| The morbidity and mortality rates of gastric cancer(GC) rank in the forefront of various malignancies in our country, but its pathogenesis remains unclear. In recent years, a large number of studies have found that: the expression or dysfunction of long non-coding RNA(lnc RNA) is closely associated with the occurrence of human diseases, but it is still unclear whether lnc RNA participates in the occurrence,developing and prognosis of gastric cancer or not. On the basis of our results obtained by the measurement of the chip, we chose lnc RNA HOXA-AS2 as research object,and further study its expression in gastric cancer tissue samples; by transfection of si RNA against lnc RNA HOXA-AS2, inhibiting its expression level in gastric cancer cell lines, and study the influences to gastric cancer during cell growth, proliferation,migration, invasion and apoptosis when lnc RNA HOXA-AS2 reduce expression; then find and identify possible downstream molecules, preliminary study its correlation with downstream HOXA4 molecules. The results of this study will lay a foundation for further research on molecular mechanism during gastric cancer's occurrence,developing and treatments.Objective : to study lnc RNA HOXA-AS2's expression in gastric cancer and its correlation with clinical and pathological information; preliminarily research lnc RNA HOXA-AS2's influence on gastric cancer cell growth, proliferation,migration, invasion and apoptosis ability, and explore possible lnc RNA HOXA-AS2 downstream molecules, making a foundation for further understanding of Lnc RNA in gastric cancer's rule and mechanism.Methods:(1) Using Human lnc RNA Expression Microarray v3.0(Arraystar), detect differential expression Lnc RNAs(in Lnc RNAs' expression, gastric cancer analyzing and paired adjacent cancer tissues) from six pairs of qualified paired gastric cancer tissues and 5cm adjacent normal tissues outside cancer; choose lnc RNA HOXA-AS2 with significant differences(differences in multiples of 3.067)(P<0.05) to enlarge sample size validation: by Quantitative Real-time PRC, we will see if the expression of lnc RNA HOXA-AS2 in tissue samples is the same with in genechip, and analyze the correlation between the expression level and clinic pathological features, serum gastrointestinal tumor markers.(2) Artificial chemically synthesize two small interfering RNA(si RNA) by analyzing full-length lnc RNA HOXA-AS2, and transfect to gastric cancer cells, to verify its validity in gastric cancer cell lines. With non-transfected si RNA gastric cancer cell as blank control and transfected disorderly encoding si RNA gastric cancer cell as negative control, through cell would healing assay, cell proliferation assay, cell invasion and apoptosis assay to analyze, knock down and inhibit the expression of lnc RNA HOXA-AS2 and check its influences in gastric cancer cell's growth,migration, proliferation, invasion and apoptosis capacity.(3)Detect the expression of lnc RNA HOXA-AS2 downstream molecule HOXA4,and analyze their impact on the correlation between gastric cancer and HOXA-AS2 expression.Results:(1)Tissue and cellular level validate expression status: Detecting 80 pairs of paired gastric cancer tissues and adjacent normal tissues by quantitative real-time PRC, we find lnc RNA HOXA-AS2 was significantly over expressed in gastric cancer tissues;compared with paired adjacent cancer tissues, the overexpression rate is67.5%(average up-regulation rate with 5.7 times). This result is the same as the one we did in Method(1) by Human lnc RNA Expression Microarray v3.0(Arraystar).Real-time quantitative PCR results showed that: compared with normal gastric epithelial cell line GES-1 cells, lnc RNA HOXA-AS2's expression in four different gastric cancer cell lines(SGC-7901, BGC-823, MGC-803, AGS) is obviously up-regulated, the average expression multiples of 3 ~ 6 times, and the difference has statistic meanings(p<0.05). Further analysis shows that the up-regulation of lnc RNA HOXA-AS2 is unrelated with gender, age, histological grading, tumor size(max diameter), lymphatic metastasis, neural invasion, the depth of tumor invasion and serum concentration(CEA ? AFP ? CA19-9)(P > 0.05), but related with vascular invasion(P<0.05).(2) Function research: separately transfect si RNA against two different sites of lnc RNA HOXA-AS2, effectively inhibited the expression of lnc RNA HOXA-AS2 in gastric cancer cells, inhibition ratio above 50%; significantly reduced the proliferation of gastric cancer BGC-823 and AGS cells, and significantly reduced the migration of gastric cancer BGC-823 and SGC-7901 cells, and reduced the invasion of SGC-7901cells; compared with untransfected blank control MOCK and negative control NC(scramble si RNA transfection), these differences have statistic significance(P<0.05).Preliminary results suggest that the inhibition of the expression of lnc RNA HOXA-AS2 has no impact to gastric cancer BGC-823 cells' apoptosis, compared with the control cells, P>0.05.(3)Preliminary research to related downstream molecules: by searching lnc RNA HOXA-AS2 downstream molecules via NCBI database, we found that Homeobox A4(HOXA4) might be the effective downstream molecule corresponding to lnc RNA HOXA-AS2. Then we detected HOXA4 m RNA's expression level in gastric cancer tissues and adjacent tissues by real-time quantitative PCR, results showed: the expression of HOXA4 m RNA in gastric cancer was significantly up-regulated, with a multiple of 3.86, and the expression level of HOXA m RNA was significantly related to the expression level of lnc RNA HOXA-AS2(r=0.648, P=0.000).Conclusion:lnc RNA HOXA-AS2 is over expressed in gastric cancer tissues and cells.The overexpression of lnc RNA HOXA-AS2 is unrelated with gender, age,histological grading, tumor size, lymphatic metastasis, neural invasion, the depth of tumor invasion and serum concentration(CEA ? AFP ? CA19-9), but related with vascular invasion. Inhibit expression of lnc RNA HOXA-AS2 in gastric cancer can inhibit growth, proliferation, migration and invasion of gastric cancer cells. HOXA4 m RNA is over expressed in gastric cancer tissues, and significantly associated with HOXA-AS2. These results indicate that: lnc RNA HOXA-AS2 may involve in gastric cancer cells' occurrence and development through its impact on gastric cancer cells' proliferation, migration and invasion; HOXA4 is possibly a downstream molecule by HOXA-AS2 function; lnc RNA HOXA-AS2 can be used as gastric cancer's molecule marker and new target in treatment. This study may lay a foundation for better understanding of Lnc RNA's role and mechanism in gastric cancer. |
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