Experimental Study Of Proliferation Of Gastric Cancer Metastasis Of MiR-9, Let-7b And Let-7g Cthrc1 Gene Regulation Affected

Gastric cancer is one of the most common cancer, especilly in Asia included Korea,Japan and China.And gastric cancer is the second leading cause of cancer-related death worldside,which is the first in China.In2008, the number of new cases of stomach cancer was989,000and the estimated death reached738,000worldside.H.pylori (HP) is condsided as the I class carcinogen of gastric cancer.MicroRNAs (miRNAs) are small non-coding RNAs of20-22nucleotides that regulate expression of target mRNAs at post transcriptional level. MiRNAs are involved in crucial biological processes, including development, differentiation, apoptosis and proliferation.Accumulating evidence suggests that alterations of miRNAs expression may play various roles in the pathogenesis of many human cancers. Some miRNAs have been shown to possess oncogenic or tumor suppressor activity.Collagen triple helix repeat protein (collagen triple helix repeat containing1,Cthrc1) gene was first found highexpresion in the process of arterial injury. Cthrcl can reduce the deposition of collagen and promote cell migration. Recently,Cthrc1was revealed overexpression in human cancers such as breast cancer and melanoma, involved in tumor invasion and metastasis, but the exact mechanism remains unclear.In the current study, we screened the miRNAs expression profile in gastric cancer using Taqman Low Density Array (TLDA) chips followed by individual quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assays in tissues and cells respectivelly.We further invesgated the mechanism of these miRNAs regulating the proliferation and metastasis of gastric cancer cell.Methods: Aberrant miRNAs in gastric cancer1.384miRNAs expression profiles in8pairs of gastric cancer tissues were analyzed using microarray assay,the obviously dyregulation miRNAs were confirmed by real-time RT PCR in the gastric cancer tissues in47cases which29HP(+) cases and18HP(-) cases.2. The expression levelsin SGC-7901and GES-1cells were further analyzed by real-time PCR,which in cells after HPstimulated were also examined.miR-9, let-7b and let-7g suppress Cthrcl expression as tumor-suppressor gene in gastric cancer1. We used three microRNA (miRNA) target prediction programs, and information from the existing literature to predict the target genes of miR-9, let-7b and let-7g. To test the Cthrcl expression in gastric cancer and adjacent normal tissues, immunohistochemistry was performed in47pairs of gastric tissue samples, and then we analyzed the clinicopathological characteristics in47gastric tumors;Then we investigated Cthrcl expression by qRT-PCR and western blot in gastric cancer cell lines.2. Transfection in vitro with mimics or inhibitors of miR-9, let-7b and let-7gwas used to observe the binding relationship of the miRNAs with Cthrc13’-UTR via luciferase reporter assay test, western blot was detected Cthrcl protein levels.3. After constructingCthrc1over-expression/RNA interference lentiviral vectors, we screened the stable Cthrcl-overexpressing and Cthrcl-knockdown gastric cancer cell lines.The cellular growth activity was measured by MTT assay.The cellular migration and invasion ability were detected by transwell assay.Results:Aberrant miRNAs in gastric cancer1. Ten miRNAs were down regulated in gastric cancer(>2folds),included let-7b,let-7g,miR-9,miR-133a,miR-133b,miR-139-5p,miR-141,miR-145, miR-204and miR-212.2. The miRNAs (miR-9, let-7b,let-7g and miR-204) were identified and consistently validated to be significantly down-regulatedin47paires of gastric cancer tissues. Meanwhile, we also found that the expression levels of miR-9,let-7b and let-7gwere decreased in H.pylori infection samples.3. The levels of miR-9,let-7b and let-7g were also siginificantly decreased in SGC-7901cells,which were especially after being infected with HP.miR-9, let-7b and let-7g suppress Cthrcl expression as tumor-suppressor gene in gastric cancer1. Using bioinformatic tools, we predicted that Cthrc1might be the most potent target gene of themiR-9, let-7b and let-7g. In addition, we found that Cthrcl expression is dramatically increased both in gastric cancer tissues and cell lines, and is also related with lymph node metastasis.2. Cthrc1was the direct target gene of miR-9, let-7b and let-7g via transfecting the miRNAs mimics, inhibitor and luciferase reporter assay.3. Cthrcl knockdown and overexpression markedly affected the proliferative ability of gastriccancer cell lines.Overexpression of Cthrcl promoted the migration and invasion abilities of GES-1cells. Whereas knockdown of Cthrcl attenuated the migration and invasion ability ofSGC-7901cells.Conclusion:1. miR-9,let-7b and let-7gwere down expressed in gastric cancer tissuesandSGC-7901cells.ThesemiRNAswere low-regulated in HP infectionsamples providing a new direction to study the relationship between HPinfection and gastric cancer.2. Cthrc1was the direct target of miR-9, let-7b and let-7g. 3. Down-regulated miR-9, let-7b and let-7g reduced the inhibition of Cthrcl,which inducedthe expression of Cthrcl and promoted themigration and invasion abilities of gastric cancer cells.