Cx37-si RNAs and subcutaneous gastric cancer tumours in miceObjective: To construct models of subcutaneous gastric cancer tumours in mice,lentiviruses targeting Cx37-si RNAs were injected effectively into the tumours, and confirm the inhibition expression of Cx37 genes.Methods: MFC cell lines were cultured in RPMI 1640 complete medium. 30 mice were subcutaneously inculated with 0.2 m L of the murine gastric cancer cell line suspension(2.5×107cells/m L) at the forelimb to establish the gastric cancer mouse model.And they were randomly divided into three groups:Cx37-si RNA group, mock-si RNA group and saline group. After the tumour inoculation, the body weights and tumour volumes were calculated. At the end of the experiment, the part of tumours were excised from the mice, immediately weighed and then stored in 10% formalin. The samples were later sectioned using a microtome, dehydrated in graded alcohol, embedded in paraffin,stained with haematoxylin and eosin, and examined using fluorescence microscope. The other part of tumors were used to extract total m RNA and protein. RT-PCR and Westernblot were performed to examine the m RNA and protein expression levels of Cx37 gene.Results: Through fluorescence microscope, GFP protein expression level was high,indicating that the lentiviruses targeting Cx37-si RNAs was successfully transfected. No significant difference was found between groups of mice body weight. The m RNA expression level of Cx37 gene in Cx37-si RNA group is significantly lower than those of mock-si RNA and saline group(P < 0.05). However, No significant difference was found between mock-si RNA and saline group. Western-blot showed that the protein expression level of Cx37 gene in Cx37-RNAi group was significantly lower than those of mocksi RNA and saline group(P < 0.05).Conclusions: Effective inhibition of Cx37 expression by small interfering RNA vector injected with Cx37-si RNA sequences can be found in subcutaneous gastric cancer tumours in mice.Section Three Effect of Cx37-si RNAs on proliferation and apoptosis in mouse subcutaneous gastric cancer tumoursObjective: To explore the effect of Cx37-si RNAs on proliferation and apoptosis in mouse subcutaneous gastric cancer tumours.Methods: According to section two, 40 female BALB/c-nu/nu mice were used to construct models of subcutaneous gastric cancer tumours and were randomly divided into four groups: blank group, Cx37-si RNA group, mock-si RNA group and saline group. Eight weeks after, CCK8 assay was used to detect cell proliferation of each group, Annexin VFITC, PI and Hoechst33342 were performed to observe the effect of RNAi for Cx37 induced early apoptosis and necrosis and the m RNA and protein expressions of Bcl-2 and Bax genes.Results: CCK8 assay showed that proliferation capability of subcutaneous gastric cancer tumours in Cx37-si RNA group was significantly lower than those of the three groups(P < 0.01). Annexin V-FITC and PI staining method showed that the apoptotic cell numbers in Cx37-si RNA group was significantly higher than those of the three groups(P <0.05). Hoechst33342 staining showed that the positive rate of pyknotic nuclei tumor cells was significantly higher than those of mock-si RNA and saline groups(P < 0.01). RT-PCR and Western-blot results showed that, the m RNA and protein expressions of Bcl-2 and Bax genes were significantly lower than those of mock-si RNA and saline groups(P < 0.01). No significant difference was found between Mock-si RNA group and saline group.Conclusions: Interference of Cx37 gene expression can effectively inhibit the proliferation and enhance the apoptotis of MFCs in mouse subcutaneous gastric cancer model.