| Gastric cancer remains one of the most common malignant tumors worldwide.It is the second most lethal disease in the world.It is also the leading cause of cancer related deaths in China,especially in Asian countries.In spite of the incidence of the tumor is lower than before,there are more than 1000000 newly diagnosed cases in the world and850000 cases of global death each year.The occurrence of gastric cancer is a complex process,including a variety of factors,a variety of steps,and the encoded and non coded genes.The risk factors of gastric cancer including Helicobacter pylori(Helicobacter pylori,H.pylori)infection,genetic factors,high salt diet,smoked food,smoking and drinking.These factors can cause normal gastric mucosa to superficial gastritis,atrophic gastritis,intestinal metaplasia,dysplasia,and ultimately the development of gastric cancer.H.pylori infection is one of the most important risk factors.As early as 1994,the WHO International Cancer Research Institute(IARC)identified H.pylori as class I carcinogen,but up to now,the exact mechanism of H.pylori causing gastric cancer is still unknown to humans.Although H.pylori was designated as carcinogen and clear H.pylori to prevent gastric cancer treatment has not been universally accepted,but modern epidemiological studies have shown that H.pylori play an important role in the development of intestinal type and diffuse type gastric cancer,some occur in up to 80% of non cardiac gastric cancer can be attributed to H.pylori.Epidemiological evaluation showed persistent infection of H.pylori for more than 10 years will lead to further development ofsuperficial gastritis and atrophic gastritis,along with the development of the disease,this will atrophy in gastric body from the pyloric gland extends to the fundic gland,in the next 10 years or longer will appear intestinal metaplasia.The infection of the H.pylori could increase the incidence of gastric cancer.H.pylori clearance can at least slow down the process of gastric atrophy or intestinal metaplasia,thereby reducing the risk factors of gastric cancer.Studies have shown that H.pylori infection can affect the expression level of mi RNAs,suggesting that H.pylori may play a role in the occurrence and development of gastric cancer by regulating gene level.However,few reports have been reported on how H.pylori mediates the occurrence of gastric cancer by mi RNAs.Mi RNAs is a small,noncoding RNA molecule,containing about 19-24 nt.Many genes encoding mi RNAs are simple copies,multiple copies,or gene clusters.Other forms exist in the silent region of the protein encoding gene or intron.They are highly conservative,temporary and organization specific.Although mi RNAs is not encoded as a protein,they can regulate the expression of genes at the post transcriptional level.By complementing with target m RNAs 3’-UTRs completely or completely,mi RNAs undergo chromatin recombination,silent specific genes,division,destruction of m RNAs,inhibition of post-transcriptional expression,etc.The expression of mi RNA regulating genes contributes to growth,differentiation,inflammation and oncology.At present,more than 24500 databases have been entered into mi R,indicating that there are more than 30000 mature mi RNAs products,and the number of mi RNAs is expected to increase in the future.The study found that more than 30% of the human genes were regulated by mi RNAs,and one mi RNA could regulate the expression of hundreds of RNA.The abnormal expression of many mi RNAs is closely related to the development of gastric cancer.Some studies have found that mi RNAs is not only a biological marker of tumor,but also a means of tumor targeting therapy.Micro RNA-222,a short mi R-222,was first found in umbilical vein endothelial cells(HUVECs)and played an important role in epithelial tumors.Studies have shown that mi R-222 has abnormal expression in a variety of tumor types.Besides,in the malignant glioma,papillary thyroid carcinoma,hepatocellular carcinoma,pancreaticcancer,breast cancer and colorectal cancer,the role of mi R-222 in cell proliferation is also affirmed.In vitro study found that mi R-222 can enhance cell proliferation and migration ability.However,a recent study found that mi R-222 could inhibit the growth of non-small cell lung cancer.In addition,in erythroleukemia,mi R-222 can inhibit the oncogene by down regulation of the c-kit gene.This suggests that the role of mi R-222 in the tumor may be paradoxical.In addition to its role in cancer,mi R-222 is related to many physiological and pathological processes,such as increasing cardiac hypertrophy and promoting the proliferation of pulmonary artery smooth muscle cells.At present,mi R-222 is seldom studied in the occurrence and development of gastric cancer,and its role and mechanism in gastric cancer are not completely clear.Starting from the clinical pathology of gastric cancer samples,this study first forecast 3-5 may target genes of mi R-222-3p through bioinformatics analysis,and then sieve H.pylori26695 positive and negeative gastric cancer tissue samples,to detect the expression of mi R-222-3p and potential target genes in H.pylori positive and negative gastric cancer tissues by q RT-PCR,to analyze the correlation between mi R-222-3p and target genes in the sample of H.pylori positive infection.Luciferase reporter assays and western blot were performed to verify the targeted relationship of mi R-222-3p and HIPK2.HIPK2 was detected by immunohistochemistry in different gastric cancer tissues,and HIPK2 sh RNA was used to interfere expression of HIPK2.For in vitro studies,CCK8 、 FACS and transwell assay were used to assess the function of mi R-222-3p and HIPK2 in gastric cancer progression.Further more,subcutaneous tumorigenesis in BALB/c nude mice were used to verify the function of mi R-222-3p and HIPK2 in vivo.Finally,to discuss the biological role of mi R-222-3p/HIPK2 signal patheway in the progression of gastric cancer infected by H.pylori.Part I The correlation between mi R-222-3p and H.pylori and clinicopathologic types in gastric cancer tissuesObjective: To investigate the correlation between mi R-222-3p and H.pylori and clinical pathological type,and verify that mi R-222-3p is involved in the pathogenic process of H.pylori26695 infection induced gastric cancer,and lay a preliminary foundation for the study of mechanism of gastric cancer.Methods: The expression of mi R-222-3p was detected by q RT-PCR method.The relationship between mi R-222-3p and clinicopthologic factors was analyzed using Spearman rank correlation analysis.Results: The expression of mi R-222-3p and gender,age,size,invasive depth and lymph node metastasis had no significant correlation(P>0.05),mi R-222-3p was significantly upregulated in Ⅲ and Ⅳ stage compared with Ⅰ and Ⅱ stage(P<0.05),which was positively correlated with TNM staging gastric carcinoma(P=0.007<0.01).mi R-222-3p was significantly upregulated in H.pylori(+)group compared with H.pylori(-)group(P<0.001).Conclusion: The expression of mi R-222-3p in gastric cancer tissue is positively correlated with H.pylori infection.The expression of mi R-222-3p in gastric cancer tissue is correlated with TNM stage of gastric cancer,which can be regarded as one of the evaluation indexes of gastric cancer in advanced stage.Part Ⅱ The targeted studies of mi R-222-3p and the function verification of target geneObjective: To investigate the noval target gene of mi R-222-3p,to verify the correlation of the expression of target gene and mi R-222-3p in gastric cancer tissues and H.pylori infection.To analyze the function of the target gene in gastric cancer cells.Methods: The expressioon of HIPK2,CPEB3 and PCDHA11 were detected by q RT-PCR.The luciferase reporter asseys experiment demonstrated the regulation of mi R-222-3p with potential target genes.Western blot analysis detected the taregeted regulation of mi R-222-3p on HIPK2.CCK8 method was used to detect the proliferation of the cells after HIPK2 sh RNA letivirus infection of SGC-7901.The cell apoptosis of SGC-7901 cells was detected by HIPK2 sh RNA letivirus infection.Transwell detected the invasion of SGC-7901 cells after the HIPK2 sh RNA letivirus infection.The expression of HIPK2 in gastric cancer tissue was detected by immunohistochemistry.Results: The expression of mi R222-3p in gastric cancer tissue was negatively correlated with HIPK2 and PCDHA11(P<0.05).There was no correlation between the expression of mi R-222-3p and CPEB3(P>0.05).The relative luciferase(Rluc/Fluc)in HIPK2-3’UTR luciferase plasmid transfection group after mi R-222-3p mimic transfection 293 cells was significantly lower than that of mimic NC(P<0.05).The expression of HIPK2 in SGC-7901 cells after transfection of mi R-222-3p mimics was significantly reduced(P<0.05).The expression of HIPK2 in SGC-7901 cells was not significantly reduced after transfection with mi R-222-3p inhibitors(P>0.05).Compared with NC sh RNA lentivirus infection,the proliferation rate of SGC-7901 cells increased significantly after HIPK2 sh RNA letivirus infection(P<0.001).The apoposis rate of SGC-7901 cells after the HIPK2 sh RNA lentivirus infection was significantly decreased(P<0.01).The invasion capacity of SGC-7901 was significantly increased after the HIPK2 sh RNA lentivirus infection(P<0.01).The HIPK2 immunohistochemical staining in H.pylori positive gastric cancer tissue was lower than that in H.pylori negative gastric cancer tissue.The expression of mi R-222-3p in tissue was negatively correlated with HIPK2(P<0.05).Conclusion: HIPK2 is a noval target gene of mi R-222-3p.Interfering with HIPK2 expression can promote the proliferation and invasion of SGC-7901 gastric cancer cell lines and inhibit its apoptosis.The expression of HIPK2 in gastric cancer tissues was negatively correlated with mi R-222-3p and H.pylori infection.Part Ⅲ The biological role of mi R-222-3p/HIPK2 axis in the progression of gastric canerObjective: To investigate the function of the mi R-222-3p in gastric cancer cells,the biological function of mi R-222-3p/HIPK2 axis in the development of gastric cancer in the two aspects of gastric cancer cells and nude mice experiment,providing a certain basis for gene therapy of gastric cancer.Methods: CCK8 method was used to detect the proliferation of the cells after mi R-222-3p OE and mi R-222-3p KD letivirus infection of SGC-7901.The cell apoptosis of SGC-7901 cells was detected by mi R-222-3p OE and mi R-222-3p KD letivirus infection.Transwell detected the invasion of SGC-7901 cells after mi R-222-3p OE and mi R-222-3p KD letivirus infection.The expressioon of mi R-222-3p was detected by q RT-PCR after LV-mi R-222-3p OE and LV-HIPK2 OE letivirus infection of SGC-7901.The expression of HIPK2 protein was detected by WB after LV-mi R-222-3p OE and LV-HIPK2 OE letivirus infection of SGC-7901.The effects of mi R-222-3p/HIPK2 axis on the growth,apoptosis and invasion of SGC-7901 were verified.The SGC-7901 cell subcutaneous inoculation was performed to establish a tumor model of the nude mouse,and the effect of mi R-222-3p/HIPK2 on the subcutaneous tumor of SGC-7901 was verified by using mi R-222-3p and HIPK2 lentivirus.Results: Functional experiments revealed that LV-mi R-222-3p OE significantly enhanced the proliferation and invasion(P<0.01),while inhibiting apoptosis of SGC-7901 cells(P<0.01),compared with the control vector of LV-NC.Conversely,LV-mi R-222-3p KD significantly inhibited the proliferation and invasion,but promoting apoptosis of SGC-7901 cells.Compared with LV-mi R NC group,the expression of mi R-222-3p in SGC-7901 cells after infected with LV-mi R-222-3p OE was significantly increased(P<0.05).Compared with LV-mi R-222-3p OE+LV-NC group,the expression of mi R-222-3p in SGC-7901 cells after infected with LV-mi R-222-3p OE+LV-HIPK2 OE was significantly decreased(P<0.05).The expression of HIPK2 in SGC-7901 cells after infected with LV-mi R-222-3p OE was significantly reduced(P<0.001).Meanwhile,western blot analysis revealed LV-mi R-222-3p OE+LV-HIPK2 OE markedly reversed downregulated HIPK2 in LV-mi R-222-3p OE group(P<0.001).Compared with LV-NC group,the viability of SGC-7901 cells after lentivirus infected with LV-mi R-222-3p OE was significantly accelerated(P<0.01).Compared with LV-mi R NC group,flow cytometry assays of apoptosis in gastric cancer cell line SGC-7901 after infected with LV-mi R-222-3p OE was significantly reduced(P<0.01).Compared with LV-NC group,the invision ability of SGC-7901 cells after infected with LV-mi R-222-3p OE was significantly increased(P<0.001).Morever,we examined the effect of co-overexpression of mi R-222-3p and HIPK2 on proliferation,invasion and apoptosis of SGC-7901 cells.All cell proliferation,invasion and apoptosis assay data indicated that the reintroduction of HIPK2 antagonized the effects of mi R-222-3p on SGC-7901 cells(P<0.001).LV-mi R-222-3p OE significantly increased tumor growth compared with control tumor(P<0.001),while the addition of HIPK2 inhibited the promoting effect of mi R-222-3p on tumor growth(P<0.001).Conclusion: mi R-222-3p target the expression of HIPK2.Overexpression of mi R-222-3p could promote the proliferation and invasion of the gastric cancer cells of SGC-7901,and inhibition of apoptosis.Reintroduction of HIPK2 antagonized the effects of mi R-222-3p.Mi R-222-3p/HIPK2 regulatory network provided a novel strategy for targeted treatment of gastric cancer. |
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