Expression Of CD44 Transcript Variant In Gastric Cancer

BACKGROUNDGastric cancer?GC?is one of the most common malignancies in the world today,causing the third major death to be associated with cancer and seriously threatening the life and health of people around the world.Studies have shown that it can promote the proliferation,migration,invasion,metastasis and tumor formation of gastric cancer cells.As a tumor promoting cancer gene,CD44 may also be a pro-cancer gene in gastric cancer.This experiment mainly used RT-Nested PCR to detecte the expression of CD44 transcript variants of gastric cancer tissue and normal mucosa adjacent to carcinoma,and interfered the expressed CD44 transcripts into plasmid,cloned and sequenced.the relationship between each CD44 transcription variant with clinical pathological features of gastric cancer were investigated.Identificated the mechanism of the occurrence and development of gastric cancer,providing experimental basis for the targeted therapy.OBJECTIVETo investigate which CD44 transcript variant was espressed in tissue from gastric cancer and adjacent normal gastric mucosa.The relationship between the expressing transcript variant with the clinical pathological features and the mechanism of the process of gastric cancer were analyzed.METHODSCollected tissue from 3 advanced gastric cancer cases and the normal gastric mucosa of the corresponding case.Designed 2 pairs of primers,basing on the mRNA sequence of CD44 gene CDS containning a total length from NCBI,for RT-NPCR,and choosed the best expression of CD44 products of RT-NPCR,used agarose gel electrophoresis to identificate amplified products,extracted and purificated.Then connected with the cloning Vector?pMD18-T Vector?.Interfered the amplified bands into to the competent E.coli,culture in plate,take the positive clones for extracting plasmid and sequencing.The sequencing results were analyzed by DNAman.The identified CD44 transcripts in the first part were used as the DNA markers in the follow-up experiment.Collected tissue from 40 gastric cancer cases and corresponding normal gastric mucosa for RT-nested PCR.The products were compared by reference to DL5000 DNA Marker and the Marker of the expressed CD44transcript variants.Observed the expression of each CD44 transcription variants of all the specimens,and the relationship between the expression of CD44transcription variants in gastric cancer tissue and normal gastric mucosa with clinical pathological features of gastric cancer were analyzed.RESULTS1.The OD260/OD280 of the total RNA of the tissue from 3 advanced gastric cancer case and normal gastric mucosa were range from 1.8 to 2.0,indicating that the purity was fine and the total RNA was not mixed with protein or other sundry materials.The results of total RNA agarose gel electrophoresis showed that the RNA fragment was complete that 28S,18S,5S were clear and28S:18S>2:1.2.Referring to the RT-nested PCR product of GAPDH,the agarose gel electrophoresis results of the PCR products of the tissue from 3 gastric carcinoma cases and normal gastric mucosa showed that the product were clear and bright that the amplificated bands were 411bp and the amplificated bands of the No.3 was obviously optimal.3.The total RNA extracted from the No.3 gastric cancer case was use as template RNA for CD44 RT-NPCR and the products were identified by agarose electrophoresis.The results showed that the products had 4 different amplificated bands that were 2000bp,1500bp,1200bp and 700bp referring to the DL5000DNA Marker.Denoted them as No.1,2,3,4 transcripts;1 band,which was680bp,of the PCR of the normal gastric mucosa was showed and denoted it as No.5 transcripts.No other obvious band.4.The sequencing results showed that the homology between No.1transcripts and CD44v4 was 99.66%,and there were 3 bases changed,namely⁹¹⁷G?A?⁹²⁴-?C?⁹⁴¹A?C;The homology between No.2 transcripts and CD44v2 was 100%;The homology between No.3 transcripts and CD44v3 was99.66%,and there were 3 base changed,namely ¹²⁶³C?G,¹³⁶²G?C,¹⁵⁶¹G?T;The homology between transcription 4 and CD44v8 was 99.44%,and there were 3 base changed,namely ⁶⁵¹C?T,⁶⁵⁸C?G,⁸⁰⁷G?C;The homology between No.5 transcripts and CD44v7 was 98.87%,and there are 5 base changed,namely 460^C?T,⁴⁹⁷A?C,⁵¹⁰C?G,⁶⁹⁰A?G,⁷⁰⁰A?T.No obvious effect was showed the expression of protein changed of the changed bases by SnapGene analyzed.No.1 transcripts was CD44v4;No.2 transcripts was CD44v2,No.3 transcripts was CD44v3;No.4 transcripts was CD44v8;No.5 transcripts was CD44v7.5.The expression rate of CD44v4 of the tissue from 40 gastric cancer cases was 47.5%?19/40?;The expression rate of CD44v2 of the tissue from 40 gastric cancer cases was 25.0%?10/40?;The expression rate of CD44v3 of the tissue from 40 gastric cancer cases was 55.0%?22/40?;The expression rate of CD44v8of the tissue from 40 gastric cancer cases was 57.5%?23/40?;The expression rate of CD44v7 of the tissue from 40 normal gastric mucosa cases adjacent to carcinoma was 52.5%?21/40?;No other amplification bands were showed in the all specimens.6.CD44v4 was relative to lymph node metastasis??²=7.278,P=0.007??the degree of differentiation??²=11.235,P=0.001??invading depth??²=9.048,P=0.003?and TNM stage??²=14.593,P=0.000?,the difference was statistically significant?P<0.05?;CD44v4 was not relative to gender??²=0.067,P=0.796??age??²=0.000,P=0.987??the tumor size?2=2.434,P=0.119?and Lauren type??²=1.520,P=0.218?,the difference was not statistically significant?P>0.05?.CD44v2 was related to the tumor size??²=11.868,P=0.001??the lymph node metastasis??²=8.000,P=0.005??the degree of differentiation??²=8.000,P=0.005?and TNM stage??²=7.519,P=0.006?,and the difference was statistically significant?P<0.05?.CD44v2 was not relative to gender??²=0.000,P=0.640??age??²=1.637,P=0.501??Lauren type??²=1.671,P=0.196?and the invading depth??²=3.333,P=0.068?,the difference was not statistically significant?P>0.05?.CD44v3 was correlated with the tumor size??²=4.835,P=0.028??Lauren type??²=5.507,P=0.019??the lymph node metastasis??²=22.653,P=0.000??the invading depth??²=7.298,P=0.007?and TNM stage??²=22.922,P=0.000?,and the difference was statistically significant?P<0.05?.CD44v3 was not relative to gender??²=0.269,P=0.604??age??²=0.123,P=0.726?and the degree of differentiation??²=0.637,P=0.412?,the difference was not statistically significant?P>0.05?.CD44v8 was related to the tumor size??²=3.913,P=0.048??lymph node metastasis??²=32.471,P=0.000?and TNM stage??²=25.765,P=0.000?,and the difference was statistically significant?P<0.05?.CD44v8 was not relative to gender??²=0.017,P=0.896??age??²=0.474,P=0.491??Lauren type??²=0.251,P=0.616??the degree of differentiation??²=1.153,P=0.283?and the invading depth??²=1.637,P=0.201?,and the difference was not statistically significant?P>0.05?.CD44v7 was related to the tumor size??²=8.338,P=0.004??lymph node metastasis??²=16.047,P=0.000?and TNM stage??²=14.593,P=0.000?,and the difference was statistically significant?P<0.05?.CD44v7 was not relative to gender??²=0.067,P=0.796??age??²=0.000,P=0.987??Lauren type??²=0.351,P=0.554??the degree of differentiation??²=1.931,P=0.165?and the invading depth??²=2.030,P=0.152?,the difference was not statistically significant?P>0.05?.CONCLUSION1.5 CD44 transcript variants were expressed:CD44v2,CD44v3,CD44v4,CD44v8 and CCD44v7 were found expressed in gastric cancer.2.CD44v2,CD44v3,CD44v4,CD44v8 and CCD44v7 may be related to the occurrence and development of gastric cancer.