Experimental Oncology ��� �������� �������� ��ecem�er� 277 Exp Oncol ���� ��� �� ������� MICROARRAY BASED GENE EXPRESSION PROFILING OF ADVANCED GALL BLADDER CANCER A. Kumar1, R. Gupta1, N. Mathur1, V.K. Iyer2, S. Thulkar1, C.P. Prasad1, P. Das2, L. Rani1, M. Maqbool1, N.K. Shukla1, S. Pal2, D. Sundar3, A. Sharma1, * 1Dr. B.R.A., Institute-Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi 110029, India 2All India Institute of Medical Sciences, New Delhi 110029, India 3Indian Institute of Technology Delhi, New Delhi 110016, India Background: Gall bladder cancer (GBC) is an aggressive cancer with specific predilection like female gender and specific geographical areas, however the molecular mechanisms and factors contributing to the clinical or biological behavior are not understood. Aim: The aim of this study was to perform a comprehensive analysis of differentially expressed genes in advanced GBC and chronic cholecys- titis (CC) cases. Materials and Methods: Microarray was planned on fresh specimens of advanced GBC and CC cases using single color cRNA based microarray technique (8X60K format; Agilent Technologies, USA). Twelve advanced GBC and four CC patients were included in the study. Results: Of the total of 1307 differentially expressed genes, 535 genes were significantly upregulated, while 772 genes were significantly downregulated in advanced GBC vs CC samples. Differentially expressed genes were associated with biological processes (55.03%), cellular components (31.48%), and molecular functions (13.49%) respectively. The important pathways or key processes affected were cell cycle, DNA replication, oxidative stress, gastric cancer pathway. Using in silico analysis tools, three differentially expressed genes i.e. TPX2, Cdc45 and MCM4 were selected (for their significant role in DNA replication and microtubule function) and were further validated in 20 advanced GBC cohort by immunohistochemistry. Significant positive association of Cdc45 and MCM4 proteins was found in advanced GBC cases (p = 0.043), suggesting the probable oncogenic role of Cdc45 and MCM4 proteins in advanced GBC. Conclusion: Our data demonstrate the potential regulation of Cdc45-MCM4 axis in advanced GBC tumors. Additionally, our study also revealed a range of differentially expressed genes (e.g. TPX2, AKURA etc.) between GBC and CC, and further validation of these genes might provide a potential diagnostic or therapeutic target in future. Key Words: gall bladder cancer, chronic cholecystitis, microarray, Cdc45, MCM4. DOI: 10.32471/exp-oncology.2312-8852.vol-42-no-4.15476 Gall �ladder cancer �GBC� is highly lethal malig- microarray technology has led to the finding of new nancy. Its etiopathogenesis and the reason for its molecules in GBC. Study �y Kim et al. [5]� though aggressive �ehaviour is poorly understood. GBCs descri�e the comprehensive gene expression profile are often diagnosed at late stages and are therapy of GBC� �ut the study was not exclusively designed resistant [1]. The incidence of GBC demonstrates for advanced and incura�le GBC. Apart from gene marked geographic variation with female predomi- expression studies� there are various independent nance; for example� it is the single largest cause reports on deregulated protein profiles in GBC pa- of cancer death for women in Chile� �ut accounts for tients. In immunohistochemical study performed only < �.5% of cancers in women in the United States. �y Li et al. [6]� authors demonstrated the a�errant Chile and Bolivia �1� to 15 persons/1������ popula- expression of Skp-� and p27Kip1- proteins there�y tion/year� have the highest incidence rate of GBC conferring aggressive �ehavior to GBC. In a similar worldwide [�]. Much of the geographic variation cor- immunohistochemical study� expression of p53� relates with the tendency to form gallstones� a well- β-catenin and survivin proteins was found to �e posi- known risk factor in GBC. Cholelithiasis refers to the tively associated with progression of GBC� compared presence of gall stones in gall �ladder that might with chronic cholecystitis �CC� [�� �]. In a recent lead to cholecystitis �after cystic duct o�struction study �y Espinoza et al. [9]� authors demonstrated from cholelithiasis�. It is a strong risk factor for GBC increased mRNA and proteins levels of mucin 5B� and is found in �� to 9�% of patients with GBC [�]. Not car�onic anhydrase 9 and claudin 1� in GBCs and much work has �een done that explain the genetic proposed these as theranostics markers. a�normalities in advanced GBC� however several Most of advanced GBC patients are inopera�le and oncogenes and tumor suppressor genes like KRAS� treated with palliative intent chemotherapy. By now BRAF� EGFR� CDKN2A and TP53 have �een found we know that there is definite �enefit of chemotherapy to �e mutated in GBC and cholangiocarcinoma [3� in advanced GBC� over �est supportive care [1�]. �]. Study of differentially expressed genes using �NA However� we still don’t know if there are certain genes/ or proteins that might provide sensitivity� resistance Submitted: April 20, 2020. or predict response to chemotherapy in GBC. That’s why *Correspondence: E-mail: [email protected] it will �e helpful if such markers can �e identified. Hence� Abbreviations used: CC — chronic cholecystitis; GBC — gall the present study was planned to generate information bladder cancer; GO — Gene Ontology; RIN — RNA integrity pertaining to gene expression profile of advanced GBC. number. 278 Experimental Oncology ��� 277–284, ���� ��ecem�er� MATERIALS AND METHODS set to 1.� and quantile normalization was implement- Sample collection. This study was conducted ed. Unpaired t-test was executed to define the genes at All India Institute of Medical Sciences� New �elhi differentially expressed �etween the two groups; after approval �y the Institute ethics committee �Ref a significance level of p < �.�5 and a fold change cut IEC/NP-��5/��1��. Period of study was from March off ± � was taken into account. Gene Ontology �GO� ��1� to Fe�ruary ��1�. A total of 1� samples of GBC tool and pathway analysis tool of gene spring were and 6 samples of CC were collected. The advanced used to predict �iological function and pathways rep- stage GBC tissues were o�tained �y core �iopsy and resented �y the differentially regulated genes [1�]. gall �ladder tissues for control were o�tained from the Immunohistochemical validation of differen- cholecystectomy specimens. Samples were collected tially expressed genes. The archival formalin-fixed� in RNAlater �Thermo Fisher Scientific� USA� and then paraffin-em�edded tissues �locks of gall �ladder pa- they were SNAP frozen using liquid nitrogen and stored tients were cut into 5 µm sections. After routine deparaf- in Trizol reagent �Invitrogen� USA� at ��� °C till further finization and rehydration steps� exogenous peroxidase processing. activity was �locked using �.1% of H�O� in methanol for RNA isolation. Total RNA was isolated from all �� min� followed �y citric acid �1� mM� �uffer antigen the samples using the mirVanaTMmiRNA isolation kit retrieval step in microwave. After �locking� slides were �Am�ion� USA�. RNA quality and quantity were checked incu�ated with primary anti�odies i.e. respectively using Nano�rop-1��� spectrophotometer and Agilent at 4 °C overnight [Primary anti�odies: MCM� �sc-2831�; �1�� Bioanalyzer� using an Agilent RNA 6��� Nano dilution: 1:1���� TPX� �sc-2715��; dilution: 1:1��� and Kit. Only samples with RNA integrity num�er �RIN� C�C�5 �sc-55569; dilution: 1:200). All primary anti- value ≥ �.� were processed for gene expression stud- �odies were procured from Santa Cruz Biotechnology ies� and these included1� GBC samples out of 1� GBC Inc. �Texas� USA�]. After O/N incu�ations� slides were and � control out of 6 samples of CC. washed and treated with CRF-Anti-Polyvalent HRP cRNA microarray and hybridization. �ou�le- Polymer Kit �SkyTek la�oratories� UT� USA� at room stranded c�NA was generated from ��� ng total RNA temperature for 6� min� followed �y developing with di- using the low input quick amp la�elling kit �Agilent amino�enzidine �availa�le with kit�. Finally� the sections Technologies� USA�� T� primer� dNTPs and Affinityscript were counterstained with hematoxylin and mounted. RNase �lock. In vitro transcription of c�NA to cRNA was Microscopic scoring. The percentage of the performed using T� RNA polymerase and NTP mix and immunostained tumor cells was determined semi- la�elled with Cyanine3 using Cy3-CTP. The la�elled quantitatively �y assessing the whole section and cRNA was purified according to manufacturer’s protocol classified into � groups: � �< 1�% positive cells�; using RNAeasy extraction kit �Qiagen� Germany�. The 1 �11���% positive cells�; � ��1���% positive cells�; efficiency of cRNA synthesis and dye incorporation was 3 ��1�6�% positive cells�; � �61���% positive cells� measured using Nano�rop-1��� spectrophotometer. and 5 �> 80% positive cells�. The intensity of staining These values were then used to calculate specific activity was further graded as 1 �a�sent/very faint�� � �strong� of Cy-3 using the following formula: Conc. of Cy3/ and 3 �very strong�. To calculate H score� scores from Conc. of cRNA X 1��� and expressed as pmol Cy3 per each section were multiplied together and a total score μg of cRNA. For hy�ridization� 6�� ng of Cy-3 la�eled greater than � was designated as a positive result. cRNA was mixed with 1� × blocking agent and fragmen- Statistical analysis. Pearson χ� or Fisher’s exact tation �uffer and incu�ated at 6� °C for exactly 3� min test was used to examine the association �etween the to fragment RNA followed �y addition of hy�ridization proteins. A p-value of less than �.�5 was considered �uffer to stop further fragmentation. The la�eled cRNA statistically significant. Statistical analysis was per- mixture was then applied to a microarray slide ��X6�K formed using IBM SPSS ��.� version. format; Agilent Technologies� USA�� assem�led in a hy- RESULTS �ridization cham�er fitted with a gasket slide and incu- Of the 1� patients of unresecta�le advanced and �ated for 1� h in a hy�ridization oven at 65 °C and 1� rpm.